≟ࡢ㑇ఏᛶ⑌ᝈ࡟࠾ࡅࡿཎᅉ㑇ఏᏊゎᯒࡢࡓࡵࡢ㑇ఏᏊ㸦'1$㸧
ࣂࣥࢡᣐⅬᙧᡂᖹᡂ  ᖺᗘ㹼ᖹᡂ  ᖺᗘ⚾❧኱Ꮫᡓ␎ⓗ◊✲ᇶ┙ᙧᡂᨭ᥼஦ᴗ
◊✲ᡂᯝሗ࿌᭩ᖹᡂ  ᖺ  ᭶Ꮫᰯἲேྡ 㯞ᕸ⋇་Ꮫᅬ
኱Ꮫྡ

㯞ᕸ኱Ꮫ

◊✲⤌⧊ྡ 㯞ᕸ኱Ꮫ⋇་Ꮫ㒊
◊✲௦⾲⪅

㜰ཱྀ 㞞ᘯ

ᖹᡂ  ᖺᗘ㹼ᖹᡂ  ᖺᗘ⚾❧኱Ꮫᡓ␎ⓗ◊✲ᇶ┙ᙧᡂᨭ᥼஦ᴗ
◊✲ᡂᯝሗ࿌᭩
◊✲ࣉࣟࢪ࢙ࢡࢺྡ㸸≟ࡢ㑇ఏᛶ⑌ᝈ࡟࠾ࡅࡿཎᅉ㑇ఏᏊゎᯒࡢࡓࡵࡢ㑇ఏᏊ㸦'1$㸧ࣂࣥࢡᣐⅬᙧᡂ┠ḟ㸸◊✲ࣉࣟࢪ࢙ࢡࢺࡢᴫせ◊✲ᡂᯝሗ࿌᭩ᴫせ◊✲ᡂᯝሗ࿌
≟ࡢ㑇ఏᛶ⑌ᝈ࡟࠾ࡅࡿ '1$ ཰㞟࣭⢭〇࣭౪⤥࠾ࡼࡧ⑌ᝈ᝟ሗࡢࢹ࣮ࢱ࣮࣋ࢫ໬㸦㜰ཱྀ㞞ᘯ㸧≟ࡢ⥳ෆ㞀࠾ࡼࡧⓑෆ㞀ࡢ㑇ఏᏊゎᯒ㸦༳∾ಙ⾜㸧





≟࡟࠾ࡅࡿࣄࢺ࡜ࡢ㐺ྜᛶ࡟㛵ࡍࡿ㑇ఏᏊゎᯒ ⳥Ỉ೺ྐ㸧





≟ࡢ⭘⒆ቯṚᅉᏊ 71)Ș 㑇ఏᏊ࡟㛵ࡍࡿ◊✲㸦ஂᮎṇᬕ㸧





≟ࡢ࢔ࣟ࣌ࢩ࢔;㸦࣏࣓ࣛࢽ࢔ࣥ⬺ẟ⑕㸧ࡢ㑇ఏᏊゎᯒ㸦ᮧୖ㈼㸧

≟ࡢ࡚ࢇ࠿ࢇࡢ㑇ఏᏊゎᯒ㸦㰻⸨ᘺ௦Ꮚ㸧





≟ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࡢ㑇ఏᏊゎᯒ㸦ᮧୖ⿱ಙ㸧





≟ࡢ ,/ 㑇ఏᏊࡢゎᯒ㸦⁪ἑ㐩ஓ㸧୰㛫ሗ࿌఍࣭㐍ᤖ≧ἣホ౯᭩

◊✲ࣉࣟࢪ࢙ࢡࢺࡢᴫせ≟ࡢ㑇ఏᛶ⑌ᝈ࡟࠾ࡅࡿཎᅉ㑇ఏᏊゎᯒࡢࡓࡵࡢ㑇ఏᏊ㸦'1$㸧ࣂࣥࢡᣐⅬᙧᡂ◊✲௦⾲⪅  㜰ཱྀ㞞ᘯ≟ࡶே࡜ྠᵝ࡟ከࡃࡢ㑇ఏᛶ⑌ᝈࡀ࠶ࡾࠊே࡜⑓ែࡀఝ࡚࠸ࡿ⑌ᝈࡶከࡃᏑᅾࡍࡿࠋ
ᮏ◊✲࡟࠾࠸࡚≟ࡢ㑇ఏᏊ '1$ ㈨※ࡢά⏝࡜ಖᏑࡢࡓࡵ࡟ࠊ⋇་⣔኱Ꮫ㝃ᒓື≀⑓㝔➼ࡢ⮫ᗋ་ࢆ୰ᚰ࡟㑇ఏ㛵㐃⑌ᝈࢆྵࡴ㠀ឤᰁᛶ⑌ᝈ≟ࡸ㝜ᛶᑐ↷࡜࡞ࡿ೺ᖖ≟࠿ࡽࡢ'1$ ཰㞟ࢆ⾜࠺ࠋ཰㞟ࡉࢀࡓ '1$ ࢆ౑⏝ࡋ࡚≟ࡢ㑇ఏ㛵㐃⑌ᝈ࡟࠾ࡅࡿཎᅉ㑇ఏᏊࡢゎᯒࢆ⾜࠺ࡇ࡜ࢆᮏ◊✲ࡢ┠ⓗ࡜ࡋࡓࠋ
ᮏࣉࣟࢪ࢙ࢡࢺࡣ㸶⤌ࡢࢡ࣮ࣝࣉ࠿ࡽᵓᡂࡉࢀ࡚࠸ࡿࠋᣓᘼෆࡀࡑࢀࡒࢀࡢࢢ࣮ࣝ
ࣉ㛗࡛࠶ࡿࠋ  ≟ࡢ㑇ఏᛶ⑌ᝈ࡟࠾ࡅࡿ '1$ ཰㞟࣭⢭〇࣭౪⤥࠾ࡼࡧ⑌ᝈ᝟ሗࡢࢹ࣮
ࢱ࣮࣋ࢫ໬㸦㜰ཱྀ㞞ᘯ㸧  ≟ࡢ⥳ෆ㞀࠾ࡼࡧⓑෆ㞀ࡢ㑇ఏᏊゎᯒ㸦༳∾ಙ⾜㸧  ≟࡟࠾ࡅࡿࣄࢺ࡜ࡢ㐺ྜᛶ࡟㛵ࡍࡿ㑇ఏᏊゎᯒ㸦⳥Ỉ೺ྐ㸧  ≟ࡢ⭘⒆ቯṚᅉᏊ 71)Ș 㑇ఏᏊ࡟㛵ࡍࡿ◊✲㸦ஂᮎṇᬕ㸧  ≟ࡢ࢔ࣟ࣌ࢩ࢔;㸦࣏࣓ࣛࢽ࢔ࣥ⬺ẟ⑕㸧ࡢ㑇ఏᏊゎᯒ㸦ᮧୖ㈼㸧  ≟ࡢ࡚ࢇ࠿ࢇࡢ㑇ఏᏊゎᯒ㸦㰻⸨ᘺ௦Ꮚ㸧  ≟ࡢ࢔ࢺࣆ࣮
ᛶ⓶⭵⅖ࡢ㑇ఏᏊゎᯒ㸦ᮧୖ⿱ಙ㸧ᖹᡂ  ᖺᗘࡲ࡛ࡣᕝཎ஭᫴ᖹㅮᖌࡀ◊✲ࢡ࣮ࣝࣉ㛗࡛࠶ࡗࡓࡀ  ᖺᗘ࠿ࡽᮧୖ⿱ಙຓᩍ࡟ኚ᭦ࡋࡓࠋ  ≟ࡢ ,/ 㑇ఏᏊࡢゎᯒ㸦⁪ἑ㐩ஓ㸧࡛࠶ࡿࠋ཰㞟ࡉࢀࡓ '1$ ᳨యࡣ  ࡢࢢ࣮ࣝࣉ࡛⢭〇࣭⟶⌮ࡉࢀ࡚ࠊࡇࡢࣉࣟࢪ
࢙ࢡࢺࡢ◊✲⪅࡛࠶ࡿྛࢢ࣮ࣝࣉ࡟ᥦ౪ࡉࢀ࡚㑇ఏᏊゎᯒࢆ⾜ࡗࡓࠋࡲࡓࠊᖹᡂ  ᖺ࠿ࡽ◊✲ࢡ࣮ࣝࣉ௨እࡢ௚ࡢ◊✲⪅࡬ࡶ '1$ ࢆ౪⤥ࡋࠊඹྠ◊✲ࢆ⾜ࡗ࡚࠸ࡿࠋ
ᾏእ࡟࠾ࡅࡿ≟ࡢ '1$ ࣂࣥࢡ࡜ࡋ࡚ࡣࠊ⡿ᅜ࡛ࡣ࣐ࢧࢳ࣮ࣗࢭࢵࢶᕤ⛉኱Ꮫ࡜ࣁࣂ࣮
ࢻ኱Ꮫࡢ %URDG,QVWLWXWH ࡜⡿ᅜᅜ❧⾨⏕◊✲ᡤ 1,+ ࡟ࡼࡿ≟ࢤࣀ࣒ࣉࣜࢪ࢙ࢡࢺࠊ
࣮ࣚࣟࢵࣃ࡛ࡣ㸯㸰࠿ᅜࡢ⋇་኱Ꮫ࡟ࡼࡿ /XSD ࣉࣟࢪ࢙ࢡࢺࡀ᭷ྡ࡛࠶ࡿࠋ᪥ᮏ࡟࠾
࠸࡚ࡣ≟ࡢ㑇ఏ㛵㐃⑌ᝈࡢ◊✲ࢆ⾜ࡗ࡚࠸ࡿ◊✲ࡣᏑᅾࡍࡿࡀࠊࡑࢀࡽࡢ◊✲⪅ࡀ㑇ఏ㛵㐃⑌ᝈࡢ '1$ ᳨యࢆඹ᭷ࡍࡿࡼ࠺࡞㑇ఏᏊࣂࣥࢡࡣ࡞࠿ࡗࡓࠋᮏࣉࣟࢪ࢙ࢡࢺ࡟ࡼࡾࠊ

௒ᚋࠊ≟ࡢ㑇ఏ㛵㐃⑌ᝈࢆྵࡴ㠀ឤᰁᛶ⑌ᝈࡢ '1$ ࡸ୍⯡㞟ᅋ≟ࡢ '1$ ࡞࡝ࡀඹ᭷࡛ࡁࠊ
᪥ᮏࡢ≟ࡢ㑇ఏ㛵㐃⑌ᝈࡢཎᅉ㑇ఏᏊࡢゎᯒࡢࡓࡵࡢ᳨యᥦ౪ࡢ୰ᚰⓗ࡞ᙺ๭ࢆᢸ࠺
ࡇ࡜ࡀ࡛ࡁࡿ࡜⪃࠼ࡽࢀࡿࠋ

1ᖹᡂ䠎䠏ᖺᗘ䡚ᖹᡂ䠎䠓ᖺᗘ䛂⚾❧኱Ꮫᡓ␎ⓗ◊✲ᇶ┙ᙧᡂᨭ᥼஦ᴗ䛃㻌◊✲ᡂᯝሗ࿌᭩ᴫせ㻌䠍㻌 Ꮫᰯἲேྡ㻌 㻌 㯞ᕸ⋇་Ꮫᅬ㻌 㻌㻌 㻌 䠎㻌 ኱Ꮫྡ㻌 㻌 㯞ᕸ኱Ꮫ㻌 㻌䠏㻌 ◊✲⤌⧊ྡ㻌 㻌 㯞ᕸ኱Ꮫ⋇་Ꮫ㒊㻌 㻌䠐㻌 䝥䝻䝆䜵䜽䝖ᡤᅾᆅ㻌 ⚄ዉᕝ┴┦ᶍཎᕷ୰ኸ༊ῡ㔝㎶ 㻝㻙㻝㻣㻙㻣㻝㻌 㻌䠑㻌 ◊✲䝥䝻䝆䜵䜽䝖ྡ㻌 ≟䛾㑇ఏᛶ⑌ᝈ䛻䛚䛡䜛ཎᅉ㑇ఏᏊゎᯒ䛾䛯䜑䛾㑇ఏᏊ䠄㻰㻺㻭䠅㻌䝞䞁䜽ᣐⅬᙧᡂ㻌䠒㻌 ◊✲ほⅬ㻌 㻌 ◊✲ᣐⅬ䜢ᙧᡂ䛩䜛◊✲㻌䠓㻌 ◊✲௦⾲⪅㻌◊✲௦⾲⪅ྡ㻌ᡤᒓ㒊ᒁྡ㻌⫋ྡ㻌㜰ཱྀ㻌 㞞ᘯ㻌

⋇་Ꮫ㒊㻌

ᩍᤵ㻌

䠔㻌 䝥䝻䝆䜵䜽䝖ཧຍ◊✲⪅ᩘ㻌 㻌 䠍䠍㻌 ྡ㻌䠕㻌 ヱᙜᑂᰝ༊ศ㻌 㻌 㻌 㻌 ⌮ᕤ䞉᝟ሗ㻌⏕≀䞉་ṑ㻌䠍䠌㻌 ◊✲䝥䝻䝆䜵䜽䝖䛻ཧຍ䛩䜛୺䛺◊✲⪅㻌
◊✲⪅ྡ㻌

㜰ཱྀ㞞ᘯ㻌

༳∾ಙ⾜㻌
⳥Ỉ೺ྐ㻌
ஂᮎṇᬕ㻌

ᮧୖ㈼㻌
㰻⸨ᘺ௦Ꮚ㻌
ᮧୖ⿱ಙ㻌ᡤᒓ䞉⫋ྡ㻌

ேᩥ䞉♫఍㻌

䝥䝻䝆䜵䜽䝖䛷䛾◊✲ㄢ㢟㻌≟䛾㑇ఏᛶ⑌ᝈ䛻䛚䛡䜛ᚤ⏕≀➨㻰㻺㻭 ཰㞟䞉⢭〇䞉౪⤥䛚䜘䜃୍䞉ᩍᤵ㻌⑌ᝈ᝟ሗ䛾䝕䞊䝍䝧䞊䝇໬㻌㝃ᒓື≀≟䛾⥳ෆ㞀䛚䜘䜃ⓑ ෆ 㞀 䛾⑓㝔䞉෸ᩍ㑇ఏᏊゎᯒ㻌ᤵ㻌క ౶ ື ≀ ≟䛻䛚䛡䜛䝠䝖䛸䛾㐺ྜᛶ䛻Ꮫ䞉ᩍᤵ㻌㛵䛩䜛㑇ఏᏊゎᯒ㻌䝥䝻䝆䜵䜽䝖䛷䛾ᙺ๭㻌≟䛾㑇ఏᛶ⑌ᝈ䛾 㻰㻺㻭⟶⌮䛚䜘䜃᝟ሗ䛾䝕䞊䝍䝧䞊䝇໬║⑌ᝈ⑕౛ 㻰㻺㻭 䝃䞁䝥䝹䛾཰㞟䛚䜘䜃◊✲䛾⥲ᣓ≟䛻䛚䛡䜛䝠䝖䛸䛾㐺ྜᛶ䛻㛵䛩䜛㑇ఏᏊゎᯒ㻌≟䛾⭘⒆ቯṚᅉᏊෆ ⛉ ➨ 䠎 䞉 ≟䛾⭘⒆ቯṚᅉᏊ㻔㼀㻺㻲㻙䃐㻕㻔㼀㻺㻲㻙䃐㻕㑇ఏᏊ䛻㛵䛩෸ᩍᤵ㻌㑇ఏᏊ䛻㛵䛩䜛◊✲㻌䜛◊✲㻌≟䛾䜰䝻䝨䝅䜰 㼄䠄䝫䝯䝷ศ Ꮚ ⏕ ≀ ≟䛾䜰䝻䝨䝅䜰㻌 㼄䠄䝫䝯䝷䝙䝙䜰䞁⬺ẟ⑕䠅䛾ཎᅉ㑇Ꮫ䞉ᩍᤵ㻌䜰䞁⬺ẟ⑕䠅䛾㑇ఏᏊゎᯒ
ఏᏊྠᐃ
እ⛉➨䠎䞉
≟䛾䛶䜣䛛䜣䛾ཎᅉ㑇≟䛾䛶䜣䛛䜣䛾㑇ఏᏊゎᯒ㻌
෸ᩍᤵ㻌
ఏᏊྠᐃ㻌
≟䛾䜰䝖䝢䞊ᛶ⓶⭵⅖䛾㑇
⾨⏕➨䠎䞉
≟ 䛾 䜰 䝖 䝢 䞊 ᛶ⓶ ⭵ ⅖
ఏᏊゎᯒ㻌
ຓᩍ㻌
䛾㑇ఏᏊゎᯒ㻌2⁪ἑ㐩ஓ㻌ື≀ᕤᏛ䞉≟䛾 㻵㻸㻙㻝㻟 㑇ఏᏊ䛾ゎᯒ㻌ᩍᤵ㻌≟䛾 㻵㻸㻙㻝㻟 㑇ఏᏊ䛾ゎᯒ㻌䠄ඹྠ◊✲ᶵ㛵➼䠅㻌

㻌

㻌

㻌

≟䛾㑇ఏᏊゎᯒἲ䛾ຓゝ㻌≟䛾㑇ఏᏊゎᯒἲ䛾ຓゝ㻌ஂಖ඘᫂㻌 㻌኱᳃ၨኴ㑻䢢኱࿴㻌 ಟ㻌

⌮໬Ꮫ◊✲
ᡤ⤫ྜ⏕࿨
་⛉Ꮫ◊✲
䝉䞁䝍䞊๪䝉
䞁䝍䞊㛗㻌
ᮾி㎰ᕤ኱
⋇་ศᏊ⑓
ែ἞⒪Ꮫ䞉ㅮᖌ㻌≟䛾 㻰㻺㻭 ཰㞟䛸䝥䝻䝆䜵䜽䝖 ≟䛾 㻰㻺㻭 ཰㞟䛸䝥䝻䝆䜈䛾ຓゝ㻌䜵䜽䝖䜈䛾ຓゝ㻌㮵 ඣ ᓥ ኱Ꮫ 䞉 ⋇ ་ ⮫ ≟䛾 㻰㻺㻭 ཰㞟䛸䝥䝻䝆䜵䜽䝖ᗋ ⑓ ⌮ 䞉 ᩍ 䜈䛾ຓゝ㻌ᤵ㻌≟䛾 㻰㻺㻭 ཰㞟䛸䝥䝻䝆䜵䜽䝖䜈䛾ຓゝ㻌㻌ᪧ㻌䢈䢛䢗䡸䢚䡦䡴䢀䛷䛾◊✲ㄢ㢟㻌ᡤᒓ䞉⫋ྡ㻌

◊✲⪅Ặྡ㻌

≟䛾䜰䝺䝹䜼䞊⑌ᝈ䛾 ᑠ ື ≀ ⮫ ᗋ 䞉 ຓᕝཎ஭᫴ᖹ㻌
㑇ఏᏊゎᯒ㻌
ᩍ㻌䠄ኚ᭦䛾᫬ᮇ䠖ᖹᡂ䠎䠑ᖺ㻌 䠐᭶㻌 䠍᪥䠅㻌㻌㻌᪂㻌ኚ᭦๓䛾ᡤᒓ䞉⫋ྡ㻌ኚ᭦䠄ᑵ௵䠅ᚋ䛾ᡤᒓ䞉⫋ྡ㻌≟ࡢ࢔ࣞࣝࢠ࣮⑌
⾨⏕➨㸰࣭ຓᩍ
ᝈࡢ㑇ఏᏊゎᯒ䝥䝻䝆䜵䜽䝖䛷䛾ᙺ๭㻌≟䛾䜰䝺䝹䜼䞊⑌ᝈ䛾㑇ఏᏊゎᯒ㻌

◊✲⪅Ặྡ㻌

䝥䝻䝆䜵䜽䝖䛷䛾ᙺ๭㻌ᮧୖ⿱ಙ≟ࡢ࢔ࣞࣝࢠ࣮⑌ᝈ
ࡢ㑇ఏᏊゎᯒ㻌㻌ᪧ㻌䢈䢛䢗䡸䢚䡦䡴䢀䛷䛾◊✲ㄢ㢟㻌ᡤᒓ䞉⫋ྡ㻌

◊✲⪅Ặྡ㻌≟䛾 㻰㻺㻭 ཰㞟䛸㑇ఏ⑓ ᮾி㎰ᕤ኱⋇་ᒾᓮ฼㑻㻌䛾ண㜵䞉἞⒪ἲ䛾㛤Ⓨ㻌 ෆ⛉䞉ᩍᤵ㻌䠄ኚ᭦䛾᫬ᮇ䠖ᖹᡂ䠎䠑ᖺ㻌 䠐᭶㻌 䠍᪥䠅㻌㻌㻌᪂㻌ኚ᭦๓䛾ᡤᒓ䞉⫋ྡ㻌ኚ᭦䠄ᑵ௵䠅ᚋ䛾ᡤᒓ䞉⫋ྡ㻌◊✲⪅Ặྡ㻌

≟ࡢ '1$ ཰㞟◊✲ᮾி㎰ᕤ኱⋇་ศᏊ⑓ែ
ࣉࣟࢪ࢙ࢡࢺ࡬ࡢ
኱᳃ၨኴ㑻
἞⒪Ꮫ࣭ㅮᖌຓゝ㻌3䝥䝻䝆䜵䜽䝖䛷䛾ᙺ๭㻌≟䛾 㻰㻺㻭 ཰㞟䛸㑇ఏ⑓䛾ண㜵䞉἞⒪ἲ䛾㛤Ⓨ㻌䝥䝻䝆䜵䜽䝖䛷䛾ᙺ๭㻌≟ࡢ '1$ ཰㞟࡜ࣉࣟࢪ࢙ࢡࢺ࡬ࡢຓゝᪧ㻌䢈䢛䢗䡸䢚䡦䡴䢀䛷䛾◊✲ㄢ㢟㻌ᡤᒓ䞉⫋ྡ㻌

◊✲⪅Ặྡ㻌

㑇ఏᛶ⑌ᝈ䛾᪤▱䛾 ᮾி኱Ꮫ䞉⋇་⑌ ᝈ ཎ ᅉ 㑇 ఏ Ꮚ 㢖 ᗘ ⮫ ᗋ ⑓ ⌮ Ꮫ 䞉 ຓ ⋢ཎᬛྐ㻌䛾ㄪᰝ㻌ᩍ㻌䠄ኚ᭦䛾᫬ᮇ䠖ᖹᡂ䠎䠒ᖺ㻌 䠐㻌 ᭶㻌 䠍㻌 ᪥䠅㻌㻌㻌᪂㻌ኚ᭦๓䛾ᡤᒓ䞉⫋ྡ㻌ኚ᭦䠄ᑵ௵䠅ᚋ䛾ᡤᒓ䞉⫋ྡ㻌◊✲⪅Ặྡ㻌≟䛾 㻰㻺㻭 ཰㞟◊✲㮵ඣᓥ኱Ꮫ䞉⋇་⮫ᗋ⑓䝥 䝻䝆 䜵 䜽䝖 䜈䛾 ຓ኱࿴㻌 ಟ㻌
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4

䝥䝻䝆䜵䜽䝖䛷䛾ᙺ๭㻌㑇ఏᛶ⑌ᝈ䛾᪤▱䛾⑌ᝈཎᅉ㑇ఏᏊ㢖ᗘ䛾ㄪᰝ㻌䝥䝻䝆䜵䜽䝖䛷䛾ᙺ๭㻌≟䛾 㻰㻺㻭 ཰㞟◊✲䝥䝻䝆䜵䜽䝖䜈䛾ຓゝ㻌䠍䠍㻌 ◊✲䛾ᴫせ䠄䈜㻌 㡯┠඲య䜢䠍䠌ᯛ௨ෆ䛷సᡂ䠅㻌䠄䠍䠅◊✲䝥䝻䝆䜵䜽䝖䛾┠ⓗ䞉ព⩏ཬ䜃ィ⏬䛾ᴫせ≟䜒ே䛸ྠᵝ䛻ከ䛟䛾㑇ఏᛶ⑌ᝈ䛜䛒䜚䚸ே䛸⑓ែ䛜ఝ䛶䛔䜛⑌ᝈ䜒ከ䛟Ꮡᅾ䛩䜛䚹ᮏ◊✲䛻䛚䛔䛶≟䛾㑇ఏᏊ㻔㻰㻺㻭㻕㈨※䛾ά⏝䛸ಖᏑ䛾䛯䜑䛻䚸⋇་⣔኱Ꮫ㝃ᒓື≀⑓㝔➼䛾⮫ᗋ་䜢୰ᚰ䛻ྛ✀㑇ఏ㛵㐃⑌ᝈ䜢ᣢ䛴≟䛾 㻰㻺㻭 ཰㞟䜢⾜䛖䚹཰㞟䛥䜜䛯 㻰㻺㻭 䜢౑⏝䛧䛶≟䛾㑇ఏ㛵㐃⑌ᝈ䛻䛚䛡䜛ཎᅉ㑇ఏᏊ䛾ゎᯒ䜢⾜䛖䛣䛸䜢ᮏ◊✲䛾┠ⓗ䛸䛩䜛䚹㻌ព⩏䛸䛧䛶䛿䚸኱Ꮫ䛾⋇་⑓㝔䛻䛚䛔䛶⮫ᗋᏛⓗ䛻ಙ㢗ᛶ䛾㧗䛔 㻰㻺㻭 ᳨య䜢཰㞟䛩䜛䛣䛸䛜䛷䛝䚸ᑗ᮶䛾≟䛾㑇ఏ㛵㐃⑌ᝈ◊✲䜢᥎㐍䛩䜛䛯䜑䛾 㻰㻺㻭 䝞䞁䜽䛾ᇶ┙䛜ᙧᡂ䛩䜛䛣䛸䛜䛷䛝䜛䚹䛥䜙䛻䛭䛾◊✲ᡂᯝ䛿≟䛸ྠ䛨䜘䛖䛺㑇ఏ㛵㐃⑌ᝈ䛜Ꮡᅾ䛩䜛ே䛾㑇ఏ㛵㐃⑌ᝈ䛾ゎ᫂䛻䜒ᙺ❧䛴䛸⪃䛘䜙䜜䜛䚹㻌㻌 ィ⏬䛾ᴫせ䛸䛧䛶ୖ㏙䛧䛯䜘䛖䛻≟䛾㑇ఏ㛵㐃⑌ᝈ䛻㛵䛩䜛⑕౛᝟ሗ䛸䝀䝜䝮 㻰㻺㻭 䛺䛹⮫ᗋ᳨య䜢䚸඲ᅜ⋇་⣔኱Ꮫ㝃ᒓື≀⑓㝔➼䛸㐃ᦠ䛧䛶཰㞟䞉ಖ⟶䛩䜛䚹䛥䜙䛻཰㞟䛥䜜䛯㻰㻺㻭 ᳨య䜢⏝䛔䛶䛣䜜䜎䛷䛻ሗ࿌䛥䜜䛶䛔䜛ே䜔䝬䜴䝇➼䛾㑇ఏ㛵㐃⑌ᝈ䛻㛵䛩䜛ཎᅉ㑇ఏᏊ䛾᝟ሗ䜢ඖ䛻 㻼㻯㻾 ἲ➼䜢⏝䛔䛶ゎᯒ䛩䜛䚹䜎䛯䚸䛣䜜䜎䛷䛻ཎᅉ㑇ఏᏊ᝟ሗ䛜䛺䛔⑌ᝈ䛻㛵䛧䛶䛿ྛ⑌ᝈ䛻䛚䛔䛶ྠ≟✀䛾⨯ᝈ≟⩌䛸೺ᖖ≟⩌䛻䛚䛔䛶䚸⥙⨶ⓗ䛺 㻿㻺㻼䠄୍ሷᇶከᆺ䠖㑇ఏᏊ㓄ิ୰䛻䠍䛴䛾 㻰㻺㻭 䛜ኚ␗䛧䛯ከᵝᛶ䛜ㄆ䜑䜙䜜䚸䛭䛾ኚ␗䛜㞟ᅋෆ䛷 㻝㻑௨ୖ䛾㢖ᗘ䛷ぢ䜙䜜䜛䜒䛾㻕ゎᯒ䜔ḟୡ௦㑇ఏᏊゎᯒ⿦⨨䛻䜘䜛ゎᯒ䛺䛹䛷ཎᅉ㑇ఏᏊ䛾ྠᐃ䜢ᐇ᪋䛩䜛䚹㑇ఏᏊゎᯒ䛻䜘䛳䛶᫂䜙䛛䛸䛺䛳䛯ཎᅉ㑇ఏᏊ䛾ᶵ⬟ゎᯒ䜢⾜䛔䚸⑌ᝈ䛾Ⓨ⏕ᶵᗎ䜢ゎ᫂䛩䜛䚹䛭䛾Ⓨ⏕ᶵᗎ䜢ඖ䛻⑌ᝈ䛾ண㜵ἲ䛚䜘䜃἞⒪ἲ䛾᳨ウ䜢⾜䛺䛔䚸䛭䛾⑌ᝈ䛾ண㜵ἲ䞉἞⒪ἲ䜢㛤Ⓨ䛩䜛䚹㻌䠄䠎䠅◊✲⤌⧊◊✲௦⾲⪅ࡢᙺ๭㸸◊✲௦⾲⪅ࡢ㜰ཱྀࡣࠊ㯞ᕸ኱Ꮫ࠾ࡼࡧ௚኱Ꮫࡸ⋇་⑓㝔࡟࠾ࡅࡿ≟ࡢ㑇ఏ㛵㐃⑌ᝈ⑕౛ࢆ㞟ࡵࠊ㣫࠸୺ࡢᢎㅙᚋࠊࡑࡢ⑓Ẽࡢ≟࠿ࡽ⾑Ύ࡜'1$ࢆ᥇ྲྀ࣭ಖ
Ꮡࡍࡿࠋࡲࡓࠊࡑࡢẚ㍑◊✲ࡢࡓࡵ࡟ࠊ୍⯡≟ࡢ'1$ࢆ᥇ྲྀ࣭ಖᏑࡍࡿࠋ◊✲⪅ࡢせᮃ࡟ἢࡗ࡚ᚲせ࡞'1$ࢆ౪⤥ࡍࡿࠋࡲࡓࠊ㑇ఏᏊゎᯒ࡟ᚲせ࡞ᢏ⾡ࡸ᝟ሗࢆྛ◊✲⪅࡟౪⤥ࡋࠊศᏊ⏕Ꮫⓗᡭἲ࡟័ࢀ࡞࠸⮫ᗋ⋇་ᖌ࡟ᑐࡋ࡚ࡣᐇ㝿ࡢᐇ㦂࣭ゎᯒࢆᡭఏ࠺ࠋࡲ
ࡓࠊྛ◊✲⪅㛫ࡢ᳨యࡸ᝟ሗࢆ௰௓ࡋ࡚◊✲⪅㛫ࡢ㐃ᦠࡀࢫ࣒࣮ࢬ࡟⾜࠺ࡼ࠺࡟ࡍࡿࠋ
ྛ◊✲⪅ࡢᙺ๭ศᢸࡸ㈐௵యไࡢ᫂☜ࡉ㸸◊✲⪅ࡈ࡜࡟◊✲ࢸ࣮࣐ࢆᣢࡕࠊᖺ㸰ᅇ◊✲⤖ᯝࡢⓎ⾲఍ࢆ⾜࠺ࠋࡑࡢ఍㆟࡟ࡣୖグࡢእ㒊ࡢඹྠ◊✲ᶵ㛵ࡢඛ⏕᪉ࡶཧຍࡋࠊホ౯࣭ࢥ࣓ࣥࢺࢆ࠸ࡓࡔ࠸ࡓࠋࡑࡢ㐍ᤖ≧ἣࢆ◊✲௦⾲⪅ࡀุ᩿ࡋࠊࢫ࣒࣮ࢬ࡟◊✲ࡀ㐍ࡴࡼ࠺࡟ຓゝ࣭ࢧ࣏࣮ࢺࢆ⾜ࡗࡓࠋ◊✲ࡢ㐍⾜࡟㛵ࡋ࡚ࡣᢸᙜࡢ◊✲⪅࡜◊✲௦⾲⪅ࡀ㈐௵ࢆᣢࡗ࡚⾜࠸ࠊ◊✲㈝ࡢᨭฟࡣྛ◊✲⪅ࡢ㐍⾜࡟ᛂࡌ࡚◊✲௦⾲⪅ࡀᨭ⤥ࡋࡓࠋ◊✲ࣉࣟࢪ࢙ࢡࢺ࡟ཧຍࡍࡿ◊✲⪅ࡢேᩘ㸸◊✲ࣉࣟࢪ࢙ࢡࢺ࡟ཧຍࡋ࡚࠸ࡿ୺࡞◊✲⪅ࡣ㸯㸮࡟グ㍕ࡋࡓ࡜࠾ࡾࠊྡ࡛࠶ࡿࠋࡑࡢ௚ࡢ◊✲༠ຊ⪅ࡀྡ㸦ෆヂ࡜ࡋ࡚㯞ᕸ኱Ꮫྡࠊᮾி኱Ꮫྡࠊி㒔኱Ꮫ㸱ྡࠊᶓ὾ᕷ❧኱Ꮫ㸰ྡࠊᡂ⫱་⒪◊✲ࢭࣥࢱ࣮ྡࠊ⛉Ꮫ㆙ᐹ◊✲ᡤ㸯ྡࠊ⌮໬Ꮫ◊✲ᡤྡࠊ࣓ࢽࢥࣥ㸯ྡࠊ࢝࣍ࢸࢡࣀ㸯ྡࠊ᳨デࢭࣥࢱ࣮࣑࢟ࣕࢵࢡ㸰ྡࠊΏ㎶ື≀⑓㝔㸯ྡ㸧࡛ྜィྡ࡛࠶ࡿࠋ
኱Ꮫ㝔⏕࣭3'㸪5$ࡢேᩘ࣭ά⏝≧ἣ㸸኱Ꮫ㝔ྡࠊ3'ྡࠊ5$ྡࡀཧຍࡋ࡚࠸ࡿࠋ
◊✲ࢳ࣮࣒㛫ࡢ㐃ᦠ≧ἣ㸸◊✲ࢳ࣮࣒ෆ࡛ࡢ㐃ᦠࡣ࠺ࡲࡃᶵ⬟ࡋ࡚࠸ࡿࠋ
࣭ஂᮎࡣ⁪ἑ◊✲ᐊ࡟ '1$ ኚ␗㒊ศࡢ '1$ ゎᯒࢆ౫㢗ࡋࡓࠋ
࣭ᮧୖ㈼ࡣᑠື≀⮫ᗋᡤᒓࡢᕝཎ஭ඛ⏕࡜༠ຊࡋ࡚⬺ẟ⑕ࡢ≟࠿ࡽࡢ⓶⭵⤌⧊ࢧࣥࣉࣝ
᥇㞟ࢆ౫㢗ࡋ࡚࠸ࡿࠋࡲࡓࠊᑠື≀⮫ᗋᡤᒓࡢ༳∾࡜༠ຊࡋ࡚ࢩ࣮ࢬ࣮≟࡟ぢࡽࢀࡿཎ5ᅉ୙᫂ࡢඛኳᛶ⥙⭷๤㞳ࡢ║⑌ᝈ࡟࠾ࡅࡿ㑇ఏᏊ␗ᖖࡢ᝟ሗ஺᥮ࢆࡋ࡚࠸ࡿࠋ⫧‶㛵㐃㑇ఏᏊከᆺゎᯒࡢࡓࡵ࡟ࠊ⳥Ỉ࠿ࡽ≉ᐃࡢ≟✀ࡢ '1$ ࢧࣥࣉࣝࡢᥦ౪ࢆ౫㢗ࡋࡓࠋ࣭㰻⸨ࡣ࡚ࢇ࠿ࢇࡢ㑇ఏᏊゎᯒ࡟࠾࠸࡚㜰ཱྀ࡟㑇ఏᏊゎᯒᢏ⾡ᣦᑟࢆ౫㢗ࡋࡓࠋ࣭⁪ἑࡣື≀ᕤᏛ◊✲ᐊᡤᒓࡢ⏣୰࿴᫂෸ᩍᤵ࡜ඹྠ࡛ࠊ㑇ఏᏊከᆺࡢゎᯒࢆ㐍ࡵࡓࠋࡉࡽ࡟ஂᮎ࡜ඹྠ࡛≟ࡢ⭘⒆ቯṚᅉᏊ 71)Ș 㑇ఏᏊࡢከᆺゎᯒ࡟ࡘ࠸ඹྠ࡛◊✲ࢆ㐍ࡵࡓࠋ≟ࡢ '1$ ᳨యࡣஂᮎࠊ㜰ཱྀ࠿ࡽ᳨యࡢ౪୚ࢆཷࡅࡓࠋ
ࡇࡢࡼ࠺࡟◊✲⌜ෆ࡛ '1$ ࡢ᳨యᮦᩱࢆ஫࠸࡟⼥㏻ࡋ࠶࠸ࠊᐇ㦂ᡭᢏࡸࢹ࣮ࢱゎᯒࡶ༠ຊࡋ࡚㐍ࡵࡓࠋ
◊✲ᨭ᥼యไ㸸ୖ㏙ࡋࡓࡼ࠺࡟◊✲⪅㛫࡛ᚓព࡞ศ㔝ࢆ⏕࠿ࡋ࡚┦⿵ⓗ࡟◊✲ࢆࢧ࣏࣮ࢺࡋ࡚࠸ࡿࠋᚲせ࡞ᶵჾ➼ࡣ◊✲௦⾲⪅࡜ศᢸ⪅ࡀヰࡋྜࡗ࡚㉎ධࢆࡋ࡚࠸ࡿࠋ
ඹྠ◊✲ᶵ㛵➼࡜ࡢ㐃ᦠ㸸⌮໬Ꮫ◊✲ᡤ䛾ஂಖඛ⏕䛿ே䛾㑇ఏ㛵㐃⑌ᝈ䛾㑇ఏᏊゎᯒ䛾ᑓ㛛ᐙ䛷䚸≟䛾㑇ఏ㛵㐃⑌ᝈ䛾ゎᯒ䛾ຓゝ䜢⾜䛳䛯䚹≟䛾ⓑෆ㞀䛾 㻿㻺㻼䠄୍ሷᇶከᆺ䠅ゎᯒ䛿ஂಖඛ⏕䛾༠ຊ䛷⌮◊䝆䜵䝛䝅䝇䛷⾜䛳䛯䚹ᮾி㎰ᕤ኱Ꮫ⋇་Ꮫ⛉䛾ᒾᓮඛ⏕䛿⋇་ෆ⛉ᩍᤵ䛷≟䛾㑇ఏ㛵㐃⑌ᝈ䛾 㻰㻺㻭 ᳨య䛾཰㞟䜔ゎᯒ䛾䜰䝗䝞䜲䝇䜢⾜䛳䛯䚹≉䛻≟䛾䜰䝺䝹䜼䞊⑌ᝈ䛾◊✲䛾ᶒጾ⪅䛷䛒䜛䚹ಶேⓗ㒔ྜ䛷ᖹᡂ 㻞㻡 ᖺ 㻟 ᭶䛻኱Ꮫ䜢㏥ᐁ䛥䜜䛯䛾䛷䚸ᖹᡂ 㻞㻡 ᖺᗘ䛛䜙ྠ䛨኱Ꮫ䛾኱᳃ඛ⏕䛻౫㢗䛧䛯䚹ᮾி኱Ꮫ⋇་Ꮫ⛉䛾⋢ཎඛ⏕䛿≟䛾㑇ఏ⑓⑌ᝈ䛾◊✲䜢⾜䛳䛶䛚䜚䚸≟䛾㑇ఏ㛵㐃⑌ᝈ䛾 㻰㻺㻭 ᳨య䛾཰㞟䜔ゎᯒ䛾䜰䝗䝞䜲䝇䜢౫㢗䛧䛯䚹ᖹᡂ 㻞㻢 ᖺ 㻠 ᭶ᮾி኱Ꮫ䛛䜙Ẹ㛫䛾⋇་⑓㝔䛻⛣䜙䜜䛯䛾䛷䚸ᖹᡂ 㻞㻢 ᖺᗘ䛛䜙㮵ඣᓥ኱Ꮫ⋇་Ꮫ⛉䛾኱࿴ඛ⏕䛻௦䜟䜚䜢䛚㢪䛔䛧䛯䚹኱࿴ඛ⏕䛿≟䞉⊧䛾㑇ఏ㛵㐃⑌ᝈ䛾◊✲䜢⾜䛳䛶䛚䜚䚸≟䛾㑇ఏ㛵㐃⑌ᝈ䛾 㻰㻺㻭 ᳨య䛾཰㞟䜔ゎᯒ䛾ຓゝ䜢⾜䛳䛯䚹ඹྠ◊✲ᶵ㛵௨እ䛸䛾ඹྠ◊✲䞉༳∾䛿║⑌ᝈ䛾ඹྠ◊✲⪅䛸䛧䛶ᶓ὾ᕷ❧኱Ꮫ䞉་Ꮫ㒊║⛉Ꮫᩍᐊ䛾Ỉᮌಙஂᩍᤵ䚸┠㯮᫂ㅮᖌ䛻ᮏ䝥䝻䝆䜵䜽䝖㛵㐃䛾䜰䝗䝞䜲䝇䜢౫㢗䛧䛶䛔䜛䚹䜎䛯䚸䝯䝙䝁䞁♫䛾௒ᏳṇᶞẶ䛻 㻰㻺㻭 ゎᯒ䜢౫㢗䛧䛶䛔䜛䚹䞉⳥Ỉ䛿ඹྠ◊✲⪅䛸䛧䛶ி㒔኱Ꮫ䛾⸨⏣࿴⏕ඛ⏕䚸ᮧᒣ⨾✑ඛ⏕䛛䜙䜸䜸䜹䝭䛾 㻰㻺㻭 䛾ᥦ౪䛻ຍ䛘䚸㑇ఏᏊゎᯒᢏ⾡ᣦᑟ䜢ཷ䛡䛶䛔䜛䚹ᅜ❧ᡂ⫱་⒪◊✲䝉䞁䝍䞊䛾῝ぢ┿ᕼඛ⏕䛛䜙䛿 㻰㻺㻭 䛾䝁䝢䞊䝘䞁䝞䞊ኚ␗䛾ゎᯒ䜢ඹྠ䛷ᐇ᪋䛧䛯䚹䜎䛯ᅜ❧ᡂ⫱་⒪◊✲䝉䞁䝍䞊䛾⛙೺୍㑻ඛ⏕䛸䛿ᰘ≟䛾ḟୡ௦ᆺ䝅䞊䜽䜶䞁䝃䞊䜢⏝䛔䛯⥙⨶ⓗ㑇ఏᏊゎᯒ䜢ᐇ᪋䛧䛯䚹⌮໬Ꮫ◊✲ᡤ䛾᱈ἑᖾ⚽ඛ⏕䛻䛿䚸⥙⨶ⓗ 㻿㻺㻼 ゎᯒ䛸⾜ື䛸䛾㛵㐃ゎᯒ䛻㛵䛧䛶䛾ຓゝ䜢౫㢗䛧䛯䚹
䞉ஂᮎ䛿≟䛾⭘⒆ቯṚᅉᏊ㻔㼀㻺㻲㻙䃐㻕㑇ఏᏊ䛻㛵䛩䜛◊✲䜢䜹䝩䝔䜽䝜䛾ඹྠ䛷⾜䛳䛶䛔䜛䚹䞉ᮧୖ㈼䛿Ꮫእඹྠ◊✲⪅䛸䛧䛶䛔䜛ி㒔኱Ꮫ኱Ꮫ㝔㎰Ꮫ◊✲⛉ᛂ⏝⏕≀Ꮫᑓᨷ䛾⯚ሙṇᖾඛ⏕䛻≟䛾⫧‶䜔⬡⫫⣽⬊ศ໬䛻㛵䛩䜛㑇ఏᏊⓎ⌧䛾᝟ሗᥦ౪䜔ᐇ㦂䛾༠ຊ䜢౫㢗䛧䛶䛔䜛䚹䜎䛯䚸⫧‶㛵㐃㑇ఏᏊከᆺゎᯒ䛾䛯䜑䛻䚸ᮾி኱Ꮫ⋇་Ꮫ⛉㎷ᮏඛ⏕䛛䜙≉ᐃ䛾≟✀䛾 㻰㻺㻭 䝃䞁䝥䝹䛾ᥦ౪䜢౫㢗䛧䛯䚹䞉㰻⸨䛿㯞ᕸ኱Ꮫ䠑ྡ䠄⚄⤒⛉◊ಟ⋇་ᖌ䠅䚸᳨デ䝉䞁䝍䞊䜻䝱䝭䝑䜽䚸Ώ㎶ື≀⑓㝔䛛䜙䚸᳨యᥦ౪䛸⮫ᗋ᝟ሗᥦ౪䜢౫㢗䛧䛯䚹䠄䠏䠅◊✲᪋タ䞉タഛ➼◊✲䛻ᚲせ䛺タഛ䛿䛣䛾ண⟬䛷㉎ධ䛧䛯䚹௨๓䛻㉎ධ䛧䛯タഛ䜒ᮏ◊✲䛻⏝䛔䛶䛔䜛䚹㻌ᖹᡂ䠎䠏ᖺᗘ䠖⺯ග䞉Ⓨග䝥䝺䞊䝖䝸䞊䝎㻌 㻝㻡㻢 ᫬㛫㻛䠍ᖺ䠄䠍ᅇ䛾౑⏝䛜䠎䠌ศ⛬ᗘ䠅㻌ᖹᡂ䠎䠐ᖺᗘ䠖䝸䜰䝹䝍䜲䝮 㻼㻯㻾 ⿦⨨㻌 㻝㻜㻡㻟 ᫬㛫㻛䠍ᖺ㻌ᖹᡂ䠎䠑ᖺᗘ䠖⺯ග㢧ᚤ㙾⿦⨨୍ᘧ㻌 㻝㻤㻜 ᫬㛫㻛䠍ᖺ㻌 㻌ᮏண⟬䛷㉎ධ䛧䛯௚䛾タഛ䛷୺䛺䜒䛾䠖෭༷㐲ᚰᶵ 㻝㻜㻜㻜 ᫬㛫㻛ᖺ䚸䝃䞊䝬䝹䝃䜲䜽䝷䞊㻔㻼㻯㻾6⿦⨨㻕㻞㻢㻜 ᫬㛫㻛ᖺ䚸䝯䝕䜱䜹䝹෭ⶶᗜ㻌 㻤㻣㻢㻜 ᫬㛫㻛䠍ᖺ 䚸㉸ప䞁䝟䜽㌿෗⿦⨨ 㻟㻜㻜 ᫬㛫㻛䠍ᖺ㻌䝣䝸䞊䝄䞊㻤㻣㻢㻜 ᫬㛫㻛䠍ᖺ䚸䝍㻌䠄䠐䠅◊✲ᡂᯝ䛾ᴫせ㻌 㻌≟䛾㑇ఏᛶ⑌ᝈ䛻䛚䛡䜛 㻰㻺㻭 ཰㞟䞉⢭〇䞉౪⤥䛚䜘䜃⑌ᝈ᝟ሗ䛾䝕䞊䝍䝧䞊䝇໬㻌㯞ᕸ኱Ꮫ䛺䛹䛾኱Ꮫᡤᒓ䛾⋇་ᖌ䜔◊✲⪅䜢୰ᚰ䛻㑇ఏ⑓䚸㑇ఏ㛵㐃⑌ᝈ䜢ྵ䜐㠀ឤᰁᛶ⑌ᝈ䛾≟䛚䜘䜃୍⯡㞟ᅋ≟䛛䜙 㻰㻺㻭 ཰㞟䜢⾜䛔䚸䠍୓ 㻡㻤㻢㻡 ᳨య䛜཰㞟䛥䜜䛯䚹཰㞟᳨య䛾┠ᶆ䛜 㻝 ୓ 㻤㻜㻜㻜 ᳨య䛺䛾䛷䚸㻤㻤㻑䛾㐩ᡂᗘ䛷䛒䜛䚹≟✀䛿 㻝㻟㻜 ✀௨ୖ䛷ୖ఩䠏≟✀䛿䜴䜵䝹䝅䝳࣭ࢥ࣮ࢠ࣮࣭࣌ࣥࣈ࣮ࣟࣈ  ౛ࠊࢳࣗ࢔ࢲࢵࢡࢫ  ౛ࠊࣉ࣮ࢻࣝ ౛䛷䛒䛳䛯䚹䜸䝇䞉䝯䝇䛾ẚ⋡䛿 㸸䛷䚸ᖹᆒᖺ௧ࡣ  ᡯ࡛࠶ࡗࡓࠋⓑෆ㞀䜢䛿䛨䜑䛸䛩䜛║⑌ᝈ䚸䛶䜣䛛䜣䜢䛿䛨䜑䛸䛩䜛⚄⤒⑌ᝈ䚸䜰䝺䝹䜼䞊⑌ᝈ䜢䛿䛨䜑䛸䛩䜛ච␿⑌ᝈ䛺䛹 㻣㻢㻞㻤 ⑕౛䛜཰㞟䛥䜜䛯䚹⌧ᅾ䚸᭱⤊ⓗ䛺デ᩿䛜Ỵᐃ䛧䛶䛔䛺䛔⑕౛䜔⑌ᝈ᝟ሗ䛜䜎䛰᮶䛶䛔䛺䛔䜒䛾䛜ከᩘ䛒䜚䚸௒ᚋ䚸⑌ᝈ᝟ሗ䜢㏣ຍ䛧䛶䛔䛟䚹୍⯡㛤ᴗ䛾⋇་ᖌ䛾ሙྜ䚸⑌ᝈデ᩿ᇶ‽䜔⬟ຊ䛻䝞䝷䝒䜻䛜䛒䜛䛯䜑䚸⑕౛᳨య䛻㛵䛧䛶䛿⋇་኱Ꮫ䛾ᩍဨ䜢୰ᚰ䛻デ⒪䞉デ᩿䛧䛶᥇ྲྀ䛧䛯䚹䛧䛛䛧䚸䜰䝖䝢䞊ᛶ⓶⭵⅖䛻㛵䛧䛶䛿䜰䝺䝹䜼䞊἞⒪ἲ䛾㐍Ṍ䛷⋇་኱Ꮫ⑓㝔䛻᮶㝔䛩䜛⑌ᝈ≟䛜㠀ᖖ䛻ᑡ䛺䛟䛺䛳䛯䛯䜑䚸㛤ᴗ⋇་ᖌ䛷䜰䝖䝢䞊ᛶ⓶⭵⅖䛾ᑓ㛛་䛻౫㢗䛧䛶ᅜ㝿ⓗ䛺デ᩿ᇶ‽䛾ᇶ䛻᳨య䜢᥇ྲྀ䛧䛯䚹㻌⑌ᝈ⑕౛䛜㞟䜑䜙䜜䚸⑌ᝈ㛵㐃㑇ఏᏊ䜢ゎᯒ䛩䜛䛸䛝䛻䚸㝜ᛶᑐ㇟䛸䛺䜛୍⯡㞟ᅋ䛛䜙᥇ྲྀ䛧䛯≟䛾 㻰㻺㻭 䛜ᚲせ䛸䛺䛳䛯䚹୍⯡㞟ᅋ≟䛾 㻰㻺㻭 䛜䛺䛔䛸⑌ᝈ㑇ఏᏊゎᯒ䛜㐍䜎䛺䛔䛯䜑䚸ᮏ䝥䝻䝆䜵䜽䝖䛷䛿䚸୍⯡㞟ᅋ䛛䜙䛾≟䛾 㻰㻺㻭 ᳨య䛾᥇ྲྀ䜒✚ᴟⓗ䛻⾜䛖䜘䛖䛻䛺䜚䚸཰㞟 㻰㻺㻭 ᳨యᩘ䛻୍⯡㞟ᅋ≟䛾᳨యᩘ䜒ຍ䛘䛯䚹㻌཰㞟ࡉࢀࡓ '1$ ᳨యࡣࡇࡢࣉࣟࢪ࢙ࢡࢺࡢ◊✲⪅࡟ᥦ౪ࡋ࡚㑇ఏ㛵㐃⑌ᝈࡢゎᯒࢆ⾜ࡗࡓࠋࡲࡓࠊ௚ࡢ◊✲⪅࡬ '1$ ࢆ౪⤥ࡋࠊඹྠ◊✲ࢆ⾜ࡗ࡚࠸ࡿࠋ 㻖 ⛉Ꮫ㆙ᐹ◊✲
ᡤ࡛⾜ࡗ࡚࠸ࡿࠕ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺከᆺゎᯒࢆ⏝࠸ࡓ≟ࡢಶయ㆑ูἲࡢ᳨ウࠖ࡟≟ࡢ'1$ ࢆᥦ౪ࡋࡓࠋࡇࢀࡣ≟ࡢ '1$ ࡣ㸪യᐖࡸ⾑⤫᭩ഇ⿦࡞࡝┤᥋ື≀ࡀ㛵ࢃࡿ஦௳࠿ࡽ㸪ᙉ┐ࡸᙉጮ࡞࡝ࡢ⌧ሙ࡟≢ே࡟௜╔ࡋࡓື≀ẟࡀ㑇␃ࡉࢀࡿ஦௳ࡲ࡛㸪ከᒱ࡟ࢃࡓࡗ࡚
㔜せ࡞ドᣐ࡞ࡿࡓࡵ࡛࠶ࡿ Ꮫ఍Ⓨ⾲  ࠋࡲࡓࠊ 㻖 ᮾி኱Ꮫ⋇་Ꮫ⛉ᆤ஭ඛ⏕࡟ࡣ
ࠕ:KROH([RPH6HTXHQFH ἲ࡟ࡼࡿ≟ࡢ㑇ఏᛶ⚄⤒⑌ᝈࡢཎᅉ㑇ఏᏊྠᐃࠖࡢ◊✲࡛ࣃࣆࣚࣥ≟ࡢ⚄⤒㍈⣴ࢪࢫࢺࣟࣇ࢕࣮ࡢ㝜ᛶᑐ↷ࡢࣃࣆࣚࣥ≟ࡢ  ᳨యࡢ '1$ ࢆ㑇ఏᏊࣂࣥࢡ࠿ࡽᥦ౪ࡋࡓ Ꮫ఍Ⓨ⾲  ࠋ㑇ఏ㛵㐃⑌ᝈ䛾◊✲⪅䛿⑕౛䛛䜙⑌ᝈ㑇ఏᏊ䛾཰㞟䜢⾜䛖䛜䚸ྠ䛨≟✀䛷㝜ᛶᑐ↷䛸䛺䜛䜒䛾䛾཰㞟䛜䛺䛔ሙྜ䛜ከ䛔䛾䛷䚸䛣䛾䜘䛖䛺㑇ఏᏊ䝞䞁䜽䛾฼⏝䜒௒ᚋ䚸⾜䛳䛶䛔䛟ணᐃ䛷䛒䜛䚹཰㞟䛥䜜䛯᳨య᝟ሗ䛿䝞䜲䜸䝞䞁䜽䛻Ⓩ㘓䛧䚸⑌ᝈ᝟ሗ䜢䝕䞊䝍䝧䞊䝇໬䛧䛯䚹௒ᚋ䚸཰㞟䛥䜜䛯≟✀䜔⑌ᝈ䛻㛵䛧䛶䛿◊✲䜾䝹䞊䝥ෆእ䛷䛾᝟ሗ䛾ඹ᭷䜢⾜䛳䛶䛔䛟䛸䛸䜒䛻䚸ୖ
㏙䛧䛯䜘䛖䛻௚䛾◊✲⪅䛜⾜䛖◊✲䛻䜒✚ᴟⓗ䛻༠ຊ䜢⾜䛖䚹㻌䞉≟䛾⥳ෆ㞀䛚䜘䜃ⓑෆ㞀䛾㑇ఏᏊゎᯒ㻌㻌 ≟䛾⥳ෆ㞀㛵㐃㑇ఏᏊ䛻䛴䛔䛶䛿ゎᯒ䜢⾜䛔䚸ㄽᩥ໬䛷䛝䛯䛜䚸ⓑෆ㞀䛾㑇ఏᏊゎᯒ䛻䛴䛔䛶䛿䜎䛰ゎᯒ୰䛷㐩ᡂᗘ䛿 㻢㻜䠂䛷䛒䜛䚹㯞ᕸ኱Ꮫ㝃ᒓື≀⑓㝔䛾║⛉䛻⤂௓䛥䜜䛯║⑌ᝈ⑕౛䜢䜒䛸䛻║⑌ᝈ䛻䛚䛡䜛ཎᅉ㑇ఏᏊ䜢᥈ồ䛧䛯䚹㻰㻺㻭 䝃䞁䝥䝹䛾║⑌ᝈ䛿䚸⋇་║⛉ᑓ㛛་䛻䜘䜛║⛉᳨ᰝ䛻䜘䛳䛶デ᩿䛥䜜䛯⑕౛䛸䛧䛯䚹䛣䛾䝥䝻䝆䜵䜽䝖䛾ཎᅉ㑇ఏᏊゎ᫂䛾⑌ᝈ䛿⤂௓᮶㝔㢌ᩘ䜢ㄪᰝ䛧䚸≟✀౫Ꮡᛶ䛜ᴟ䜑䛶㧗䛔䜒䛾䛸䛧䛶ゎᯒ䛧䛯䚹㯞ᕸ኱Ꮫ㝃ᒓື≀⑓㝔䛾║⛉䛻⤂௓䛥䜜䛯 㻠㻢㻣㻜 ⑕౛䛛䜙䚸║⑌ᝈ⑕౛䛾 㻰㻺㻭 䝃䞁䝥䝹䜢཰㞟䛧䚸ᘏ䜉 㻞㻢㻡㻡 䝃䞁䝥䝹䜢཰㞟䛧䛯䚹║⑌ᝈ䛻䛚䛡䜛ཎᅉ㑇ఏᏊ䜢᥈ồ䛩䜛┠ⓗ䛷䚸⑕౛䛾⑌ᝈ⩌䜢ㄪ䜉䛯䚹䛭䛾⤖ᯝ䚸㻠㻢㻣㻜 ⑕౛䛾䛖䛱䚸ⓑෆ㞀䛜⣙ 㻞㻝䠂䚸⥳ෆ㞀䛜⣙䠓䠂䜢♧䛧䛶䛔

7䛯䚹䛣䛾䠎䛴䛾⑌ᝈ䛻ዲⓎ≟✀䛜Ꮡᅾ䛩䜛䛣䛸䛜䜟䛛䛳䛯䚹㻔㻖㻟㻕ⓑෆ㞀䛿║⑌ᝈ⨯ᝈ䜰䝯䝸䜹䞁䞉䝁䝑䜹䞊䞉䝇䝟䝙䜶䝹䛾 㻢㻣㻚㻣㻑䛷ㄆ䜑䜙䜜䚸䜎䛯⥳ෆ㞀䛿║⑌ᝈ⨯ᝈᰘ≟䛾 㻠㻞㻚㻥㻑䛷ㄆ䜑䜙䜜䛯䠄ㄽᩥ 㻝㻝䠅䚹䛭䛣䛷䚸䜰䝯䝸䜹䞁䞉䝁䝑䜹䞊䞉䝇䝟䝙䜶䝹䛷䛿ⓑෆ㞀ཎᅉ㑇ఏᏊ䜢䚸ᰘ≟䛷䛿⥳ෆ㞀ཎᅉ㑇ఏᏊ䛾ゎ᫂䜢⾜䛳䛯䚹㻌䜰䝯䝸䜹䞁䞉䝁䝑䜹䞊䞉䝇䝟䝙䜶䝹䛾ⓑෆ㞀䛾Ⓨ⑕䛻䛴䛔䛶䚸䛩䛷䛻ሗ࿌䛥䜜䛶䛔䜛䝇䝍䝑䝣䜷䞊䝗䝅䝱䇷䞉䝤䝹䞉䝔䝸䜰䛸䝪䝇䝖䞁䞉䝔䝸䜰䛾ⓑෆ㞀㛵㐃㑇ఏᏊ 㻴㻿㻲㻠 㑇ఏᏊ䛾ኚ␗䛻䛴䛔䛶䚸䜰䝯䝸䜹䞁䞉䝁䝑䜹䞊䞉䝇䝟䝙䜶䝹䛾ⓑෆ㞀⑕౛ 㻞㻡 㢌䛷㑇ఏᏊゎᯒ䜢⾜䛳䛯䛜 㻴㻿㻲㻠 㑇ఏᏊ䛾ኚ␗䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䚹ḟ䛻 㻰㻺㻭 䜰䝺䞊䜢⏝䛔䛯 㻿㻺㻼䠄୍ሷᇶከᙧ䠖㑇ఏᏊ㓄ิ୰䛻䠍 䛴 䛾 㻰㻺㻭 䛜 ኚ ␗ 䛧 䛯 ከ ᵝ ᛶ 䠅 䛾 ⥙ ⨶ ⓗ 㑇 ఏ Ꮚ ゎ ᯒ 䜢 ⾜ 䛳 䛯 䚹 㻝㻡 ␒ ᰁ Ⰽ య ୖ 䛾㼞㼟㻞㻞㻠㻞㻤㻠㻡㻠㻔䜶䞁䝗䝉䝸䞁ཷᐜయ䝍䜲䝥 㻭 㑇ఏᏊ㻕䛸 㻟㻝 ␒ᰁⰍయୖ䛾 㼞㼟㻞㻟㻢㻤㻡㻡㻣㻡 䛜Ⓨ⑕䛸䛾㛵㐃䜢♧၀䛩䜛⤖ᯝ䛜ᚓ䜙䜜䛯䚹䜶䞁䝗䝉䝸䞁ཷᐜయ䝍䜲䝥 㻭 㑇ఏᏊ䛿䠓䛴䛾䜶䜽䝋䞁䛛䜙䛺䜚䚸㼞㼟㻞㻞㻠㻞㻤㻠㻡㻠 䛿䜶䜽䝋䞁䠍䛸䜶䜽䝋䞁䠎䛾㛫䛾䜲䞁䝖䝻䞁㡿ᇦ䛻Ꮡᅾ䛩䜛䚹䜰䝯䝸䜹䞁䞉䝁䝑䜹䞊䞉䝇䝟䝙䜶䝹䛾ⓑෆ㞀≟㝧ᛶ౛ 㻞㻝 ౛䚸㝜ᛶ౛ 㻡 ౛䛾䜶䞁䝗䝉䝸䞁ཷᐜయ䝍䜲䝥 㻭 㑇ఏᏊ䛾඲㑇ఏᏊ㓄ิ䛾ゎᯒ䜢⾜䛳䛯䚹䛧䛛䛧䚸䜶䜽䝋䞁㒊ศ䛻䛚䛔䛶௚䛾ኚ␗䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䚹䛣䛾䜶䞁䝗䝉䝸䞁ཷᐜయ䝍䜲䝥 㻭 㑇ఏᏊ䛾䜶䜽䝋䞁㒊ศ䛾㑇ఏᏊኚ␗䛜ⓑෆ㞀Ⓨ⑕䛻㛵୚䛩䜛ྍ⬟ᛶ䛿ప䛔䛸⪃䛘䜙䜜䛯䚹㻌㻔㻖㻠㻕≟䛾⥳ෆ㞀䛾㑇ఏᏊゎᯒ䛾 㻰㻺㻭 ᳨య䛸䛧䛶䚸ᰘ≟ 㻠㻣 㢌䚸೺ᖖ≟ 㻟㻠 㢌䜢⏝䛔䛯䚹䛭䛾⤖ᯝ䚸㻿㻾㻮㻰㻝 㑇ఏᏊ䛾 㻞 䛴䛾᪂䛧䛔ኚ␗ 㼞㼟㻞㻞㻜㻝㻤㻡㻝㻟 䛸 㼞㼟㻞㻞㻜㻝㻤㻡㻝㻠 䛜≟⥳ෆ㞀䛾Ⓨ⑕㛵㐃䜢♧၀䛩䜛⤖ᯝ䛜ᚓ䜙䜜䛯㻔㞧ㄅㄽᩥ 㻠㻢䚸Ꮫ఍Ⓨ⾲ 㻟㻝㻕䚹≟⥳ෆ㞀䛾ཎᅉ㛵㐃㑇ఏᏊ䛿䚸䛩䛷䛻䝡䞊䜾䝹≟䛷 㻭㻰㻭㻹㼀㻿㻝㻜 㑇ఏᏊ䛾 㻿㻺㻼㻡㻢㻜㻥㻣㻜㻢㻡 䛜ሗ࿌䛥䜜䛶䛔䜛䛜䚸䛣䛾 㻿㻺㻼 䛸⥳ ෆ 㞀 ᰘ ≟ 䛸 䛾 㛵 㐃 䛿 䜏 䜙 䜜 䛺 䛛 䛳 䛯 䚹 䜎 䛯 䚸 㻔㻖㻡㻕㻿㻾㻮㻰 䠍 㑇 ఏ Ꮚ 䛾 㼞㼟㻞㻞㻜㻝㻤㻡㻝㻟 䛸㼞㼟㻞㻞㻜㻝㻤㻡㻝㻠 䛜ᰘ≟䛾㣫⫱ᆅ䛻౫Ꮡ䛧䛶䛔䜛ྍ⬟ᛶ䛜ྰᐃ䛷䛝䛺䛔䛣䛸䛛䜙䚸䜘䜚ᗈᇦ䛻㣫⫱䛧䛶䛔䜛ᰘ≟䜢ᑐ㇟䛸䛧䛯ㄪᰝ䜢⾜䛳䛯䚹䛭䛾⤖ᯝ䚸ᰘ≟⥳ෆ㞀䛸䛾㛵㐃䛜 㻿㻾㻮㻰䠍㑇ఏᏊ䛾 㼞㼟㻞㻞㻜㻝㻤㻡㻝㻟 䛷≉䛻ᙉ䛟ㄆ䜑䜙䜜䚸ᰘ≟⥳ෆ㞀䛻䛚䛡䜛 㻿㻾㻮㻰㻝 㑇ఏᏊ䛾ኚ␗䛾෌⌧ᛶ䛜☜ㄆ䛷䛝䛯㻔Ꮫ఍Ⓨ⾲ 㻝㻜㻕䚹㻌≟䛻䛚䛡䜛䝠䝖䜈䛾㐺ྜᛶ䛻㛵䛩䜛㑇ఏᏊゎᯒ㻌䝠䝖䛸䛾㐺ᛂᛶ䚸䛩䛺䜟䛱♫఍ᛶ䛻㛵䛩䜛㑇ఏᏊྠᐃ䜢ヨ䜏䚸䠍䠅♫఍ᛶ䛻㛵䛩䜛≟✀ᕪ䜢㑇ఏⓗ᥎⛣䛾୰䛻ぢฟ䛧䛯䠄㐩ᡂ 㻝㻜㻜㻑䠅䚸䠎䠅≟䛾ᐙ␆໬䛾㐣⛬䛷䛣䛾䜘䛖䛺♫఍ᛶ䛻䜘䜛㑅ᢥᅽ䛜ᙉ䛛䛳䛯䛣䛸䜢♧၀䛩䜛䝕䞊䝍䜢ぢฟ䛧䛯䠄㐩ᡂ 㻝㻜㻜㻑䠅䚸䠏䠅䝠䝖䜈䛾♫఍ㄆ▱⬟ຊ䛻≟✀ᕪ䜢ぢฟ䛧䚸䛭䜜䛻㛵㐃䛩䜛㑇ఏᏊ䜢」ᩘྠᐃ䛧䛯䠄㐩ᡂ 㻥㻜㻑䠅䚸䛣䛸䛛䜙䜋䜌┠ⓗ䜢㐩䛧䛯䚹㻌᪥ᮏཬ䜃⡿ᅜ䛻䛚䛡䜛୍⯡䛾㣫䛔୺ཬ䜃䝤䝸䞊䝎䞊䜢ᑐ㇟䛸䛧䛯≟䛾⾜ື≉ᛶ䛻㛵䛩䜛ㄪᰝ䜢ᐇ᪋䛧䛯䚹≟✀䜢᪤䛻ሗ࿌䛥䜜䛶䛔䛯㑇ఏศᒱᅗ䜢ඖ䛻 㻤 䛴䛾䜾䝹䞊䝥䛻ศ㢮䛧䚸ᅉᏊศᯒཬ䜃ᖹ⾜ศᯒ䜢⾜䛔䚸ྛᅉᏊ䛾ᖹᆒ್䜢ᅉᏊᚓⅬ䛸䛧䛶䚸⾜ື≉ᛶ䛾≟✀䜾䝹䞊䝥ẚ㍑䛻⏝䛔䛯䚹䛭䛾⤖ᯝ䚸䠄㻖㻢䠅ཎጞⓗ䛺≟䜾䝹䞊䝥䛾≟䛿䛹䛾≟✀䜾䝹䞊䝥䜘䜚䜒䝠䝖䜈䛾ឡ╔䛜ప䛔䛣䛸䛜᫂䜙䛛䛸䛺䛳䛯䠄㼜㻨㻜㻚㻜㻡䠅㻔㞧ㄅㄽᩥ 㻝㻞㻕䚹ཎጞⓗ䛺≟䛾ឡ╔䛿䛭䛾௚䛾䛹䛾≟✀䜾䝹䞊䝥䜘䜚䜒ప䛟䚸ඛ⾜◊✲䛷▱䜙䜜䛶䛔䜛㑇ఏศᒱᅗ䛸୍⮴䛧䛯䚹䛭䛾௚䛾≟✀䜾䝹䞊䝥䛷䛿䚸䝽䞊䜻䞁䜾䜾䝹䞊䝥䛿䚸ぢ▱䜙䛼ே䛻ᑐ䛩䜛ᜍᛧ཯ᛂ䚸ぢ▱䜙䛼≟䛻ᑐ䛩䜛ᜍᛧ཯ᛂ䚸㠀♫఍ⓗ่⃭䛻ᑐ䛩䜛ᜍᛧ཯ᛂ䚸㣫䛔୺䛻ᑐ䛩䜛ᨷᧁᛶ䚸άⓎᗘ䛜ప䛔䛣䛸䛺䛹䛜᫂䜙䛛䛸䛺䛳䛯䚹㻌ᵝ䚻䛺≟✀䛾≟䜢⏝䛔䚸≟䛾ㄆ▱⬟ຊ䛾≟✀ᕪ䜢ゎỴ୙ྍ⬟ㄢ㢟ཬ䜃ᣦᕪ䛧஧⪅㑅ᢥㄢ㢟䛻䜘䜚ホ౯䛧䛯䚹ゎỴ୙ྍ⬟ㄢ㢟䛷䛿䚸ཎጞⓗ䛺≟䛿䚸᭱ึ䛻䝠䝖䜢ぢ䜛䜎䛷䛾᫬㛫䛜㛗䛟䚸䝠䝖䜢ぢ䛶䛔䜛᫬㛫䛜▷䛟䚸䝠䝖䜢ぢ䜛ᅇᩘ䛸஺஫จど䛾ᅇᩘ䛜ᑡ䛺䛛䛳䛯䠄㼜㻨㻜㻚㻜㻝䠅䚹ᣦᕪ䛧஧⪅㑅ᢥㄢ㢟䛷䛿䚸ཎጞⓗ䛺≟䛾ᡂ⦼䛿䛭䛾௚䛾≟✀䜾䝹䞊䝥䛸ྠ➼䛷䛒䜚䚸≟✀䜾

8䝹䞊䝥䛻䜘䜛᭷ព䛺ᕪ䛿ぢ䜙䜜䛺䛛䛳䛯䚹㻌♫఍ᛶ⬟ຊ䛻㛵䜟䜛㑇ఏᏊ䛸䛧䛶䚸䝯䝷䝜䝁䝹䝏䞁䠎ཷᐜయ䠄㻹㻯㻞㻾䠅䛸䜸䜻䝅䝖䝅䞁䚸䜸䜻䝅䝖䝅䞁ཷᐜయ䛾㑇ఏᏊ䜢㑅ᢥ䛧䛯䚹䛥䜙䛻䝀䝜䝮䝽䜲䝗ゎᯒ䛾⤖ᯝ䛛䜙 㼃㻮㻿㻯㻾㻝㻣䠄䜴䜱䝸䜰䝮䝈⑕ೃ⩌㛵㐃㑇ఏᏊ䠅䜢㑅ᢤ䛧䚸䛣䜜䜙䛻㛵㐃䛩䜛㑇ఏᏊ䛾ከᆺ䜢ㄪ䜉䚸≟✀ᕪ䜔⾜ືᐇ㦂䛾⤖ᯝ䛸䛾㛵㐃ᛶ䜢ㄪ䜉䛯䚹≟䛾㐍໬ೃ⿵㑇ఏᏊ䛷䛒䜛 㼃㻮㻿㻯㻾㻝㻣 䛻䛚䛔䛶䚸ཎጞⓗ䛺≟䛸୍⯡ⓗ䛺≟✀䛸䛷ฟ⌧㢖ᗘ䛾␗䛺䜛୍ሷᇶከᆺ䛜᳨ฟ䛥䜜䚸㼀 ᆺ䛾㑇ఏᏊᆺ䜢ᣢ䛴ಶయ䛷䛿䚸ᣦᕪ䛧஧⪅㑅ᢥㄢ㢟䛾ど⥺䠇ᣦᕪ䛧䠇䝍䝑䝢䞁䜾䛾ㄢ㢟䛾ṇ⟅ᩘ䛜㧗䛔⤖ᯝ䛸䛺䛳䛯䠄㼜㻨㻜㻚㻜㻡䠅䚹㻹㻯㻞㻾 䛻䛚䛔䛶䚸ཎጞⓗ䛺≟䛸୍⯡ⓗ䛺≟✀䛸䛷ฟ⌧㢖ᗘ䛾␗䛺䜛୍ሷᇶከᆺ䛜᳨ฟ䛥䜜䚸㻭 ᆺ䛾㑇ఏᏊᆺ䜢ᣢ䛴ಶయ䜢୍⯡ⓗ䛺≟✀ෆ䛷ẚ㍑䛧䛯⤖ᯝ䚸ᣦᕪ䛧஧⪅㑅ᢥㄢ㢟䛾ど⥺䠇ᣦᕪ䛧䠇䝍䝑䝢䞁䜾ㄢ㢟䚸ど⥺䠇ᣦᕪ䛧ㄢ㢟䚸ᣦᕪ䛧ㄢ㢟䛾ṇ⟅ᩘ䛜ప䛔⤖ᯝ䛸䛺䛳䛯䠄㼜㻨㻜㻚㻜㻡䠅䚹㻻㼀䠄䜸䜻䝅䝖䝅䞁㑇ఏᏊ䠅䛻䛚䛔䛶䚸ཎጞⓗ䛺≟䛸୍⯡ⓗ䛺≟✀䛸䛷ฟ⌧㢖ᗘ䛾␗䛺䜛୍ሷᇶከᆺ䛸཯᚟ᩘከᆺ䛜᳨ฟ䛥䜜䚸㻭㻛㻭 ᆺ䛾୍ሷᇶከᆺ䜢ᣢ䛴ಶయ䛷䛿䚸㻭㻛㻯 ᆺ䛾㑇ఏᏊᆺ䜢ᣢ䛴ಶయ䜘䜚䜒䚸ゎỴ୙ྍ⬟ㄢ㢟䛾䝠䝖䜢ぢ䛶䛔䜛᫬㛫䚸䝠䝖䜢ぢ䜛ᅇᩘ䚸஺஫จど䛾ᅇᩘ䛻䛚䛔䛶䝠䝖䜢ぢ䛺䛔⤖ᯝ䛸䛺䛳䛯䠄㼜㻨㻜㻚㻜㻡䠅䚹㻔㻖㻣㻕䠄䜸䜻䝅䝖䝅䞁ཷᐜయ㑇ఏᏊ䠅䛻䛚䛔䛶䛿䚸ཎጞⓗ䛺≟䛸୍⯡ⓗ䛺≟✀䛸䛷ฟ⌧㢖ᗘ䛾␗䛺䜛୍ሷᇶከᆺ䛜᳨ฟ䛥䜜䛯䜒䛾䛾㻔㞧ㄅㄽᩥ 㻝㻠㻕䚸⾜ືᐇ㦂䛾⤖ᯝ䛸䛿㛵㐃ᛶ䛜ぢ䜙䜜䛺䛛䛳䛯䚹㻌䜸䜸䜹䝭䚸⛅⏣≟䚸ᰘ≟䚸ཎጞⓗ䛺≟䜾䝹䞊䝥௨እ䛾≟䛾䜰䝭䝷䞊䝊䝁䝢䞊ᩘ䜢ㄪᰝ䛧䛯䚹䜸䜸䜹䝭䛾䜰䝭䝷䞊䝊䝁䝢䞊ᩘ䛿 㻞 䝁䝢䞊⛬ᗘ䛷䛒䛳䛯䚹䜎䛯䚸䜸䜸䜹䝭䜈䛾㑇ఏⓗ㏆䛥䛛䜙ண᝿䛧䛯䛸䛚䜚䚸⛅⏣≟䛾䝁䝢䞊ᩘ䛿䜸䜸䜹䝭 䜘䜚䜒ከ䛔䛜୍⯡ⓗ䛺≟✀䜘䜚ᑡ䛺䛛䛳䛯䠄㼜㻨㻜㻚㻜㻝䠅䚹㻔㻖㻤㻕≟䛸ྠ䛨䛟ཎጞⓗ䛺≟䜾䝹䞊䝥䛻ྵ䜎䜜䜛ᰘ≟䛾䝁䝢䞊ᩘ䛿䚸୍⯡ⓗ䛺≟✀䛸ྠ⛬ᗘ䛷䛒䜚䚸ᰘ≟䛾䜸䜸䜹䝭䜈䛾㑇ఏⓗ㏆䛥䛸཯䛧䛯⤖ᯝ䛸䛺䛳䛯䚹⦖ᩥᰘ䛾䝁䝢䞊ᩘ䛿䚸ᰘ≟䜘䜚ᑡ䛺䛟䠄㼜㻨㻜㻚㻜㻝䠅䚸⛅⏣≟䜘䜚ከ䛛䛳䛯䠄㼜㻨㻜㻚㻜㻡䠅㻔㞧ㄅㄽᩥ 㻝㻡㻕䚹㻌≟⭘⒆ቯṚᅉᏊ㻔㼀㻺㻲㻙䃐㻕㑇ఏᏊ䛻㛵䛩䜛◊✲㻌㼀㻺㻲㻙䃐䛾㑇ఏᏊ䛷䛒䜛 㼀㻺㻲㻭 㑇ఏᏊ䛻䛚䛔䛶䝬䜲䜽䝻䝃䝔䝷䜲䝖ኚ␗䠄䠍䛛䜙䠑ሷᇶ䛾㓄ิ䛜⧞䜚㏉䛩㡿ᇦ䠅䜢☜ㄆ䛧䛯䚹≟䛾ච␿௓ᅾᛶ⾑ᑠᯈῶᑡ䚸䝸䞁䝟⭘䚸ᝏᛶ㯮Ⰽ⭘䛷䛭䛾ኚ␗䛾㑇ఏᏊゎᯒ䜢ᐇ᪋䛧䛯䛜䚸≉␗ⓗ䛺 㻿㻺㻼 䛾ゎᯒ䛻䛿⮳䜙䛺䛛䛳䛯䚹௒ᚋ䚸≟䛾௚䛾ච␿௓ᅾᛶ⾑ᾮ⑌ᝈ䛚䜘䜃⭘⒆ᛶ⑌ᝈ䛻䛴䛔䛶ゎᯒ୰䛷㐩ᡂᗘ䛿 㻠㻜䠂䛷䛒䜛䚹㻌䝠䝖䛾་Ꮫ㡿ᇦ䛷䛿 㼀㻺㻲㻙䃐䛿ᵝ䚻䛺ච␿௓ᅾᛶ⑌ᝈ䛷䚸䛭䛾␗ᖖ䛜ㄆ䜑䜙䜜䛶䛔䜛䚹ᮏ◊✲䛷䛿≟䛾 㼀㻺㻲㻙䃐䛾㑇ఏᏊ䛷䛒䜛 㼀㻺㻲㻭 㑇ఏᏊ䛻䛚䛔䛶䝬䜲䜽䝻䝃䝔䝷䜲䝖ኚ␗䛾ゎᯒ䜢ヨ䜏䛯䚹≟䛾㑇ఏⓗ䝸䝇䜽ᅉᏊ䜢≉ᐃ䛩䜛┠ⓗ䛷䚸䜎䛪ṇᖖ≟䛾 㼀㻺㻲㻙䃐䛾㑇ఏᏊ䛷䛒䜛㼀㻺㻲㻭 㑇ఏᏊ䛾ሷᇶ㓄ิ䜢ゎᯒ䛩䜛䛣䛸䛸䛧䛯䚹䝡䞊䜾䝹䚸䝏䝽䝽䚸䝭䝙䝏䝳䜰䝎䝑䜽䝇䚸䝖䜲䝥䞊䝗䝹䚸䝁䞊䜼䞊䛚䜘䜃ᰘ≟䚸ྛ 㻞㻜 㢌௨ୖ䛛䜙䛺䜛ྜィ 㻝㻟㻥 㢌䛾 㻰㻺㻭 ヨᩱ䜢⏝䛔䚸㻼㻯㻾 ⏘≀䛿 㻟㻝㻟㻜㻌 㻳㼑㼚㼑㼠㼕㼏㻌 㼍㼚㼍㼘㼥㼦㼑㼞㻌 䜢⏝䛔䛯㑇ఏᏊᆺุᐃ䛻౪䛧䛯䚹㻔㻖㻥㻕➨䠍䜲䞁䝖䝻䞁ෆ䛻 㻠 ሷᇶ䜢༢఩䛸䛩䜛䝬䜲䜽䝻䝃䝔䝷䜲䝖㻔㼠㼑㼠㼞㼍㻙㼚㼡㼏㼘㼑㼛㼠㼕㼐㼑㻌 㼞㼑㼜㼑㼍㼠㼟㻧௨ᚋ 㼀㻺㻾㻕ኚ␗䜢ぢฟ䛧䛯㻔㞧ㄅㄽᩥ 㻟㻞㻕䚹䛥䜙䛻➨䠍䜲䞁䝖䝻䞁ෆ䛾 㼀㻺㻾 ከᆺ䜢ุᐃ䛩䜛䛯䜑䛻䚸⣙ 㻝㻞㻜 ሷᇶᑐ䛾⏘≀䛜ᚓ䜙䜜䜛䝥䝷䜲䝬䞊䜢タィ䛧䛯䚹≟ 㼀㻺㻲㻭 㑇ఏᏊ䛾➨䠍䜲䞁䝖䝻䞁䛻 㻳㻭㻭㼀 䜢཯᚟༢఩䛸䛩䜛 㼀㻺㻾 䛜Ꮡᅾ䛧䚸⧞䜚㏉䛧ᩘከᆺ䛜Ꮡᅾ䛩䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛯䚹ㄪᰝ䛧䛯 㻝㻟㻥 ಶయ䛛䜙㻌 㻡 ᅇ䠈㻢ᅇ䠈㻣 ᅇ䠈㻤 ᅇᆺ䛾 㻠 ᑐ❧㑇ఏᏊ䛜᳨ฟ䛥䜜䛯䚹䝡䞊䜾䝹✀䛛䜙䛿 㻡 ᅇᆺ䛛䜙 㻤 ᅇᆺ䜎䛷඲䛶䛾ᑐ❧㑇ఏᏊ䛜᳨ฟ䛥䜜䛯䛜䚸௚䛾 㻡 ရ✀䛛䜙䛿 㻤 ᅇᆺ䛿᳨ฟ䛥䜜䛺䛛䛯䚹䝦䝔䝻᥋ྜᗘ䛾ᮇᚅ್䠄㻴㼑䠅䛚䜘䜃ከᆺ᝟ሗ㔞㻔㻼㻵㻯㻕䛿䚸ရ✀䛻䜘䛳䛶䚸䛭䜜䛮䜜 㻜㻚㻟㻤㻥㻌㻙㻌㻜㻚㻣㻠㻥㻌 䛸㻌 㻜㻚㻟㻟㻟㻌㻙㻌㻜㻚㻢㻤㻞 䛾㛫䛷ᕪ␗䛜ㄆ䜑䜙䜜䛯䚹䛣䛾䝬䜲䜽䝻䝃䝔䝷䜲䝖䝬䞊䜹䞊䛿䚸ከᆺᛶ䛻ᐩ䜏ྠ᫬䛻ᆺุᐃ䛜ᐜ᫆䛷䛒䜛䛯䜑䚸≟ 㼀㻺㻲㻭 㑇ఏᏊ䛾䝷䞁䝗䝬䞊䜽䛸䛧䛶䚸ఏ㐩୙ᖹ⾮䝔䝇䝖䛺䛹䛾⑌ᝈ┦㛵ゎᯒ䛻฼⏝ྍ⬟䛷䛒䜛䛸ண᝿䛥䜜䛯䚹㻌䛣䜜䜙䛾䝬䜲䜽䝻䝃䝔䝷䜲䝖䛿䚸≟䛾ච␿௓ᅾᛶ⾑ᾮ⑌ᝈ䛚䜘䜃⭘⒆ᛶ⑌ᝈ䛾デ᩿ᛂ⏝䛻9᭷ຊ䛸⪃䛘᥈⣴䜢⾜䛳䛯䚹ච␿௓ᅾᛶ⑌ᝈ䛷䛿䚸᭱⤊ⓗ䛻 㻟㻢 ౛䛾≟䛾ච␿௓ᅾᛶ⾑ᑠᯈῶᑡ䛷ゎᯒ䜢ᐇ᪋䛧䛯䛜䚸≉␗ⓗ䛺 㻿㻺㻼 䛾ゎᯒ䛻䛿⮳䜙䛺䛛䛳䛯䚹䛥䜙䛻䚸䝸䞁䝟⭘ 㻝㻜 ౛䛚䜘䜃ᝏᛶ㯮Ⰽ⭘䛻䛚䛔䛶 㼀㻺㻲㻭 㑇ఏᏊ䛾䝬䜲䜽䝻䝃䝔䝷䜲䝖ゎᯒ䜢ᐇ᪋䛧䛶䛔䛯䛜䚸ኚ␗䛿ぢฟ䛰䛩䛣䛸䛿ᅔ㞴䛷≉␗ⓗ䛺 㻿㻺㻼 䛾ゎᯒ䛻䛿⮳䜙䛺䛛䛳䛯䚹䛣䛾ཎᅉ䛸䛧䛶䚸ච␿௓ᅾᛶ
⾑ᾮ⑌ᝈ䛿䛭䜒䛭䜒ẕ㞟ᅋ䛜ᑡ䛺䛟䚸䛛䛴☜ᐃデ᩿せ௳䜢‶䛯䛩䜒䛾䛜ᑡ䛺䛳䛯䛣䛸䛜ᣲ䛢䜙䜜䜛䚹䜎䛯⭘⒆ᛶ⑌ᝈ䛻䛚䛔䛶䛿䚸஧ḟデ⒪᪋タ䛻᮶㝔䛩䜛䝸䞁䝟⭘䛚䜘䜃ᝏᛶ㯮Ⰽ⭘䛾᳨యᩘ䛜ῶᑡഴྥ䛷䛒䜛䛣䛸䜒ᣲ䛢䜙䜜䜛䚹䛣䛾䜘䛖䛻ゎᯒᩘ䛜䛤䛟ᑡᩘ䛷䛒䜛䛯䜑䚸ኚ␗䛾᭷↓䜢☜ㄆ䛷䛝䛺䛔䛣䛸䛸䝥䝷䜲䝬䞊䛾タィ⮬య䛻ၥ㢟䛜䛒䛳䛯ྍ⬟ᛶ䛜䛒䜚䚸௒ᚋ᳨ウ䛩䜛ᚲせ䛜䛒䜛䜒䛾䛸⪃䛘䜙䜜䛯㻌≟䛾䜰䝻䝨䝅䜰㻌 㼄䠄䝫䝯䝷䝙䜰䞁⬺ẟ⑕䠅䛾㑇ఏᏊゎᯒ㻌㻌 ≟䛾䜰䝻䝨䝅䜰㻌 㼄䠄䝫䝯䝷䝙䜰䞁⬺ẟ⑕䠅䛾㑇ఏᏊゎᯒ䛻䛚䛔䛶᭱᭷ຊೃ⿵㑇ఏᏊ䛸䛧䛶䚸㻸㼑㼒㻝 ཬ䜃 㻰㼘㼤㻟 䛜ᚓ䜙䜜䛯䛜䚸⑌ᝈ≟䛸೺ᖖ≟䛻䛚䛡䜛㑇ఏኚ␗䛻䛴䛔䛶䛿⌧ᅾ䚸ゎᯒ୰䛷㐩ᡂᗘ䛿 㻣㻜䠂䛷䛒䜛䚹㻌䝫䝯䝷䝙䜰䞁䛺䛹≉ᐃ䛾≟✀䛷ぢ䜙䜜䜛ᡂᖺⓎ⑕ᆺ㠀⅖⑕ᛶ㠀ᥙ⑛ᛶ䛷䚸ඛኳᛶ䛾⬺ẟ⑌ᝈ䛷䛒䜛 㻭㼘㼛㼜㼑㼏㼕㼍㻌㼄 䛾⑌ᝈ㛵㐃ཎᅉ㑇ఏᏊ䜢ྠᐃ䛩䜛䛣䛸䜢➨୍䛾┠ⓗ䛸䛧䚸䛭䛾ᡂᯝ䜢᪂䛯䛺἞⒪ἲ䠄἞⒪⸆䛾㛤Ⓨ䠅䜔೺඲䛺Ⓨẟ䞉⫱ẟ䜈ᛂ⏝䛩䜛䛣䛸䜢᭱⤊┠ⓗ䛸䛩䜛䚹㐣ཤ䛻䚸೺ᖖ≟䛸 㻭㼘㼛㼜㼑㼏㼕㼍㻌 㼄 ⑕䝫䝯䝷䝙䜰䞁≟䛾⓶⭵䛛䜙ᢳฟ䛧䛯 㻾㻺㻭 䜢⏝䛔䛶䚸㻰㻺㻭 䝬䜲䜽䝻䜰䝺䜲䛻䜘䜛⥙⨶ⓗ㑇ఏᏊⓎ⌧ẚ㍑ゎᯒ䜢ᐇ᪋䛧䚸㻭㼘㼛㼜㼑㼏㼕㼍㻌 㼄 ⑕䛷᭷ព䛺Ⓨ⌧ῶᑡ䛜ぢ䜙䜜䛯」ᩘ䛾ཎᅉೃ⿵㑇ఏᏊ䜢᥎ᐃ䛧䛯䚹䛣䜜䜙䛾㑇ఏᏊ䛾ཝᐦ䛺ᐃ㔞ⓗⓎ⌧ゎᯒ䜢㼞㼑㼍㼘㻙㼠㼕㼙㼑㻌㻾㼀㻙㻼㻯㻾 ἲ䜢⏝䛔䛶䚸ᩘከ䛟䛾ಶయ䠄⑌ᝈ≟䜔೺ᖖ≟䛺䛹䠅䛻䛴䛔䛶ᐇ᪋䛧䛯䚹䛭䛾⤖ᯝ䚸᭱᭷ຊೃ⿵㑇ఏᏊ䛸䛧䛶䚸㻸㼑㼒㻝 ཬ䜃 㻰㼘㼤㻟 䛜ᚓ䜙䜜䛯䚹㻰㼘㼤㻟 㑇ఏᏊ⏘≀䛿ẟໟ䛾ศ໬䜔ẟ䛾࿘ᮇ䛻㛵୚䛩䜛䝍䞁䝟䜽㉁䛾 㻝 䛴䛷䛒䜚䚸㻸㼑㼒㻝 㑇ఏᏊ⏘≀䛿 㻰㼘㼤㻟 㑇ఏᏊⓎ⌧䜢ಁ㐍䛩䜛㌿෗ᅉᏊ䛷䛒䜛䛣䛸䛜䝬䜴䝇䛷▱䜙䜜䛶䛔䜛䚹≟ 㻰㼘㼤㻟 㑇ఏᏊ䛾ᵝ䚻䛺㛗䛥䛾䝥䝻䝰䞊䝍䞊㡿ᇦ䜢䜒䛴䝺䝫䞊䝍䞊䝧䜽䝍䞊ཬ䜃≟ 㻸㼑㼒㻝 㑇ఏᏊ䛾Ⓨ⌧⣔䝧䜽䝍䞊䜢స〇䛧䛶䚸䝺䝫䞊䝍䞊䜰䝑䝉䜲⣔䜢ᵓ⠏䛧䛯䚹㻸㼑㼒㻝 㑇ఏᏊ䛾䝥䝻䝰䞊䝍䞊㡿ᇦ䜢䜒䛴䝺䝫䞊䝍䞊䝧䜽䝍䞊䛻䜘䜛䝺䝫䞊䝍䞊䜰䝑䝉䜲⣔䜒ᵓ⠏䛧䛯䚹䛣䜜䜙䛾ホ౯⣔䜢⏝䛔䛶䚸Ⓨẟ䞉⫱ẟ㛵㐃㑇ఏᏊ䛾Ⓨ⌧ไᚚᶵᵓ䛻㛵䛩䜛ศᏊ䞉⣽⬊⏕≀Ꮫⓗศᯒ䜢䛩䜛䛸䛸䜒䛻䚸㑇ఏᏊⓎ⌧䜢ಁ㐍䛩䜛≀㉁䛾᥈⣴䜢⾜䛳䛶䛔䜛䚹㻸㼑㼒㻝 ㌿෗ᅉᏊ䛜ᛂ⟅䛩䜛㓄ิ௨እ䛻䜒䚸㻰㼘㼤㻟 㑇ఏᏊ䛾Ⓨ⌧䜢ไᚚ䛩䜛ᛂ⟅䜶䝺䝯䞁䝖䛜 㻰㼘㼤㻟 㑇ఏᏊ䛾䝥䝻䝰䞊䝍䞊㡿ᇦ䛻Ꮡᅾ䛩䜛䛣䛸䛜䜟䛛䛳䛯䚹䜎䛯䚸⑌ᝈ≟䛸೺ᖖ≟䛻䛚䛡䜛 㻰㼘㼤㻟 㑇ఏᏊ䠄䝥䝻䝰䞊䝍䞊㡿ᇦ䜢ྵ䜐䠅䛾ሷᇶ㓄ิ䛾┦㐪䜢ㄪ䜉䛶䛔䜛䚹㻌≟䛾䛶䜣䛛䜣䛾㑇ఏᏊゎᯒ㻌㻌 ≟䛾䛶䜣䛛䜣䛾㑇ఏᏊゎᯒ䛻䛚䛔䛶⥙⨶ⓗ䛺඲䝀䝜䝮㛵㐃ゎᯒ䜢⾜䛔䚸䠐䛴䛾 㻿㻺㻼 䜢ぢฟ䛧䛯䚹㐩ᡂᗘ䛸䛧䛶䛿 㻠㻜㻑䛷䛒䜛䚹≉Ⓨᛶ䛶䜣䛛䜣䛿䚸ே䛸ྠᵝ䛻≟䛻䛚䛔䛶᭱䜒୍⯡ⓗ䛺⚄⤒⑌ᝈ䛷䛒䜛䛜䚸⑓ᅉ䛿䜋䛸䜣䛹ศ䛛䛳䛶䛔䛺䛔䚹㑇ఏⓗせᅉ䛜␲䜟䜜䛶䛔䜛䚹ᮏ⑌ᝈ䛿ཎᅉ䛜୙᫂䛺䛯䜑᰿἞⒪ἲ䛜䛺䛟䚸䜋䛸䜣䛹䛾≟䛷䚸ᑐ⑕⒪ἲ䛸䛧䛶䛾䛶䜣䛛䜣䜢ᢚ䛘䜛⸆䛾ᢞ୚䛜⏕ᾭᚲせ䛸䛺䜛䚹ᮏ◊✲䛻䛚䛔䛶䚸≟䛾≉Ⓨᛶ䛶䜣䛛䜣䛾ཎᅉ㑇ఏᏊ䜢ྠᐃ䛧䚸⑓ែᶵᵓ䜢᫂䜙䛛䛻䛩䜛䛣䛸䛿䚸ཎᅉ⒪ἲ㛤Ⓨ䛾⣒ཱྀ䛸䛺䜚䚸⋇་Ꮫ䛾Ⓨᒎ䛻኱䛝䛟ᐤ୚䛷䛝䜛䛸⪃䛘䜛䚹䛥䜙䛻䛭䛾᝟ሗ䛿䚸䝠䝖䛾䛶䜣䛛䜣་⒪䛻ᑐ䛧䛶䜒᭷⏝ᛶ䛜㧗䛔䛸⪃䛘䜛䚹㻌㯞ᕸ኱Ꮫ㝃ᒓື≀⑓㝔⚄⤒⛉䛻᮶㝔䛧䚸≉Ⓨᛶ䛶䜣䛛䜣䛸デ᩿䛥䜜䛯≟䛾⾑ᾮ䝃䞁䝥䝹䛾཰㞟䛸⮫ᗋ᝟ሗ䛾཰㞟䜢⾜䛳䛯䚹Ⓨస᫬䛾ᚩೃ䛾ヲ⣽䛺ၥデ䛸䝡䝕䜸ゎᯒ䜢⾜䛖䛸䛸䜒䛻䚸ྍ⬟䛺㝈䜚⬻Ἴ᳨ᰝ䜒⾜䛔䚸䛭䜜䛮䜜䛾⑕౛䛻䛴䛔䛶䚸Ⓨసᆺศ㢮䠄Ⓨస䛾⾲⌧ᆺ䛾ศ㢮䠅䜢⾜䛳䛯䚹ᵝ䚻䛺≟✀䛾䝃䞁䝥䝹䛜཰㞟䛥䜜䛯䛜䚸䛭䛾୰䛷䚸≉Ⓨᛶ䛶䜣䛛䜣䛜ዲⓎ䛧䛶䛔䜛༢୍≟✀䛻╔┠䛧䚸⨯ᝈ≟䛸㠀⨯ᝈ≟䛾 㻰㻺㻭 䜢⏝䛔䛶䚸㻿㻺㻼㻌 䜰䝺䞊䛷⥙⨶ⓗ䛺඲䝀䝜䝮㛵㐃ゎᯒ㻌 㻔㻳㼃㻭㻿㻕㻌 䜢⾜䛳䛯䚹㠀⨯ᝈ≟䛾 㻰㻺㻭 䛿㑇ఏᏊ䝞䞁䜽䛻ಖ⟶䛥䜜䛶䛔䜛⨯ᝈ≟✀10䛸ྠ୍≟✀䛾᳨య䜢⏝䛔䛯䚹㻌㻳㼃㻭㻿 䛻䜘䛳䛶⤠䛳䛯䚸䛶䜣䛛䜣Ⓨ⑕䛸᭷ព䛺㛵㐃ᛶ䜢♧䛩 㻿㻺㻼 䛻䛴䛔䛶䚸᪂䛯䛻᳨య䜢཰㞟䛧☜ㄆᐇ㦂䜢⾜䛳䛯䚹᪂䛯䛺᳨య䛿䚸㯞ᕸ኱Ꮫ㝃ᒓື≀⑓㝔⚄⤒⛉᮶㝔⑕౛䛻ຍ䛘䚸䛶䜣䛛䜣䛾デ᩿䜔デ⒪䛻㛗䛡䛯௚䛾」ᩘ䛾᪋タ䛛䜙䜒཰㞟䛧䛯䚹㻌≉Ⓨᛶ䛶䜣䛛䜣䛸デ᩿䛧䛯ィ 㻝㻠㻟 㢌䛛䜙 㻰㻺㻭 䝃䞁䝥䝹䛜ᚓ䜙䜜䛯䚹ᑐ㇟䛸䛧䛯≟✀䛾≉Ⓨᛶ䛶䜣䛛䜣⨯ᝈ≟ 㻝㻜 㢌䛸㠀⨯ᝈ≟ 㻞㻞 㢌䛾䝃䞁䝥䝹䛻䛴䛔䛶䚸䜲䝹䝭䝘♫䛾 㻯㼔㼕㼜㻌㼍㼞㼞㼍㼥 䜢⏝䛔䛶 㻝㻣 ୓ಶ䛾 㻿㻺㻼 䜢⥙⨶ⓗ䛻ゎᯒ䛧䛯䛸䛣䜝䚸≉Ⓨᛶ䛶䜣䛛䜣⨯ᝈ≟⩌䛷ኚ␗䛾ฟ⌧㢖ᗘ䛾㧗䛔䠐䛴䛾 㻿㻺㻼 䜢ㄆ䜑䛯䚹䛣䜜䜙䛾≟䛻䛚䛡䜛䛶䜣䛛䜣Ⓨసᆺ䛿䚸ᙉ┤䞉㛫௦Ⓨస 㻟 㢌䚸↔Ⅼᛶ㐠ືⓎస 㻟 㢌䚸⾜ືⓎస 㻟 㢌䚸⮬ᚊ⚄⤒Ⓨస 㻝 㢌䛷䛒䛳䛯䚹≉Ⓨᛶ䛶䜣䛛䜣⨯ᝈ≟⩌䛷ኚ␗䛾ฟ⌧㢖ᗘ䛜㧗䛛䛳䛯 㻿㻺㻼 䛾ᗄ䛴䛛䛿ྠ୍ᰁⰍయୖ䛾୍㒊䛾㡿ᇦ䛻Ꮡᅾ䛧䚸㧗䛔䜸䝑䝈ẚ䜢♧䛧䛯䚹䛣䛾㡿ᇦ䛻䛿⬻䛾ᶵ⬟䛻㛵䜟䜛㑇ఏᏊ䚸䝠䝖䛾䛶䜣䛛䜣䛾ཎᅉ䛸䛺䜛㑇ఏᏊ䚸䛥䜙䛻≟䛾⑕ೃᛶ䛶䜣䛛䜣䛾㛵㐃㑇ఏᏊ䛸ྠ䛨ാ䛝䜢ᣢ䛴㑇ఏᏊ䛜ྵ䜎䜜䛶䛔䛯䚹䛣䜜䜙ೃ⿵㑇ఏᏊ䛾୍䛴䛻䛴䛔䛶䛿䚸≟䛾⬻⤌⧊䛛䜙ᢳฟ䛧䛯 㼙㻾㻺㻭 䜢⏝䛔䛶 㻾㼀㻙㻼㻯㻾 䜢⾜䛔䚸≟䛷Ⓨ⌧䛧䛶䛔䜛䛣䛸䜢☜ㄆ䛧䛯䚹䛥䜙䛻䚸ྠᵝ䛾 㻳㼃㻭㻿 䛻䜘䜛᳨⣴䜢௚≟✀䛾䛶䜣䛛䜣⨯ᝈ≟䛸㠀⨯ᝈ≟䛻䛴䛔䛶⾜䛳䛯䛜䚸ྠ䛨 㻿㻺㻼 䛻䛚䛡䜛䛶䜣䛛䜣䛸䛾㛵㐃ᛶ䛿௚≟✀䛷䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䚹㻌㻳㼃㻭㻿 䛻䛶䛶䜣䛛䜣䛸䛾㛵㐃ᛶ䜢ᣢ䛴 㻿㻺㻼 䛜᫂䜙䛛䛻䛺䛳䛯≟✀䛻䛚䛔䛶䚸᪂つ᳨య䛾཰㞟䜢⾜䛔䚸᪂つ⨯ᝈ≟ 㻥 㢌䛸㠀⨯ᝈ≟ 㻝㻟 㢌䛾᳨య䛜཰㞟䛥䜜䛯䚹᳨యᩘ䛜䛥䜙䛻ቑ䛘䛯䛾䛱䛻䚸Ⓨసᆺ䛾ゎᯒ䛸䚸䝀䝜䝮 㻰㻺㻭 䜢䝔䞁䝥䝺䞊䝖䛸䛧䛶䚸ೃ⿵㑇ఏᏊ䛻Ꮡᅾ䛩䜛 㻿㻺㻼 㡿ᇦ䛾 㻼㻯㻾 䜢⾜䛖䛣䛸䛻䜘䜛☜ㄆᐇ㦂䜢ᐇ᪋ணᐃ䛷䛒䜛䚹㻌≟䛾䜰䝖䝢䞊ᛶ⓶⭵⅖䛾㑇ఏᏊゎᯒ㻌ᰘ≟䛻䛚䛔䛶⥙⨶ⓗ䛺඲䝀䝜䝮㛵㐃ゎᯒ䜢⾜䛳䛯䛸䛣䜝䚸䠎䛴䛾㡿ᇦ䛻㞟୰䛧䛶 㻿㻺㻼 䛜ㄆ䜑䜙䜜䛯䚹⌧ᅾ䚸䛭䛾㡿ᇦ䛻⤠䛳䛶≟䛾䜰䝖䝢䞊ᛶ⓶⭵⅖䛻㛵㐃䛩䜛㑇ఏᏊ䛾ゎᯒ䛸௚䛾≟✀䛷䛾㑇ఏᏊゎᯒ䜢⾜䛳䛶䛚䜚䚸㐩ᡂᗘ䛸䛧䛶䛿 㻢㻜㻑䛷䛒䜛䚹㻌≟䛾䜰䝖䝢䞊ᛶ⓶⭵⅖㻔㼏㻭㻰㻕䛿⎔ቃ䜔㣗≀୰䛻ྵ䜎䜜䜛ᢠཎ䜈䛾㐣ᩄ཯ᛂ䛷䛒䜚䚸ே䛾䜰䝖䝢䞊ᛶ⓶⭵⅖䛸⑓ែ䛜㢮ఝ䛧䛶䛔䜛䚹㻔㻖㻝㻜㻕䛭䛾䜰䝺䝹䜼䞊䛾デ᩿䛾䛯䜑䛻ཎᅉ䜰䝺䝹䝀䞁䛾᳨ᰝἲ䛾㛤Ⓨ䜢⾜䛔䚸ᮏ◊✲䛻౑⏝䛩䜛≟䛾䜰䝺䝹䜼䞊䛾デ᩿䛻⏝䛔䛯㻔㞧ㄅㄽᩥ㻢㻤㻘㻤㻠㻕䚹䜎䛯䚸㼏㻭㻰 䛿ᐙ᪘ᛶ⣲ᅉ䛰䛡䛷䛺䛟≟✀䛻䜘䜚Ⓨ⏕⋡䛜␗䛺䜛䛣䛸䛛䜙䚸ே䛸ྠᵝ䚸⑌⑓䛸㑇ఏⓗ⫼ᬒ䛜ᐦ᥋䛻㛵୚䛧䛶䛔䜛䛣䛸䛜␲䜟䜜䜛䚹䛭䛣䛷䚸㼏㻭㻰 䛾Ⓨ⏕⋡䛜㧗䛔ᰘ≟䚸䝣䝺䞁䝏䝤䝹䝗䝑䜽䚸䝭䝙䝏䝳䜰䝎䝑䜽䝇䝣䞁䝖䚸䝖䜲䝥䞊䝗䝹䛾 㻠 ≟✀䛷䛒䜚䚸ྠ䛨ᆅᇦ䛷ᐊෆ㣫⫱䛥䜜䛶䛔䜛≟䛻㝈ᐃ䛧䛶䝃䞁䝥䝹䛾཰㞟䜢⾜䛳䛯䚹㼏㻭㻰 䛾デ᩿䛻䛴䛔䛶䛿 㻲㼍㼢㼞㼛㼠 䛾デ᩿ᇶ‽㻔㼂㼑㼠㼑㼞㼕㼚㼍㼞㼥㻌 㻰㼑㼞㼙㼍㼠㼛㼘㼛㼓㼥㻘㻌 㻞㻜㻜㻥㻕䛻‽䛨䛶ྠ䛨⋇་ᖌ䛻䜘䛳䛶デ᩿䜢⾜䛳䛯䚹䜎䛯䚸㼏㻭㻰 䛷䛿䛺䛔೺ᖖ≟䛿ᖺ㱋䚸⑓≧䚸᪤ Ṕ䜢䜒䛸䛻デ᩿䜢⾜䛔䚸䝃䞁䝥䝹䛾཰㞟䜢⾜䛳䛯䚹䛭䛾⤖ᯝ䚸㼏㻭㻰 Ⓨ⑕≟䛿䚸ᰘ≟ 㻠㻡 㢌䚸䝭䝙䝏䝳䜰䝎䝑䜽䝇䝣䞁䝖 㻟㻟 㢌䚸䝖䜲䝥䞊䝗䝹 㻟㻜 㢌䚸䝣䝺䞁䝏䝤䝹䝗䝑䜽 㻞㻜 㢌䛾ィ 㻝㻟㻜 㢌䛛䜙 㻰㻺㻭 䝃䞁䝥䝹䜢ᚓ䜛䛣䛸䛜䛷䛝䛯䚹䜎䛯䚸೺ᖖ≟䛿ᰘ≟ 㻞㻜 㢌䚸䝭䝙䝏䝳䜰䝎䝑䜽䝇䝣䞁䝖 㻠㻝 㢌䚸䝖䜲䝥䞊䝗䝹 㻝㻢 㢌䚸䝣䝺䞁䝏䝤䝹䝗䝑䜽 㻞 㢌䛾ィ 㻣㻥 㢌䛛䜙 㻰㻺㻭 䝃䞁䝥䝹䜢ᚓ䜛䛣䛸䛜䛷䛝䛯䚹䛥䜙䛻䚸㼏㻭㻰 Ⓨ⑕≟䛻䛚䛔䛶䝎䝙䜰䝺䝹䝀䞁≉␗ⓗ 㻵㼓㻱 ᢠయ᳨ᰝ䜢⾜䛳䛯䛸䛣䜝䚸ᰘ≟䛷䛿 㻣㻞㻚㻡㻑䛸௚䛾≟✀䜘䜚㧗䛔ᢠయಖ᭷⋡㻔䝣䝺䞁䝏䝤䝹䝗䝑䜽㻦㻡㻡㻚㻢㻑㻘㻌 䝭䝙䝏䝳䜰䝎䝑䜽䝇䝣䞁䝖㻦㻡㻜㻚㻜㻑㻘㻌 䝖䜲䝥䞊䝗䝹㻦㻞㻜㻑㻕䛷䛒䛳䛯䚹㻌㼏㻭㻰 䛾ཎᅉ㑇ఏᏊ䜢⥙⨶ⓗ䛻᥈⣴䛩䜛䛯䜑䚸ᰘ≟䛾䝃䞁䝥䝹䜢⏝䛔䛶䝎䝙䜰䝺䝹䝀䞁≉␗ⓗ 㻵㼓㻱 㝧ᛶ䛾 㼏㻭㻰 Ⓨ⑕≟ 㻞㻥 㢌䛸 㻵㼓㻱 㝜ᛶ䛷 㼏㻭㻰 ᮍⓎ⑕≟ 㻝㻥 㢌䛻䛚䛔䛶 㻰㻺㻭 䜰䝺䞊䜢⏝䛔䛯 㻿㻺㻼 䛾⥙⨶ⓗ㑇ఏᏊゎᯒ䜢⾜䛳䛯䚹䛭䛾⤖ᯝ䚸 㼏㻭㻰 Ⓨ⑕≟䛷㧗㢖ᗘㄆ䜑䜙䜜䛯 㻿㻺㻼䛿➨ 㻞㻥 ␒ᰁⰍయ䛷䛿 㻢㻜㻜㼗㼎㼜 ௨ෆ䛻䚸➨ 㻤 ␒ᰁⰍయ䛷䛿 㻥㻜㼗㼎㼜 ௨ෆ䛻㝈ᒁ䛧䛶ㄆ䜑䜙䜜䚸䛭䛾䛖䛱䜂䛸䛴䛿➨ 㻞㻥 ␒ᰁⰍయ䛻䝁䞊䝗䛥䜜䛶䛔䜛ᶵ⬟୙᫂䛺 㻾㻮㻹㻝㻞㻮 㑇ఏᏊ䛾䜶䜽䝋䞁ୖ11䛾 㻿㻺㻼 䛷䛒䜛䛣䛸䛜᫂䜙䛛䛸䛺䛳䛯䚹䜎䛯䚸➨ 㻤 ␒ᰁⰍయ䛾 㻝㻝 䛾 㻿㻺㻼 䛿⣽⬊Ṛ䛻㛵୚䛩䜛㻵㻼㼀㻷㻝 㑇ఏᏊୖ䛾䜲䞁䝖䝻䞁㡿ᇦ䛻఩⨨䛧䛶䛔䛯䚹ᮏ◊✲䛷䛿䜰䝺䝹䜼䞊䛻㛵㐃䛩䜛㑇ఏᏊୖ䜎䛯䛿㏆ഐ䛾 㻿㻺㻼 䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䛜䚸㻌 㼏㻭㻰 Ⓨ⑕≟䛷㧗㢖ᗘ䛻ㄆ䜑䜙䜜䛯 㻿㻺㻼 䛾ୖ఩ 㻡 䛴䛿඲䛶➨ 㻞㻥 ␒ᰁⰍయ䛾㝈ᒁ䛧䛯఩⨨䛻Ꮡᅾ䛩䜛䛣䛸䛛䜙䚸➨ 㻞㻥 ␒ᰁⰍయୖ䛻 㼏㻭㻰䛻㛵㐃䛩䜛㑇ఏᏊ㡿ᇦ䛜Ꮡᅾ䛩䜛䛣䛸䛜ᙉ䛟♧၀䛥䜜䛯䚹䜎䛯䚸䛭䜜䜙䛾 㻿㻺㻼 ㏆ഐ䛻䛿㻌 ᶵ⬟䛜୙᫂䛺 㻸㻻㻯㻝㻜㻜㻢㻤㻡㻢㻜㻡 䜒Ꮡᅾ䛩䜛䛣䛸䛛䜙䚸㼏㻭㻰 䛻㛵㐃䛩䜛㑇ఏᏊ䛷䛒䜛ྍ⬟ᛶ䜒⪃䛘䜙䜜䜛䚹㼏㻭㻰 Ⓨ⑕≟䛻㛵㐃䛩䜛㑇ఏᏊ䛾ྠᐃ䛻䛿⮳䜙䛺䛛䛳䛯䛜䚸㼏㻭㻰 䜈䛾㛵୚䛜␲䜟䜜䜛䝀䝜䝮㡿ᇦ䜢᥎ᐃ䛩䜛䛣䛸䛜䛷䛝䜛䛸⪃䛘䜙䜜䜛䚹㻌≟䛾䜰䝖䝢䞊ᛶ⓶⭵⅖䛻⑌ᝈཎᅉ㑇ఏᏊ䛾ゎᯒ䛾䛯䜑䛾ᇶ♏ⓗ䛺◊✲䛾䠍䛴䛸䛧䛶≟䛾㻯㻰㻝㻠 䛾㑇ఏᏊ䛿䜽䝻䞊䝙䞁䜾䜢⾜䛔䚸㻌 㻯㻰㻝㻠 䛾඲㛗㓄ิ䜢ึ䜑䛶Ỵᐃ䛧䛯䚹㻯㻰㻝㻠 䛿༢⌫䛾⣽⬊⾲㠃䛻୺䛻Ⓨ⌧䛩䜛䚸䜾䝷䝮㝜ᛶ⳦䛾⣽⬊ቨᵓᡂ䝍䞁䝟䜽䛷䛒䜛䝸䝫䝫䝸䝃䝑䜹䝷䜲䝗䠄㻸㻼㻿䠅䛾ㄆ㆑䛻㛵䜟䜛䜒䛾䛷䛒䜛䚹㻯㻰㻝㻠 䛾䝥䝻䝰䞊䝍䞊㡿ᇦ䛾ኚ␗䛿ႍᜥ䚸䜰䝖䝢䞊ᛶ⓶⭵
⅖䚸㰯⭍䝫䝸䞊䝥䛸䛾㛵ಀᛶ䛜ே䛷᫂䜙䛛䛸䛺䛳䛶䛔䜛䚹≟䛾 㻯㻰㻝㻠 䛿 㻞㻥㻜 ಶ䛾䜰䝭䝜㓟䛛䜙䛺䜚䚸∵䛸 㻣㻡㻑䚸⨺䛸 㻣㻢㻑䚸ே䛸 㻣㻞㻑䚸䝲䜼䛸 㻢㻤㻑䛾┦ྠᛶ䛷䛒䛳䛯䚹䛣䜜䛻䜘䜚䚸௒ᚋ䚸㑇ఏᏊከᆺ䛾ゎᯒ䜢㐍䜑䜛䛣䛸䛜䛷䛝䜛䚹㻌≟䛾 㻵㻸㻙㻝㻟 㑇ఏᏊゎᯒ㻌≟䛾⭘⒆Ⓨ⑕䝸䝇䜽䛾ೃ⿵䛸䛧䛶 㻵㻸㻙㻝㻟 㑇ఏᏊ䛾ከᆺ䛸䛾㛵㐃䛿ぢฟ䛫䛺䛛䛳䛯䛜䚸 㻵㻸㻝㻟㑇ఏᏊ䛾 㻡㼿㞄᥋㡿ᇦ䛻Ꮡᅾ䛩䜛 㻿㻺㻼 䛸䜰䝖䝢䞊ᛶ⓶⭵⅖䛸䛾㛵㐃䛜ㄆ䜑䜙䜜䚸㐩ᡂᗘ䛿㻣㻜䠂䛷䛒䛳䛯䚹㻵㻸㻙㻝㻟 㑇ఏᏊ䜢䝜䝑䜽䜰䜴䝖䛧䛯䝬䜴䝇䛷䛿 㻰㻹㻮㻭㻙㼀㻼㻭 ㄏⓎ⣔䛾 㻞 ẁ㝵⓶⭵⭘⒆ᙧᡂ䝰䝕䝹䛻䛚䛔䛶⭘⒆ᙧᡂ䛜ஹ㐍䛩䜛䛸ሗ࿌䛥䜜䛶䛔䜛䠄㻾㼛㼠㼔㼑㻌㼑㼠㻌㼍㼘㻚㻘㻞㻜㻝㻟䠅䚹䛭䛣䛷䚸≟䛾⭘⒆Ⓨ⑕䝸䝇䜽䛾ೃ⿵䛸䛧䛶 㻵㻸㻙㻝㻟 㑇ఏᏊ䛾ከᆺ䜢᳨⣴䛧䛯䚹䝁䝸䞊䚸䝅䜵䝑䝖䝷䞁䝗䞉䝅䞊䝥䝗䝑䜾䚸䝪䞊䝎䞊䞉䝁䝸䞊㻌 䚸䝷䝤䝷䝗䞊䝹䞉䝺䝖䝸䞊䝞䞊㻌 䚸䝂䞊䝹䝕䞁䞉䝺䝖䝸䞊䝞䞊䚸ᰘ≟䚸䝅䝧䝸䜰䞁䞉䝝䝇䜻䞊䚸䝅䞊䝈䞊䚸䝉䝑䝍䞊䚸䝫䝯䝷䝙䜰䞁䚸⛅⏣≟䚸䝡䞊䜾䝹䚸䝭䝙䝏䝳䜰䝎䝑䜽䝇䝣䞁䝖䚸䝆䝱䞊䝬䞁䞉䝅䜵䝟䞊㻌 䝗䛚䜘䜃⏥ᩫ≟䛛䜙䛺䜛ྜィ 㻟㻠 ಶయ䜢ᑐ㇟䛻䚸㻵㻸㻙㻝㻟 㑇ఏᏊ䛾㛤ጞ䝁䝗䞁䜘䜚⣙ 㻝㻚㻡㼗㼎㼜 ୖὶ䛛䜙⤊ጞ䝁䝗䞁䜘䜚 㻝㼗㼎㼜 ୗὶ䜎䛷䛾䚸⣙ 㻡㼗㼎㼜 䛾ሷᇶ㓄ิ䜢Ỵᐃ䛧䛯䚹䛭䛾⤖ᯝ䚸≟ 㻝㻝 ␒ᰁⰍయ䛾㓄ิ䠄㻺㻯㼋㻜㻜㻢㻡㻥㻟䠅䜢ᇶ‽䛸䛧䛶䚸㼓㻚㻞㻜㻥㻡㻣㻜㻣㻡㻌 㻭㻳㻳㼀㻳㻳㻳㻯㻭䠖䠷 㻝 䠹 䠚 䠷 㻞 䠹 䚸 㼓㻚㻞㻜㻥㻡㻣㻟㻤㻜㻌 㻳㻪㻯 䚸 㼓㻚㻞㻜㻥㻡㻣㻠㻞㻡㻌 㻭㻪㻳 䚸 㼓㻚㻞㻜㻥㻡㻣㻡㻤㻜㻌 㻳㻪㻭 䚸 㼓㻚㻞㻜㻥㻡㻣㻢㻞㻢㻌 㻯㻪㼀 䚸㼓㻚㻞㻜㻥㻡㻣㻤㻟㻝㻌 㻭㻪㻯䚸㼓㻚㻞㻜㻥㻡㻣㻤㻟㻢㻌 㻳㻪㻯䚸㼓㻚㻞㻜㻥㻡㻤㻝㻣㻜㻌 㻯㻪㼀䚸㼓㻚㻞㻜㻥㻡㻤㻟㻣㻤㻌 㻳㻪㻭䚸㼓㻚㻞㻜㻥㻡㻤㻣㻣㻠㻌 㻯㻪㼀䚸㼓㻚㻞㻜㻥㻡㻤㻤㻟㻜㻌 㻯㻪㻭䚸㼓㻚㻞㻜㻥㻡㻤㻥㻠㻜㻌 㻳㻪㻯䚸㼓㻚㻞㻜㻥㻢㻜㻜㻤㻞㻌 㻭㻪㻳䚸㼓㻚㻞㻜㻥㻢㻜㻥㻤㻥㻌 㻳㻪㻯䚸䛚䜘䜃 㼓㻚㻞㻜㻥㻢㻝㻡㻥㻤㻌㼀㻪㻯 䛾 㻝㻡 䜹ᡤ䛾ከᆺ䜢᳨ฟ䛧䛯䚹䛣䛾䛖䛱 㼓㻚㻞㻜㻥㻢㻜㻜㻤㻞㻌 㻭㻪㻳 䛿䚸➨䠏䜶䜽䝋䞁䛻Ꮡᅾ䛧䚸㻵㻸㻝㻟䛾 㻢㻟 ␒┠䛾䜰䝭䝜㓟䜢䝖䝺䜸䝙䞁䛛䜙䜰䝷䝙䞁䛻⨨᥮䛥䛫䜛䜒䛾䛷䛒䛳䛯䚹㻼㼛㼘㼥㼜㼔㼑㼚㻙㻞䠄㼔㼠㼠㼜㻦㻛㻛㼓㼑㼚㼑㼠㼕㼏㼟㻚㼎㼣㼔㻚㼔㼍㼞㼢㼍㼞㼐㻚㼑㼐㼡㻛㼜㼜㼔㻞㻛䠅䜢⏝䛔䛯᥎ᐃ䛛䜙䜰䝭䝜㓟⨨᥮䛻䜘䛳䛶䝍䞁䝟䜽㉁䛾㧗ḟᵓ㐀䛜኱䛝䛟ኚ໬䛩䜛䛣䛸䛜♧䛥䜜䛯䚹䛣䜜䜙䛾ኚ␗䛸ங⭢⭘⒆䛾㛵㐃ゎᯒ䜢ᐇ᪋䛧䛯䛜᭷ព䛺┦㛵䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䚹㻌䜎䛯䚸㻵㻸㻝㻟 䛿䚸䝠䝖䛷䛿䜰䝺䝹䜼䞊ᛶ⑌ᝈ䛸䛾㛵㐃䛜ሗ࿌䛥䜜䛶䛔䜛䛣䛸䛛䜙䚸䝞䜲䜸䝞䞁䜽䛻䛚䛔䛶཰㞟䛥䜜䛯䜰䝖䝢䞊ᛶ⓶⭵⅖䜢ᣢ䛴ᰘ≟䜢ᑐ㇟䛻┦㛵ゎᯒ䜢ᐇ᪋䛧䛯䚹㼓㻚㻞㻜㻥㻢㻜㻜㻤㻞㻌㻭㻪㻳䠄㼜㻚㻢㻟㻌㼀㼔㼞㻪㻭㼘㼍䠅ከᆺ䛷䛿䚸䜰䝖䝢䞊ᛶ⓶⭵⅖䛾ᝈ␆ 㻝㻜 ಶయ䛷䛿䚸㻳 ᆺᑐ❧㑇ఏᏊ䛾㢖ᗘ䛿㻚㻜㻚㻜㻡䚸㠀ᝈ␆ 㻝㻜 ಶయ䛷䛿 㻜㻚㻝㻜 䛷䛒䜚䚸ኚ␗ᆺᑐ❧㑇ఏᏊ䛾㢖ᗘ䛜ప䛔䛯䜑┦㛵ゎᯒ䛿ᐇ᪋䛷䛝䛺䛛䛳䛯䚹㼓㻚㻞㻜㻥㻡㻣㻢㻞㻢㻌 㻯㻪㼀㻌 㻔㼞㼟㻤㻥㻣㻟㻞㻥㻤㻕ከᆺ䛷䛿䚸ᝈ␆䠄㻯㻯 ᆺ 㻠 ಶయ䚸㻯㼀 ᆺ 㻟 ಶయ䚸㼀㼀 ᆺ 㻟 ಶయ䠅䛸㠀ᝈ␆ 㻟㻟 ಶయ䠄㻯㻯 ᆺ 㻠 ಶయ䚸㻯㼀 ᆺ 㻞㻝 ಶయ䚸㼀㼀 ᆺ 㻤 ಶయ䠅䛷䛒䛳䛯䚹ᝈ␆䛸㠀ᝈ␆㛵䛾㑇ఏᏊᆺ㢖ᗘ䛿䚸䜹䜲஧஌್䛜 㻟㻚㻥㻟㻥䠄䡌䠙㻜㻚㻜㻠㻣䠅䛷᭷ព䛻␗䛺䛳䛶䛔䛯䚹䛣䛾᫬䚸㑇ఏᏊᆺ 㻯㻯 ᆺ䜢䜰䝖䝢䞊ᛶ⓶⭵⅖䛾䝸䝇䜽ᆺ䛸௬ᐃ䛩䜛䛸䜸䝑䝈ẚ䛿 㻠㻚㻤㻟㻟䠄㻥㻡䠂ಙ㢗༊㛫䛿 㻝㻚㻜㻝㻤䠉㻞㻟㻚㻠㻡㻡䠅䛸䛺䛳䛯䚹䠄㻖㻝㻝䠅䛣䜜䛻䜘䜚ᰘ≟䛷䛿䚸㻵㻸㻝㻟 㑇ఏᏊ䛾 㻡㼿㞄᥋㡿ᇦ䛻Ꮡᅾ䛩䜛 㻿㻺㻼䠄㼞㼟㻤㻥㻣㻟㻞㻥㻤䠅䛿䚸䜰䝖䝢䞊ᛶ⓶⭵⅖䛾Ⓨ⑕⋡䝸䝇䜽䛸㛵㐃䛩䜛஦䛜♧၀䛥䜜䛯

12

㻔Ꮫ఍Ⓨ⾲䠍㻕䚹⌧ᅾ䚸ู䛾≟✀䛻ᑐ䛧䛶䜒┦㛵ゎᯒ䜢㐍䜑䛶䛔䜛䚹㻌䠘ඃ䜜䛯ᡂᯝ䛜䛒䛜䛳䛯Ⅼ䠚㻌ᾏእ䛻䛚䛡䜛≟䛾㑇ఏᏊ䝞䞁䜽䛸䛧䛶䛿䚸⡿ᅜ䛷䛿 㻺㻵㻴䚸䝝䝞䞊䝗኱Ꮫ䛸 㻹㻵㼀 䛾 㻮㼞㼛㼍㼐㻌㻵㼚㼟㼠㼕㼠㼡㼠㼑 䛻䜘䜛≟䛾䝀䝜䝮䝥䝸䝆䜵䜽䝖䚸䝶䞊䝻䝑䝟䛷䛿䠍䠎䛛ᅜ䠎䠌䛾⋇་኱Ꮫ䛻䜘䜛 㻸㼡㼜㼍 䛜᭷ྡ䛷䛒䜛䚹᪥ᮏ䛻䛚䛔䛶䛿≟䛾㑇ఏ㛵㐃⑌ᝈ䛾◊✲䜢⾜䛳䛶䛔䜛◊✲䛿Ꮡᅾ䛩䜛䛜䛭䜜䜙䛾◊✲⪅䛜⑌ᝈ㑇ఏᏊ䜢ඹ᭷䛩䜛䜘䛖䛺 㻰㻺㻭 䝞䞁䜽䛿䛺䛛䛳䛯䚹ᮏ䝥䝻䝆䜵䜽䝖䛻䜘䜚䚸䛣䜜䛛䜙≟䛾㑇ఏ㛵㐃⑌ᝈ≟䛾 㻰㻺㻭 䜔୍⯡㞟ᅋ≟䛾 㻰㻺㻭 䛺䛹䛜ඹ᭷䛷䛝䚸௒ᚋ䚸᪥ᮏ䛾≟䛾㑇ఏ㛵㐃⑌ᝈ䛾ཎᅉ㑇ఏᏊ䛾ゎᯒ䛾䛯䜑䛾᳨యᥦ౪䛾୰ᚰⓗ䛺ᙺ๭䜢ᢸ䛖䛣䛸䛜䛷䛝䜛䛸⪃䛘䜙䜜䜛䚹䜎䛯䚸䝛䝁䛾 㻰㻺㻭 䝞䞁䜽䛿ᅜ㝿ⓗ䛻䜒ᮍⓎ㐩䛷䛒䜛䚹ᮏ䝥䝻䝆䜵䜽䝖䛻䜘䜚䚸䝛䝁䛾 㻰㻺㻭 䝞䞁䜽䛾タ❧䛾䛝䛳䛛䛡䛜䛷䛝䛯䚹㻌≟䛾⥳ෆ㞀䛻䛚䛔䛶 㻿㻾㻮㻰䠍㑇ఏᏊ䛸䛾㛵㐃ᛶ䜢ぢฟ䛧䛯䚹䛣䛾 㻿㻾㻮㻰䠍㑇ఏᏊ䛾ኚ␗䛿ே䛾ṇᖖ║ᅽ⥳ෆ㞀䛾㛵㐃㑇ఏᏊ䛸䛧䛶䜒ㄆ䜑䜙䜜䛶䛚䜚䚸䛣䛾≟䛾⥳ෆ㞀䛜ே䛾䝰䝕䝹䛸䛧䛶䜒᭷⏝䛷䛒䜛ྍ⬟ᛶ䛜♧၀䛥䜜䛯䚹㻌≟䛻䛚䛡䜛䝠䝖䛾㐺ྜᛶ䛾ほⅬ䛛䜙䚸䛭䛾୰䛷᪥ᮏ≟䛾䜒䛴㑇ఏⓗ≉ᛶ䜢᫂䜙䛛䛻䛧䛯䚹᪥ᮏ≟䛾⾜ື≉ᛶ䛻㛵䜟䜛㑇ఏᏊ⩌䜢ㄪᰝ䛧䛯䛸䛣䜝䚸䜸䜸䜹䝭䛸ྠᵝ䛾䜒䛾䜢ᣢ䛴䛣䛸䛜♧䛥䜜䚸♫఍ᛶ䛛䜙≟䛾㐍໬䜢᫂䜙䛛䛻䛧䛴䛴䛒䜛䚹㻌㻌 ≟䛾 㼀㻲㻺㻙䃐䛾䝬䜲䜽䝻䝃䝔䝷䜲䝖䛻䛴䛔䛶䛿䛣䜜䜎䛷䛻ሗ࿌䛜䛺䛟䚸䛣䛾▱ぢ䛜ึ䜑䛶䛾ሗ࿌䛷䛒䛳䛯䚹㻌
㻭㼘㼛㼜㼑㼏㼕㼍㻌㼄 ⑕䛻㛵䛩䜛ཎᅉ㑇ఏᏊ䛿䛣䜜䜎䛷䛻ሗ࿌䛥䜜䛶䛚䜙䛪䚸௒ᅇ䚸ೃ⿵㑇ఏᏊ䜢⤠䜚㎸䜐䛣䛸䛜䛷䛝䚸≟⏤᮶䛾䛭䛾㑇ఏᏊ䛾Ⓨ⌧䜢ホ౯䛩䜛䜰䝑䝉䜲⣔䜢ᵓ⠏䛧䛯䚹㻌䛶䜣䛛䜣ೃ⿵㑇ఏᏊ䜢⤠䜚㎸䜣䛰≟✀䛿䚸ୡ⏺ⓗ䛻䜒୍⯡ⓗ䛺≟✀䛷䛒䜚䚸⾲⌧ᆺ䛻䛴䛔䛶䛿䚸≟䛻䜘䛟ぢ䜙䜜䜛䝍䜲䝥䛾䛶䜣䛛䜣䛷䛒䛳䛯䚹䛣䜜䛻ᑐ䛧䚸⌧ᅾ䜎䛷䛻ሗ࿌䛾䛒䜛≟䛾䛶䜣䛛䜣䛾㛵㐃㑇ఏᏊ䛿䚸䠎䛴䛸䜒ẚ㍑ⓗ≉Ṧ䛺≟✀䛻䛚䛡䜛≉Ṧ䛺䝍䜲䝥䛾䛶䜣䛛䜣䛷䛒䜛䚹䛧䛯䛜䛳䛶䚸ᡃ䚻䛾ᡂᯝ䜢䜒䛸䛻䛧䛯䛣䜜䛛䜙䛾◊✲䛿䚸䜘䜚ᗈ䛟⋇་⒪䛻㈉⊩䛩䜛䜒䛾䛸⪃䛘䜙䜜䜛䚹㻌ೃ⿵ 㻿㻺㻼 㑇ఏᏊ䛛䜙ཎᅉ㑇ఏᏊ䛜≉ᐃ䛥䜜䜜䜀䚸㼏㻭㻰 ዲⓎ≟✀䛸䛺䜚䛖䜛≟䜢デ᩿䛧䚸䝤䝸䞊䝕䜱䞁䜾䛻⏝䛔䛺䛔䜘䛖䛻デ᩿䛩䜛䛣䛸ྍ⬟䛷䛒䜛䚹㻌≟䛾 㻝㻝 ␒ᰁⰍయ 㻵㻸㻙㻝㻟 㑇ఏᏊ䛾䝥䝻䝰䞊䝍䞊㡿ᇦ 㼓㻚㻞㻜㻥㻡㻣㻜㻣㻡 䛻䛚䛔䛶㼇㻭㻳㻳㼀㻳㻳㻳㻯㻭㼉䜢༢఩䛸䛩䜛཯᚟ከᆺ䜢Ⓨぢ䛧䛯䚹㻝㻜㻜㻜 ಶయศ䛾≟ 㻰㻺㻭 䜢཰㞟䛷䛝䛯䛣䛸䛛䜙䚸ከᆺゎᯒ䛻᭷ຠ䛺䝸䝋䞊䝇䜢ᚓ䜛䛣䛸䛜䛷䛝䛯䚹㻌䠘ၥ㢟Ⅼ䠚㻌
≟䛾⑌ᝈ䛾 㻰㻺㻭 ཰㞟䛾యไ䛿ᩚ䛔䚸䛭䛾༑ศ䛺⑌ᝈ᝟ሗ䜒ᚓ䜙䜜䛯䛜䚸ᑓ㛛䛻䛭䛾᳨య䜔䝕䞊䝍⟶⌮䜢⾜䛖ᖖ໅䛾⫋ဨ䛜ᮍᩚഛ䛷䛒䜛䚹௒ᚋ䚸䛣䛾䝥䝻䝆䜵䜽䝖䛾 㻰㻺㻭 䝞䞁䜽䜢ᜏஂⓗ䛺኱Ꮫ䛾᪋タ䛸䛧䛶㐠⏝䛩䜛䛣䛸䛜ㄢ㢟䛸⪃䛘䛶䛔䜛䚹䜎䛯䚸⌧ᅾ䚸᪥ᮏㄒ䛾䝩䞊䝮䝨䞊䝆䛧䛛సᡂ䛧䛶䛺䛔䚹௒ᚋ䚸ⱥㄒ䛾䝩䞊䝮䝨䞊䝆䜢సᡂ䛧䛶ᾏእ䛾◊✲⪅䛻䜒᝟ሗ䜢౪⤥䛧䚸ᾏእ䛾◊✲⪅䛸䜒᝟ሗ䜔ඹྠ◊✲䛜⾜䛘䜛యไ䜢ᩚ䛘䛯䛔䚹㻌║⑌ᝈ⑕౛䛾 㻰㻺㻭 䝃䞁䝥䝹䛾཰㞟䛜䚸║⛉ᑓ㛛་䛜デ᩿䛥䜜䛯⑕౛䛻㝈ᐃ䛥䜜䛯䛣䛸䛷䚸䜘䜚ከ䛟䛾䝃䞁䝥䝹䛜཰㞟䛷䛝䛺䛛䛳䛯䚹䜎䛯䚸䛭䛾཰㞟䛻ᇶ䛵䛟ゎᯒ䛜䚸║⛉䛾ᑓ㛛▱㆑䜢ᚲせ䛸䛧䛯䛣䛸䛷䚸║⛉Ꮫⓗゎᯒ䛻せ䛩䜛ேᮦ☜ಖ䛿䜋䛸䜣䛹䛷䛝䛺䛛䛳䛯䚹㻌≟䛻䛚䛡䜛䝠䝖䛾㐺ྜᛶ䛾ほⅬ䛛䜙䚸᪥ᮏ≟䛾䜒䛴㑇ఏⓗ≉ᛶ䜢᫂䜙䛛䛻䛧䛯䚹᪥ᮏ≟䛾⾜ື≉ᛶ䛻㛵䜟䜛㑇ఏᏊ⩌䜢ㄪᰝ䛧䛯䛸䛣䜝䚸䜸䜸䜹䝭䛸ྠᵝ䛾䜒䛾䜢ᣢ䛴䛣䛸䛜♧䛥䜜䚸♫఍ᛶ䛛䜙≟䛾㐍໬䜢᫂䜙䛛䛻䛧䛴䛴䛒䜛䚹㻌ᵓ⠏䛧䛯䝺䝫䞊䝍䞊䜰䝑䝉䜲⣔䛾཯ᛂᛶ䛿䚸⏝䛔䜛ᇵ㣴⣽⬊ᰴ䛻䜘䛳䛶ᙳ㡪䜢ཷ䛡䜛䚹䛔䛟䛴䛛䛾ᇵ㣴⣽⬊ᰴ䜢᳨ウ䛧䚸ẚ㍑ⓗ཯ᛂᛶ䛾Ⰻ䛔⣽⬊䜢㑅ᢥ䛧䛯䛜䚸༑ศ䛸䛿ゝ䛘䛪䚸䜘䜚

13

Ⓨ⌧ᛂ⟅䛾ඃ䜜䛯⣽⬊ᰴ䜢฼⏝䛧䛯䜰䝑䝉䜲⣔䛻ᨵⰋ䛩䜛ᚲせ䛜䛒䜛䚹㻌㑇ఏᏊኚ␗䛸䛶䜣䛛䜣Ⓨ⑕䛾㛵㐃ᛶ䜢ゎᯒ䛩䜛䛣䛸䛸䚸ཎᅉ㑇ఏᏊ䛾ᶵ⬟ゎᯒ䛸Ⓨ⑕䛸䛾㛵㐃䛾᳨ウ䜢⾜䛖䛣䛸䛜䚸㑇ఏᏊྠᐃ䛾䛯䜑䛻ᐇ᪋䛩䜉䛝㡯┠䛸䛧䛶ṧ䛳䛶䛔䜛䚹㻌ೃ⿵ 㻿㻺㻼 㑇ఏᏊ䛜 㼏㻭㻰 䛾⑓ែ䛻㛵୚䛧䛶䛔䜛䛛୙᫂䛷䛒䜛䛯䜑䚸㻿㻺㻼 䛸⑓ែ䛾㛵㐃ᛶ䛻䛴䛔䛶ゎᯒ䛩䜛ᚲせ䛜䛒䜛䚹㻌䠘ホ౯యไ䠚㻌
ྛ◊✲⪅䛿ᖺ䠎ᅇ䛾◊✲఍㆟䛷༙ᖺศ䛾◊✲ᡂᯝ䜢Ⓨ⾲䛧䛶䛔䜛䚹䛣䛾఍㆟䛻䛿ඹྠ◊✲ᶵ㛵䛾⌮໬Ꮫ◊✲ᡤ䛾ஂಖ඘᫂ඛ⏕䚸㮵ඣᓥ኱Ꮫ䛾኱࿴ඛ⏕䚸ᮾி㎰ᕤ኱Ꮫ䛾኱᳃ඛ⏕䜒䛤ฟᖍ䛔䛯䛰䛝ホ౯䜔䝁䝯䞁䝖䜢䛔䛯䛰䛔䛯䚹䛥䜙䛻௚䛾ศᢸ◊✲⪅䛛䜙䛾䝁䝯䞁䝖䜒ཧ⪃䛻䛧䛶䚸◊✲௦⾲⪅䛜ホ౯䛧䛶䛔䜛䚹◊✲㈝䛿᭱ప㝈ᚲせ䛺◊✲㈝䜢ศ㓄䛧䛶䚸䛒䛸䛿䛭䛾ホ౯䛻ᛂ䛨䛶㑇ఏᏊゎᯒ䛺䛹㈝⏝䛻඘䛶䜛䚹䛥䜙䛻䛭䛾ホ౯䜢ᐈほⓗ䚸බᖹ䛻䛩䜛䛯䜑䛻እ㒊ホ౯ጤဨ䛸䛧䛶䚸ᮾி኱Ꮫ⋇་Ꮫ⛉㻌 ㎷ᮏ㻌 ඖᩍᤵ䚸໭ᾏ㐨኱Ꮫ⋇་Ꮫ⛉㻌 ✄ⴥ㻌 ╬ᩍᤵ䚸㓗㎰Ꮫᅬ኱Ꮫ⋇་Ꮫ⛉㻌 ໭ᮧ㻌 ᾈᩍᤵ䛻䛚㢪䛔䛧䛶୰㛫ホ౯఍䜢⾜䛔䚸ᮏ䝥䝻䝆䜵䜽䝖䛾ྛ◊✲⪅䛜◊✲ᡂᯝ䛾Ⓨ⾲䜢⾜䛔䚸ホ౯䞉䝁䝯䞁䝖䜢䛔䛯䛰䛔䛯䚹㻌䠘◊✲ᮇ㛫⤊஢ᚋ䛾ᒎᮃ䠚㻌ᮏ䝥䝻䝆䜵䜽䝖⤊஢ᚋ䜒≟䛾 㻰㻺㻭 䝞䞁䜽䛻䛚䛡䜛 㻰㻺㻭 ᳨య䛾཰㞟䜢⾜䛖ணᐃ䛷䛒䜛䚹㻌཰㞟䛧䛯║⑌ᝈ⑕౛ 㻰㻺㻭 䝃䞁䝥䝹䛿䚸ᮍ䛰䜋䛸䜣䛹ᡭ௜䛛䛪䛷䚸ရ✀౫Ꮡᛶ║⑌ᝈ䛾᥈ồ䜢௒ᚋ䜒⥅⥆䛧䛶⾜䛖䚹䜎䛯䚸ⓑෆ㞀䛚䜘䜃⥳ෆ㞀䛾㑇ఏᏊ䛾୍㒊䜢ぢฟ䛧䛯䛜䚸䛭䛾ᶵ⬟ⓗ䛺ᙺ๭䛜䜎䛰୙᫂䛾䛯䜑䚸◊✲䜢⥅⥆䛧䛶䛭䛾㑇ఏᏊ䛾ᶵ⬟䛾ゎᯒ䜢⾜䛖䚹㻌ᰘ≟䜢ᮦᩱ䛻䚸♫఍ᛶ䛾㐍໬䛻䛛䛛䜟䜛㑇ఏᏊ䜢䚸ḟୡ௦ᆺ䝅䞊䜽䜶䞁䝃䞊䛷ᢳฟ䛧䛴䛴䛒䜛䚹䛣䜜䜙䛾㑇ఏᏊ䛸⾜ື䛸䛾㛵㐃ゎᯒ䜢ᐇ᪋䛩䜛ணᐃ䛷䛒䜛䚹㻌㻌 ≟䛾䜰䝻䝨䝅䜰 㼄 䛾ཎᅉ䛸䛺䜛ೃ⿵㑇ఏᏊ䜢ぢฟ䛧䚸䛭䛾㑇ఏᏊⓎ⌧䛾ホ౯⣔䜒ᵓ⠏䛧䛯䚹䛧䛛䛧䚸ᶵ⬟ⓗᙺ๭䛿୙᫂䛷䛒䜚䚸Ⓨ⑕䝯䜹䝙䝈䝮䛾ゎ᫂䜔ཎᅉ㑇ఏᏊ䛸䛧䛶䛾≉ᐃ䛻䛿⮳䛳䛶䛚䜙䛪䚸䜎䛯䛭䛾㑇ఏᏊⓎ⌧䜢ᅇ᚟䛩䜛ලయⓗ䛺≀㉁䜒ぢ䛴䛛䛳䛶䛔䛺䛔䚹ೃ⿵㑇ఏᏊ䛸⑌ᝈ䛸䛾㛵ಀ䛻䛴䛔䛶⮫ᗋᛂ⏝䛜ྍ⬟䛸䛺䜛䝕䞊䝍䛜ᚓ䜙䜜䜛䜘䛖䛻௒ᚋ䜒◊✲䜢⥅⥆䛩䜛䚹䜎䛯䚸䝬䜲䜽䝻䝏䝑䝥ḟୡ௦䝅䞊䜿䞁䝃䞊䜢฼⏝䛧䛶䚸䛥䜙䛻ከ䛟䛾≟䛾䜰䝻䝨䝅䜰 㼄⑌ᝈೃ⿵㑇ఏᏊ䜢Ⓨぢ䛧䚸Ⓨ⑕ᶵᵓ䛾ゎ᫂䜈䛾㈉⊩䛸㑇ఏᏊデ᩿ἲ䛾㛤Ⓨ䛻ྲྀ⤌䜐䚹㻌㻌 ≟䛾≉Ⓨᛶ䛶䜣䛛䜣䛾ೃ⿵㑇ఏᏊ䜢⤠䜚㎸䜣䛰䛜䚸ྠᐃ䜎䛷䛿⮳䛳䛶䛔䛺䛔䛯䜑䚸◊✲䜢⥅⥆䛧䛶䚸㑇ఏ⤫ィᏛⓗᡭἲ䛻䜘䛳䛶㑇ఏᏊኚ␗䛸䛶䜣䛛䜣Ⓨ⑕䛾㛵㐃ᛶ䜢ゎᯒ䛧䚸䛥䜙䛻ཎᅉ㑇ఏᏊ䛾ᶵ⬟ゎᯒ䛸Ⓨ⑕䛸䛾㛵㐃䛾᳨ウ䜢⾜䛖䚹Ⓨసᆺ䛸㑇ఏᏊኚ␗䛸䛾㛵㐃ᛶ䜒ㄪ䜉䜛ணᐃ䛷䛒䜛䚹䛭䛾䛯䜑䛻௒ᚋ䜒◊✲䜢⥅⥆䛩䜛䚹㻌䠘◊✲ᡂᯝ䛾๪ḟⓗຠᯝ䠚㻌㻌 ᮧୖ䛜ᮏ◊✲䛷ᵓ⠏䛧䛯⣽⬊䝺䝧䝹䛷䛾㑇ఏᏊⓎ⌧ホ౯⣔䛿⬺ẟ⑕䜢ᑐ㇟䛸䛧䛯⸆๣㛤
Ⓨ䛾䛯䜑䛾ೃ⿵≀㉁䝇䜽䝸䞊䝙䞁䜾䜔స⏝ᶵᗎゎ᫂䛻㈉⊩䛷䛝䜛ྍ⬟ᛶ䛜䛒䜛䚹௻ᴗ䛸䛾ඹྠ◊✲䜒㐍䜑䛶䛔䜛䚹䛥䜙䛻ᮏ◊✲䜢㐍䜑䜛୰䛷◊✲⪅㛫䛷䛾≟䛾㑇ఏ㛵㐃⑌ᝈ䛾᝟ሗ஺᥮䛜άⓎ䛻䛺䜚䚸䝅䞊䝈䞊⥙⭷๤㞳䞉◪Ꮚయኚᛶ⑕䛾ཎᅉ㑇ఏᏊ᥈⣴䛸㑇ఏᏊデ᩿ἲ䛾㛤Ⓨ䚸≟䛾⫧‶య㉁䛻㛵୚䛩䜛㑇ఏᏊ䛾ከᆺゎᯒ䚸ኚᛶᛶ⬨㧊⑕䛸䜶䞊䝷䝇䞉䝎䞁䝻䝇⑕ೃ⩌䛾㛵㐃ೃ⿵㑇ఏᏊ䛾᥈⣴◊✲䛻䜒᪂䛯䛻╔ᡭ䛧䛶䛚䜚䚸䛣䜜䜙䜢ᘬ䛝⥆䛝⾜䛖䚹㻌≉チ⏦ㄳ㻌༳∾ಙ⾜䠄௦⾲䠅䛿≟䛾⥳ෆ㞀䜢デ᩿䛩䜛᪉ἲཬ䜃䜻䝑䝖䠄≉㢪 㻞㻜㻝㻝㻙㻝㻡㻞㻣㻠㻡䠅䚸ᾏእฟ㢪䠄㻞㻜㻝㻞 ᖺ䚸㻼㻯㼀㻛㻶㻼㻞㻜㻝㻞㻛㻜㻢㻣㻝㻣㻟䠅ཬ䜃䠄㻞㻜㻝㻟 ᖺ䚸➨ 㻝㻠㻛㻞㻟㻞㻚㻝㻜㻣 ྕ䚸㼁㻿䠅㻌㰻⸨ᘺ௦Ꮚ㻌 ᶓ᳃⛱䛂䛶䜣䛛䜣Ⓨస䛾䝰䝙䝍䝸䞁䜾䝅䝇䝔䝮ཬ䜃䝰䝙䝍䝸䞁䜾᪉ἲ䛃≉チබ㛤 㻞㻜㻝㻠㻙㻞㻝㻣㻢㻠㻥㻌14㻌䠍䠎㻌 䜻䞊䝽䞊䝗䠄ᙜヱ◊✲ෆᐜ䜢䜘䛟⾲䛧䛶䛔䜛䛸ᛮ䜟䜜䜛䜒䛾䜢䠔㡯┠௨ෆ䛷グ㍕䛧䛶䛟䛰䛥䛔䚹䠅㻌䠄䠍䠅㻌 ≟㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 䠄䠎䠅㻌 㻌 㑇ఏ⑓㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 䠄䠏䠅㻌 㑇ఏᏊ㻌 㻌 㻌 㻌 㻌 㻌 㻌䠄䠐䠅㻌 㑇ఏᏊゎᯒ㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 䠄䠑䠅㻌 㻌 㑇ఏᏊከᙧ㻔㻿㻺㻼㻕㻌 㻌 㻌 㻌 㻌 㻌 㻌 䠄䠒䠅㻌 䝞䜲䜸䝞䞁䜽㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌䠄䠓䠅㻌 㑇ఏᏊ㈨※㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 䠄䠔䠅㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌㻌䠍䠏㻌 ◊✲Ⓨ⾲䛾≧ἣ䠄◊✲ㄽᩥ➼බ⾲≧ἣ䚹༳ๅ୰䜒ྵ䜐䚹䠅㻌ୖグ䚸䠍䠍㻔䠐㻕䛻グ㍕䛧䛯◊✲ᡂᯝ䛻ᑐᛂ䛩䜛䜒䛾䛻䛿䠆䜢௜䛩䛣䛸䚹㻌㻌䠘㞧ㄅㄽᩥ䠚㻌
1)
Ohkita M, Nagasawa M, Kazutaka M, Kikusui T.: Owners' direct gazes increase dogs'
attention-getting behaviors. Behav Processes. in press. ᰝㄞ᭷
2)
Miyaji K, Okamoto N, Saito S, Yasueda H, Takase Y, Shimakura H, Saito S, Sakaguchi
M. :Cross-reactivity between major IgE core epitopes on Cry j 2 allergen of Japanesecedar pollen and relevant sequences on Cha o 2 allergen of Japanese cypress pollen.
Allergol Int in press. ᰝㄞ᭷
3)
Shigehisa R, Uchiyama J, Kato S, Takemura-Uchiyama I, Yamaguchi K, Miyata R,
Ujihara T, Sakaguchi Y, Okamoto N, Shimakura H, Daibata M, Sakaguchi M,
Matsuzaki S.: Characterization of Pseudomonas aeruginosa phage KPP21 belonging tofamily Podoviridae genus N4-like viruses isolated in Japan. Microbiol Immunol in press.
ᰝㄞ᭷4)
Kawarai, S, Fujimoto, A, Nozawa, G, Kanemaki, N, Madarame, H, Shida, T, Kiuchi, A.
Evaluation of weekly bathing in allergic dogs with methicillin-resistant Staphylococcal
colonization. Jpn J Vet Res in press ᰝㄞ᭷
5)
Katakawa Y, Funaba M, Murakami M.: Smad8/9 is regulated through the BMP pathway.
J Cell Biochem [Epub ahead of print] 2016. ᰝㄞ᭷
6)Kida R, Yoshida H, Murakami M, Shirai M, Hashimoto O, Kawada T, Matsui T, Funaba
M.: Direct action of capsaicin in brown adipogenesis and activation of brown adipocytes.
Cell Biochem Funct 34, 34-41, 2016. ᰝㄞ᭷
7)
Shigehis R, Uchiyama J, Kato S, Takemura-Uchiyama I, Yamaguchi K, Miyata R,
Ujihara T, Sakaguchi Y, Okamoto N, Shimakura H, Daibata M, Sakaguchi M,
Matsuzaki, S.:㻌 Characterization of Pseudomonas aeruginosa phage KPP21 belonging tofamily Podoviridae genus N4-like viruses isolated in Japan. Microbiol Immunol, inpress.㻌 ᰝㄞ᭷
8)Takai T, Okamoto Y, Okubo K, Nagata M, Sakaguchi M, Fukutomi Y, Saito A, Yasueda
H, Masuyama, K.䠖 Japanese Society of Allergology task force report on standardizationof house dust mite allergen vaccines. Allergol Int 64,181-186, 2015.㻌 ᰝㄞ᭷
9)Mineshige T, Kawarai S, Yauchi T, Segawa K, Neo S, Sugahara G, Kamiie J, Hisasue M,
Shirota K.㻌 Cutaneous epitheliotropic T-cell lymphoma with systemic dissemination ina dog. J Vet Diagn Invest. 2016 Mar 7. ᰝㄞ᭷10) Horimoto T, Gen F, Murakami S, Iwatsuki-Horimoto K, Kato K, Hisasue M, Sakaguchi
M, Nidom CA, Kawaoka Y. Cats as a potential source of emerging influenza virus
infections. Virologica Sinica 30,221-223,2015.㻌 ᰝㄞ᭷11) (㻖3)༳∾ಙ⾜䚸ᕷᕝ㝧୍ᮁ䚸ᕝཎ஭᫴ᖹ䚸ⴠྜ⚽἞䠊㯞ᕸ኱Ꮫ║⛉䛻᮶㝔䛧䛯⥳ෆ

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㞀⑕౛䛾㝶క⑕䛾ศ㢮䚸᪥ᮏ⋇་ᖌ఍㞧ㄅ䚸68,55-58,㻌 2015䠊ᰝㄞ᭷(㻖6)Tonoike A, Nagasawa M, Mogi K, Serpell J, Ohtsuki H, Kikusui T. Comparison ofowner-reported behavioral characteristics among genetically clustered breeds of dog(Canis familiaris). Sci Rep Dec 18;5:17710.ᰝㄞ᭷
Nagasawa M, Mogi K, Serpell J, Kikusui T. Comparison of behavioral characteristics ofdogs in the United States and Japan. J Vet Med Sci, in press㻌 ᰝㄞ᭷(㻖7)Tonoike A, Terauchi G, Inoue-Murayama M, Nagasawa M, Mogi K, Kikusui T.㻌 The
Frequency Variations of the Oxytocin Receptor Gene Polymorphisms among Dog
Breeds. J Azabu Uni 27,11-18, 2015㻌 ᰝㄞ᭷(㻖8)Tonoike A, Hori Y, Inoue-Murayama M, Konno A, Fujita K, Miyado M, Fukami M,
Nagasawa M, Mogi K, Kikusui T.㻌 Copy number variations in the amylase gene inJapanese native dog breeds. Anim Genet 46:580-3. doi: 10.1111/age.12344,2015 㻌 ᰝㄞ᭷Nagasawa M, Mitsui S, En S, Ohtani N, Ohta M, Sakuma Y, Onaka T, Mogi K, Kikusui
T: Oxytocin-gaze positive loop and the coevolution of human-dog bonds. Science 348,
333-6, 2015.㻌 ᰝㄞ᭷
Romero T, Nagasawa M, Mogi K, Hasegawa T, Kikusui T.: Intranasal administration ofoxytocin promotes social play in domestic dogs. Commun Integra Biol 112, E311-320,
2015.㻌 ᰝㄞ᭷
Kawarai S, Hisasue M, Matsuura S, Ito T, Inoue Y, Neo S, Fujii Y, Madarame H, Shirota
K, Tsuchiya R. Canine pemphigus foliaceus with concurrent immune-mediatedthrombocytopenia. Am Anim Hosp Assoc 51,56-63, 2015. ᰝㄞ᭷
Nishino Y, Murakami M, Funaba M.:Expression and role of the TGF-ȕ IDPLO\ LQ JOLDO
cells infected with Borna disease virus. Microbes Infect [Epub ahead of print] 2015. ᰝㄞ᭷Kasai T, Kato Y, Saegusa S, Murakami M.:Distribution of major staphylococcalca
cassette chromosome mec types and exfoliative toxin genes in Staphylococcus
pseudintermedius strains from dogs with superficial pyoderma. J Azabu Uni [in print]
2015.ᰝㄞ᭷
Yamada A, Kodo Y, Murakami M, Kuroda M, Aoki T, Fujimoto T, Arai K.:Hybrid originof gynogenetic clones and the introgression of their mitochondrial genome into sexual
diploids through meiotic hybridogenesis in the loach, Misgurnus anguillicuadatus. J
Exp Zool A Ecol Genet Physiol 323,593-606,2015. ᰝㄞ᭷
Shirai M, Nomura R, Kato Y, Murakami M, Kondo C, Takahashi S, Yamasaki Y,
Matsumoto-Nakano M, Arai N, Yasuda H, Nakano K, Asai F.: Short communication:
Distribution of Porphyromonas gulae fimA genotypes in oral specimens from dogs withmitral regurgitation. Res Vet Sci 102,49-52,2015. ᰝㄞ᭷
Kanamori Y, Murakami M, Matsui T, Funaba M.: Role of TPA-responsive element in
hepcidin transcription induced by the bone morphogenetic protein pathway. Biochem
Biophys Res Commun 466, 162-6,2015. ᰝㄞ᭷
Murakami M, Ohi M, Ishikawa S, Shirai M, Horiguchi H, Nishino Y, Funaba M.
Adaptive expression of uncoupling protein 1 in the carp liver and kidney in response tochanges in ambient temperature. Comp Biochem Physiol Part A, 185, 142-9,2015. ᰝㄞ᭷Okubo T, Hayashi D, Yaguchi T, Fujita Y, Sakaue M, Suzuki T, Tsukamoto A,㻌
Murayama O, Lynch J, Miyazaki Y, Tanaka K, Takizawa T.: Differentiation of rat㻌
adipose tissue-derived stem cells into neuron-like cells by valproic acid, a㻌 histone

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deacetylase inhibitor. Exp Anim [Epub ahead of print] , PMID: 26411320,2015.ᰝㄞ᭷
Yuan Y, Kitamura-Muramatsu Y, Saito S, Ishizaki H, Nakano M, Haga S, Matoba K,
Ohno A, Murakami H, Takeshima SN, Aida Y. Detection of the BLV provirus from nasal
secretion and saliva samples using BLV-CoCoMo-qPCR-2: Comparison with blood
samples from the same cattle. Virus Res 210:248-54,2015. ᰝㄞ᭷ᮧୖ⿱ಙ䚸㛫㝧Ꮚ: HIV-1 䜰䜽䝉䝃䝸䞊⺮ⓑ㉁ Vpr 䛸ᐟ୺ᅉᏊ. ⮫ᗋච␿䞉䜰䝺䝹䜼䞊⛉. 63:484-488,2015. ᰝㄞ↓
Horimoto T, Gen F, Murakami S, Iwatsuki-Horimoto㻌 K, Kato㻌 K, Akashi H, Hisasue M,
Sakaguchi M, Kawaoka Y, Maeda K: Serological evidence of infection of dogs withhuman influenza viruses in Japan. Vet Rec 174, 96, 2014. ᰝㄞ᭷㧗஭ᩄᮁ, ᒸᮏ⨾Ꮥ, ኱ஂಖබ⿱, Ọ⏣┿, 㜰ཱྀ㞞ᘯ, ⚟෠཭㤿, 㰻⸨᫂⨾, Ᏻᯞᾈ, ቑᒣᩗ♸:䝎䝙䜰䝺䝹䝀䞁䝽䜽䝏䞁ᶆ‽໬䛻㛵䛩䜛᪥ᮏ䜰䝺䝹䜼䞊Ꮫ఍䝍䝇䜽䝣䜷䞊䝇ሗ࿌.䜰䝺䝹䜼䞊㻌 63,1229-40,2014.㻌 ᰝㄞ᭷
Romero T, Nagasawa M, Mogi K, Hasegawa T, Kikusui T.: Oxytocin promotes social
bonding in dogs. Proc Natl Acad Sci U S A, 111, 9085-90, 2014.㻌 ᰝㄞ᭷
Nagasawa M, Shimozawa A, Mogi K, Kikusui T.: N-acetyl-D-mannosamine treatment
alleviates age-related decline in place-learning ability in dogs. J Vet Med Sci 76, 757-61,
2014.㻌 ᰝㄞ᭷( 9)Watanabe M, Tanaka K, Takizawa T, Segawa K, Neo S, Tsuchiya R, Murata M,
Murakami M, Hisasue M.:Characterization of a canine tetranucleotide microsatellite
marker located in the first intron of the tumor necrosis factor alpha gene.㻌 J Vet Med Sci
76,119-22. 2014. ᰝㄞ᭷
Kamiie J, Shimoyama N, Aihara N, Hisasue M, Naya Y, Ogihara K, Shirota K
4XDQWLWDWLYHDQDO\VLVRI&'İLQDFORQHGFDQLQHO\PSKRPDFHOOOLQHE\VHOHFWHGUHDFWLRQ
monitoring assay.㻌 Biosci Biotechnol Biochem 78,271-5,2014. ᰝㄞ᭷
Kawarai S, Matsuura M, Yamamoto S, Kiuchi A, Kanemaki N, Madarame H, Sirota K: A
case of cutaneous sterile pyogranuloma/granuloma syndrome in a Maltese. J Am Anim
Hosp Assoc, 50:278-283, 2014 ᰝㄞ᭷
Watanabe S, Ito J, Baba T, Hiratsuka T, Kuse K, Ochi H, Anai Y, Hisasue M, Tsujimoto
H, Nishigaki K. Notch2 transduction by feline leukemia virus in a naturally infectedcat.㻌 J Vet Med Sci 76,553-7, 2014. ᰝㄞ᭷
Kurihara Y, Suzuki T, Sakaue M, Murayama O, Miyazaki Y, Onuki A, Aoki T, Saito M,
Fujii Y, Hisasue M, Tanaka K, Takizawa T.㻌 Valproic acid, a histone deacetylase
inhibitor, decreases proliferation of and induces specific neurogenic differentiation ofcanine adipose tissue-derived stem cells.㻌 J Vet Med Sci 76:15-23, 2014. ᰝㄞ᭷
Kanamori Y., Murakami M., Matsui T. and Funaba M.: The regulation of hepcidin
expression by serum treatment: requirements of the BMP response element and STATand AP-1-binding sites. Gene 551, 119-126,2014. ᰝㄞ᭷
Kanamori Y, Murakami M, Matsui T, Funaba M.:Hepcidin expression in liver cells:
evaluation of mRNA levels and transcriptional regulation.Gene 546, 50-5,2014. ᰝㄞ᭷
Yamaguchi S, Sano A, Hiruma M, Murata M, Kaneshima T, Murata Y, Takahashi H,
Takahashi S, Takahashi Y, Chibana H, Touyama H, Nguyen Thi Thanh Ha, Nakazato Y,
Uehara Y, Hirakawa M, Imura Y, Terashima Y, Kawamoto Y, Takahashi K,Sugiyama K,
Hiruma M, Murakami M, Hosokawa A, Uezato H.: Isolation of Dermatophytes andRelated Species from Domestic Fowl (Gallus gallus domesticus). Mycopathologia178,
135-43,2014. ᰝㄞ᭷

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Nishita T, Yatsu J, Murakami M, Kamoshida S, Orito K, Ichihara N, Arishima K, Ochiai
H.: Isolation and sequencing of swine carbonic anhydrase VI, an enzyme expressed in
the swine kidney. BMC Res Notes 7, 116, 2014.ᰝㄞ᭷
Asai K, Funaba M, Murakami M.:Enhancement of RANKL-induced MITF-E expression
and osteoclastogenesis by TGF-ȕ&HOO%LRFKHP)XQFW-9, 2014. ᰝㄞ᭷
Murata M, Murakami M.:Two distinct mtDNA lineages among captive African penguinsin Japan. J Vet Med Sci 76, 559-63,2014. ᰝㄞ᭷
Ogawa M, Uchida K, Isobe K, Saito M, Harada T, Chambers JK, Nakayama H.: Globoid
cell leukodystrophy (Krabbe’s disease) in a Japanese domestic cat. Neuropathology
34,190-6, 2014. ᰝㄞ᭷
Utsugi S, Saito M, Shelton D. Resolution of polyneuropathy in a hypothyroid dog
following thyroid supplementation. J Am Anim Hosp Assoc 50,345-9, 2014.㻌 ᰝㄞ᭷
Miyaji K, Yurimoto T, Saito A, Yasueda H, Takase Y, Shimakura H, Okamoto N, Kiuchi
A, Saito S, Sakaguchi M: Analysis of conformational and sequential IgE epitopes on the
major allergen Cry j 2 of Japanese cedar pollen in humans by using monoclonal
antibodies for Cry j 2. J Clin Immunol 33, 977-83, 2013.㻌 ᰝㄞ᭷(㻖4)Kanemaki N, Tchedre KT, Imayasu M, Kawarai S, Sakaguchi M, Yoshino A, Itoh N,
Meguro A, Mizuki N. Dogs and humans share a common susceptibility gene SRBD1 forglaucoma risk. PLoS One 8, e74372, 2013. ᰝㄞ᭷
Nagasawa M, Kawai E, Mogi K, Kikusui T.: Dogs show left facial lateralization uponreunion with their owners. Behav Processes 98, 112-6, 2013㻌 ᰝㄞ᭷
Nagasawa M, Shibata Y, Yonezawa A, Morita T, Kanai M, Mogi K, Kikusui T.: Thebehavioral and endocrinological development of stress response in dogs. Dev Psychobiol
56, 726-33, 2013.㻌 ᰝㄞ᭷
Fujimoto A, Neo S, Ishizuka C, Kato T, Segawa K, Kawarai S, Ogihara K, Hisasue M,
Tsuchiya R.㻌 Identification of cell surface antigen expression in canine hepatocellular
carcinoma cell lines. J Vet Med Sci 75,831-5,2013. ᰝㄞ᭷
Kato T, Hisasue M, Segawa K, Fujimoto A, Makiishi E, Neo S, Yasuno K, Kobayashi R,
Tsuchiya R.: Accumulation of xenotransplanted canine bone marrow cells in
12'6&,'ȖF QXOO  PLFH ZLWK DFXWH KHSDWLWLV LQGXFHG E\ &&O 㻌 J Vet Med Sci
75,847-55, 2013. ᰝㄞ᭷
Murata M, Takahashi H, Takahashi S, Takahashi Y, Chibana H, Murata Y, Sugiyama K,
Kaneshima T, Yamaguchi S, Miyasato H, Murakami M, Kano R, Hasegawa A, Uezato H,
Hosokawa A, Sano A.: Isolation of Microsporum gallinae from a fighting cock (Gallus
gallus domesticus) in Japan. Med Mycol 51, 144-9, 2013. ᰝㄞ᭷
Shibuya E, Murakami M, Kondo M, Kamei Y, Tomonaga S, Matsui T, Funaba
M.:Downregulation of Pgc-Į H[SUHVVLRQ E\ WHD OHDYHV DQG WKHLU E\-products. Cell
Biochem Funct 32,236-40,2013. ᰝㄞ᭷
Suenaga M, Kurosawa N, Asano H, Kanamori Y, Umemoto T, Yoshida H, Murakami M,
Kawachi H, Matsui T, Funaba M.:Bmp4 expressed in preadipocytes is required for the
onset of adipocyte differentiation. Cytokine 64, 138-45,2013. ᰝㄞ᭷
Dong J, Murakami M, Fujimoto T, Yamaha E, Arai K.: Genetic characterization of theprogeny of a pair of the tetraploid silver crucian carp Carassius auratus langsdorfii. FishSci 79, 935-41,2013.ᰝㄞ᭷
Iida N, Fukushima H, Hiroi M, Yagi M, Kanda T, Murakami M, Sugiyama K.:
Development of duplex SYBR green real-time PCR for rapid and simultaneous detectionof 16 specific genes of 16 major foodborne bacteria. Jpn J Food Microbiol (᪥ᮏ㣗ရᚤ

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Yoshida H, Kanamori Y, Asano H, Hashimoto O, Murakami M, Kawada T, Matsui T,
Funaba M.:Regulation of brown adipogenesis by the Tgf-ȕ IDPLO\ ,QYROYHPHQW RI
Srebp1c in Tgf-ȕ- and Activin-induced inhibition of adipogenesis. Biochim Biophys
Acta 1830, 5027–35,2013. ᰝㄞ᭷
Ohnishi M, Okatani AT, Harada K, Sawada T, Marumo K, Murakami M, Sato R, Esaki
H, Shimura K, Kato H, Uchida N, Takahashi T.:Genetic characteristics of CTX-M-Type
extended-spectrum-ȕ-lactamase (ESBL)-producing enterobacteriaceae involved in
mastitis cases on Japanese dairy farms, 2007 to 2011. J Clin Microbiol 51, 3117-22,2013.
ᰝㄞ᭷
Ohnishi M, Okatani AT, Esaki H, Harada K, Sawada T, Murakami M, Marumo K, Kato
Y, Sato R, Shimura K, Hatanaka N, Takahashi T.:Herd prevalence of Enterobacteriaceae
producing CTX-M-type and CMY- ȕ-lactamases among Japanese dairy farms. J Appl
Microbiol 115, 282-9,2013. ᰝㄞ᭷Hirai N, Shirai M, Kato Y, Murakami M, Nomura R, Yamasaki Y, Takahashi S,
Matsumoto-Nakano M, Nakano K, Asai F.:Correlation of age with distribution of
periodontitis-related bacteria in Japanese dogs. J Vet Med Sci 75, 999-1001,2013. ᰝㄞ᭷Murakami M, Shirai M, Ooishi R, Tsuburaya A, Asai K, Hashimoto O, Ogawa K,
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Biochem Genet 51, 202-10,2013. ᰝㄞ᭷
Tsuboi M, Uchida K, Ide T, Ogawa M , Inagaki T, Tamura S, Saito M, Chambers JK,
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75,327-35, 2013.ᰝㄞ᭷Aida Y, Murakami H, Takahashi M, Takeshima S.: Mechanisms of pathogenesis inducedby bovine leukemia virus (BLV) as a model for human T-cell leukemia virus (HTLV)”
Front. Microbiol 4:328, 2013.ᰝㄞ᭷⸨ᮧṇே,㜰ཱྀ㞞ᘯ:≟䛾䜰䝖䝢䞊ᛶ⓶⭵⅖䛻ᑐ䛩䜛 8 ᪥㛫䝥䝻䝖䝁䞊䝹䜢⏝䛔䛯ᛴ㏿
ῶឤస⒪ἲ䛾Ᏻ඲ᛶ. ⋇་䜰䝖䝢䞊䞉䜰䝺䝹䜼䞊䞉ච␿Ꮫ఍ㄅ㻌 2,14-18,2013.㻌 ᰝㄞ᭷
Kubota S, Miyaji K, Shimo Y, Shimakura H, Takase Y, Okamoto N, Kiuchi A., Fujimura
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Kawakami M, Narumoto O, Mastuo Y, Horiguchi K, Yamashita N, Sakaguchi M, Lipp
M, Nagase T, Yamashita N: The role of CCR 7 in allergic airway inflammation inducedby house dust mite exposure. Cell Immunol 275:24-32, 2012.㻌 ᰝㄞ᭷
Narumoto O, Matsuo Y, Sakaguchi M, Shoji S, Yamashita N, Shubert D, Abe K,
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airway inflammation and remodeling. Exp Mol Pathol 93, 18-25, 2012. ᰝㄞ᭷
Miyaji K, Suzuki A, Shimakura H, Takase Y, Kiuchi A, Fujimura M, Kurita G, Tsujimoto
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vaccines in Japan. Vet Immunol Immunopathol 145, 447-52, 2012.㻌 ᰝㄞ᭷( 10)Tsukui T, Sakaguchi M, Kurata K, Maeda S, Ohmori K, Masuda K, Tsujimoto H,
Iwabuchi S: Measurement for canine IgE using canine recombinant high affinity IgEUHFHSWRUĮFKDLQ )Fİ5,Į   -9HW0HG6FL,851-6, 2012.㻌 ᰝㄞ᭷
Nagasawa M, Mogi K, Kikusui T: Continued Distress among Abandoned Dogs in

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Fukushima. Sci Rep 2, 724, 2012.㻌 ᰝㄞ᭷
Nagasawa M, Yatsuzuka A, Mogi K, Kikusui T: A new behavioral test for detecting
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Nagasawa M, Okabe S, Mogi K, Kikusui T: Oxytocin and mutual communication inmother-infant bonding. Front Hum Neurosci 6, 31, 2012.
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Yamada T, Tsuchiya R. Effects of prostaglandin E1 on the preparation of plateletconcentrates in dogs. J Vet Intern Med 26,370-6,2012. ᰝㄞ᭷
Haishima A, Murakami M, Ikeda T, Inoue K, Kamiie J, Shirota K.: Detection of Bcl-2
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Tanaka M, Izawa T, Kuwamura M, Nakao T, Maezono Y, Ito S, Murata M, Murakami M,
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Nishikawa O, Arishima K, Kobayashi T, Shirai M, Murakami M, Sakaue M,Yamamoto
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microsomal enzymes in male rat offspring. J Vet Med Sci 74, 247-53, 2012. ᰝㄞ᭷
Yamasaki Y, Nomura R, Nakanoa K, Inaba H, Kuboniwa M, Hirai N, Shirai M, Kato Y,
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F.:Distribution and molecular characterization of Porphyromonas gulae㻌 carrying a new
fimA genotype. Vet Microbiol 161, 196-205,2012. ᰝㄞ᭷
Nomura R, Shirai M, Kato Y, Murakami M, Nakano K, Hirai N, Mizusawa T, Naka S,
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Porphyromonas gulae clinical isolates from Japanese dogs. J Vet Med Sci 74,
885-91,2012. ᰝㄞ᭷
Ishii Y, Takizawa T, Iwasaki H, Fujita Y, Murakami M, Groppe JC, Tanaka K.:
Nucleotide polymorphisms in the canine Noggin gene and their distribution among dog(Canis lupus familiaris) breeds. Biochem Genet 50, 12-8,2012. ᰝㄞ᭷
Ooishi R, Shirai M, Funaba M, Murakami M. (2012) Microphthalmia-associatedtranscription factor is required for mature myotube formation. Biochim Biophys Acta
1820, 76–83,2012. ᰝㄞ᭷
Mizukami K, Kawamichi T, Koie H, Tamura S, Matsunaga S, Imamoto S, Saito M,
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K, Kang KG, Matsuda H.: Daily intake of Jeju groundwater improves the skin conditionof the model mouse for human atopic dermatitis. J Dermatol 40,193-200, 2013.
኱࿴ ಟ102) Kohyama M, Yabuki A, Kawasaki Y, Kawaguchi H, Miura N, Kitano Y, Onitsuka T,
Rahman MM, Miyoshi N, Yamato O.: GM2 gangliosidosis variant 0 (Sandhoff disease)in a mixed-breed dog. J Am Anim Hosp Assoc 51,396-400,2015. ᰝㄞ᭷103) Rahman MM, Yabuki A, Kohyama M, Mitani S, Mizukami K, Uddin MM, Chang HS,
Kushida K, Kishimoto M, Yamabe R, Yamato O.: Real-time PCR genotyping assay forGM2 gangliosidosis variant 0 in toy poodles and the mutant allele frequency in Japan. J
Vet Med Sci 76, 295–9, 2014. ᰝㄞ᭷104) Mizukami K, Yabuki A, Kawamichi T, Chang HS, Rahman MM, Uddin MM, Kohyama
M, Yamato O.: Real-time PCR genotyping assay for canine trapped neutrophil syndrome
and high frequency of the mutant allele in Border collies. Vet J 195, 260–1, 2013.

22

㻌
䠘ᅗ᭩䠚㻌
1. 㰻⸨ᘺ௦Ꮚ㸸≟࡜⊧ࡢᢠ࡚ࢇ࠿ࢇ⸆ࡢ౑࠸᪉ࠕ࡚ࢇ࠿ࢇ἞⒪࢞࢖ࢻࣈࢵࢡࠖ ࣇ࢓࣮࣒ࣉࣞࢫࠊᮾிࠊin press.
2. 㛗㇂ᕝ኱㍜ࠊᯞᮧ୍ᘺࠊ㰻⸨ᘺ௦Ꮚ㸦┘ಟࠊඹⴭ㸧㸸≟࡜⊧ࡢ⚄⤒⑓Ꮫྛㄽ⦅,⥳᭩ᡣࠊᮾிࠊ2015.
3. 㜰ཱྀ㞞ᘯ.ື≀࢔ࣞࣝࢤࣥࠕ࠶࡞ࡓࡢࡲࢃࡾ࡟₯ࡴ㌟㏆࡞࢔ࣞࣝࢤࣥࠖ㸦㇂ཱྀṇᐇࠊ⚟ᐩ཭㤿┘ಟ㸧ࠊ࣓ࢹ࢖࢝ࣝࣞࣅ࣮ࣗ♫ࠊᮾிࠊp14-17,2015.
4. 㰻⸨ᘺ௦Ꮚ㸦ඹⴭ㸧:⋇་ෆ⛉Ꮫ➨㸰∧ࠕ⚄⤒⑌ᝈࡢ᳨ᰝ࡜デ᩿ࠖᩥⱥᇽฟ∧,ᮾிࠊ2014.
5. 㜰ཱྀ㞞ᘯ:㠀≉␗ⓗ⏕య㜵ᚚ㸦⮬↛ච␿㸧ࠕ⋇་ᚤ⏕≀᳨ᰝᏛࠖ㸦⚟ᡤ⛅㞝௚┘ಟ㸧㏆௦ฟ∧ࠊᮾிࠊp41-4,2014.
6. 㜰ཱྀ㞞ᘯ㸸࣌ࢵࢺ. ࠕ྾ධᛶ࢔ࣞࣝࢤࣥࡢྠᐃ࡜ᑐ⟇ࠖ㸦㇂ཱྀṇᐇࠊ⚟ᐩ཭㤿┘ಟ㸧ࠊ࣓ࢹ࢖࢝ࣝࣞࣅ࣮ࣗ♫ࠊᮾிࠊp34-7,2014.
7. 㜰ཱྀ㞞ᘯ:CpG ࣡ࢡࢳࣥ. ࠕ࢔ࣞࣝࢠ࣮⑌ᝈࡢච␿⒪ἲ࡜ศᏊᶆⓗ἞⒪ࠖ
㸦㏆⸨┤⨾⦅㞟㸧デ᩿࡜἞⒪♫ࠊᮾிࠊp110-4,2013.
8. 㜰ཱྀ㞞ᘯ:⎔ቃ࢔ࣞࣝࢠ࣮࢔ࢻࣂ࢖ࢨ࣮ヨ㦂 බᘧࢸ࢟ࢫࢺ(㜰ཱྀ㞞ᘯ௚┘ಟ)ࠊ᪥ᮏ
⬟⋡༠఍࣐ࢿࢪ࣓ࣥࢺࢭࣥࢱ࣮ࠊᮾிࠊ2013.
9. 㜰ཱྀ㞞ᘯ,᳃⏣ⱥ฼ࠊ⏣ཎཱྀᬛኈ㸸ᚤ⏕≀Ꮫࠊㅮㄯ♫ࠊᮾிࠊ2013.
10. Sakaguchi M, Inouye S.: Gelatin allergies associated with diphtheria-tetanus-acellular
pertussis (DTaP) vaccines. "Trends in Diphtheria Research." in Wheeler B.S. (ed).
Nova Science Publishers, New York, USA, p63-79, 2011.
11. 㜰ཱྀ㞞ᘯ:ච␿୙඲࡜⮬ᕫච␿ࠕ⋇་ᚤ⏕≀Ꮫࠖ㸦ぢୖ ᙭┘ಟ㸧ᩥỌᇽࠊᮾிࠊ
p363-366,2011.
12. ᑠ᪉᐀ḟ┘ಟ䠈ᩬ┠ᗈ㑻䠈ᕝཎ஭᫴ᖹ䠄ඹⴭ䠅䠖䜰䝺䝹䜼䞊䜟䜣䛣䛾ᬽ䜙䛧䛸䝺䝅䝢ⓒ⛉䚸ㄔᩥᇽ᪂ග♫䚸ᮾி䚸2011.
㻌䠘Ꮫ఍Ⓨ⾲䠚㻌
1. (㻖11) ⏣୰࿴᫂, ⁪ἑ㐩ஓ, ᒣᮏᮍဏ, ᒸᮏ᠇᫂, ᓥ಴⚽຾, 㜰ཱྀ㞞ᘯ: ࢖ࢾ IL-13
㑇ఏࡢከᆺ࡜ᰘ≟࡟࠾ࡅࡿ࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡜ࡢ㛵㐃ㄪᰝ. ᪥ᮏ␆⏘Ꮫ఍➨ 121 ᅇ኱఍, Ṋⶶ㔝ࠊ3 ᭶ 2016 ᖺ
2. ෆᒣ῟ᖹࠊෆᒣఀ௦ࠊ➉ෆ ၨ᫭ࠊ㜰ཱྀ⩏ᙪࠊ㜰ཱྀ㞞ᘯࠊᯇᓮ ⱱᒎ: άᛶᆺࣆࣟࣜ
⳦ࣇ࢓࣮ࢪ KHP30 ࡢ≉ᚩ௜ࡅ࡜ࡑࡢ₯అឤᰁᛶࡢゎᯒ.➨ 89 ᅇ᪥ᮏ⣽⳦Ꮫ఍ࠊ኱
㜰ࠊ3 ᭶ 2016 ᖺ
3. 㑣㡲ᕝᛅᘺࠊෆᒣ῟ᖹࠊ㕥ᮌோேࠊᐑ⏣⋹ዉࠊᒣཱྀ⍆⤮ࠊᖹᒣ㝯୍㑻ࠊෆᒣఀ௦ࠊ
Ặཎ㝯Ꮚࠊ㜰ཱྀ⩏ᙪࠊ㜰ཱྀ㞞ᘯࠊᯇ㷂ⱱᒎ㸸⥳⮋⳦ PAO1 ᰴ࡜ࣇ࢓࣮ࢪ KPP22 ࡢ▷ᮇ㛫㐍໬ⓗ㌷ᣑ➇㉮ࡢゎᯒ. ➨ 89 ᅇ᪥ᮏ⣽⳦Ꮫ఍ࠊ኱㜰ࠊ3 ᭶ 2016 ᖺ
4. Ỉ㇂᱁அࠊᚋ⸨⿱Ꮚࠊⶶ⏣ᆂ࿃ࠊ⸨ཎள⣖ࠊ㜰ཱྀ㞞ᘯࠊ኱㔝⪔୍ࠊ㎷ᮏඖ㸸࢖ࢾࡢࣜࣥࣃ⭘⑕౛࡟࠾ࡅࡿ⾑Ύ୰ྍ⁐ᛶ࢖ࣥࢱ࣮ࣟ࢖࢟ࣥ-2 ཷᐜయȘ㙐ࡢ ᐃ. ➨ 12ᅇ᪥ᮏ⋇་ෆ⛉Ꮫ࢔࢝ࢹ࣑࣮Ꮫ⾡኱఍ࠊᶓ὾ࠊ2 ᭶ 2016 ᖺ
5. ⓑ஭⚽἞ࠊ㜰ཱྀ㞞ᘯ㸸ࢲࢽ࢔ࣞࣝࢤࣥ࡟ᑐࡍࡿᐙᗞෆᑐ⟇ࡢ㠀⮫ᗋ◊✲ࢆ୰ᚰࡋࡓホ౯.➨ 52 ᅇ᪥ᮏᑠඣ࢔ࣞࣝࢠ࣮Ꮫ఍ࠊዉⰋࠊ11 ᭶ 2015 ᖺ6.
(㻖)᪫ ឡ㸪⸨஭ᏹ἞㸪ᒸᮏ᠇᫂㸪❑⏣ ⪽㸪㛵ཱྀ࿴ṇ㸪㜰ཱྀ㞞ᘯ㸪Ṋෆࡺ࠿ࡾ㸪ᯇ⏣⚽᫂㸸࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺከᆺゎᯒࢆ⏝࠸ࡓ࢖ࢾࡢಶయ㆑ูἲࡢ᳨ウ㸬➨ 2123ᅇ᪥ᮏἲ⛉Ꮫᢏ⾡Ꮫ఍㸪᯽ࠊ11 ᭶ 2015 ᖺ
7. ᮧୖ⿱ಙ䚸஧㝵ᇽ⣪ᜨ䚸బ⸨♩୍㑻䚸ሯᮏ೺ྖ䠖㻌 ∵ⓑ⾑⑓䜴䜲䝹䝇 G4 㑇ఏᏊḞᦆᰴ䛾䜴䜲䝹䝇⏘⏕㔞. ➨ 63 ᅇ᪥ᮏ䜴䜲䝹䝇Ꮫ⾡㞟఍䚸⚟ᒸ䚸11 ᭶ 2015 ᖺ
8. 㕥ᮌ༓⿱䚸ᡞ㧘ᬕ⳯䚸஧㝵ᇽ⣪ᜨ䚸బ⸨♩୍㑻䚸ሯᮏ೺ྖ䚸ᮧୖ⿱ಙ: BLV ឤᰁᛶศᏊ䜽䝻䞊䞁䜢⏝䛔䛯䜴䜲䝹䝇⏘⏕㔞䛸䝀䝜䝮ኚ␗䛾ゎᯒ. ➨ 63 ᅇ᪥ᮏ䜴䜲䝹䝇Ꮫ⾡㞟఍䚸⚟ᒸ䚸11 ᭶ 2015 ᖺ
9. ෆᒣ῟ᖹ㸪㜰ཱྀ㞞ᘯ㸪ᯇᓮⱱᒎ㸸ࣆࣟࣜ⳦ࣇ࢓࣮ࢪࡢⓎぢ࡜ࡑࡢゎᯒ㸬➨ 68 ᅇ᪥ᮏ⣽⳦Ꮫ఍୰ᅜ࣭ᅄᅜᨭ㒊⥲఍㸪ᒸᒣ㸪10 ᭶ 2015 ᖺ
10. (㻖5)Ota N, Kanemaki N, Tsujita H, Kobayashi Y, Abe M, Takimoto Y, Imayasu M, Meguro
A, Mizuki N. Associations of single-nucleotide polymorphisms of SRBD1 gene 64 withglaucoma in Shiba-inu dog: A meta-analysis in six Japanese veterinary hospitals. ACVO
Annual meeting, Coeur d’Alene, ID, USA, Oct, 2015.
11. ᓥ಴⚽຾ࠊ㰻⸨ᣅࠊ⸨ᮧṇே ࠊᒸᮏ᠇᫂ࠊෆᒣ῟ᖹࠊ㜰ཱྀ㞞ᘯ㸸㣗≀᭷ᐖ཯ᛂࢆ
♧ࡍ≟࡟࠾ࡅࡿ༸ⓑᢠཎ࡟ᑐࡍࡿ IgE ཯ᛂᛶࡢ᳨ウ㸬➨ 158 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊ༑࿴
⏣ࠊ㸷᭶ 2015 ᖺ
12. ᖹᒣ 㝯୍㑻ࠊ㕥ᮌ ோேࠊෆᒣ ῟ᖹࠊᯇ஭ ┿⌮ࠊ㕥ᮌ 㔛࿴ࠊᰘᒣ ᜨ࿃ࠊ㜰ཱྀ 㞞ᘯࠊᮌෆ ᫂⏨: ࢖ࢾ࡟࠾ࡅࡿ᪂つཱྀ⭍ෆᖖᅾ⣽⳦ࡢྠᐃ࡜඲ࢤࣀ࣒㓄ิゎᯒ㸬 ➨158 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊ༑࿴⏣ࠊ㸷᭶ 2015 ᖺ
13. ෆᒣ῟ᖹࠊᯇ஭⚽ோࠊⰼᮌ⚽᫂ࠊᯇᓮⱱᒎࠊ㜰ཱྀ㞞ᘯ:⣽⳦ឤᰁ⑕࡟࠾ࡅࡿデ᩿ἲ
࡜἞⒪ἲࡢ㛤Ⓨ㸸ࣂࢡࢸࣜ࢜ࣇ࢓࣮ࢪࡢ฼⏝㸬➨ 158 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊ༑࿴⏣ࠊ㸷
᭶ 2015 ᖺ
14. ኱ஂಖ೔Ꮚ䚸㉥ụ䛛䜣䛺䚸ᮧୖ㈼䠖ᅜෆ䜿䞊䝥䝨䞁䜼䞁䛾ಶయ㆑ู䛚䜘䜃∗Ꮚ㚷ᐃ䛻䛚䛡䜛䝬䜲䜽䝻䝃䝔䝷䜲䝖 DNA 䝬䞊䜹䞊䛾᭷⏝ᛶ䠊➨ 158 ᅇ᪥ᮏ⋇་Ꮫ఍Ꮫ⾡㞟఍䚸༑࿴⏣䚸9 ᭶ 2015 ᖺ
15. Nomura N, Saito M, Hasegawa D, Watanabe N. Uchida K, Okuno S, Nakai M, Orito㻌 K.
Clinical efficacy and safety of zonisamide monotherapy in dogs with newly diagnosed
idiopathic epilepsy. 2015 ACVIM Forum, Indianapolis, June, 2015.
16. ▼஭ᓫྐ, ᪂಴㞝୍, ⣽ᮌᩗ♸, ὠஂ஭฼ᏹ, 㜰ཱྀ㞞ᘯ, ୕Ꮿ೺௓, 㛗℩㝯ⱥ, ᒣୗ┤⨾㸸 ႍᜥ⑓ែ࡟࠾ࡅࡿẼ㐨ୖ⓶⣽⬊ࡢάᛶ໬࡜᪂つࢡࣝࢡ࣑ࣥㄏᑟయ࡟ࡼࡿไ
ᚚ࡟ࡘ࠸࡚ࡢゎᯒ.➨ 55 ᅇ᪥ᮏ࿧྾ჾᏛ఍ࠊᮾிࠊ4 ᭶ 2015 ᖺ
17. (㻖2)ᆤ஭ㄔஓ, Ώ㑔Ꮫ, ஧⎼࿴⨾, ྜྷぢዉὠᏊ, ຍ⸨᫂ஂ, 㜰ཱྀ㞞ᘯ, ኱࿴ಟ,᷸⏣࿴ဢ , ࢳ ࢙ ࣥ ࣂ ࣮ ࢬ ࢪ ࢙ ࣮ ࣒ ࢬ , Ⳣ 㔝 ⣧ ኵ , ෆ ⏣ ࿴ ᖾ , ୰ ᒣ ⿱ அ 㸸 Whole Exome
Sequence ἲ࡟ࡼࡿ࢖ࢾ㑇ఏᛶ⚄⤒⑌ᝈࡢཎᅉ㑇ఏᏊྠᐃࠋ➨ 11 ᅇ⋇་ෆ⛉࢔࢝ࢹ࣑࣮ࠊᶓ὾ࠊ2 ᭶ 19 ᪥ 2014 ᖺ
18. እụள⣖Ꮚ䚸Ọ⃝⨾ಖ䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: ㄆ▱⬟ຊ䛾≟✀㛫ẚ㍑䛸㛵㐃㑇ఏᏊ䛾᥈⣴䛻䜘䜛䜲䝚䛾㐍໬䛻㛵䛩䜛◊✲.䛂ඹឤᛶ䛾㐍໬䞉⚄⤒ᇶ┙䛃➨ 2 ᅇ㡿ᇦ఍㆟䚸ᮾ኱ᑎ⥲ྜᩥ໬䝉䞁䝍䞊䚸1 ᭶ 2015 ᖺ
19. ኱໭☐䚸Ọ⃝⨾ಖ䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: 䜲䝚䛻䛚䛔䛶䝠䝖䛛䜙䛾ど⥺䛿ሗ㓘䛻䛺䜛䛛䠛䛂ඹឤᛶ䛾㐍໬䞉⚄⤒ᇶ┙䛃➨ 2 ᅇ㡿ᇦ఍㆟䚸ᮾ኱ᑎ⥲ྜᩥ໬䝉䞁䝍䞊䚸1 ᭶ 2015ᖺ20. Ọ⃝⨾ಖ䚸⳥Ỉ೺ྐ: ཰ᩡ㐍໬䛜䜒䛯䜙䛧䛯䝠䝖䞊䜲䝚㛫䛾⤎ᙧᡂ䛸䜸䜻䝅䝖䝅䞁䛾ᙺ
๭. 䛂ඹឤᛶ㡿ᇦ䛃䛂⮬ᕫไᚚ⢭⚄㡿ᇦ䛃ྜྠⱝᡭ⫱ᡂ䝅䞁䝫䝆䜴䝮䚸ᮾி་⛉ṑ⛉኱Ꮫ䚸12 ᭶ 2014 ᖺ
21. ⳥Ỉ೺ྐ: ே䛸≟䛾ඹ⏕㻌 ᚰ䛾䛴䛺䛜䜚. ᪥ᮏ⚄⤒ᾘ໬ჾ⑓Ꮫ఍䚸Ꮫ⾡⥲ྜ䝉䞁䝍䞊䞉24୍ᶫㅮᇽ䚸11 ᭶ 2014 ᖺ
22. ཎ⏣᫴ࠊⓑ஭⚽἞ࠊ㉺ᮏ▱኱ࠊ㜰ཱྀ㞞ᘯ,ᕤ⸨ẚ➼ᚿ㸸⿕ẟ࠾ࡼࡧၚᾮࡢ୧⪅࡬ࡢឤసࡀㄆࡵࡽࢀࡓࢪࣖࣥ࢞ࣜ࢔ࣥࣁ࣒ࢫࢱ࣮࡟ࡼࡿ࢔ࢼࣇ࢕ࣛ࢟ࢩ࣮ࡢ 1 ౛.➨㸲㸲ᅇ᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍ࠊ௝ྎࠊ11 ᭶ 2014 ᖺ
23. బ⸨⨾⣖䚸ᐑᓮ㝧Ꮚ䚸㟷ᮌ༟☻䚸⸨⏣ᖾᘯ䚸ᅭᑿᣅஓ䚸㰺⸨ᘺ௦Ꮚ䚸⸨஭ὒᏊ䚸ஂᮎṇᬕ䚸⏣୰࿴᫂䚸⁪ἑ㐩ஓ:䜲䝚⬡⫫⤌⧊ᖿ⣽⬊(ASC)䛾⣽⬊ቑṪ䛸⚄⤒ศ໬䛻ཬ䜌䛩䝞䝹䝥䝻㓟䛸ప㓟⣲䛾ᙳ㡪.㻌 ➨ 87 ᅇ᪥ᮏ⏕໬Ꮫ఍䚸ᅜ❧ி㒔ᅜ㝿఍㤋䚸10 ᭶ 2014 ᖺ
24. ᰘ⏣᭙䚸Ọ⃝⨾ಖ䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: 䜲䝚䛻䛚䛡䜛䝇䝖䝺䝇ෆศἪ䛾Ⓨ㐩≉ᛶ䛸⾜ື䛸䛾㛵㐃. ➨ 157 ᅇ᪥ᮏ⋇་Ꮫ఍Ꮫ⾡㞟఍䚸ᮐᖠ䚸9 ᭶ 2014 ᖺ
25. ⛅ྜྷ ுேࠊஂᮎ ṇᬕࠊᅵᒇு㸸ཎⓎᛶ㛛⬦పᙧᡂ࡜デ᩿ࡋࡓ≟ 30 ⑕౛࡟࠾ࡅࡿ
⮫ᗋ⑓⌮Ꮫⓗ᳨ᰝᡤぢࡢ᳨ウ. ➨ 157 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊᮐᖠࠊ9 ᭶ 2014 ᖺ
26. 㯮⏣┿㐨䚸ᮧୖ㈼䚸⸨ᮏ㈗ྐ䚸Ⲩ஭ඞಇ䠖཯᚟㓄ิ䜢䝥䝻䞊䝤䛸䛧䛯 FISH 䛻䜘䜛ᰁⰍయ㆑ู䛸䜽䝻䞊䞁䝗䝆䝵䜴䛾㞧✀㉳※ゎ᫂䠊ᖹᡂ 26 ᖺᗘ᪥ᮏỈ⏘Ꮫ఍⛅Ꮨ኱఍䚸⚟ᒸ䚸9
᭶ 2014 ᖺ
27. ᮧ⏣೔Ꮚ䚸ட஭᪩⣖䚸ᮧୖ㈼䠖䝣䞁䝪䝹䝖䝨䞁䜼䞁ᒓ 3 ✀䛾䝨䞁䜼䞁䛾᰾ᆺศᯒ䛻䜘䜛≉ᚩ௜䛡䠊➨ 157 ᅇ᪥ᮏ⋇་Ꮫ఍Ꮫ⾡㞟఍䚸ᮐᖠ䚸9 ᭶ 2014 ᖺ
28. ∦ᕝඃᏊ䚸⯚ሙṇᖾ䚸ᮧୖ㈼䠖BMP 䛻䜘䜛 Smad8 Ⓨ⌧ㄪ⠇䝯䜹䝙䝈䝮䛾ゎᯒ䠊➨ 157
ᅇ᪥ᮏ⋇་Ꮫ఍Ꮫ⾡㞟఍䚸ᮐᖠ䚸9 ᭶ 2014 ᖺ
29. Ọ⃝⨾ಖ䚸ᰘ⏣᭙䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: The development of endocrine stress responsein early socialization period is associated with that in adulthood in dogs. ᪥ᮏື≀ᚰ⌮Ꮫ఍➨ 74 ᅇ኱఍䚸≟ᒣᅜ㝿ほග䝉䞁䝍䞊“䝣䝻䜲䝕”䚸7 ᭶ 2014 ᖺ
30. 㜰ཱྀ㞞ᘯࠊⓑ஭⚽἞㸸ᐊෆ⎔ቃ࢔ࣞࣝࢤࣥ.➨ 23 ᅇ᪥ᮏ⮫ᗋ⎔ቃ་Ꮫ఍ࠊி㒔ࠊ6
᭶ 2014 ᖺ
31. (㻖)Imayasu M, Kanemaki N, Meguro A, Mizuki N. A novel single nucleotide polymerism
asocial woth glaucoma in Shiba-inu and Shih-Tzu dogs, ARVO Annual Meeting, Orlando,
FL, USA, May, 2014.
32. ▼஭ᓫྐ, ᪂಴㞝୍, ⓑ஭⚽἞, 㜰ཱྀ㞞ᘯ, Ჴඖ᠇୍, ᐊ஭ṇᚿ, 㛗℩㝯ⱥ, ᒣୗ┤⨾: IRF3 ࡢዲ㓟⌫ᛶẼ㐨⅖⑕⑓ែ࡟࠾ࡅࡿᙺ๭.➨ 26 ᅇ᪥ᮏ࢔ࣞࣝࢠ࣮Ꮫ఍᫓Ꮨ⥲఍ࠊ5 ᭶ 2014 ᖺ
33. 㕥ᮌ㻌 㯞㔛Ꮚ䚸㯮ᓮ㻌 ᫭ᙪ䚸㣕⏣㻌 ၨ㍜䚸㟷ᮌ㻌 ᗣኵ䚸ᯘ㔝㻌 ῟䚸㎷ᮏ㻌 ඖ䚸㜰ཱྀ㻌 㞞ᘯ䠖≟䛾䜰䝖䝢䞊ᛶ⓶⭵⅖䛻䛚䛡䜛᪂つங㓟⳦ Lactobacillus crispatus KT-11 〇๣䛾ᢞ୚ヨ㦂.➨䠍䠓ᅇ᪥ᮏ⋇་⓶⭵⛉Ꮫ఍Ꮫ⾡኱఍,䛥䛔䛯䜎䚸3 ᭶ 2014 ᖺ
34. 㜰ཱྀ㞞ᘯ, ὠஂ஭฼ᗈ, ኱㝮ᑛྐ, ㎷ᮏඖ:≟ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࡢ࢔ࣞࣝࢤࣥࢆ⏝࠸ࡓච␿⒪ἲ࡟ࡘ࠸࡚.➨ 10 ᅇ⋇་ෆ⛉࢔࢝ࢹ࣑࣮ࠊᶓ὾ࠊ2 ᭶ 2014 ᖺ
35. ༳∾ಙ⾜ࠊᕝཎ஭᫴ᖹࠊ㜰ཱྀ㞞ᘯ㸸࢖ࢾࡢࣂ࢖࢜ࣂࣥࢡࣉࣟࢪ࢙ࢡࢺ࡟࠾ࡅࡿ࢖ࢾ
⥳ෆ㞀ឤཷᛶ㑇ఏᏊࡢ◊✲.➨㸯㸮ᅇ⋇་⮫ᗋ㑇ఏ◊✲఍ࠊኳ❺ࠊ12 ᭶ 2013 ᖺ
36. 㔝㷂῟ኵࠊ㕥ᮌ┤ஓࠊᡂ⏣Ὀ❶ࠊⓑ஭⚽἞ࠊ㜰ཱྀ㞞ᘯ㸸ᕷ㈍࣐ࢫࢡ࡟࠾ࡅࡿ✵Ẽởᰁ≀㉁ࡢ㝖ཤ≉ᛶ࡟ࡘ࠸࡚ ࡑࡢ 1 ࢞ࢫ≧≀㉁㸦࢔ࣥࣔࢽ࢔ࠊVOC㸧
㸬2013 ᖺᗘ࿧྾ಖㆤ࡟㛵ࡍࡿ◊✲Ⓨ⾲఍ࠊᮾிࠊ12 ᭶ 2013 ᖺ
37. ∦ᕝඃᏊ䚸ᇼཱྀᫀ⚽䚸⯚ሙṇᖾ䚸ᮧୖ㈼䠖BMP 䛻䜘䜛 Smad8 Ⓨ⌧ㄪ⠇䠊➨ 36 ᅇ᪥ᮏศᏊ⏕≀Ꮫ఍ᖺ఍䚸⚄ᡞ䚸12 ᭶ 2013 ᖺ
38. ὸ஭ஂ⨾Ꮚ䚸⯚ሙṇᖾ䚸ᮧୖ㈼䠖TGF-ȕ 䛻䜘䜛 RANKL ㄏᑟᛶ Mitf-E 䛾Ⓨ⌧䛸◚㦵⣽⬊ศ໬䜈䛾ᙳ㡪䠊➨ 36 ᅇ᪥ᮏศᏊ⏕≀Ꮫ఍ᖺ఍䚸⚄ᡞ䚸12 ᭶ 2013 ᖺ
39. Kawarai S, Fujimoto A, Nozawa G, Kanemaki N, Madarame H, Shida T, Kiuchi A:

25

Decreasing methicillin-resistant Staphylococcal colonization in allergic dog skin byshampoo.The Asian Meeting of Animal Medicine Specialties. Bogor,Indonesia, Dec, 2013.
40. ⓑ஭⚽἞䚸ᡂ⏣Ὀ❶䚸ᯇᮏ⹒ୡ䚸ᰘ⏣ᙲ䚸㔝ᓮ῟ኵ䚸㜰ཱྀ㞞ᘯ䠖䝬䝇䜽╔⏝䛻䜘䜛䝎䝙䜰䝺䝹䝀䞁྾ධண㜵ຠᯝ䛾᳨ウ.➨ 63 ᅇ᪥ᮏ䜰䝺䝹䜼䞊Ꮫ఍⛅ᏘᏛ⾡኱఍,ᮾி䚸11
᭶ 2013 ᖺ
41. ᮧୖ⿱ಙ䚸ⴗཎᜤ஧䚸㕥ᮌ೺⿱䚸ᇽ๓┤䚸㛫㝧Ꮚ: 䝥䝻䝔䜸䝭䜽䝇ἲ䛻䜘䜚ྠᐃ䛧䛯᪂つVpr ⤖ྜᅉᏊ PRMT5/7 䛻䜘䜛䜴䜲䝹䝇」〇ไᚚᶵᵓ. ➨ 27 ᅇ᪥ᮏ䜶䜲䝈Ꮫ⾡㞟఍䚸
11 ᭶ 2013 ᖺ
42. ᮧୖ⿱ಙ䚸ⴗཎᜤ஧䚸㕥ᮌ೺⿱䚸ᇽ๓┤䚸㛫㝧Ꮚ: ᪂つ Vpr ⤖ྜᅉᏊ䛾䝥䝻䝔䜸䝭䜽䝇ἲ䛻䜘䜛ྠᐃ䛚䜘䜃䜴䜲䝹䝇⏘⏕䜈䛾ᙳ㡪. ➨ 61 ᅇ᪥ᮏ䜴䜲䝹䝇Ꮫ఍Ꮫ⾡㞟఍䚸⚄ᡞ䚸11 ᭶ 2013 ᖺ
43. እụள⣖Ꮚ䚸Ọ⃝⨾ಖ䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: ≟䛾♫఍ㄆ▱䛻㛵䜟䜛㑇ఏᏊ䛾᥈⣴.
᪂Ꮫ⾡㡿ᇦ◊✲䛂ඹឤᛶ䛾㐍໬⚄⤒ᇶ┙䛃 ➨ 1 ᅇ㻌 ື≀⾜ື䛸䜸䜻䝅䝖䝅䞁◊✲఍㆟䚸
⇕ᾏ䚸11 ᭶ 2013 ᖺ
44. Murata M, Murakami M.: Characterization of DNA markers isolated from the African
penguin (Spheniscus demersus) by using RDA and MASA techniques., 6th Asian Meetingon Zoo and Wildlife Medicine/Conservation, Singapore, Oct, 2013.
45. ᮧୖᘯṇ䚸⣽ᕝ⪷▮䚸ᕝཎ஭᫴ᖹ䚸ᩬ┠ᗈ㑻䠖ῶឤస⒪ἲ䜢ᐇ᪋䛧䛯≟䜰䝖䝢䞊ᛶ⓶⭵⅖䛾 2 ౛. ᪥ᮏᑠື≀⋇་Ꮫ఍ᮾ໭ᆅ༊኱఍. ⚟ᓥ䚸10 ᭶ 2013 ᖺ
46. ᓥ಴⚽຾ࠊᒸᒣኴ㑻ࠊቑ⏣೺୍ࠊὠஂ஭฼ᗈࠊ⸨ᮧṇேࠊ㜰ཱྀ㞞ᘯ㸸࢖ࢾࡢ⾑Ύ୰࢔ࣞࣝࢤࣥ≉␗ⓗ IgE ᢠయ㔞࡜࢔ࣞࣝࢠ࣮཯ᛂ㜈್⃰ᗘ࡜ࡢẚ㍑.➨ 156 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊᒱ㜧ࠊ9 ᭶ 2013 ᖺ
47. እụள⣖Ꮚ䚸ᑎෆ㇦䚸Ọ⃝⨾ಖ䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ. 䜲䝚䛾䝠䝖䛸䛾䝁䝭䝳䝙䜿䞊䝅䝵䞁⬟ຊ䛾ရ✀ᕪ䠈㛵㐃㑇ఏᏊ䛾᥈⣴. ᪥ᮏື≀ᚰ⌮Ꮫ఍➨ 73 ᅇ኱఍䚸⟃Ἴ኱Ꮫ䚸9 ᭶
2013 ᖺ㻌48. ∦ᒣ┿ᕼ䚸Ọ⃝⨾ಖ䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: య఩䜔᝟ື䛾ኚ໬䛜䜲䝚䛾ᚰᢿኚື䛻ཬ䜌䛩ᙳ㡪. ᪥ᮏື≀ᚰ⌮Ꮫ఍➨ 73 ᅇ኱఍䚸⟃Ἴ኱Ꮫ䚸9 ᭶ 2013 ᖺ㻌
49. ᑎෆ㇦䚸Ọ⃝⨾ಖ䚸እụள⣖Ꮚ䚸ᆏ⏣᪥㤶⌮䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: 䜲䝚䛾䝠䝖䛸䛾ど⥺䝁䝭䝳䝙䜿䞊䝅䝵䞁䛾≟✀ᕪ. ᪥ᮏື≀ᚰ⌮Ꮫ఍➨ 73 ᅇ኱఍䚸⟃Ἴ኱Ꮫ䚸9 ᭶ 2013ᖺ㻌50. ᑠᕝ⨾㔛䚸ᅭྐ⥴⌮䚸Ọ⃝⨾ಖ䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ䚸: 䜸䜻䝅䝖䝅䞁ᢞ୚䛻䜘䜛䝠䝖䜈䛾ど⥺ቑᙉ䛾᪥ᮏ≟✀䛸ὒ≟✀䛾ẚ㍑. ᪥ᮏື≀ᚰ⌮Ꮫ఍➨ 73 ᅇ኱఍䚸⟃Ἴ኱Ꮫ䚸9
᭶ 2013 ᖺ㻌
51. Ώ㎶ᚁ, ⏣୰࿴᫂, ⁪ἑ㐩ஓ, ℩ᕝ࿴ோ, ᰿ᑿḈᏊ, ᅵᒇு, ᮧ⏣೔Ꮚ,ᮧୖ㈼, ஂᮎṇᬕ㸸࢖ࢾ⭘⒆ቯṚᅉᏊ(TNFA)㑇ఏᏊࡢ➨㸯࢖ࣥࢺࣟࣥ࡟Ꮡᅾࡍࡿ 4 ሷᇶ཯᚟࣐࢖
ࢡࣟࢧࢸࣛ࢖ࢺࡢ≉ᚩ. ➨ 156 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊᒱ㜧ࠊ9 ᭶ 2013 ᖺ
52. ᰘ⏣᭙䚸Ọ⃝⨾ಖ䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: 䜲䝚䛻䛚䛡䜛䝇䝖䝺䝇ෆศἪ䛾Ⓨ㐩≉ᛶ䛸⾜ື䛾㛵㐃. ᪥ᮏ⾜ື⚄⤒ෆศἪ◊✲఍䠄JSBN䠅䚸㮵ඣᓥᕷ䝺䜲䞁䝪䞊ᱜᓥ䚸7 ᭶ 2013 ᖺ
53. 㜰ཱྀ㞞ᘯ:ᐇ㦂ື≀࢔ࣞࣝࢠ࣮࡜⎔ቃᅉᏊ.➨ 50 ᅇ᪥ᮏᐇ㦂ື≀⎔ቃ◊✲఍,ᮾி,7
᭶ 2013 ᖺ
54. 㔠᳃⪀ᖹ䚸ᮧୖ㈼䚸ᯇ஭ᚭ䚸⯚ሙṇᖾ䠖⫢⣽⬊ᇵ㣴⣔䛸䝦䝥䝅䝆䞁㑇ఏᏊⓎ⌧䠊➨ 30 ᅇ᪥ᮏᚤ㔞ᰤ㣴⣲Ꮫ఍Ꮫ⾡㞟఍䚸ி㒔䚸6 ᭶ 2013 ᖺ
55. Kanemaki N, Tchedre KT, Imayasu M, Meguro A, Mizuki N. Identification of S1 RNAbinding domain-1 SRBD1 as a major gene determing glaucoma in dogs. ARVO Annual
Meeting, Seattle, WA, USA, May, 2013.

26

56. 㜰ཱྀ㞞ᘯ: ≟ࡢࣂ࢖࢜ࣂࣥࢡࣉࣟࢪ࢙ࢡࢺ. ➨ 155 ᅇ᪥ᮏ⋇་Ꮫ఍,ᮾி,3 ᭶ 2013
ᖺ
57. Kikusui T, Mogi K, Nagasawa M: Sociobehavioral study of human-dog interactions: anapproach from neuroendocrinology. IIAS Research Conference 2012 “Evolutionary
Origins of Human Mind”, International Institute for Advanced Studies, Kyoto, Japan,
December, 2012
58. Nagasawa M, En S, Mogi K, Kikusui T Reciprocal communication and neuroendocrine
response in human-dog interaciotns. IIAS Research Conference 2012 “Evolutionary
Origins of Human Mind”, International Institute for Advanced Studies, Kyoto, Japan,
December, 201259. ⳥Ỉ೺ྐ: ே䛸≟䛾ඹ⏕㐍໬㻌 ʊ㻌 䛺䛬≟䛿ே䛸ඹ䛻⏕䛝䜛䜘䛖䛻䛺䛳䛯䛾䛛. ➨ 33 ᅇື≀⮫ᗋ་Ꮫ఍ᖺḟ኱఍䚸኱㜰ᅜ㝿఍㆟ሙ䚸11 ᭶ 2012 ᖺ
60. ⚄ᓮ⨾⋹, ⓑ஭⚽἞, ⓙᕝᜨᏊ ᶫᮏ▱ᖾࠊ㜰ཱྀ㞞ᘯࠊ┾ቨ㑳, 㣤ᓥⱱᏊ:࠾ዲࡳ↝ࡁ⢊࡟⦾Ṫࡋࡓࢳࣜࢲࢽ㢮࡟ࡼࡿ༶᫬ᆺ࢔ࣞࣝࢠ࣮ࡢẕᏊ౛. ➨ 62 ᅇ᪥ᮏ࢔ࣞࣝࢠ
࣮Ꮫ఍⛅ᏘᏛ⾡኱఍. ኱㜰,11 ᭶ 2012 ᖺ
61. ⓑ஭⚽἞ࠊᡂ⏣Ὀ❶ࠊᯇᮏ⹒ୡࠊᰘ⏣ᙲࠊ㔝ᓮ῟ኵࠊ㜰ཱྀ㞞ᘯ: ࣐ࢫࢡ╔⏝࡟ࡼࡿࢲࢽ࢔ࣞࣝࢤࣥ྾ධண㜵ຠᯝࡢ᳨ウ. ➨ 62 ᅇ᪥ᮏ࢔ࣞࣝࢠ࣮Ꮫ఍⛅ᏘᏛ⾡኱఍.
኱㜰,11 ᭶ 2012 ᖺ
62. ᓥ಴⚽຾ࠊ࿘⸨᫂⨾ࠊቑ⏣೺୍ࠊᮌෆ᫂⏨ࠊ㜰ཱྀ㞞ᘯ:ங㓟⳦ࢆ⏝࠸ࡓ㣗≀࢔ࣞࣝࢠ࣮ࡢ᪂つ἞⒪ἲࡢ㛤Ⓨ㸸࢖ࢾ࡟࠾ࡅࡿ⑌ᝈࣔࢹࣝ࡜ࡋ࡚ࡢ㣗≀࢔ࣞࣝࢠ࣮≟ࡢ᳨ウ.➨ 87 ᅇ㯞ᕸ⋇་Ꮫ఍ࠊ┦ᶍཎࠊ11 ᭶ 2012 ᖺ
63. ᒸᮏ᠇᫂ࠊ▼୸ᾈ㟹ࠊᮌෆ᫂⏨ࠊ⸨ᮧṇேࠊ㜰ཱྀ㞞ᘯ㸸࢔ࢺࣆ࣮ᛶ⓶⭵⅖≟࡟࠾ࡅࡿ Malassezia pachydermatis ࡢឤస≧ἣ. ➨ 87 ᅇ㯞ᕸ⋇་Ꮫ఍ࠊ┦ᶍཎࠊ11 ᭶ 2012
ᖺ64. ᐑᆅ୍ᶞࠊᮌෆ᫂⏨ࠊ⸨ᮧṇேࠊᰩ⏣࿃㑻ࠊ㜰ཱྀ㞞ᘯ㸸᪥ᮏ࡟࠾ࡅࡿ≟ΰྜ࣡ࢡࢳ
ࣥ᥋✀ᚋ๪཯ᛂ࡟㛵ࡍࡿ኱つᶍ࡞␿Ꮫㄪᰝ. ➨ 87 ᅇ㯞ᕸ⋇་Ꮫ఍ࠊ┦ᶍཎࠊ11 ᭶
2012 ᖺ
65. ᮡᒣ࿴ᑑ䚸ᑠஂಖ⪷Ꮚ䚸ෆ⏣㈗኱䚸ᮧୖ㈼䠖Chaetomium globosum ཬ䜃 Microsporum
canis 䛾 PCR 䛻䜘䜛㚷ูデ᩿䛾᳨ウ.㻌 ➨ 87 ᅇ㯞ᕸ⋇་Ꮫ఍䚸┦ᶍཎ䚸11 ᭶ 2012 ᖺ
66. 㜰ཱྀ㞞ᘯ:≟ࡢ⑌ᝈゎᯒࡢࡓࡵࡢ㑇ఏᏊࣂࣥࢡ. ➨ 6 ᅇᐙ␆㹂㹌㸿す㒓ࢩ࣏ࣥࢪࣗ
࣮࣒,⚟ᓥࠊ10 ᭶ 2012 ᖺ
67. ᑎෆ㇦䚸Ọ⃝⨾ಖ䚸እụள⣖Ꮚ䚸ᆏ⏣᪥㤶㔛䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: 䜲䝚䛾䝠䝖䛻ᑐ䛩䜛♫఍ⓗㄆ▱⬟ຊ䛾≟✀ᕪ. ᪥ᮏ့ங㢮Ꮫ఍ 2012 ᖺᗘ኱఍䚸┦ᶍཎ䚸9 ᭶ 2012 ᖺ
68. 㜰ཱྀ㞞ᘯ:ᖏ㟁ᚤ⢏ᏊỈࠕࢼࣀ࢖࣮ࠖ࡟ࡼࡿ࣌ࢵࢺ࢔ࣞࣝࢤࣥ୙ά໬ࡢ᳨ウ.᪥ᮏ⮫ᗋ⋇་Ꮫࣇ࢛࣮࣒ࣛࠊᮾிࠊ9 ᭶ 2012 ᖺ
69. ᇼᮏὈ௓ࠊ⋞ᩥᏹࠊᒾ㝃◊Ꮚࠊຍ⸨೺ኴ㑻ࠊஂᮎṇᬕࠊ㜰ཱྀ㞞ᘯࠊ᫂▼༤⮧ࠊఀ⸨ኖࠊἙᒸ⩏⿱ࠊ㕥ᮌ࿴⏨ࠊ๓⏣೺: ࢃࡀᅜࡢ့ஙື≀࡟࠾ࡅࡿ࢖ࣥࣇ࢚ࣝࣥࢨ࢘࢖
ࣝࢫឤᰁ. ➨ 154 ᅇ᪥ᮏ⋇་Ꮫ఍,ᒾᡭ,9 ᭶ 2012 ᖺ
70. ཎᗣᘯ䚸୰ᒸඃᕼ䚸⸨஭♸㍤䚸⸨⏣㞝኱䚸ᒣᮏᮍဏ䚸ᐑᓮ㝧Ꮚ䚸⏣୰࿴᫂䚸⁪ἑ㐩ஓ䠖䜲䝚⬡⫫⤌⧊⏤᮶㛫ⴥ⣔ᖿ⣽⬊䛾⚄⤒ศ໬䛻ཬ䜌䛩䝠䝇䝖䞁⬺䜰䝉䝏䝹໬㓝⣲㜼ᐖ๣䛾ᙳ㡪䠊➨ 154 ᅇ᪥ᮏ⋇་Ꮫ఍Ꮫ⾡㞟఍䠈ᒾᡭ䠈9 ᭶ 2012 ᖺ
71. ⣽ᕝ⪷▮䚸ᕝཎ஭᫴ᖹ䚸ὠஂ஭฼ᗈ䚸ஂᮎṇᬕ䚸༳∾ಙ⾜䚸ᩬ┠ᗈ㑻䚸ᅵᒇு䚸ᑠ᪉᐀ḟ:ᅜ⏘䝁䝘䝠䝵䜴䝠䝎䝙ᢠཎᾮ䛾᭷⏝ᛶ䛻㛵䛩䜛෌⪃. ➨ 154 ᅇ᪥ᮏ⋇་Ꮫ఍Ꮫ⾡㞟27

఍. ᒾᡭ䚸9 ᭶ 2012 ᖺ
72. ℩ᕝ࿴ோ, ⸨ᮏ࠶ࡺࡳ, ຍ⸨ᓫ, ᰿ᑿḈᏊ, ஂᮎṇᬕ, ᅵᒇு:࢖ࢾࡢ㍺⾑⏝⾑ᑠᯈ〇๣෭ⶶಖᏑࡢ᳨ウ.➨ 154 ᅇ ᪥ᮏ⋇་Ꮫ఍ࠊᒾᡭࠊ9 ᭶ 2012 ᖺ
73. ⸨ᮏ࠶ࡺࡳ, ▼ሯࡕ࡞ࡘ, ὸ㔝ᑗே, ∾▼ᜨ฼, ຍ⸨ᓫ, ℩ᕝ࿴ோ, ▼ᕝṊྐ, 㟷ᮌ༟☻, ᕝཎ஭᫴ᖹ, ᰿ᑿḈᏊ, ஂᮎṇᬕ, ᅵᒇு:࢖ࢾ⬡⫫⤌⧊⏤᮶㛫㉁⣽⬊࠿ࡽ⫢⣽⬊ᵝ⣽⬊࡬ࡢศ໬ㄏᑟ࡜ᶵ⬟ゎᯒ. ➨ 154 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊᒾᡭࠊ9 ᭶ 2012 ᖺ
74. ᰿ᑿḈᏊ, ∾▼ᜨ฼, ⸨ᮏ࠶ࡺࡳ, ຍ⸨ᓫ, 㕥ᮌ኱௓, ௦⏣Ḡ஧, Ώ㎶ᚁ, ஂᮎ ṇᬕ, ᅵᒇு:࢖ࢾ㦵㧊⣽⬊࠿ࡽศ໬ㄏᑟࡋࡓ⫢ᵝ⣽⬊ࡢ㛗ᮇᇵ㣴ࡢヨ.➨ 154 ᅇ ᪥ᮏ⋇་Ꮫ఍ࠊᒾᡭࠊ9 ᭶ 2012 ᖺ
75. ຍ⸨ᓫ, ஂᮎṇᬕ, ℩ᕝ࿴ோ, ⸨ᮏ䛒䜖䜏, ᰿ᑿḈᏊ, ᑠᯘு௓, ᅵᒇு:NOG 䝬䜴䝇䜢฼⏝䛧䛯䜲䝚㦵㧊⣽⬊䛾㞀ᐖ⫢䜈䛾 Homing స⏝䛾ゎᯒ. ➨ 154 ᅇ ᪥ᮏ⋇་Ꮫ఍ࠊ
ᒾᡭࠊ9 ᭶ 2012 ᖺ
76. Mogi K, Nagasawa M, Shibata Y, Morita T, Yonezawa A, Yabana Y, Kikusui T: The
relationship between stress response of guide dogs and their temperament traits inperipubatal period. 3rd Canine Science Forum, Barcelona, Spain, July, 2012.
77. Nagasawa M, En S, Mogi K, Kikusui T: Reciprocal communication and neuroendocrine
response in human-dog interactions. 3rd Canine Science Forum, Barcelona, Spain, July,
2012.
78. Tsuchihashi N, Kawai E, Nagasawa M, Mogi K, Kikusui T: Dogs show right facial
lateralization to stressful stimuli. 3rd Canine Science Forum, Barcelona, Spain, July, 2012.
79. Ọ⃝⨾ಖ䚸ᅭྐ⥴⌮䚸ᑠᕝ⨾㔛䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: 䜸䜻䝅䝖䝅䞁ᢞ୚䛻䜘䜛䜲䝚䛾㣫䛔୺䜈䛾ὀど⾜ື䛾ቑᙉ. ᪥ᮏື≀ᚰ⌮Ꮫ኱఍➨ 72 ᅇ኱఍䚸㛵すᏛ㝔኱Ꮫ䚸5 ᭶
2012 ᖺ
80. ᡂᮏ἞㸪ᯇᑿ⏤⣖Ꮚ㸪㜿㒊࿴✑ࠊ㜰ཱྀ㞞ᘯࠊ㛗℩㝯ⱥ㸪ᒣୗ┤⨾㸸ዲ୰⌫ᾐ₶ࢆక࠺ႍᜥࣔࢹࣝࢆ⏝࠸ࡓẼ㐨⅖⑕ࡢไᚚࡢゎᯒ.➨㸳㸰ᅇ᪥ᮏ࿧྾ჾᏛ఍, ⚄ᡞࠊ4 ᭶
2012 ᖺ
81. 㣤㔝⍞㈗䚸すᮏඃᏊ䚸⸨ᮏ䛒䜖䜏䚸㔝⃝※ኴ䚸ᕝཎ஭᫴ᖹ䚸ಙ⏣༟⏨䚸ᮌෆ᫂⏨: ≟䛾䜰䝺䝹䜼䞊ᛶ⓶⭵⅖䛾⑕≧⥔ᣢ䛻䛚䛡䜛䝬䜲䜽䝻䝞䝤䝹䜢⏝䛔䛯䝅䝱䞁䝥䞊⒪ἲ䛾᭷ຠᛶ䛾᳨ウ. ᪥ᮏື≀┳ㆤᏛ఍㛵すᆅ༊➨ 5 ᅇ౛఍. ኱㜰䚸2012 ᖺ 3 ᭶
82. 㜰ཱྀ㞞ᘯ 㸸≟ࡢࣂ࢖࢜ࣂࣥࢡࣉࣟࢪ࢙ࢡࢺࡢヨࡳ. ᖹᡂ 23 ᖺᗘ᪥ᮏ⋇་ᖌ఍⋇་Ꮫ⾡Ꮫ఍,ᮐᖠࠊ2 ᭶ 2012 ᖺ
83. Fujimura T, Yonekura S, Horiguchi S, Sakaguchi M, Taniguchi M, Okamoto Y.㸸
Candidate for response monitoring or prognostic biomarkers in two-year sublingual
immunotherapy for Japanese cedar pollinosis. 22th World Allergy Congress, Cancun,
Mexico, Dec, 2011.
84. ཎ⏣᫴ࠊⓑ஭⚽἞ࠊ㜰ཱྀ㞞ᘯ㸸࠾ዲࡳ↝ࡁ⢊ࠊࢱࢥ↝ࡁ⢊࡟ΰධࡋࡓࢲࢽ࡟ࡼࡿ࢔
ࣞࣝࢠ࣮཯ᛂࡢⓎ⑕ᶵᗎ࡟㛵ࡍࡿ⪃ᐹ ➨ 61 ᅇ᪥ᮏ࢔ࣞࣝࢠ࣮Ꮫ఍⥲఍ࠊᮾிࠊ11
᭶ 2011 ᖺ.
85. ⳥Ỉ೺ྐ: 䝨䝑䝖䛾ၥ㢟⾜ື䜈䛾ྲྀ䜚⤌䜏䛂Ẽ㉁䠄ᛶ᱁䠅䜢⌮ゎ䛧䛶デᐹ䜢㛤ጞ䛩䜛䚹䡚ᝈ␆䛾Ẽ㉁䛚䜘䜃ၥ㢟⾜ື䛾ᢕᥱ䛻᭷⏝䛺 C-barq䛃. ➨ 32 ᅇື≀⮫ᗋ་Ꮫ఍ᖺḟ኱
఍. 䜾䝷䞁䜻䝳䞊䝤኱㜰,11 ᭶ 2011 ᖺ
86. Nagasawa M, Kikusui T.: Reciprocal communication and endocrine response in
human-dog interactions. Workshop on the Biology of Prosocial Behavior. Emory
University. Oct, 201128

87. Ἑྜ⤮⨾䚸Ọ⃝⨾ಖ䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: 䜲䝚䛾⾲᝟䛻䛚䛡䜛᝟ື⾲ฟ䛾ഃᛶ໬. 䠐Ꮫ఍ྜྠ኱఍䛂Animal 2011䛃. ៞᠕⩏ሿ኱Ꮫ୕⏣䜻䝱䞁䝟䝇. 9 ᭶ 2011 ᖺ
88. 24.ᑎෆ㇦䚸Ọ⃝⨾ಖ䚸ᆏ⏣᪥㤶㔛䚸ⱱᮌ୍Ꮥ䚸⳥Ỉ೺ྐ: 䜲䝚䛾䝠䝖䛾♫఍ⓗ䝆䜵䝇䝏䝱䞊⌮ゎ䛾≟✀ᕪ. 䠐Ꮫ఍ྜྠ኱఍䛂Animal 2011䛃. ៞᠕⩏ሿ኱Ꮫ୕⏣䜻䝱䞁䝟䝇. 9
᭶ 2011 ᖺ
89. Ọ⃝⨾ಖ䚸⳥Ỉ೺ྐ䚸ⱱᮌ୍Ꮥ䚸ᑎෆ㇦䚸Ἑྜ⤮⨾: ≀య㑅ᢥㄢ㢟䛻䛚䛡䜛ᰘ≟䛾≉ᛶ.䠐Ꮫ఍ྜྠ኱఍䛂Animal 2011䛃. ៞᠕⩏ሿ኱Ꮫ୕⏣䜻䝱䞁䝟䝇. 9 ᭶ 2011 ᖺ
90. ⳥Ỉ೺ྐ: 䝠䝖䛸≟䜢⤎䛠 ʊ⾜ື䛛䜙ぢ䛯 2 ⪅䛾㛵ಀʊ බ㛤䝅䞁䝫䝆䜴䝮䛂≟䜢Ꮫ䜆䚸≟䛻Ꮫ䜆䛃. 䠐Ꮫ఍ྜྠ኱఍䛂Animal 2011䛃. ៞᠕⩏ሿ኱Ꮫ୕⏣䜻䝱䞁䝟䝇. 9 ᭶ 2011 ᖺ
91. ᯇᾆ♸௓䚸㧗ᶫு஧䚸ᕝཎ஭᫴ᖹ䚸ᒣཱྀᛅ⩏䚸ᩬ┠ᗈ㑻䚸௦⏣Ḡ஧. ≟䛻䛚䛡䜛↓⳦ᛶ⫗ⱆ⭘ᛶ⓶⭵⅖䛾୍౛. 㛵ᮾ䞉ᮾிྜྠᆅ༊⋇་ᖌ኱఍.⚄ዉᕝ䚸9 ᭶ 2011 ᖺ
92. ཎᗣᘯ䚸୰ᒸඃᕼ䚸⸨஭♸㍤䚸⸨⏣㞝኱䚸ᐑᓮ㝧Ꮚ䚸⏣୰࿴᫂䚸⁪ἑ㐩ஓ䠖䜲䝚⬡⫫⤌⧊⏤᮶㛫ⴥ⣔ᖿ⣽⬊䛾⣽⬊ቑṪ䛚䜘䜃ከศ໬⬟䛻ཬ䜌䛩䝠䝇䝖䞁⬺䜰䝉䝏䝹໬㓝⣲㜼ᐖ๣䛾ᙳ㡪䠊㻌 ➨ 152 ᅇ᪥ᮏ⋇་Ꮫ఍Ꮫ⾡㞟఍䠈ሜ䠈㻌 9 ᭶ 2011 ᖺ
93. ᓥ಴⚽຾ࠊ࿘⸨᫂⨾ࠊஂಖ⏣⩧ኴࠊᐑᆅ୍ᶞࠊ㧗℩᭷ຍ㔛ࠊᮌෆ᫂⏨ࠊ㜰ཱྀ㞞ᘯ:㑇ఏᛶࡀ␲ࢃࢀࡿ㣗≀࢔ࣞࣝࢠ࣮≟࡟࠾ࡅࡿᐙ⣔ࡢㄪᰝ. ➨ 152 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊሜࠊ9 ᭶ 2011 ᖺ
94. Tsukui T, Fukui M, Ohsumi T, Maeda S, Chimura N, Sakaguchi M, Tsujimoto, H.
Immunotherapy using pullulan-conjugated recombinant house dust mite allergen (Der f2-P). Asian Meeting of Animal Medicine Specialties, Bangkok, Thailand, May , 2011.
95. Kawarai S, Takahashi R, Madarame H, Shirota K. A case of granulomatous dermatitis in adog. Asian Meeting of Animal Medicine Specialties. Bangkok , Thailand, May, 2011.
96. ᡂᮏ἞, ᯇᑿ⏤⣖Ꮚ, 㜿㒊࿴✑, ⓑ஭⚽἞, 㜰ཱྀ㞞ᘯ, 㛗℩㝯ⱥ, ᒣୗ┤⨾㸸 ႍᜥ⑓ែ࡟࠾ࡅࡿẼ㐨ୖ⓶⣽⬊ࡢάᛶ໬࡜᪂つࢡࣝࢡ࣑ࣥㄏᑟయ࡟ࡼࡿไᚚ࡟ࡘ࠸࡚ࡢ
ゎᯒ.➨ 51 ᅇ᪥ᮏ࿧྾ჾᏛ఍ࠊᮾிࠊ4 ᭶ 2011 ᖺ
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䠘◊✲ᡂᯝ䛾බ㛤≧ἣ䠚䠄ୖグ௨እ䠅㻌䝅䞁䝫䝆䜴䝮䞉Ꮫ఍➼䛾ᐇ᪋≧ἣ䚸䜲䞁䝍䞊䝛䝑䝖䛷䛾බ㛤≧ἣ➼㻌䠘᪤䛻ᐇ᪋䛧䛶䛔䜛䜒䛾䠚㻌ᮏ䝥䝻䝆䜵䜽䝖䛻䛴䛔䛶䛿㛵㐃䛧䛯Ꮫ఍䞉◊✲఍䛷䛾㛵㐃䝅䞁䝫䝆䜴䝮➼䛷䛾ᣍᚅㅮ₇䛷ᮏ䝥䝻䝆䜵䜽䛾⤂௓䜔ᡂᯝ䛾Ⓨ⾲䜢⾜䛳䛶䛝䛯䠄Ꮫ఍Ⓨ⾲ 㻡㻜㻘㻢㻜㻘㻣㻢䠅䚹䛥䜙䛻䝩䞊䝮䝨䞊䝆䜢స ᡂ 䛧 䚸 ᮏ 䝥 䝻 䝆 䜵 䜽 䝖 䛾 ෆ ᐜ 䜔 ◊ ✲ ᡂ ᯝ 䛻 䛴 䛔 䛶 බ 㛤 䛧 䛶 䛝 䛯䠄㼔㼠㼠㼜㻦㻛㻛㼣㼣㼣㻚㼍㼦㼍㼎㼡㻙㼡㻚㼍㼏㻚㼖㼜㻛㼟㼓㼗㻛㻜㻞㻚㼔㼠㼙㼘䠅䚹䛥䜙䛻཰㞟≧ἣ䛻䛴䛔䛶䛿཰㞟≟✀➼䛻䛴䛔䛶බ㛤䛩䜛䚹㻌
䠘䛣䜜䛛䜙ᐇ᪋䛩䜛ணᐃ䛾䜒䛾䠚㻌ᮏ䝥䝻䝆䜵䜽䝖䛾ᡂᯝ䛾䜎䛸䜑䛸䛧䛶᮶ᖺᗘ䛻䛣䛾䝥䝻䝆䜵䜽䝖䛻ཧຍ䛧䛶◊✲⪅䛾ᡂᯝ䜢
Ⓨ⾲䛩䜛䝅䞁䝫䝆䜴䝮䜢⾜䛖ணᐃ䛷䛒䜛䚹䛥䜙䛻ᮏ䝥䝻䝆䜵䜽䝖䛾䝩䞊䝮䝨䞊䝆䛷䚸௒ᚋ䚸཰㞟䛥䜜䛯⑌ᝈ䝸䝇䝖䛻䛴䛔䛶䜒බ㛤䛩䜛䚹䛥䜙䛻ⱥᩥ䛷䛾䝩䞊䝮䝨䞊䝆䜒సᡂ䚸ᾏእ䜈䛾᝟ሗබ㛤䜒⾜䛖ணᐃ䛷䛒䜛䚹䛣䜜䛻䜘䛳䛶䚸ᾏእ䛸䛾◊✲䜒ᖜᗈ䛟⾜䛔䛯䛔䚹䜎䛯䚸ᰘ≟䛾඲䝀䝜䝮䝅䞊䜽䜶䞁䝇䝕䞊䝍䛾බ㛤䜢⾜䛖ணᐃ䛷䛒䜛䚹㻌㻌䠍䠐㻌 䛭䛾௚䛾◊✲ᡂᯝ➼㻌ஂᮎ䛿䜹䝩䝔䜽䝜♫䛸䚸䝬䜲䜽䝻䝃䝔䝷䜲䝖ゎᯒ䛻䜘䜛≟䛾⭘⒆デ᩿䜈ᛂ⏝䜢◊✲䛧䛶䛔䜛㻌ᮧୖ䛿ᰴᘧ఍♫∵㉺⏕⌮Ꮫ◊✲ᡤ䛜ື≀⏝೺ᗣ⿵ຓ㣗ရ䛸䛧䛶〇㐀䞉㈍኎䛧䛶䛔䜛 㻾㻒㼁䛻䛿 㻭㼘㼛㼜㼑㼏㼕㼍㻌 㼄 䛻ᑐ䛧䛶๻ⓗ䛺Ⓨẟ⫱ẟຠᯝ䜢♧䛩䛣䛸䛜䛒䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛䚹ᙜヱ௻ᴗ䛸༠ຊ䛧䛶䛭䛾స⏝ᶵᗎ䛾ゎ᫂䜢⾜䛳䛶䛔䜛䚹㻌㰻⸨䛿୰ኸ䝅䝇䝔䝮ᢏ◊㻛䝉䝹䞉䝁䞊䝫䝺䞊䝅䝵䞁䛸䚸≟䛾䛶䜣䛛䜣Ⓨస᥈▱䝅䝇䝔䝮䛾䝅䝇䝔䝮㛤Ⓨ䜢⾜䛳䛶䛔䜛䚹䜎䛯䚸≉チฟ㢪䜢⾜䛳䛶䛔䜛ຍ㏿ᗘ䜢฼⏝䛧䛯≟䛾䛶䜣䛛䜣ุู⏝䜰䝹䝂䝸䝈䝮䜢⏝䛔䛶䚸䝇䝬䞊䝖䝣䜷䞁䛺䛹䜈䝸䜰䝹䝍䜲䝮䛷䛶䜣䛛䜣Ⓨస䛾㏻▱䚸ᫎീ䛷䛾䝰䝙䝍䝸䞁䜾䛜ྍ⬟䛺䝅䝇䝔䝮䛾㛤Ⓨ䛸⮫ᗋ᳨ド䜈ྥ䛡䛯䝅䝇䝔䝮඲య䛾ㄪᩚ䜢⾜䛳䛶䛔䜛䚹㻌ᮧୖ㈼䛿≟䛾⫧‶䛻㛵㐃䛩䜛ೃ⿵㑇ఏᏊ䛾ኚ␗䜢ㄪ䜉䛯䚹䝬䜴䝇䛸䝠䝖䛻䛚䛔䛶⫧‶䛸㛵㐃䛩䜛㑇ఏᏊ䛸䛧䛶䚸䛭䜜䛮䜜 㻭㼘㼗㻣㻔㻭㼢㼏㼢㼞㻝㼏㻕䛸 㻭㻸㻷㻟㻔㻮㻹㻼㻾㻝㻭㻕䛾ኚ␗䛜ሗ࿌䛥䜜䛶䛔䜛䚹䛣䜜䜙䛾㑇ఏᏊ䛻䛴䛔䛶䚸≟✀䛻䜘䜛ኚ␗䛻䛴䛔䛶᳨⣴䛧䛯䚹䜲䝚䛾 㻭㼘㼗㻣 䛸 㻭㼘㼗㻟 㑇ఏᏊ䛾䛭䜜䛮䜜䛻䛴䛔䛶䚸᥎ᐃ䛥䜜䜛ኚ␗㒊఩䜢⡆౽䛻᳨ฟ䛷䛝䜛 㻼㻯㻾䌦㻾㻲㻸㻼 ἲ䜢⪃᱌䛧䚸㻡㻝 ≟✀㻝㻢㻝 ಶయ䛸 㻠㻜 ≟✀ 㻥㻟 ಶయ䛻䛴䛔䛶ㄪ䜉䛯䛜㓄ิኚ␗䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䚹㻌㻌䠍䠑㻌 䛂㑅ᐃ᫬䛃ཬ䜃䛂୰㛫ホ౯᫬䛃䛻௜䛥䜜䛯␃ព஦㡯䛸䛭䜜䜈䛾ᑐᛂ㻌䠘䛂㑅ᐃ᫬䛃䛻௜䛥䜜䛯␃ព஦㡯䠚㻌ヱᙜ䛺䛧㻌䠘䛂㑅ᐃ᫬䛃䛻௜䛥䜜䛯␃ព஦㡯䜈䛾ᑐᛂ䠚㻌ヱᙜ䛺䛧㻌䠘䛂୰㛫ホ౯᫬䛃䛻௜䛥䜜䛯␃ព஦㡯䠚㻌ヱᙜ䛺䛧㻌䠘䛂୰㛫ホ౯᫬䛃䛻௜䛥䜜䛯␃ព஦㡯䜈䛾ᑐᛂ䠚㻌
ヱᙜ䛺䛧㻌

30

㻝㻠㻝㻜㻜㻝
㻿㻝㻝㻜㻝㻜㻞㻟

ἲே␒ྕ䝥䝻䝆䜵䜽䝖␒ྕ䠍䠒䚷᪋タ䞉⿦⨨䞉タഛ䞉◊✲㈝䛾ᨭฟ≧ἣ䠄ᐇ⦼ᴫせ䠅ෆᖺᗘ䞉༊ศ ᨭฟ㢠 ἲ䚷ே
ᖹ ᪋ タ
ᡂ
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ᡂ
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ヂ

ඹྠ◊
ཷク
✲ᶵ㛵
ᐤ௜㔠 䛭䛾௚䠄
◊✲➼
㈇ᢸ

⚾䚷Ꮫ
ຓ䚷ᡂ

㈇䚷ᢸ

䠄༓෇䠅

䠅

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◊✲㈝

㻝㻟㻟㻘㻠㻟㻥

㻣㻝㻘㻜㻢㻤

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⥲
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䈜 ᭱⤊ᖺᗘ䛿ணᐃ㢠䚹

31

ഛ䚷⪃

ἲே␒ྕ

㻝㻠㻝㻜㻜㻝

䠍䠓䚷᪋タ䞉⿦⨨䞉タഛ䛾ᩚഛ≧ἣ㻌㻌䠄⚾Ꮫຓᡂ䜢ཷ䛡䛯䜒䛾䛿䛩䜉䛶グ㍕䛧䛶䛟䛰䛥䛔䚹䠅䛀᪋䚷䚷タ䛁㻌㻌䠄⚾Ꮫຓᡂ䜢ཷ䛡䛶䛔䛺䛔䜒䛾䜒ྵ䜑䚸౑⏝䛧䛶䛔䜛᪋タ䜢䛩䜉䛶グ㍕䛧䛶䛟䛰䛥䛔䚹䠅᪋䚷タ䚷䛾䚷ྡ䚷⛠ᩚഛᖺᗘ◊✲᪋タ㠃✚◊ ✲ ᐊ ➼ ᩘ ౑⏝⪅ᩘ

஦ᴗ⤒㈝

䠄༓෇䠅
⿵ຓ୺య

⿵ຓ㔠㢠

䛺䛧

䈜 ⚾Ꮫຓᡂ䛻䜘䜛⿵ຓ஦ᴗ䛸䛧䛶⾜䛳䛯᪂ቑ⠏䛻䜘䜚䚸ᩚഛ๓䛸ẚ㍑䛧䛶ቑຍ䛧䛯㠃✚䟝䛀⿦⨨䞉タഛ䛁䚷䠄⚾Ꮫຓᡂ䜢ཷ䛡䛶䛔䛺䛔䜒䛾䛿䚸୺䛺䜒䛾䛾䜏䜢グ㍕䛧䛶䛟䛰䛥䛔䚹䠅⿦⨨䞉タഛ䛾ྡ⛠

ᩚഛᖺᗘ

ᆺ䚷䚷␒

ྎ䚷䚷ᩘ

✌ാ᫬㛫ᩘ㻛ᖺ

஦ᴗ⤒㈝

䠄༓෇䠅
⿵ຓ୺య

⿵ຓ㔠㢠

䠄◊✲⿦⨨䠅䚷䛺䛧䠄◊✲タഛ䠅
෭༷㐲ᚰᶵ

㻴㻞㻟

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䠍ྎ

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䡄

㻢㻞㻜

䡷䡬䢋䢕䡷䡮䡴䢓䞊㻔㻼㻯㻾⏝㻕㻴㻞㻟㼀㻼㻙㻢㻜㻜

㻝ྎ

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䝯䝕䜱䜹䝹෭ⶶᗜ

㻴㻞㻟㻹㻼㻾㻙㻡㻝㻠

㻝ྎ

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䡄

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䡄

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㻜

⺯ග䞉Ⓨග䝥䝺䞊䝖䝸䞊䝎䞊୍ᘧ㻴㻞㻟㻮㼀㻙㻿㻹㻭㼀㻮㻸䝟䝽䞊䝇䜻䝱䞁㻹㼄

䠍ྎ

㻝㻡㻢

䡄

㻡㻘㻠㻢㻜

㻟㻘㻢㻠㻜

䝸䜰䝹䝍䜲䝮㻼㻯㻾⿦⨨୍ᘧ㻴㻞㻠㻣㻡㻜㻜䇲㻜㻝

㻝ྎ

㻝㻘㻜㻡㻟

䡄

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⺯ග㢧ᚤ㙾⿦⨨୍ᘧ㻴㻞㻡㻵㼄㻤㻟㻼㻝㻙㻞㻠㻲㻸㻛㻼㻴

㻝ྎ

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䡄

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䝍䞁䝟䜽㉁㌿෗⿦⨨㻴㻞㻢㻝㻣㻜㻠㻝㻡㻜㻶㻝

㻝ྎ

㻟㻜㻜

䡄

㻞㻠㻥

䠄᝟ሗฎ⌮㛵ಀタഛ䠅
䚷䛺䛧

32

㻜
㻜

⚾Ꮫຓᡂ
ᩥ㒊⛉Ꮫ┬
⚾Ꮫຓᡂ
㻟㻘㻥㻞㻜
ᩥ㒊⛉Ꮫ┬
⚾Ꮫຓᡂ
㻟㻘㻠㻢㻢
ᩥ㒊⛉Ꮫ┬
㻜

ἲே␒ྕ
䠍䠔䚷◊✲㈝䛾ᨭฟ≧ἣ
ᖺ䚷䚷䚷ᗘ
㻌ᖹᡂ
ᑠ㻌㻌⛉㻌㻌┠㻝㻠㻝㻜㻜㻝
䠄༓෇䠅

㻞㻟

ᖺᗘ

✚䚷䚷⟬䚷䚷ෆ䚷䚷ヂ୺䚷䛺䚷౑䚷㏵㔠䚷䚷㢠୺䚷䚷䛺䚷䚷ෆ䚷䚷ᐜᩍ䚷䚷䚷䚷⫱䚷䚷䚷䚷◊䚷䚷䚷䚷✲䚷䚷䚷䚷⤒䚷䚷䚷䚷㈝䚷䚷䚷䚷ᨭ䚷䚷䚷䚷ฟ㻝㻠㻘㻜㻤㻝 ヨ⸆䞉ᐇ㦂ჾල㻝㻠㻘㻜㻤㻝 䜹䝇䝍䝮䝥䝷䜲䝬䞊䚸ὀᑕ㔪䚸䝯䝇䝅䝸䞁䝎䞊䚸㐲ỿ⟶䚸⸆ໟ⣬䜋䛛㻜㻜㻡㻜 㐠㏦㈝㻡㻜 ⾑ᾮ䝃䞁䝥䝹㍺㏦㈝䜋䛛
㻝㻞㻤 ㄽᩥᢞ✏ᩱ㻝㻞㻤 䠄♫䠅᪥ᮏ⋇་Ꮫ఍㻞㻡㻢 ⡿ᅜ㻯㼔㼑㼟㼠Ꮫ఍㻞㻤㻡 Ꮫ఍ཧຍ㈝⏝㻞㻥 ⚟ᓥ┴⋇་ᖌ఍ゎᯒᩱ䞉ⱥᩥᰯṇᩱ㻞㻘㻡㻜㻟 㻰㻺㻭䝅䞊䜽䜶䞁䝇ゎᯒ䚸䜲䝚䝃䜲䝖䜹䜲䞁 ᐃ䠈ⱥᩥᰯṇᩱ
㻞㻘㻤㻜㻟
ሗ㓘ᩱ
㻟㻜㻜 ⾑ᾮ䝃䞁䝥䝹᥇㞟㻝㻝㻠 䛭䛾௚㻝㻝㻠 Ꮫ఍ཧຍ㈝䚸኱ุ䝥䝸䞁䝍䞊౑⏝ᩱ㻝㻣㻘㻠㻢㻝
䜰䝹䚷䚷䚷䝞䚷䚷䚷䜲䝖䚷䚷䚷㛵䚷䚷䚷ಀ䚷䚷䚷ᨭ䚷䚷䚷ฟ㻞㻘㻞㻣㻣 ◊✲⿵ຓ䞉䝕䞊䝍ᩚ⌮㻞㻘㻞㻣㻣 ᫬⤥䚷㻝㻘㻟㻜㻜෇䠈ᖺ㛫᫬㛫ᩘ䚷㻝㻘㻣㻡㻞᫬㛫ᨭ䚷ฟ䚷㢠ᾘ䚷⪖䚷ရ䚷㈝ග䚷⇕䚷Ỉ䚷㈝
㏻ಙ㐠ᦙ㈝
༳ๅ〇ᮏ㈝
᪑㈝஺㏻㈝
ሗ㓘䞉ጤクᩱ䛭䛾௚ィ
ே௳㈝ᨭฟ
䠄වົ⫋ဨ䠅
ᩍ⫱◊✲⤒㈝ᨭฟ
ィᐇேᩘ䚷㻞ே㻜㻜㻞㻘㻞㻣㻣タ㻌ഛ㻌㛵㻌ಀ㻌ᨭ㻌ฟ䠄䠍ಶཪ䛿䠍⤌䛾౯᱁䛜䠑䠌䠌୓෇ᮍ‶䛾䜒䛾䠅㻠㻘㻣㻤㻞 ഛရ㻠㻘㻣㻤㻞 㻮㼕㼛㻸㼛㼓㼕㼏䚷㻯㼛㼞㼑䝅䝇䝔䝮䚸䝣䝸䞊䝈㉸ప ᵴ䜋䛛
㻜
㻜
㻠㻘㻣㻤㻞
◊䚷䚷䚷✲䚷䚷䚷䝇䝍䚷䚷䚷䝑䚷䚷䚷䝣㛵䚷䚷䚷ಀ䚷䚷䚷ᨭ䚷䚷䚷ฟ
㻜
㻜
㻜
㻜
㻜
㻜
㻜

ᩍ⫱◊✲⏝ᶵჾഛရ

ᅗ䚷䚷䚷䚷᭩
ィ䝸䝃䞊䝏䞉䜰䝅䝇䝍䞁䝖䝫䝇䝖䞉䝗䜽䝍䞊
◊✲ᨭ᥼᥎㐍⤒㈝

ィ

䠄༓෇䠅
㻞㻠

ᖺ䚷䚷䚷ᗘ

㻌ᖹᡂ

ᑠ㻌㻌⛉㻌㻌┠ᨭ䚷ฟ䚷㢠ᾘ䚷⪖䚷ရ䚷㈝ග䚷⇕䚷Ỉ䚷㈝
㏻ಙ㐠ᦙ㈝
༳ๅ〇ᮏ㈝
᪑㈝஺㏻㈝
ሗ㓘䞉ጤクᩱ䛭䛾௚ィ
ே௳㈝ᨭฟ
䠄වົ⫋ဨ䠅
ᩍ⫱◊✲⤒㈝ᨭฟ
ィᩍ⫱◊✲⏝ᶵჾഛရ

ᅗ䚷䚷䚷䚷᭩
ィ䝸䝃䞊䝏䞉䜰䝅䝇䝍䞁䝖䝫䝇䝖䞉䝗䜽䝍䞊
◊✲ᨭ᥼᥎㐍⤒㈝ィᖺᗘ✚䚷䚷⟬䚷䚷ෆ䚷䚷ヂ୺䚷䛺䚷౑䚷㏵㔠䚷䚷㢠୺䚷䚷䛺䚷䚷ෆ䚷䚷ᐜᩍ䚷䚷䚷䚷⫱䚷䚷䚷䚷◊䚷䚷䚷䚷✲䚷䚷䚷䚷⤒䚷䚷䚷䚷㈝䚷䚷䚷䚷ᨭ䚷䚷䚷䚷ฟ㻝㻞㻘㻜㻜㻠 ヨ⸆䞉ᐇ㦂ჾල㻝㻞㻘㻜㻜㻠 㻰㻺㻭඲⾑䝉䝑䝖䠈䢋䡮䡴䢗䡽䡩䡬䢈䢚䠈䝨䝥䝏䝗ྜᡂ䜋䛛
㻜
㻜
㻥 㐠㏦㈝
㻥 Ꮿ㓄౽
㻡㻝㻡 ༳ๅ〇ᮏ㈝㻡㻝㻡 䝞䜲䜸䝞䞁䜽䝸䞊䝣䝺䝑䝖సᡂ㈝㻞㻤 ᪑㈝㻞㻤 䝅䜵䝹䝍㻙ಖㆤື≀䝃䞁䝥䝸䞁䜾䠄⚟ᓥ䠅ゎᯒᩱ㻢㻥㻣 㻰㻺㻭䝅䞊䜽䜶䞁䝇ゎᯒ
㻤㻜㻜
ⱥᩥᰯṇᩱ
㻝㻜㻟 ⱥᩥᰯ㜀ಟ⧋㈝㻝㻡㻤 䝸䜰䝹䝍䜲䝮㻼㻯㻾⿦⨨ಟ⌮䠈䝢䝨䝑䝖䝬䞁ಟ⌮㻞㻜㻠䛭䛾௚㻠㻢 Ꮫ఍ཧຍ㈝䚸㧗㏿㐨㊰㏻⾜ᩱ䚸㥔㌴ሙ௦㻝㻟㻘㻡㻢㻜
䜰䝹䚷䚷䚷䝞䚷䚷䚷䜲䝖䚷䚷䚷㛵䚷䚷䚷ಀ䚷䚷䚷ᨭ䚷䚷䚷ฟ㻥㻘㻝㻟㻡 ◊✲⿵ຓ䞉䝕䞊䝍ᩚ⌮㻥㻘㻝㻟㻡 ᫬⤥䚷㻝㻘㻟㻜㻜෇䠈ᖺ㛫᫬㛫ᩘ䚷㻣㻘㻜㻞㻣᫬㛫ᐇேᩘ䚷㻠ே㻜㻜㻥㻘㻝㻟㻡タ㻌ഛ㻌㛵㻌ಀ㻌ᨭ㻌ฟ䠄䠍ಶཪ䛿䠍⤌䛾౯᱁䛜䠑䠌䠌୓෇ᮍ‶䛾䜒䛾䠅㻝㻘㻟㻥㻥 ഛရ㻝㻘㻟㻥㻥 ඲⮬ື㟁ẼὋື䡹䡿䡬䡸䡪䢙㻞㻢 ᅗ᭩㻞㻢 ὒ᭩䜋䛛
㻝㻘㻠㻞㻡◊䚷䚷䚷✲䚷䚷䚷䝇䝍䚷䚷䚷䝑䚷䚷䚷䝣㛵䚷䚷䚷ಀ䚷䚷䚷ᨭ䚷䚷䚷ฟ
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33

ἲே␒ྕ

㻝㻠㻝㻜㻜㻝
䠄༓෇䠅

㻞㻡

ᖺ䚷䚷䚷ᗘ

㻌ᖹᡂ

ᑠ㻌㻌⛉㻌㻌┠ᨭ䚷ฟ䚷㢠ᖺᗘ✚䚷䚷⟬䚷䚷ෆ䚷䚷ヂ୺䚷䛺䚷౑䚷㏵㔠䚷䚷㢠୺䚷䚷䛺䚷䚷ෆ䚷䚷ᐜᩍ䚷䚷䚷䚷⫱䚷䚷䚷䚷◊䚷䚷䚷䚷✲䚷䚷䚷䚷⤒䚷䚷䚷䚷㈝䚷䚷䚷䚷ᨭ䚷䚷䚷䚷ฟ㻝㻣㻘㻞㻟㻞 ヨ⸆䞉ᐇ㦂ჾල㻝㻣㻘㻞㻟㻞 㻰㻺㻭ྜᡂ䠈ヨ⸆䠈䝥䝷䝇䝏䝑䜽〇ရ䠈䝗䝑䜽䝣䞊䝗䜋䛛㻜㻜㻝㻜 㐠㏦㈝㻝㻜 Ꮿ㓄౽䠈᣺㎸ᡭᩘᩱ㻜㻜㻜㻜ゎᯒᩱ䞉ⱥᩥᰯṇᩱ㻝㻘㻜㻣㻜 㻰㻺㻭䝅䞊䜽䜶䞁䝇ゎᯒ䠈ⱥᩥᰯ㜀㻝㻘㻥㻥㻠䝞䜲䜸䝞䞁䜽㻴㻼㛤タ㻥㻞㻠 䝞䜲䜸䝞䞁䜽㻴㻼㛤タጤク㈝㻝㻜㻤 䛭䛾௚㻝㻜㻤 Ꮫ఍ཧຍ㈝㻝㻥㻘㻟㻠㻠
䜰䝹䚷䚷䚷䝞䚷䚷䚷䜲䝖䚷䚷䚷㛵䚷䚷䚷ಀ䚷䚷䚷ᨭ䚷䚷䚷ฟ㻠㻘㻤㻣㻤 ◊✲⿵ຓ䞉䝕䞊䝍ᩚ⌮㻠㻘㻤㻣㻤 ᫬⤥䚷㻝㻘㻟㻜㻜෇䠈ᖺ㛫᫬㛫ᩘ䚷㻟㻘㻣㻡㻞᫬㛫ᾘ䚷⪖䚷ရ䚷㈝ග䚷⇕䚷Ỉ䚷㈝
㏻ಙ㐠ᦙ㈝
༳ๅ〇ᮏ㈝
᪑㈝஺㏻㈝
ሗ㓘䞉ጤクᩱ䛭䛾௚ィ
ே௳㈝ᨭฟ
䠄වົ⫋ဨ䠅
ᩍ⫱◊✲⤒㈝ᨭฟ
ィᐇேᩘ䚷㻞ே㻜㻜㻠㻘㻤㻣㻤タ㻌ഛ㻌㛵㻌ಀ㻌ᨭ㻌ฟ䠄䠍ಶཪ䛿䠍⤌䛾౯᱁䛜䠑䠌䠌୓෇ᮍ‶䛾䜒䛾䠅㻡㻟㻣 ഛရ㻡㻟㻣 䢈䢛䢓䡮䢌䢙䡴䢚䡹䡿䡬䡸䡪䢙䠈⏕≀㢧ᚤ㙾䜋䛛
㻠㻝 ᅗ᭩
㻠㻝 ὒ᭩
㻡㻣㻤
◊䚷䚷䚷✲䚷䚷䚷䝇䝍䚷䚷䚷䝑䚷䚷䚷䝣㛵䚷䚷䚷ಀ䚷䚷䚷ᨭ䚷䚷䚷ฟ
㻜
㻜
㻜
㻜
㻜
㻜
㻜

ᩍ⫱◊✲⏝ᶵჾഛရ

ᅗ䚷䚷䚷䚷᭩
ィ䝸䝃䞊䝏䞉䜰䝅䝇䝍䞁䝖䝫䝇䝖䞉䝗䜽䝍䞊
◊✲ᨭ᥼᥎㐍⤒㈝

ィ

䠄༓෇䠅
㻞㻢

ᖺ䚷䚷䚷ᗘ

㻌ᖹᡂ

ᑠ㻌㻌⛉㻌㻌┠ᨭ䚷ฟ䚷㢠ᾘ䚷⪖䚷ရ䚷㈝ග䚷⇕䚷Ỉ䚷㈝
㏻ಙ㐠ᦙ㈝
༳ๅ〇ᮏ㈝
᪑㈝஺㏻㈝
ሗ㓘䞉ጤクᩱ䛭䛾௚ィ
ே௳㈝ᨭฟ
䠄වົ⫋ဨ䠅
ᩍ⫱◊✲⤒㈝ᨭฟ
ィᩍ⫱◊✲⏝ᶵჾഛရ

ᅗ䚷䚷䚷䚷᭩
ィ䝸䝃䞊䝏䞉䜰䝅䝇䝍䞁䝖䝫䝇䝖䞉䝗䜽䝍䞊
◊✲ᨭ᥼᥎㐍⤒㈝ィᖺᗘ✚䚷䚷⟬䚷䚷ෆ䚷䚷ヂ୺䚷䛺䚷౑䚷㏵㔠䚷䚷㢠୺䚷䚷䛺䚷䚷ෆ䚷䚷ᐜᩍ䚷䚷䚷䚷⫱䚷䚷䚷䚷◊䚷䚷䚷䚷✲䚷䚷䚷䚷⤒䚷䚷䚷䚷㈝䚷䚷䚷䚷ᨭ䚷䚷䚷䚷ฟ㻞㻜㻘㻟㻟㻢 㻰㻺㻭ྜᡂ䠈ヨ⸆䠈䝥䝷䝇䝏䝑䜽〇ရ䠈䝗䝑䜽䝣䞊䝗䜋䛛㻞㻜㻘㻟㻟㻢 ヨ⸆䞉ᐇ㦂ჾල㻜㻜㻣㻡 㐠㏦㈝㻣㻡 Ꮿ㓄౽㻜㻜㻤㻢㻡 ᪑㈝㻤㻢㻡 እ㒊ホ౯ጤဨ᪑㈝䠈䝃䞁䝥䝹཰㞟䛾䛯䜑ሗ㓘ᩱ㻢㻥㻥 ⾑Ύ᥇ྲྀ䠈䝕䞊䝍ᩚ⌮㻞㻘㻜㻡㻢ゎᯒᩱ䞉ⱥᩥᰯṇᩱ㻝㻘㻟㻡㻣 㻰㻺㻭䝅䞊䜽䜶䞁䝇ゎᯒ䠈ⱥᩥᰯ㜀䠈䢆䢚䡮䡱䢆䢚䢙䡴䡷䡬䢆䢚䡬⟶⌮ጤク㈝㻢㻜 ㈤೉ᩱ
㻢㻜 ᪋タ฼⏝ᩱ㻞㻟㻘㻟㻥㻞
䜰䝹䚷䚷䚷䝞䚷䚷䚷䜲䝖䚷䚷䚷㛵䚷䚷䚷ಀ䚷䚷䚷ᨭ䚷䚷䚷ฟ㻠㻘㻥㻤㻣 ◊✲⿵ຓ䞉䝕䞊䝍ᩚ⌮㻠㻘㻥㻤㻣 ᫬⤥䚷㻤㻣㻜䡚㻝㻘㻟㻜㻜෇䠈ᖺ㛫᫬㛫ᩘ䚷㻟㻘㻤㻠㻠᫬㛫ᐇேᩘ䚷㻟ே㻜㻜㻠㻘㻥㻤㻣タ㻌ഛ㻌㛵㻌ಀ㻌ᨭ㻌ฟ䠄䠍ಶཪ䛿䠍⤌䛾౯᱁䛜䠑䠌䠌୓෇ᮍ‶䛾䜒䛾䠅㻝㻘㻢㻜㻟 ഛရ㻝㻘㻢㻜㻟 䢆䢚䡮䡱䢎䡿䢚䡤䡲䢕䢈䢔䡬䡷䢚䡬䠈䢆䢚䡮䡱䢋䢕䡽䡴䡬䢓䡬䜋䛛㻝㻤 ᅗ᭩㻝㻤 ⬻䛸⚄⤒䛾ぢ᪉䜋䛛㻝㻘㻢㻞㻝◊䚷䚷䚷✲䚷䚷䚷䝇䝍䚷䚷䚷䝑䚷䚷䚷䝣㛵䚷䚷䚷ಀ䚷䚷䚷ᨭ䚷䚷䚷ฟ
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34

ἲே␒ྕ

㻝㻠㻝㻜㻜㻝
䠄༓෇䠅

㻞㻣

ᖺ䚷䚷䚷ᗘ

㻌ᖹᡂ

ᑠ㻌㻌⛉㻌㻌┠ᨭ䚷ฟ䚷㢠ᾘ䚷⪖䚷ရ䚷㈝ග䚷⇕䚷Ỉ䚷㈝
㏻ಙ㐠ᦙ㈝
༳ๅ〇ᮏ㈝
᪑㈝஺㏻㈝
ሗ㓘䞉ጤクᩱ䛭䛾௚ィ
ே௳㈝ᨭฟ
䠄වົ⫋ဨ䠅
ᩍ⫱◊✲⤒㈝ᨭฟ
ィᩍ⫱◊✲⏝ᶵჾഛရ

ᅗ䚷䚷䚷䚷᭩
ィ䝸䝃䞊䝏䞉䜰䝅䝇䝍䞁䝖䝫䝇䝖䞉䝗䜽䝍䞊
◊✲ᨭ᥼᥎㐍⤒㈝ィᖺᗘ✚䚷䚷⟬䚷䚷ෆ䚷䚷ヂ୺䚷䛺䚷౑䚷㏵㔠䚷䚷㢠୺䚷䚷䛺䚷䚷ෆ䚷䚷ᐜᩍ䚷䚷䚷䚷⫱䚷䚷䚷䚷◊䚷䚷䚷䚷✲䚷䚷䚷䚷⤒䚷䚷䚷䚷㈝䚷䚷䚷䚷ᨭ䚷䚷䚷䚷ฟ㻝㻢㻘㻣㻤㻜 ヨ⸆䞉ᐇ㦂ჾල㻝㻢㻘㻣㻤㻜 䡲䡹䡼䢍䢈䢛䢓䡮䢋䡬䠈ヨ⸆䠈䝥䝷䝇䝏䝑䜽〇ရ䠈䢉䢛䡫䢀䡸䡬䡾䜋䛛㻜㻜㻝㻞㻢 㐠㏦㈝㻝㻞㻢 Ꮿ㓄౽㻝㻤㻠 ㄽᩥᢞ✏ᩱ㻝㻤㻠 䜸䞊䝥䞁䜰䜽䝉䝇ฟ∧ᥖ㍕ᩱ㻤㻢㻣 ᪑㈝㻤㻢㻡 䝃䞁䝥䝹཰㞟䛾䛯䜑䠈Ꮫ఍ཧຍሗ㓘ᩱ㻝㻘㻝㻠㻣 ⾑Ύ᥇ྲྀ䠈䝕䞊䝍ᩚ⌮㻠㻘㻞㻟㻝ゎᯒᩱ䞉ⱥᩥᰯṇᩱ㻟㻘㻜㻤㻠 㻰㻺㻭䝅䞊䜽䜶䞁䝇ゎᯒ䠈ⱥᩥᰯ㜀㻥㻥 䛭䛾௚㻥㻥 Ꮫ఍ཧຍ㈝䠈㈤೉ᩱ䜋䛛㻞㻞㻘㻞㻤㻣
䜰䝹䚷䚷䚷䝞䚷䚷䚷䜲䝖䚷䚷䚷㛵䚷䚷䚷ಀ䚷䚷䚷ᨭ䚷䚷䚷ฟ㻢㻘㻠㻣㻤 ◊✲⿵ຓ䞉䝕䞊䝍ᩚ⌮㻢㻘㻠㻣㻤 ᫬⤥䚷㻤㻥㻜䡚㻝㻘㻟㻡㻜෇䠈ᖺ㛫᫬㛫ᩘ䚷㻠㻘㻥㻥㻥᫬㛫ᐇேᩘ䚷㻝㻠ே㻜㻜㻢㻘㻠㻣㻤タ㻌ഛ㻌㛵㻌ಀ㻌ᨭ㻌ฟ䠄䠍ಶཪ䛿䠍⤌䛾౯᱁䛜䠑䠌䠌୓෇ᮍ‶䛾䜒䛾䠅㻝㻘㻝㻟㻠 ഛရ㻝㻘㻝㻟㻠 㻯㻻㻞䡮䢙䡳䡩䢉䢚䡬䡼䡬䠈⸆⏝෭ⶶ䡸䡪䡬䡵䡬䡹䜋䛛㻝㻜㻝 ᅗ᭩㻝㻜㻝 ὒ᭩
㻝㻘㻞㻟㻡◊䚷䚷䚷✲䚷䚷䚷䝇䝍䚷䚷䚷䝑䚷䚷䚷䝣㛵䚷䚷䚷ಀ䚷䚷䚷ᨭ䚷䚷䚷ฟ
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35

≟ࡢ㑇ఏᛶ⑌ᝈ࡟࠾ࡅࡿ '1$ ཰㞟࣭⢭〇࣭౪⤥࠾ࡼࡧ⑌ᝈ᝟ሗࡢࢹ࣮ࢱ࣮࣋ࢫ໬
㜰ཱྀ㞞ᘯࠊᒸᮏ᠇᫂ࠊᓥ಴⚽຾㸦㯞ᕸ኱Ꮫᚤ⏕≀Ꮫ➨㸯㸧༳∾ಙ⾜㸦㯞ᕸ኱Ꮫ㝃ᒓື≀ࠊ⳥Ỉ೺ྐ㸦㯞ᕸ኱Ꮫక౶ື≀㸧ࠊஂᮎṇᬕ㸦㯞ᕸ኱Ꮫ⑓㝔㸧ᮧୖ㈼㸦㯞ᕸ኱ᏛศᏊ⏕≀㸧ෆ⛉➨㸰㸧ࠊ㰻⸨ᘺ௦Ꮚ㸦㯞ᕸ኱Ꮫእ⛉➨㸰㸧ࠊ⁪ἑ㐩ஓࠊ⏣୰࿴᫂㸦㯞ᕸ኱Ꮫື≀ᕤᏛ㸧㻌◊✲せ᪨㑇ఏ⑓ࡸ㑇ఏ㛵㐃⑌ᝈࢆྵࡴ㠀ឤᰁᛶ⑌ᝈ ≟࠾ࡼࡧ୍⯡㞟ᅋ≟࠿ࡽ '1$ ཰㞟ࢆ⾜࠸ ᳨యࡀ཰㞟ࡉࢀࠊࡑࢀࡒࢀࡢ≟ࡣࡑࡢᇶᮏ᝟ሗ࡜࡜ࡶ࡟⑌ᝈ᝟ሗࡶ᪥ᮏ⋇་Ꮫ఍⑌ᝈྡ⏝ㄒ㞟࡟㒊㢮࡟ᇶ࡙࠸࡚ࢹ࣮ࢱ࣮࣋ࢫ໬ࡉࢀࡓࠋࡇࢀࡽࡢ཰㞟ࡉࢀࡓ '1$ ᳨యࡣࡇࡢ
ࣉࣟࢪ࢙ࢡࢺࡢ◊✲⪅࡟ᥦ౪ࡋ࡚ྛㄢ㢟࡟࠾࠸࡚㑇ఏᏊゎᯒࢆ⾜ࡗࡓࠋࡲࡓࠊࡇࡢࣉࣟࢪ
࢙ࢡࢺ௨እࡢ◊✲⪅࡬ '1$ ࢆ౪⤥ࡋࠊᵝࠎ࡞ඹྠ◊✲ࢆ⾜ࡗࡓࠋ⋇་኱Ꮫ⑓㝔࡛ࡢከࡃࡢ
⑕౛᳨య࡛ࡣ⾑Ύ ⾑₢ ࡶేࡏ࡚ಖᏑࡋࡓࠋ⾑Ύࡣ㑇ఏ⑌ᝈࢆࡣࡌࡵࠊ௚ࡢᵝࠎ࡞⑌ᝈࡢ
Ⓨ⏕ᶵᗎࡢゎᯒࡸឤᰁ⑕࡟࠾ࡅࡿ⾑Ύ␿Ꮫ࡞࡝ࢆ◊✲ࡍࡿ࡜ࡁ࡟㠀ᖖ࡟ᙺ❧ࡘᮦᩱ࡛࠶ࡿ
࡜⪃࠼ࡽࢀࡿࠋ௒ᚋࡶ '1$ ཰㞟ࡣ⥅⥆ࡋ࡚⾜࠸ࠊࡇࡢ㑇ఏᏊࣂࣥࢡࡣ⋇་Ꮫࡢ⮫ᗋ◊✲ࡢ
Ⓨᒎ࡟኱࠸࡟ᐤ୚ࡍࡿ࡜⪃࠼ࡽࢀࡿࠋ◊✲┠ⓗ ≟ࡶே࡜ྠᵝ࡟ከࡃࡢ㑇ఏᛶ⑌ᝈࡀ࠶ࡾࠊே࡜⑓ែࡀఝ࡚࠸ࡿ⑌ᝈࡶከࡃᏑᅾࡍࡿࠋᮏ
◊✲࡟࠾࠸࡚≟ࡢ㑇ఏᏊ '1$ ㈨※ࡢά⏝࡜ಖᏑࡢࡓࡵ࡟ࠊ⋇་⣔኱Ꮫ㝃ᒓື≀⑓㝔➼ࡢ⮫ᗋ་ࢆ୰ᚰ࡟ᵝࠎ࡞㑇ఏ㛵㐃⑌ᝈࢆྵࡴ㠀ឤᰁᛶ⑌ᝈ≟ࡸ୍⯡㞟ᅋ࡜ࡋ࡚ࡢ≟࡞࡝࠿ࡽ'1$ ཰㞟ࢆ⾜࠺ࠋ཰㞟ࡉࢀࡓ '1$ ࡣࡑࡢ≟ࡢ⑌ᝈ᝟ሗ࡜࡜ࡶ࡟ࢹ࣮ࢱ࣮࣋ࢫ໬ࡋ࡚≟ࡢ㑇ఏ㛵㐃⑌ᝈ࡟࠾ࡅࡿཎᅉ㑇ఏᏊࡢゎᯒࡸࡑࡢ௚ࡢ≟ࡢ㑇ఏᏊࢆ฼⏝ࡋࡓ◊✲ࢆ⾜࠺ࡇ࡜ࢆᮏ
◊✲ࡢ┠ⓗ࡜ࡍࡿࠋ
ᮦᩱ࡜᪉ἲ㯞ᕸ኱Ꮫࠊᮾி኱Ꮫࠊᮾி㎰ᕤ኱Ꮫࠊ㮵ඣᓥ኱Ꮫࠊ᪥ᮏ኱Ꮫ࡞࡝ࡢᡤᒓࡢ⋇་ᖌࡸ㑇ఏ⑓◊✲⪅࡞࡝࠿ࡽ㑇ఏ㛵㐃⑌ᝈࢆྵࡴ㠀ឤᰁᛶ⑌ᝈ≟࠾ࡼࡧ୍⯡㞟ᅋ≟࠿ࡽ '1$ ࢆ཰㞟ࡋࡓࠋ཰㞟ࡉࢀࡓ⑌ᝈࡣࠊ᫂ࡽ࠿࡞㑇ఏ⑌ᝈ࠿ࡽఱࡽ࠿ࡢ㑇ఏⓗ࡞せᅉࡀ㛵୚ࡍࡿྍ⬟ᛶࡀ࠶ࡿࡶ⑌ᝈࡲ࡛ᖜᗈࡃ཰㞟ࡋࡓࠋ⋇་኱Ꮫ⑓㝔࡛ࡣ཰㞟ᅔ㞴࡞⑌ᝈ࡟࠾࠸࡚ࡣ୍⯡⋇་⑓㝔࠿ࡽ '1$ ࢆ཰㞟ࡋࡓࠋࡑࡢ≟ࡢᇶᮏ᝟ሗ࠾ࡼࡧ⑌ᝈ᝟ሗࡣ㯞ᕸ኱Ꮫ࡟࠾࠸࡚ࢹ࣮ࢱ࣮࣋

ࢫ໬ࡉࢀࡓࠋࡲࡓࠊ୍㒊ࡢ᳨య࡟࠾࠸࡚ࡣ⾑₢ࡲࡓࡣ⾑Ύࡶ& ࡟ಖᏑࡉࢀࡓࠋࡑࢀ
ࡒࢀࡢ⑌ᝈࡣ᪥ᮏ⋇་Ꮫ఍ࡢ⑌ᝈྡ⏝ㄒ㞟࡟ᇶ࡙࠸࡚ศ㢮ࡋࡓࠋ⤖ᯝ࠾ࡼࡧ⪃ᐹ㑇ఏ㛵㐃⑌ᝈࢆྵࡴ㠀ឤᰁᛶ⑌ᝈ≟࠾ࡼࡧ୍⯡㞟ᅋ≟࠿ࡽ '1$ ཰㞟ࢆ⾜࠸ࠊ㸯୓  ᳨36యࡀ཰㞟ࡉࢀࡓࠋ≟✀ࡣ  ✀௨ୖ࡛ୖ఩㸱≟✀ࡣ࢙࢘ࣝࢩ࣭ࣗࢥ࣮ࢠ࣮࣭࣌ࣥࣈ࣮ࣟࣈ
 ౛ࠊࢲࢵࢡࢫ  ౛ࠊࣉ࣮ࢻࣝ  ౛࡛࠶ࡗࡓ㸦⾲㸯㸧ࠋ࢜ࢫ࣭࣓ࢫࡢẚ⋡ࡣ 㸸࡛ࠊᖹᆒᖺ௧ࡣ  ᖺ࡛࠶ࡗࡓࠋ

ࠋ⑌ᝈ⑕౛ࡣ᪥ᮏ⋇་Ꮫ఍ࡢ⑌ᝈྡ⏝ㄒ㞟ࡢ኱ศ㢮࡟ᇶ࡙࠸࡚኱ࡁࡃศࡅࡓ㸦⾲㸰㸧ⓑෆ㞀ࢆࡣࡌࡵ࡜ࡍࡿ║⑌ᝈࠊ࡚ࢇ࠿ࢇࢆࡣࡌࡵ࡜ࡍࡿ⚄⤒⑌ᝈࠊ࢔ࣞࣝࢠ࣮⑌ᝈࢆࡣࡌ
ࡵ࡜ࡍࡿච␿⑌ᝈ࡞࡝㠀ឤᰁᛶ⑌ᝈ 㻣㻢㻞㻤 ⑕౛ࡀ཰㞟ࡉࢀࡓࠋࡑࢀࡒࢀࡢ⑌ᝈෆヂ࡟ࡘ࠸࡚ࡣ⾲ 㻟 ࠿ࡽ⾲ 㻝㻣 ࡲ࡛࡟♧ࡋࡓࠋ⌧ᅾࠊ᭱⤊ⓗ࡞デ᩿ࡀỴᐃࡋ࡚࠸࡞࠸⑕౛ࡸ⑌ᝈ᝟ሗࡀࡲ
ࡔ᮶࡚࠸࡞࠸ࡶࡢࡀከᩘ࠶ࡾࠊ௒ᚋࠊ⑌ᝈ᝟ሗࢆ㏣ຍࡋ࡚࠸ࡃࠋ୍⯡㛤ᴗࡢ⋇་ᖌࡢሙྜࠊ
⑌ᝈデ᩿ᇶ‽ࡸ⬟ຊ࡟ࣂࣛࢶ࢟ࡀ࠶ࡿࡓࡵࠊ⑕౛᳨య࡟㛵ࡋ࡚ࡣ⋇་኱Ꮫࡢᩍဨࢆ୰ᚰ࡟
デ⒪࣭デ᩿ࡋ࡚᥇ྲྀࡋࡓࠋࡋ࠿ࡋࠊ࢔ࣞࣝࢠ࣮⑌ᝈ࡛࠶ࡿ࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡟㛵ࡋ࡚ࡣ࢔ࣞࣝࢠ࣮἞⒪ἲࡢ㐍Ṍ࡛⋇་኱Ꮫ⑓㝔࡟᮶㝔ࡍࡿ⑌ᝈ≟ࡀ㠀ᖖ࡟ᑡ࡞ࡃ࡞ࡗࡓࡓࡵࠊᮏࣉ
ࣟࢪ࢙ࢡࢺ࡛ࡣಶே⑓㝔ࡢ㛤ᴗ⋇་ᖌ࡛࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࢆᑓ㛛ࡍࡿ⓶⭵⛉ᑓ㛛་࡟౫㢗ࡋ࡚ᅜ㝿ⓗ࡞デ᩿ᇶ‽ࡢᇶ࡟᳨యࢆ᥇ྲྀࡋࡓࠋ㻌⑌ᝈ⑕౛ࡀ㞟ࡵࡽࢀࠊ⑌ᝈ㑇ఏᏊࢆゎᯒࡍࡿ࡜ࡁ࡟ࠊ㝜ᛶᑐ㇟࡜࡞ࡿ୍⯡㞟ᅋ࠿ࡽࡢ᥇
ྲྀࡋࡓ≟ࡢ '1$ ࡀᚲせ࡜࡞ࡗࡓࠋ୍⯡㞟ᅋ≟ࡢ '1$ ࡀ࡞࠸࡜⑌ᝈ㑇ఏᏊゎᯒࡀ㐍ࡲ࡞࠸ࡓࡵࠊᮏࣉࣟࢪ࢙ࢡࢺ࡛ࡣࠊ୍⯡㞟ᅋ࠿ࡽࡢ≟ࡢ '1$ ᳨యࡢ᥇ྲྀࡶ✚ᴟⓗ࡟⾜࠺ࡼ࠺࡟࡞ࡾࠊ
཰㞟 '1$ ᳨యᩘ࡟୍⯡㞟ᅋ≟ࡢ᳨యᩘࡶຍ࠼ࡓࠋ
཰㞟ࡉࢀࡓ '1$ ᳨యࡣࡇࡢࣉࣟࢪ࢙ࢡࢺࡢ◊✲⪅࡟ᥦ౪ࡋ࡚㑇ఏ㛵㐃⑌ᝈࡢ㑇ఏᏊゎᯒࢆ⾜ࡗࡓࠋࡲࡓࠊࣉࣟࢪ࢙ࢡࢺ◊✲ࢢ࣮ࣝࣉእࡢ௚ࡢ◊✲⪅࡬ '1$ ࢆ౪⤥ࡋࠊඹྠ◊✲ࢆ⾜ࡗ࡚࠸ࡿࠋ⛉Ꮫ㆙ᐹ◊✲ᡤ࡛⾜ࡗ࡚࠸ࡿࠕ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺከᆺゎᯒࢆ⏝࠸ࡓ≟ࡢಶయ㆑ูἲࡢ᳨ウࠖ࡟≟ࡢ '1$ ࢆᥦ౪ࡋࡓࠋࡇࢀࡣ≟ࡢ '1$ ࡣ㸪യᐖࡸ⾑⤫᭩ഇ⿦࡞࡝┤᥋ື≀ࡀ㛵ࢃࡿ஦௳࠿ࡽ㸪ᙉ┐ࡸᙉጮ࡞࡝ࡢ⌧ሙ࡟≢ே࡟௜╔ࡋࡓື≀ẟࡀ㑇␃ࡉࢀࡿ஦௳
ࡲ࡛㸪ከᒱ࡟ࢃࡓࡗ࡚㔜せ࡞ドᣐ࡞ࡿࡓࡵ࡛࠶ࡿࠋࡲࡓࠊ ᮾி኱Ꮫ⋇་Ꮫ⛉ᆤ஭ඛ⏕࡟ࡣ
ࠕ:KROH([RPH6HTXHQFH ἲ࡟ࡼࡿ≟ࡢ㑇ఏᛶ⚄⤒⑌ᝈࡢཎᅉ㑇ఏᏊྠᐃࠖࡢ◊✲࡛ࣃࣆࣚࣥ≟ࡢ⚄⤒㍈⣴ࢪࢫࢺࣟࣇ࢕࣮ࡢ㝜ᛶᑐ↷ࡢࣃࣆࣚࣥ≟ࡢ  ᳨యࡢ '1$ ࢆ㑇ఏᏊࣂࣥࢡ࠿
ࡽᥦ౪ࡋࡓࠋ㑇ఏ⑌ᝈࡢ◊✲⪅ࡣ⑕౛࠿ࡽ⑌ᝈ㑇ఏᏊࡢ཰㞟ࢆ⾜࠺ࡀࠊྠࡌ≟✀࡛㝜ᛶᑐ↷࡜࡞ࡿࡶࡢࡢ཰㞟ࡀ࡞࠸ሙྜࡀከ࠸ࡢ࡛ࠊࡇࡢࡼ࠺࡞㑇ఏᏊࣂࣥࢡࡢ฼⏝ࡶ௒ᚋࠊ⾜ࡗ࡚࠸ࡃணᐃ࡛࠶ࡿࠋ
⋇་኱Ꮫ⑓㝔࡛ࡢከࡃࡢ⑕౛᳨య࡛ࡣ⾑Ύ㻔⾑₢㻕ࡶేࡏ࡚ಖᏑࡋࡓࠋࡇࢀࡣᑗ᮶ࠊ㑇ఏ㛵㐃⑌ᝈ➼⑌ᝈࡢཎᅉ㑇ఏᏊࡀ᫂ࡽ࠿࡟࡞ࡗࡓ᫬࡟ࡑࡢ㑇ఏ⑓ࡢཎᅉ࡜࡞ࡿᶵᗎࢆゎᯒࡍࡿ࡜ࡁ࡟ࡇࢀࡽࡢ⾑Ύࡣᙺ❧ࡘྍ⬟ᛶࡀ࠶ࡿࡓࡵ࡟㻙㻤㻜Υ࡛ಖᏑࢆ⾜ࡗ࡚࠸ࡿࠋࡲࡓࠊே࡛ࡣ
ᅜ❧ឤᰁ⑕◊✲ᡤࡢ⾑Ύࣂࣥࢡࡢࡼ࠺࡟ᵝࠎ࡞㞟ᅋࡢ⾑ΎࢆಖᏑࡍࡿࢩࢫࢸ࣒ࡀ࠶ࡿࡀࠊ
᪥ᮏᅜෆ࡛ࡣ≟ࡢ⾑ΎࢆከᩘಖᏑࡍࡿࢩࢫࢸ࣒ࡀ࡞࠿ࡗࡓࠋࡇࢀࡣ㑇ఏ⑓ࢆࡣࡌࡵࠊ௚ࡢ
ᵝࠎ࡞⑌ᝈࡢⓎ⏕ᶵᗎࡢゎᯒࡸឤᰁ⑕࡟࠾ࡅࡿ⾑Ύ␿Ꮫ࡞࡝ࢆ◊✲ࡍࡿ࡜ࡁ࡟㠀ᖖ࡟ᙺ❧ࡘᮦᩱ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ≟࡟࠾ࡅࡿ࢖ࣥࣇ࢚ࣝࣥࢨឤᰁࡢ⾑Ύ␿Ꮫㄪᰝࢆ⾜࠸ࠊ≟ࡶேࡢ࢖ࣥࣇ࢚ࣝࣥࢨࡢឤᰁࡀ㉳ࡇࡗ࡚࠸ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ㻌37ᮾி኱Ꮫື≀་⒪ࢭࣥࢱ࣮࠿ࡽࡣ୍⯡ⓗ࡞⑕౛ࡶྵࡵ࡚⑌ᝈ⑕౛ࡢࢿࢥࡢ '1$ ࡜⾑₢ࡀ཰㞟ࢆ⾜ࡗ࡚࠾ࡾࠊ⌧ᅾ࡛  ౛ࡀࣂ࢖࢜ࣂࣥࢡ࡟㞟ࡲࡗ࡚࠸ࡿࠋࢿࢥࡢ㑇ఏ⑓ࡢ◊✲ࢆ⾜ࡗ࡚࠸ࡿ㮵ඣᓥ኱Ꮫࡢ኱࿴ඛ⏕࠿ࡽࡶ  ᳨యࡢ '1$ ᝟ሗࡢᥦ౪ࢆཷࡅ࡚࠸ࡿࠋࢿࢥࡶ
ከࡃࡢ㑇ఏ⑌ᝈࡀᏑᅾࡍࡿࡀࠊゎᯒ࡟ᚲせ࡞᳨యᩘࡀ୙༑ศ࡞ࡓࡵࠊࡇࢀࡲ࡛◊✲ࡀ࠶ࡲࡾ㐍ࢇ࡛࠸࡞࠿ࡗࡓࠋࡲࡓࠊ୍⯡ⓗ࡞⑌ᝈࡢ◊✲ࡶ≟࡟ẚ࡭㐜ࢀ࡚࠸ࡿࠋࡑࡢࡓࡵࠊᮾி
኱Ꮫࡸ㮵ඣᓥ኱Ꮫࡢᙉ࠸せᮃࡶ࠶ࡾࠊࡇࢀࡽࡢ '1$ ᳨యࢆᇶ࡟ࢿࢥࡢ㑇ఏᏊࣂࣥࢡࡶጞࡵ
ࡿணᐃ࡛࠶ࡿࠋࡲࡓࠊᮾி኱Ꮫື≀་⒪ࢭࣥࢱ࣮ࡢ᳨య࡟ࡘ࠸࡚ࡣ⾑₢ࡶ᥇ྲྀࡉࢀ࡚࠾ࡾࠊ
௒ᚋࠊ≟࡜ྠᵝ࡟⑌ᝈࡢⓎ⑕ᶵᗎࡸឤᰁ⑕ࡢ⾑Ύ␿Ꮫ࡞࡝ࡢࢿࢥࡢ⑌ᝈ◊✲࡟ᖜᗈࡃ౑⏝࡛ࡁࡿྍ⬟ᛶࡀ࠶ࡿࠋ
཰㞟ࡉࢀࡓ᳨య᝟ሗࡣࣂ࢖࢜ࣂࣥࢡ࡟Ⓩ㘓ࡋࠊ⑌ᝈ᝟ሗࢆࢹ࣮ࢱ࣮࣋ࢫ໬ࡋࡓࠋ௒ᚋࠊ
཰㞟ࡉࢀࡓ≟✀ࡸ⑌ᝈ࡟㛵ࡋ࡚ࡣ◊✲ࢢ࣮ࣝࣉෆእ࡛ࡢ᝟ሗࡢඹ᭷ࢆ⾜ࡗ࡚࠸ࡃ࡜࡜ࡶ࡟ࠊ
ୖ㏙ࡋࡓࡼ࠺࡟◊✲ࢢ࣮ࣝࣉእࡢ◊✲⪅ࡀ⾜࠺◊✲࡟ࡶ✚ᴟⓗ࡟༠ຊࢆ⾜࠺ࠋ38⾲㸯 㑇ఏᏊࣂࣥࢡ࡟཰㞟ࡉࢀࡓ≟✀≟✀                       㢌ᩘ࢙࢘ࣝࢩ࣭ࣗࢥ࣮ࢠ࣮࣭࣌ࣥࣈ࣮ࣟࢡ
࣑ࢽࢳࣗ࢔ࢲࢵࢡࢫ
ࢺ࢖ࣉ࣮ࢻࣝᰘ≟            㸯㸶㸰㸵                㸯㸳㸴㸵                    㸯㸯㸵㸰࣮࣎ࢲ࣮࣭ࢥ࣮ࣜ㞧✀    㸰㸰㸱㸴             㸶㸷㸲                     㸵㸳㸷ࣛࣈࣛࢻ࣮࣭ࣝࣞࢺ࣮ࣜࣂ࣮         㸴㸷㸲ࢳ࣡࣡                      㸴㸴㸱ࢩ࣮ࢬ࣮                 㸴㸱㸲ࣅ࣮ࢢࣝ                 㸲㸯㸵࣮ࣚࢡࢩ࣮࣭ࣕࢸࣜ࢔             㸱㸳㸮ࢦ࣮ࣝࢹ࣭ࣥࣞࢺ࣮ࣜࣂ࣮⌋ᓥ         㸱㸯㸶                     㸰㸷㸲࣏࣓ࣛࢽ࢔ࣥ                 㸰㸶㸶ࣃࣆࣚࣥ                 㸰㸳㸷࣐ࣝࢳ࣮ࢬ                 㸰㸳㸵ࢥࢵ࣮࣭࢝ࢫࣃࢽ࢚ࣝ             㸰㸲㸲ࢩ࢙ࢵࢺࣛࣥࢻ࣭ࢩ࣮ࣉࢻࢵࢢ
ࣇࣞࣥࢳ࣭ࣈࣝࢻࢵࢢ         㸰㸲㸮             㸰㸱㸱࣑ࢽࢳࣗ࢔࣭ࢩࣗࢼ࢘ࢨ࣮         㸰㸰㸮࢟ࣕࣂࣜ࢔࣭࢟ࣥࢢ࣭ࢳ࣮ࣕࣝࢬ࣭ࢫࣃࢽ࢚ࣝ
ࣃࢢ 㸰㸮㸯                     㸯㸵㸶ࢪࣕࢵࢡ࣭ࣛࢵࢭ࣭ࣝࢸࣜ࢔         㸯㸳㸯ࢪ࣮࣐࣭ࣕࣥࢩ࢙ࣃ࣮ࢻ࣭ࢻࢵࢢ          㸯㸲㸳࣑ࢽࢳࣗ࢔࣭ࣆࣥࢩ࣮ࣕ               㸶㸵ࣁࢫ࣮࢟                  㸶㸳࣎ࢫࢺ࣭ࣥࢸࣜ࢔              㸶㸳࣮࢜ࢫࢺࣛࣜ࢔࣭ࣥࣛࣈࣛࢻ࣮ࢗࢻࣝ⛅⏣      㸶㸯                      㸴㸷࢚࢘ࢫࢺ࣭ࣁ࢖ࣛࣥࢻ࣭࣍࣡࢖ࢺ࣭ࢸࣜ࢔       㸴㸵ࢢ࣮ࣞࣁ࢘ࣥࢻ                   㸳㸷


39

ࢧ࣮ࣝ࢟

  㸳㸲ࣂ࣮ࢽ࣮ࢬ࣭࣐࢘ࣥࢸ࣭ࣥࢻࢵࢢ 㸳㸯ࢩ࣋ࣜ࢔࣭ࣥࣁࢫ࣮࢟

㸲㸴

ࢥ࣮ࣜ

  㸲㸳࣌࢟ࢽ࣮ࢬ  㸲㸮໭ᾏ㐨  㸱㸷࣡࢖࣮࣭ࣖࣇ࢛ࢵࢡࢫ࣭ࢸࣜ࢔ 㸱㸷ࢢ࣮ࣞࢺ࣭ࣆࣞࢽ࣮ࢬ

㸱㸴ࣅࢩ࣭ࣙࣥࣇ࣮ࣜࢮ

㸱㸱࣏࢖ࣥࢱ࣮  㸱㸯ࣂࢭࣥࢪ࣮  㸰㸶ࣇࣛࢵࢺࢥ࣮ࢸࢵࢻ࣭ࣞࢺ࣮ࣜࣂ࣮

 㸰㸵

࣎ࢡࢧ࣮

  㸰㸵⏥ᩫ  㸰㸴ࢫࣆࢵࢶ  㸰㸴ࣈࣝࢻࢵࢢ  㸰㸲ࢣ࢔࣭ࣥࢸࣜ࢔  㸰㸱ࢳࣥ  㸰㸱ࢲ࣓ࣝࢩ࢔ࣥ  㸰㸰ᅄᅜ  㸰㸯⌰⌫  㸰㸮ࢻ࣮࣐࣋ࣝࣥ  㸯㸶࣮࢜ࣝࢻ࣭࢖ࣥࢢࣜࢵࢩ࣭ࣗࢩ࣮ࣉࢻࢵࢡ 㸯㸴ࣀ࣮ࣇ࢛࣮ࢡ࣭ࢸࣜ࢔

㸯㸳ࢢ࣮ࣞࢺ࣭ࢹ࣮ࣥ

㸯㸲࢔࢖ࣜࢵࢩ࣭ࣗࢭࢵࢱ࣮

㸯㸱࢔ࣇ࣭࢞ࣥࣁ࢘ࣥࢻ

㸯㸱࢖ࣥࢢࣜࢵࢩ࣭ࣗࢫࣉ࣮࣭ࣜࣥ࢞ࢫࣃࢽ࢚ࣝ  㸯㸰㸯㸰ࢫࢥࢸ࢕ࢵࢩ࣭ࣗࢸࣜ࢔࣑࢜࢜࢝  㸯㸰࢘࢕࣌ࢵࢺ  㸯㸯࢙࢘ࣝࢩ࣭ࣗࢸࣜ࢔

㸯㸯࢚࢔ࢹ࣮࣭ࣝࢸࣜ࢔

㸯㸯

ࢧ࢚ࣔࢻ

  㸯㸯40ࣈࣜࢱࢽ࣮࣭ࢫࣃࢽ࢚ࣝ

㸯㸯ࣈࣝࢸࣜ࢔  㸯㸯࣎ࣝࢰ࢖  㸯㸯ࣟࢵࢺ࣡࢖࣮ࣛ  㸯㸯ࢭࣥࢺ࣭ࣂ࣮ࢼ࣮ࢻ 㸷ࢳࣕ࢘ࢳࣕ࢘ 㸷࢖ࣥࢢࣜࢵࢩ࣭ࣗࢭࢵࢱ࣮  㸶

ࢳࣕ࢖ࢽ࣮ࢬ࣭ࢡࣞࢫࢸࢵࢻ࣭ࢻࢵࢡ  㸶

ࢳ࣮ࣕࣝࢬ࣭ࢫࣃࢽ࢚ࣝ 㸶ࢺ࢖࣭࣐ࣥࢳ࢙ࢫࢱ࣮࣭ࢸࣜ࢔  㸶

࣎ࣟࢽ࣮ࢬ

 㸶

⣖ᕞ

 㸵

ࢽ࣮ࣗࣇ࢓ࣥࢻࣛࣥࢻ 㸵ࢪࣕ࢖࢔ࣥࢺ࣭ࢩࣗࢼ࢘ࢨ࣮  㸳

ࣂࢭࢵࢺ࣭ࣁ࢘ࣥࢻ 㸳࣋ࣝࢪ࢔࣭ࣥࢩ࢙ࣃ࣮ࢻ࣭ࢻࢵࢡ࣭ࢱ࣮ࣅࣗࣞࣥ

㸳㸯㸯㸮ࡑࡢ௚ࡑࡢ௚㸸㸲㢌௨ୗࡢࡶࡢࡣࡑࡢ௚࡜ࡋ࡚ࡲ࡜ࡵࡓࠋ

41⾲㸰 㑇ఏᏊࣂࣥࢡ࡟࠾ࡅࡿ⑌ᝈ㸦኱ศ㢮㸧ศ㢮
ศ㢮

㢌ᩘ

║⛉⑌ᝈ

 㸱㸮㸷㸮⚄⤒⑌ᝈ  㸶㸴㸳ᾘ໬ჾ⑌ᝈ  㸴㸳㸰⓶⭵⑌ᝈ  㸴㸮㸴⫢⮚࣭⫹㐨࣭⮅እศἪ⑌ᝈ

㸳㸮㸮⾑ᾮ࣭ච␿⣔⑌ᝈ  㸱㸰㸷Ἢᒀჾ⑌ᝈ  㸰㸶㸵ෆศἪ࣭௦ㅰᛶ⑌ᝈ  㸰㸳㸮ᚠ⎔ჾ⑌ᝈ  㸰㸲㸶⏕Ṫჾ࣭ஙᡣ⑌ᝈ  㸰㸰㸶㦵㛵⠇⑌ᝈ  㸰㸯㸵࿧྾ჾ⑌ᝈ  㸯㸷㸷ཱྀ⭍ෆ⑌ᝈ  㸯㸮㸷

➽⑌ᝈ

㸱㸱

⾜ື⑌ᝈ

 㸯㸳

ィ 

  㸵㸴㸰㸶42⾲㸱 ᚠ⎔ჾ⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮ศ㢮                㢌ᩘൔᖗᘚ⭷⑌ᝈ            㸴㸷⾑⟶⫗⭘            㸰㸳ൔᡣᘚ㛢㙐୙඲             㸯㸷୕ᑤᘚ㛢㙐୙඲             㸯㸵⫵㧗⾑ᅽ⑕            㸯㸳୕ᑤᘚ㏫ὶ            㸯㸮ື⬦⟶㛤Ꮡ                㸴ࣜࣥࣃ⟶ᣑᙇ             㸴⾑ᰦሰᰦ⑕             㸳኱ື⬦ᘚ㏫ὶ             㸱ᚰᐊ୰㝸Ḟᦆ             㸱⫵ື⬦ᘚ⊃✽             㸱ᚰ⮚ࢱ࣏ࣥࢼ࣮ࢹ
ᾏ⥥≧⾑⟶⭘         㸱             㸱୰⓶⭘                  㸱⫧኱ᆺᚰ➽⑕             㸰኱ື⬦ᘚ㛢㙐୙඲         㸰⣽⳦ᛶᚰෆ⭷⅖              㸰ື㟼⬦ࢁ࠺             㸰ᶓ⣠➽⫗⭘             㸰⾑⟶⭘                  㸰ࣜࣥࣃ⟶⫗⭘             㸰⭈ᛶ㧗⾑ᅽ             㸰ྑᚰ୙඲             㸯ᕥᚰ୙඲             㸯ᚰ୙඲                  㸯ୖᐊ㢖ᢿ             㸯ᚰᐊᮇእ཰⦰             㸯ከ※ᛶᚰᐊᮇእ཰⦰         㸯Ὕ೵Ṇ                  㸯࢔࣑ࣟ࢖ࢻ⑕             㸯



43


ᣑᙇᆺᚰ➽⑕             㸯ൔᖗᘚ㏫ὶ             㸯⫵ື⬦ᘚ㏫ὶ             㸯ᚋ኱㟼⬦Ḟᦆ             㸯ඛኳᛶᚰ⑌ᝈ             㸯ൔᖗᘚ␗ᙧᡂ             㸯኱ື⬦ᘚ⊃✽             㸯኱ື⬦ᘚୗ⊃✽              㸯኱㟼⬦⑕ೃ⩌             㸯ᚰ⭷⅖                  㸯ᚰ⭷୰⓶⭘             㸯኱ື⬦ᑠయ⭘⒆              㸯⾑ᰦ⑕                  㸯㟼⬦⾑ᰦ⑕             㸯⫗ⱆ⭘ᛶࣜࣥࣃ⟶⅖ࣜࣥࣃ⟶⭘         㸯             㸯㧗⾑ᅽ                  㸯ࡑࡢ௚                 㸯㸵ࡑࡢ௚⑌ᝈྡ⏝ㄒ㞟࡟࡞࠸⑌ᝈࡸศ㢮ࡀ㞴ࡋ࠸ࡶࡢࡣࡑࡢ௚࡟ࡲ࡜ࡵࡓࠋ
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44

⾲㸲 ཱྀ⭍ෆ⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮




ศ㢮

                㢌ᩘṑ⫗⅖                 㸯㸷᰿ᑤ࿘ᅖ⮋⒆

            㸯㸳ṑ࿘⅖                 㸯㸱ཱྀ⭍ෆᝏᛶ㯮Ⰽ⭘         㸵ၚᾮ⭢ᄞ⬊             㸳ཱྀ⭍㰯⭍⒦             㸲⥺⥔⭘ᛶ࢚ࣉ࣮ࣜࢫၚᾮ⭢⅖

         㸱             㸱࢚ࢼ࣓ࣝୖ⓶⭘              㸱ཱྀ⵹⿣                  㸱ၚᾮ⢓ᾮᄞ⬊             㸰ṑཎᛶᄞ⬊             㸰ṑཎᛶ⭘⒆             㸰㢡㛵⠇⅖             㸯࢚ࣉ࣮ࣜࢫ             㸯㦵ᙧᡂᛶ࢚ࣉ࣮ࣜࢫ         㸯ᕧ⣽⬊ᛶ࢚ࣉ࣮ࣜࢫ         㸯ၚᾮ⭢⭘⒆             㸯ྵṑᛶᄞ⬊             㸯࢚ࢼ࣓ࣝୖ⓶⥺⥔⫗⭘⥺⥔⫗⭘         㸯             㸯ࡑࡢ௚                 㸰㸮
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45

⾲㸳 ࿧྾ჾ⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮ศ㢮  㢌ᩘ㰯⭍ෆ⭘⒆

 㸱㸯

Ẽ⟶⹫⬺

 㸱㸯

⫵⭘⒆

  㸯㸱

៏ᛶ㰯⅖

 㸯㸰

Ẽ⟶ᨭ⅖

 㸯㸯

㌾ཱྀ⵹㐣㛗⑕  㸴

ㄗᄟᛶ⫵⅖  㸴

ႃ㢌㯞⑷

  㸳⭢⒴ 㸳▷㢌✀Ẽ㐨⑕ೃ⩌  㸲

㛫㉁ᛶ⫵⅖  㸲

㰯⭍ෆ␗≀  㸱

⬚Ỉ㈓␃

  㸱ங⣃⬚ 㸱ᶓ㝸⭷࣊ࣝࢽ࢔  㸱

Ẽ⟶ᨭ⫵⅖  㸱

⫵⾑ᰦሰᰦ⑕  㸰

ႃ㢌⅖

 㸯

ႃ㢌⭘⒆

  㸯Ẽ⟶⭘⒆

  㸯

↓Ẽ⫵

 㸯

⫵Ỉ⭘

 㸯

Ẽ⟶ᨭ⭘⒆  㸯⫵ⴥ᤬㌿  㸯

Ẽ⬚

 㸯

₃ᩯ⬚

 㸯

Ẽ⟶ᨭᣑᙇ⑕ࡑࡢ௚  㸯  㸲㸲46⾲㸴 ᾘ໬ჾ⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮




ศ㢮

                㢌ᩘ㣗≀࢔ࣞࣝࢠ࣮            㸯㸰㸯⺮ⓑ႙ኻᛶ⭠⑕             㸷㸷ᾘ໬ჾᆺࣜࣥࣃ⭘        㸴㸯៏ᛶ⭠⅖            㸲㸱⅖⑕ᛶ⭠⑌ᝈ            㸱㸲៏ᛶ⫶⅖            㸰㸱

⅖⑕ᛶ⤖┤⭠࣏࣮ࣜࣉ        㸰㸱⭠ࣜࣥࣃ⟶ᣑᙇ⑕        㸰㸰఍㝜࣊ࣝࢽ࢔            㸰㸰⭠⭢⒴                 㸯㸲㰡ᚄ࣊ࣝࢽ࢔            㸯㸰⦭ྜ⣒㛵㐃⫗ⱆ⭘ᕧ኱㣗㐨⑕        㸯㸰            㸯㸮ࣜࣥࣃ⌫ᙧ㉁⣽⬊ᛶ⭠⅖          㸶ᢠ⳦๣཯ᛂᛶ⭠⑕         㸶≉Ⓨᛶᕧ኱㣗㐨⑕         㸵⫶⅖

                 㸴࣏࣮ࣜࣉ             㸴⫗ⱆ⭘ᛶ⭠⅖             㸳⫶⭢⒴                  㸲ဗ㢌㯞⑷             㸱⤖⭠⅖                  㸱ᖹ⁥➽⭘             㸱ᖹ⁥➽⫗⭘             㸱㣗㐨⅖                  㸰㏫ὶᛶ㣗㐨⅖             㸰㣗㐨⊃✽             㸰㣗㐨⭘⒆             㸰ⴎ⦰ᛶ⫶⅖             㸰⫶᤼ฟ㐜ᘏ             㸰⫶⭘⒆                  㸰


47


࢝ࣝࢳࣀ࢖ࢻ⭘⒆

         㸰༑஧ᣦ⭠⅖             㸰࢝ࢱࣝᛶ⭠⅖             㸰ዲ㓟⌫ᛶ⭠⅖             㸰៏ᛶ⤖⭠⅖             㸰⭠㛢ሰ                  㸰ᾘ໬⟶㛫㉁⭘⒆              㸰⮖࣊ࣝࢽ࢔             㸰⫠㛛ᄞ⅖             㸰⫠㛛࿘ᅖ⒦             㸰⭠✸Ꮝ                  㸰ဗ㢌⅖                  㸯㣗㐨᠁ᐊ             㸯⫹Ồ჎ྤ⑕ೃ⩌              㸯⫗ⱆ⭘ᛶ⫶⅖             㸯ᗃ㛛⊃✽             㸯⫶ᣑᙇ                  㸯⫶ᣑᙇ᤬㌿⑕ೃ⩌         㸯ዲ㓟⌫ᛶ⤖⭠⅖              㸯⤌⧊⌫ᛶ₽⒆ᛶ⤖⭠⅖

         㸯ᑠ⭠ෆ⣽⳦㐣๫ቑṪ         㸯㐣ᩄᛶ⭠⑕ೃ⩌              㸯ᾘ໬ჾᆺ⫧‶⣽⬊⭘┤⭠㛢ሰ         㸯             㸯┤⭠⬺                  㸯⭡ቨ࣊ࣝࢽ࢔⭢⒴

             㸯                 㸯ࡑࡢ௚                 㸲㸷
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48

⾲㸵 ⫢⮚࣭⫹㐨࣭⮅እศἪ⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮
ศ㢮

ࡢ࡭㢌ᩘ

⮅⅖

  㸵㸳㛛⬦యᚠ⎔ࢩࣕࣥࢺ
⫢⅖
ཎⓎᛶ㛛⬦పᙧᡂ
⫹⟶⅖

 㸳㸲
  㸱㸳 㸱㸱  㸰㸶⫹ᄞ⢓ᾮᄞ⭘ 㸰㸲⫢⤖⠇ᛶ㐣ᙧᡂ

 㸰㸱

⫹▼⑕

  㸯㸷⫢⣽⬊⭢⭘

 㸯㸶

⫢⣽⬊⒴

 㸯㸵

⫹ᄞ⅖

  㸯㸳✵⬊ᛶ⫢㞀ᐖ 㸯㸱⫢ᚤᑠ⾑⟶␗ᙧᡂ 㸯㸯ࢫࢸࣟ࢖ࢻㄏⓎᛶ⫢⑕  㸶

㛛⬦㧗⾑ᅽ⑕  㸵

⮅እศἪ୙඲⑕  㸵

୰ẘᛶ⫢യᐖ  㸴

⫢ᄞ⬊⫢እ⫹⟶㛢ሰ 㸳  㸲

⫢୙඲

 㸱

⫢⮋⒆

 㸱

⫢◳ኚ

 㸱

⫢ᛶ⬻⑕

  㸱⫢⥺⥔⑕  㸰

ᛴᛶ⫹ᄞ⅖  㸰⫹⟶⒴ 㸰㛛⬦పᙧᡂ  㸰

⫢࢝ࣝࢳࣀ࢖ࢻ  㸯

⮅⮚⒴

 㸯

⮅ᄞ⬊

 㸯

ࡑࡢ௚

  㸵㸳49

⾲㸶 Ἢᒀჾ⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮
ศ㢮

ࡢ࡭㢌ᩘ

ᒀ▼⑕

  㸳㸵⭤⬔⅖

  㸲㸶៏ᛶ⭈⮚⑓ 㸲㸳⭤⬔⛣⾜ୖ⓶⒴

 㸰㸲

⭈⤖▼⑕

 㸯㸵

⭈⣽⬊⒴

  㸳Ỉ⭈⑕ 㸳ᛴᛶ⭈୙඲  㸳

ࢿࣇ࣮ࣟࢮ⑕ೃ⩌  㸲

⣒⌫య⭈⑕  㸲

⭈୙඲
ከⓎᛶᄞ⬊⭈⭈ⱆ⭘ 㸲  㸱 㸱៏ᛶ⭈୙඲  㸱

⭈࿘ᅖഇᄞ⬊  㸰

␗ᡤᛶᒀ⟶  㸰

ࢩࢫࢳࣥᒀ⑕  㸰

⣒⌫య⭈⅖  㸯⭈␗ᙧᡂ  㸯

⭈│⭈⅖

  㸯

ᛴᛶ⭈㞀ᐖ  㸯

ᒀ⟶⤖▼⑕  㸯ᒀ㐨⅖

 㸯

ᒀ㊰㛢ሰ⑕  㸯

ୖ఩㐠ືࢽ࣮ࣗࣟࣥᛶ᤼ᒀ㞀ᐖ  㸯

ୗ㒊㐠ືࢽ࣮ࣗࣟࣥᛶ᤼ᒀ㞀ᐖ  㸯

࣍ࣝࣔࣥ཯ᛂᛶᒀኻ⚗  㸯

⭢⒴

 㸯

⭈᱾ሰ

 㸯

ࡑࡢ௚

  㸲㸯50

⾲㸷 ෆศἪ࣭௦ㅰᛶ⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮ศ㢮  㢌ᩘ⢾ᒀ⑓  㸲㸮ࢡࢵࢩࣥࢢ⑓  㸱㸮

⏥≧⭢⒴

 㸰㸳

ୗᆶయᛶ๪⭈⓶㉁ᶵ⬟ஹ㐍⑕

 㸰㸱

࢔ࢪࢯࣥ⑓

 㸰㸯

⏥≧⭢ᶵ⬟పୗ⑕

 㸯㸵

㧗⬡⾑⑕

 㸯㸳

๪⭈⭘⒆

 㸯㸱

ୖ⓶ᑠయᶵ⬟ஹ㐍⑕  㸶

ୗᆶయᛶࢡࢵࢩࣥࢢ⑕ೃ⩌  㸵

࢖ࣥࢫࣜࣀ࣮࣐  㸴

㧗࢝ࣝࢩ࣒࢘⾑⑕  㸱

ୖ⓶ᑠయ⭢⭘  㸱ప⾑⢾⑕  㸱ୗᆶయ⭘⒆  㸰

ୗᆶయᕧ኱⭢⭘  㸰๪⭈⭢⒴

  㸰

๪⭈⓶㉁ᶵ⬟పୗ⑕  㸰

ᝏᛶ㧗࢝ࣝࢩ࣒࢘⾑⑕  㸯

పࣜࣥ⾑⑕  㸯

ୗᆶయ⭢⭘  㸯ࣛࢺࢣᄞ⬊  㸯ᒀᔂ⑕ 㸯ᢠ฼ᒀ࣍ࣝࣔࣥ୙㐺ྜศἪ⑕ೃ⩌  㸯

⏥≧⭢ᶵ⬟ஹ㐍⑕  㸯

ཎⓎᛶ⏥≧⭢ᶵ⬟పୗ⑕  㸯

⥆Ⓨᛶୖ⓶ᑠయᶵ⬟ஹ㐍⑕  㸯

㧗⾑⢾

 㸯

ࡑࡢ௚

  㸯㸶51⾲㸯㸮 ⚄⤒⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮
ศ㢮
࡚ࢇ࠿ࢇ

  㢌ᩘ  㸰㸰㸰᳝㛫ᯈ࣊ࣝࢽ࢔ 㸷㸯ኚᛶᛶ⬨㧊⑕ 㸵㸮⬨㧊✵Ὕ⑕ 㸱㸷Ỉ㢌⑕ኚᙧᛶ⬨᳝⑕  㸱㸰
 㸯㸳

୰⪥⅖

  㸯㸳㧊⭷⭘  㸯㸲᳝㛫ᯈ⑌ᝈ 㸯㸲ᑿഃᚋ㢌㒊ወᙧ⑕ೃ⩌㸯㸱⫗ⱆ⭘ᛶ㧊⭷⬻⬨㧊⅖

 㸯㸱㤿ᑿ⑕ೃ⩌ 㸯㸱ቯṚᛶⓑ㉁⬻⅖ 㸯㸰㢦㠃⚄⤒㯞⑷ 㸯㸰㹅㹋㸯࢞ࣥࢢࣜ࢜ࢩࢻ࣮ࢩࢫ 㸯㸯⎔᳝㍈᳝୙Ᏻᐃ⑕

 㸯㸯

๓ᗞ㞀ᐖ

 㸯㸯

ቯṚᛶ㧊⭷⬻⅖

 㸯㸮࣍ࣝࢿࣝ⑕ೃ⩌  㸷

ᝏᛶᮎᲈ⚄⤒㠧⭘  㸶

඲㌟ᛶ᣺ᡓ⑕ೃ⩌  㸵

ㄆ▱ᶵ⬟୙඲⑕ೃ⩌  㸴

⬻᱾ሰ

 㸳

㞴⫈

 㸳

࢟࢔ࣜᵝወᙧ  㸲

㹆㹟㹬㹱㹣㹬 㹇ᆺ㹇㹔㹂㹂  㸲

᳝㛫ᯈ⬨᳝⅖  㸲⬨㧊᱾ሰ  㸲

ᅄୣయᄞ⬊  㸱

ቯṚᛶ㧊⭷⬻⅖ 

  㸱

⬻ฟ⾑

 㸱

52


ඛኳᛶỈ㢌⑕             㸰㢌⵹ෆࡃࡶ⭷ᄞ⬊⚄⤒㠧⭘         㸰             㸰ࢫࢸࣟ࢖ࢻ཯ᛂᛶ㧊⭷⅖࣮ື⬦⅖      㸰࣑࢜ࢡࣟࢽ࣮࡚ࢇ࠿ࢇ
ࣛࣇ࢛ࣛ⑓         㸰             㸰ࢼࣝࢥࣞࣉࢩ࣮              㸰ᑿഃ㢁᳝⬨㧊⑕              㸰㐍⾜ᛶ⬨㧊㌾໬⑕         㸰≉Ⓨᛶ୕ཫ⚄⤒㯞⑷

         㸰ࣛ࢖ࢯࢰ࣮࣒⵳✚⑓         㸯Ꮝ⬻⑕                  㸯⚄⤒⭺⭘             㸯㒊ศⓎస             㸯࣑࢜ࢡ࣮ࣟࢾࢫ              㸯㢌㒊እയ             㸯⬻ᾋ⭘                  㸯⬻ື㟼⬦ወᙧ             㸯㹆㹟㹬㹱㹣㹬 㹇㹇ᆺ㹇㹔㹂㹂⬨᳝⭘⒆

     㸯             㸯⥺⥔㌾㦵ሰᰦ⑕              㸯ෆ⪥⅖                  㸯ࡑࡢ௚                㸯㸳㸮
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53

⾲㸯㸯 ➽⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮




ศ㢮

              㢌ᩘ㔜⑕➽↓ຊ⑕             㸷ከⓎᛶ➽⅖             㸳࿪ᄮ➽⅖             㸱๪⭈⓶㉁ᶵ⬟ஹ㐍⑕ᛶ➽⑕➽⥭ᙇ⑕     㸯             㸯ࡑࡢ௚                 㸯㸲
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54

⾲㸯㸰 㦵㛵⠇⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮




ศ㢮

              㢌ᩘ㛵⠇⅖                 㸴㸮ࣞࢵࢢ࣭࣌ࣝࢸࢫ⑓⫤␗ᙧᡂ        㸰㸰            㸯㸵㦵⫗⭘                 㸯㸴⤌⧊⌫⫗⭘            㸯㸴ỗ㦵⅖                  㸷㛵⠇࣐ࣜ࢘ࢳ             㸶ኚᙧᛶ㛵⠇⑕             㸳⁥⭷⣽⬊⫗⭘             㸲

㦵⅖

                 㸱๓༑Ꮠ㠌ᖏ㒊ศ᩿⿣         㸱㛵⠇⑕                  㸱ච␿௓ᅾᛶ㛵⠇⑌ᝈ         㸱⫝㛵⠇ᙧᡂ␗ᖖ              㸱ᡂ㛗ᯈ᪩ᮇ㛢㙐              㸰ෆഃୖ㢛⒵ྜ୙඲         㸰㌾㦵⫗⭘             㸰㦵ᙧᡂ୙඲⑕             㸰

ኚᙧ⒵ྜ             㸯㦵㧊⅖                  㸯㦵⭘                 㸯ከⴥ≧㦵㌾㦵⫗⭘㦵㌾㦵⭘         㸯             㸯ࡑࡢ௚                 㸱㸰
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55

⾲㸯㸱 ⾑ᾮ࣭ච␿⣔⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮




ศ㢮

              㢌ᩘ


ࣜࣥࣃ⭘

           㸯㸮㸮ච␿௓ᅾᛶ⾑ᑠᯈῶᑡ⑕         㸲㸵㦵㧊␗ᙧᡂ⑕ೃ⩌        㸲㸴ච␿௓ᅾᛶ⁐⾑ᛶ㈋⾑        㸲㸰᧛✀ᛶ⾑⟶ෆจᅛ         㸶㦵㧊⥺⥔⑕             㸶෌⏕୙Ⰻᛶ㈋⾑              㸴⾑཭⑓                  㸴┿ᛶ㉥⾑⌫ቑຍ⑕         㸳඲㌟ᛶ࢚ࣜࢸ࣐ࢺ࣮ࢹࢫ          㸳㕲Ḟஈᛶ㈋⾑             㸲㉥ⱆ⌫⒮             㸲ⓑ⾑⑓                  㸲៏ᛶࣜࣥࣃᛶⓑ⾑⑓ከⓎᛶ㦵㧊⭘         㸲             㸲៏ᛶ⑌ᝈ࡟క࠺㈋⾑         㸱ࣁ࢖ࣥࢶᑠయᛶ⁐⾑ᛶ㈋⾑     㸰ࣇ࢛ࣦࣥ࢕ࣞࣈࣛࣥࢻ⑓          㸰⁐⾑ᛶ㍺⾑཯ᛂ              㸯ࢱ࣐ࢿࢠ୰ẘ             㸯኱ື⬦⾑ᰦሰᰦ⑕㦵㧊ቯṚ             㸯ᮏែᛶ⾑ᑠᯈ⾑⑕⾑ᑠᯈ↓ຊ⑕         㸯         㸯             㸯ච␿௓ᅾᛶዲ୰⌫ῶᑡ⑕          㸯㠀෌⏕ᛶ㈋⾑             㸯៏ᛶ㦵㧊ᛶⓑ⾑⑓         㸯ࡑࡢ௚                 㸰㸮

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56

⾲㸯㸲 ⓶⭵⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮




ศ㢮

              ࡢ࡭㢌ᩘ࢔ࢺࣆ࣮ᛶ⓶⭵⅖

       㸰㸯㸲⫧‶⣽⬊⭘            㸶㸲ᝏᛶ㯮Ⰽ⭘            㸶㸱ᡥᖹୖ⓶⒴            㸰㸯⾑⟶࿘⓶⭘            㸯㸷⬡⫫⭘⑕            㸯㸵≉Ⓨᛶ↓⳦ᛶ⤖⠇ᛶ⬡⫫⧊⅖

    㸯㸴་ཎᛶࢡࢵࢩࣥࢢ⑕ೃ⩌         㸯㸲⫠㛛࿘ᅖ⭢⭘             㸳⪥ᇈ⭢⒴             㸳⓶⭵ᙧ㉁⣽⬊⭘              㸳⫧‶⣽⬊⭘⒆

             㸳⓶⭵▼⅊ỿ╔⑕              㸲⬺ẟ⑕㹖             㸲⫠㛛࿘ᅖ⭢⒴             㸲⓶⭵⫧‶⣽⬊⑕              㸲≟⓶⭵⤌⧊⌫⭘              㸱⬡⭢⭘                  㸱⬡⭢ୖ⓶⭘             㸱ከᙧ⣚ᩬ             㸰⾲⓶㐣ㄗ⭘             㸰ⴠⴥ≧ኳ⑁⒔

             㸰⬡⭢⒴                  㸰⾲⓶ྥᛶࣜࣥࣃ⭘         㸰ẟⱆ⭘                  㸰ẟໟୖ⓶⭘             㸰࢔ࣞࣝࢠ࣮ᛶ᥋ゐ⓶⭵⅖          㸯࢔࣏ࢡࣜࣥᄞ⭘              㸯ῐⰍ⿕ẟ⬺ẟ⑕              㸯㛫᧿⑈                  㸯≬≟⑓࣡ࢡࢳࣥ᥋✀ᚋ⬡⫫⧊⅖     㸯



57

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ࢡࣜ࢜ࣇ࢕ࣈࣜࣀ࣮ࢤࣥ⾑⑕     㸯⬡⭢⅖                  㸯ᑜᖖᛶኳ⑁⒔

             㸯ⶼ㯞⑈                  㸯ࣃࢱ࣮ࣥ⬺ẟ⑕              㸯㰯㊑ゅ໬⑕             㸯ࣅࢱ࣑ࣥ㸿཯ᛂᛶ⓶⭵⑕          㸯⾲ᅾᛶቯṚᛶ⓶⭵⅖

         㸯⾲⓶ᄞ⬊             㸯㢮⾲⓶ᄞ⭘             㸯⓶⭵⾑⟶⅖             㸯ࣜࣥࣃ⭘⑕             㸯ࣜࣥࣃ⭘ᵝ⫗ⱆ⭘⑕         㸯ࡑࡢ௚                 㸴㸯
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58

⾲㸯㸳 ║⛉⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮






ศ㢮

                㢌ᩘⓑෆ㞀                㸵㸴㸱⥙⭷ኚᛶ           㸰㸳㸱ࣈࢻ࢘⭷⅖           㸰㸱㸵⥳ෆ㞀                㸰㸰㸯ゅ⭷⅖                㸯㸳㸰Ỉᬗయ⬺⮻           㸯㸯㸵⥙⭷๤㞳           㸯㸯㸯⹿ᙬⴎ⦰            㸶㸴ゅ⭷ࢪࢫࢺࣟࣇ࢕࣮
ゅ⤖⭷⅖

        㸴㸱            㸳㸷◪Ꮚయ࣊ࣝࢽ࢔             㸱㸶
ࢹࢫ࣓⒗

            㸱㸰▖Ꮝ⭷㑇ṧ            㸰㸶║▛ෆ཯            㸰㸴

⬦⤡⥙⭷⅖            㸰㸴ゅ⭷₽⒆

            㸰㸰๓ᡣฟ⾑            㸰㸯

⤖⭷⅖                 㸯㸷⥙⭷ⴎ⦰            㸯㸷⹿ᙬ⒵╔            㸯㸶᰾◳໬⑕            㸯㸵ゅ⭷✸Ꮝ            㸯㸴║⌫⒮                 㸯㸴⹿ᙬᚋ⒵╔            㸯㸴ゅ⭷ศ㞳⑕            㸯㸲஝ᛶゅ⤖⭷⅖            㸯㸲ᙉ⭷⅖                 㸯㸲◪Ꮚయΰ⃮            㸯㸱ど⚄⤒⅖            㸯㸰⥙⭷⑕                 㸯㸯⹿ᙬ⅖                 㸯㸮




59

ᫍ≧◪Ꮚయ⑕

 㸯㸮

ゅ⭷ኚᛶ

  㸷ὶᾦ⑕ 㸷␗ᡤᛶ╰ẟ  㸶ゅ⭷ᾋ⭘  㸶║▛ୗᆶ  㸶║ෆฟ⾑  㸶║⌫✺ฟ  㸵ど⚄⤒ங㢌పᙧᡂ  㸵

⥙⭷⅖

 㸵ゅ⭷ෆ⓶ࢪࢫࢺࣟࣇ࢕࣮  㸴

⹿ᙬ␗Ⰽ⑕  㸴ୖᙉ⭷⅖

  㸴╰ẟ㔜⏕  㸴╰ẟ஘⏕  㸴

࢔ࣞࣝࢠ࣮ᛶ⤖⭷⅖

  㸳

⤖ᬗᛶᐇ㉁ᛶࢪࢫࢺࣟࣇ࢕࣮  㸳

ᩳど

 㸳

◪Ꮚయ⅖

  㸳

✺Ⓨᛶᚋኳᛶ⥙⭷ኚᛶ  㸳ᑠ║⌫ 㸲◪Ꮚయฟ⾑  㸲

Ỉᬗయ೫఩  㸲▐⭷✺ฟ  㸱║▛Ḟᦆ  㸰⹿ᙬ๓⒵╔  㸰⹿ᙬᄞ⭘  㸰

ࢥ࣮ࣜ║␗ᖖ  㸰

Ⰽ⣲ᛶゅ⭷⅖  㸰

➨୕║▛⭢⬺ฟ  㸰

ࡪ࡝࠺⭷⓶⭵⑕ೃ⩌  㸰

㍯㒊㯮Ⰽ⭘  㸰

㢮⓶⭘

 㸰

ゅ⭷ቯṚ

  㸯

60





║᣺

                 㸯∵║                 㸯⌫ᚋ⮋⒆             㸯⤖⠇ᛶୖᙉ⭷⅖              㸯⹿ᙬ⾑⟶᪂⏕             㸯⹿ᙬⰍ⣲ỿ╔             㸯ዲ㓟⌫ᛶ➽⅖             㸯ど⚄⤒ⴎ⦰             㸯ᑠ║▛⿣             㸯◪Ꮚయື⬦㑇ṧ              㸯㗹యኚᛶ             㸯Ⰽ⣲ᛶࡪ࡝࠺⭷⅖

         㸯➨୍ḟ◪Ꮚయ㐣ᙧᡂ㑇ṧ          㸯ࣃࣥࢾࢫ             㸯⾲ᅾᛶⅬ≧ゅ⭷⅖         㸯↓ᏍᾦⅬ             㸯↓Ỉᬗయ║             㸯⥙⭷␗ᙧᡂ             㸯⥙⭷Ⰽ⣲ୖ⓶ࢪࢫࢺࣟࣇ࢕࣮     㸯⼥ゎᛶゅ⭷₽⒆              㸯ᾦᑠ⟶㛢ሰ             㸯ᾦᄞ⅖                  㸯ࡑࡢ௚                㸲㸱㸲


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61

⾲㸯㸴 ⏕Ṫჾ࣭ஙᡣ⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮




ศ㢮

              ࡢ࡭㢌ᩘ





ங⭢⭘⒆



            㸴㸳ங⭢⒴                 㸰㸳๓❧⭢⫧኱⑕            㸯㸷๓❧⭢⒴

            㸯㸰₯ᅾ⢭ᕢ            㸯㸯ᏊᐑỈ⑕            㸯㸯๓❧⭢ᄞ⬊             㸷๓❧⭢⅖             㸴๓❧⥺⮋⒆             㸳Ꮚᐑᖹ⁥➽⭘             㸳༸⬊ᄞ⭘             㸳ࢭࣝࢺࣜ⣽⬊⭘              㸲ங⭢⅖                  㸲๓❧⭢⭘⒆             㸱㯤య㑇ṧ             㸱⢭ᕢ⅖                  㸰⢭ୖ⓶⭘             㸰」ྜᆺங⭢⭘             㸰ங⭢⭢⭘             㸰ഐ๓❧⭢ᄞ⬊             㸯༸ᕢ⥙ᄞ⬊             㸯ഇዷፎ                  㸯ࡑࡢ௚                 㸱㸮
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62


⾲㸯㸵 ⾜ື⑌ᝈ࡟࠾ࡅࡿ⑌ᝈศ㢮


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ศ㢮

              㢌ᩘᡤ᭷ᛶᨷᧁ⾜ື              㸱ᜍᛧᛶᨷᧁ⾜ື              㸰ศ㞳୙Ᏻ             㸰⫥➃⯍ᛶ⓶⭵⅖              㸯
ᑿ㏣࠸⾜ື             㸯ࡑࡢ௚                  㸴63

◊✲Ⓨ⾲
㸯.ㄽᩥⓎ⾲1)

Miyaji K, Okamoto N, Saito S, Yasueda H, Takase Y, Shimakura H, Saito S,
Sakaguchi M.: Cross-reactivity between major IgE core epitopes on Cry j 2 allergenof Japanese cedar pollen and relevant sequences on Cha o 2 allergen of Japanesecypress pollen. Allergol Int in press.

2)

Shigehisa R, Uchiyama J, Kato S, Takemura-Uchiyama I, Yamaguchi K, Miyata R,
Ujihara T, Sakaguchi Y, Okamoto N, Shimakura H, Daibata M, Sakaguchi M,
Matsuzaki S.: Characterization of Pseudomonas aeruginosa phage KPP21
belonging to family Podoviridae genus N4-like viruses isolated in Japan.
Microbiol Immunol in press.

3)

Takai T, Okamoto Y, Okubo K, Nagata M, Sakaguchi M, Fukutomi Y, Saito A,
Yasueda H, Masuyama K.: Japanese Society of Allergology task force report on
standardization of house dust mite allergen vaccines.

Allergol Int 64,181-186,

2015
4)

Horimoto T, Gen F, Murakami S, Iwatsuki-Horimoto K, Kato K, Hisasue M,
Sakaguchi M, Nidom CA, Kawaoka Y.: Cats as a potential source of emerging
influenza virus infections. Virologica Sinica 30,221-223, 2015

5)

Horimoto T, Gen F, Murakami S, Iwatsuki-Horimoto K, Kato K, Akashi H,
Hisasue M, Sakaguchi M, Kawaoka Y, Maeda K.: Serological evidence of infection ofdogs with human influenza viruses in Japan. Vet Rec 174, 96, 20146)㧗஭ᩄᮁ, ᒸᮏ⨾Ꮥ, ኱ஂಖබ⿱, Ọ⏣┿, 㜰ཱྀ㞞ᘯ, ⚟෠཭㤿, 㰻⸨᫂⨾, Ᏻᯞᾈ, ቑᒣᩗ♸:ࢲࢽ࢔ࣞࣝࢤࣥ࣡ࢡࢳࣥᶆ‽໬࡟㛵ࡍࡿ᪥ᮏ࢔ࣞࣝࢠ࣮Ꮫ఍ࢱࢫࢡࣇ࢛࣮ࢫሗ
࿌.࢔ࣞࣝࢠ࣮ 63,1229-1240,2014.

7)㜰ཱྀ㞞ᘯ:࢔ࣞࣝࢠ࣮࡟ᑐࡍࡿච␿⒪ἲ㸸ῶឤస⒪ἲ࡜᪂ࡋ࠸ච␿⒪ἲࡢ㛤Ⓨ. J-Vet
27, 8-14, 2014

8)

Kanemaki N, Tchedre KT, Imayasu M, Kawarai S, Sakaguchi M, Yoshino A, Itoh N,
Meguro A, Mizuki N. Dogs and humans share a common susceptibility gene SRBD1
for glaucoma risk. PLoS One 8, e74372, 2013.

9)

Miyaji K, Yurimoto T, Saito A, Yasueda H, Takase Y, Shimakura H, Okamoto N,
Kiuchi A, Saito S, Sakaguchi M.: Analysis of conformational and sequential IgE
epitopes on the major allergen Cry j 2 of Japanese cedar pollen in humans by using
monoclonal antibodies for Cry j 2. J Clin Immunol 33, 977-983, 2013.

10) ཎ⏣᫴, ⓑ஭⚽἞, 㜰ཱྀ㞞ᘯ㸸 ࠾ዲࡳ↝ࡁ⢊, ࢱࢥ↝ࡁ⢊࡟ΰධࡋࡓࢲࢽ࡟ࡼࡿ࢔ࣞࣝ
ࢠ࣮࡟ࡼࡿ║▛⾑⟶ᛶᾋ⭘ࡢ㸯౛. ⓶⭵⮫ᗋ 55,1127-1133, 2013.
11) ⸨ᮧṇே,㜰ཱྀ㞞ᘯ:≟ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡟ᑐࡍࡿ 8 ᪥㛫ࣉࣟࢺࢥ࣮ࣝࢆ⏝࠸ࡓᛴ㏿64

ῶឤస⒪ἲࡢᏳ඲ᛶ. ⋇་࢔ࢺࣆ࣮࣭࢔ࣞࣝࢠ࣮࣭ච␿Ꮫ఍ㄅ 2,14-18,2013.
12) ⓑ஭⚽἞ࠊ㜰ཱྀ㞞ᘯ㸸ᐊෆ⎔ቃ࢔ࣞࣝࢤࣥࡢホ౯.࢔ࣞࣝࢠ࣮ࡢ⮫ᗋ 33,51-56, 2013.
13) Kubota S, Miyaji K, Shimo Y, Shimakura H, Takase Y, Okamoto N, Kiuchi A.,
Fujimura M., Fujimura T, DeBoer J D, Tsukui T, Sakaguchi M: IgE reactivity toa Cry j 3, an allergen of Japanese cedar pollen in dogs with canine atopic dermatitis.
Vet Immunol Immunopathol 149:132-135, 2012.
14) Kawakami M, Narumoto O, Mastuo Y, Horiguchi K, Yamashita N, Sakaguchi M,
Lipp M, Nagase T, Yamashita N: The role of CCR 7 in allergic airway inflammationinduced by house dust mite exposure. Cell Immunol 275:24-32, 2012.
15) Narumoto O, Matsuo Y, Sakaguchi M, Shoji S, Yamashita N, Shubert D, Abe K,
Horiguchi K,

Yamashita N: Suppressive effects of a pyrazole derivative of

curcumin on airway inflammation and remodeling. Exp Mol Pathol 93, 18-25, 2012.
16) Miyaji K, Suzuki A, Shimakura H, Takase Y, Kiuchi A, Fujimura M, Kurita G,
Tsujimoto H, Sakaguchi M: Large-scale survey of adverse reactions to canine
non-rabies combined vaccines in Japan. Vet Immunol Immunopathol 145: 447-452,
201217) Tsukui T, Sakaguchi M, Kurata K, Maeda S, Ohmori K, Masuda K, Tsujimoto H,
Iwabuchi S: Measurement for canine IgE using canine recombinant high affinity,J(UHFHSWRUǂFKDLQ )Fǆ5,ǂ   -9HWMed Sci 74, 851-856, 201218) Kaburaki Y, Fujimura T, Kurata K, Matsuda K, Toda M, Yasueda H, Chida K,
Kawarai S, Sakaguchi M: Induction of Th1 immune responses to Japanese cedarpollen allergen in mice immunized with Cry j 1 conjugated with CpG
oligodeoxynucleotide. Comp Immunol Microbiol Infect Dis 34,157-161, 2011
19) Fujimura T, Yonekura S, Horiguchi S, TaniguchiY , Saito A, Yasueda H, Inamine
A, Nakayama T, Takemori T, Taniguchi M, Sakaguchi M, Okamoto Y.: Increase ofregulatory T cells and the ratio of specific IgE to total IgE are potential therapeuticor prognostic biomarkers in two-year sublingual immunotherapy for Japanesecedar pollinosis. Clin Immunol 139,65-74, 2011.
20) Kawarai S, Sato K, Horiguchi A, Kurata K, Kiuchi A, Tsujimoto H, Sakaguchi M.:
Potential immunological adjuvant of ‘K’-type CpG- oligodeoxynucleotides enhanced
the cell proliferation and IL-6 mRNA expression in canine B cells. J Vet Med Sci 73,
177-184, 2011.
21) Okayama T, Matsuno Y, Yasuda N, Tsukui T, Suzuta Y, Koyanagi M, Sakaguchi
M, Tsujimoto H, Ishii Y, Olivry T, Masuda K.: Establishment of a quantitative
ELISA for the measurement of an allergen-specific IgE in dogs using anti-IgE
antibody cross-reactive to mouse and dog IgE. Vet Immunol Immunopathol 139,65

99-106, 2011.
22) 㜰ཱྀ㞞ᘯ㸸࢔ࣞࣝࢠ࣮⑌ᝈ࡟ᑐࡍࡿ᰿἞ⓗච␿⒪ἲ࡟࠾ࡅࡿ࢔ࢪࣗࣂࣥࢺࢆ⏝࠸ࡓ࢔ࣞࣝࢤࣥ࣡ࢡࢳࣥ. ႍᜥ 24,58-62, 201123) ⓑ஭⚽἞ࠊ㜰ཱྀ㞞ᘯ㸸ᐊෆ࢔ࣞࣝࢤࣥࡢ ᐃ.✵ẼΎί 48, 17-23, 201124) 㜰ཱྀ㞞ᘯ:ࢫࢠⰼ⢊⑕࡟࠾ࡅࡿ⮬↛Ⓨ⑕ື≀࡜≟ࡢࣂ࢖࢜ࣂࣥࢡࣉࣟࢪ࢙ࢡࢺ. 㛵すᐇ㦂ື≀◊✲఍఍ሗ 33, 64-67, 201125) 㜰ཱྀ㞞ᘯ: ࢔ࣞࣝࢠ࣮⑌ᝈࡢ DNA ච␿⒪ἲ. ࢔ࣞࣝࢠ࣮ 60, 1591-1597, 2011㸰.Ꮫ఍Ⓨ⾲1)ෆᒣ῟ᖹࠊෆᒣఀ௦ࠊ➉ෆ ၨ᫭ࠊ㜰ཱྀ⩏ᙪࠊ㜰ཱྀ㞞ᘯࠊᯇᓮ ⱱᒎ: άᛶᆺࣆࣟࣜ⳦ࣇ
࢓࣮ࢪ KHP30 ࡢ≉ᚩ௜ࡅ࡜ࡑࡢ₯అឤᰁᛶࡢゎᯒ.➨ 89 ᅇ᪥ᮏ⣽⳦Ꮫ఍ࠊ኱㜰ࠊ3 ᭶2016 ᖺ

2)㑣㡲ᕝᛅᘺࠊෆᒣ῟ᖹࠊ㕥ᮌோேࠊᐑ⏣⋹ዉࠊᒣཱྀ⍆⤮ࠊᖹᒣ㝯୍㑻ࠊෆᒣఀ௦ࠊẶཎ㝯Ꮚࠊ㜰ཱྀ⩏ᙪࠊ㜰ཱྀ㞞ᘯࠊᯇ㷂ⱱᒎ㸸⥳⮋⳦ PAO1 ᰴ࡜ࣇ࢓࣮ࢪ KPP22 ࡢ▷ᮇ㛫㐍໬ⓗ㌷ᣑ➇㉮ࡢゎᯒ. ➨ 89 ᅇ᪥ᮏ⣽⳦Ꮫ఍ࠊ኱㜰ࠊ3 ᭶ 2016 ᖺ

3)Ỉ㇂᱁அࠊᚋ⸨⿱Ꮚࠊⶶ⏣ᆂ࿃ࠊ⸨ཎள⣖ࠊ㜰ཱྀ㞞ᘯࠊ኱㔝⪔୍ࠊ㎷ᮏඖ㸸࢖ࢾࡢࣜࣥࣃ⭘⑕౛࡟࠾ࡅࡿ⾑Ύ୰ྍ⁐ᛶ࢖ࣥࢱ࣮ࣟ࢖࢟ࣥ-2 ཷᐜయȘ㙐ࡢ ᐃ. ➨ 12 ᅇ᪥ᮏ⋇་ෆ⛉Ꮫ࢔࢝ࢹ࣑࣮Ꮫ⾡኱఍ࠊᶓ὾ࠊ2 ᭶ 2016 ᖺ

4)ⓑ஭⚽἞ࠊ㜰ཱྀ㞞ᘯ㸸ࢲࢽ࢔ࣞࣝࢤࣥ࡟ᑐࡍࡿᐙᗞෆᑐ⟇ࡢ㠀⮫ᗋ◊✲ࢆ୰ᚰࡋࡓホ౯. ➨ 52 ᅇ᪥ᮏᑠඣ࢔ࣞࣝࢠ࣮Ꮫ఍ࠊዉⰋࠊ11 ᭶ 2015 ᖺ

5)᪫ ឡ㸪⸨஭ᏹ἞㸪ᒸᮏ᠇᫂㸪❑⏣ ⪽㸪㛵ཱྀ࿴ṇ㸪㜰ཱྀ㞞ᘯ㸪Ṋෆࡺ࠿ࡾ㸪ᯇ⏣⚽᫂㸸࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺከᆺゎᯒࢆ⏝࠸ࡓ࢖ࢾࡢಶయ㆑ูἲࡢ᳨ウ㸬➨ 21 ᅇ᪥ᮏἲ⛉Ꮫᢏ⾡Ꮫ఍㸪᯽ࠊ11 ᭶ 2015 ᖺ6)ෆᒣ῟ᖹ㸪㜰ཱྀ㞞ᘯ㸪ᯇᓮⱱᒎ㸸ࣆࣟࣜ⳦ࣇ࢓࣮ࢪࡢⓎぢ࡜ࡑࡢゎᯒ㸬➨ 68 ᅇࡢ᪥ᮏ⣽⳦Ꮫ఍୰ᅜ࣭ᅄᅜᨭ㒊⥲఍㸪ᒸᒣ㸪10 ᭶ 2015 ᖺ

7)ᓥ಴⚽຾ࠊ㰻⸨ᣅࠊ⸨ᮧṇே ࠊᒸᮏ᠇᫂ࠊෆᒣ῟ᖹࠊ㜰ཱྀ㞞ᘯ㸸㣗≀᭷ᐖ཯ᛂࢆ♧ࡍ≟࡟࠾ࡅࡿ༸ⓑᢠཎ࡟ᑐࡍࡿ IgE ཯ᛂᛶࡢ᳨ウ㸬➨ 158 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊ༑࿴⏣ࠊ9
᭶ 2015 ᖺ

8)ᖹᒣ 㝯୍㑻ࠊ㕥ᮌ ோேࠊෆᒣ ῟ᖹࠊᯇ஭ ┿⌮ࠊ㕥ᮌ 㔛࿴ࠊᰘᒣ ᜨ࿃ࠊ㜰ཱྀ 㞞ᘯࠊᮌෆ ᫂⏨: ࢖ࢾ࡟࠾ࡅࡿ᪂つཱྀ⭍ෆᖖᅾ⣽⳦ࡢྠᐃ࡜඲ࢤࣀ࣒㓄ิゎᯒ㸬 ➨ 158 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊ༑࿴⏣ࠊ9 ᭶ 2015 ᖺ

9)ෆᒣ῟ᖹࠊᯇ஭⚽ோࠊⰼᮌ⚽᫂ࠊᯇᓮⱱᒎࠊ㜰ཱྀ㞞ᘯ:⣽⳦ឤᰁ⑕࡟࠾ࡅࡿデ᩿ἲ࡜἞⒪ἲࡢ㛤Ⓨ㸸ࣂࢡࢸࣜ࢜ࣇ࢓࣮ࢪࡢ฼⏝㸬➨ 158 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊ༑࿴⏣ࠊ9 ᭶ 2015ᖺ10) ▼஭ᓫྐ, ᪂಴㞝୍, ⣽ᮌᩗ♸, ὠஂ஭฼ᏹ, 㜰ཱྀ㞞ᘯ, ୕Ꮿ೺௓, 㛗℩㝯ⱥ, ᒣୗ┤⨾㸸 ႍᜥ⑓ែ࡟࠾ࡅࡿẼ㐨ୖ⓶⣽⬊ࡢάᛶ໬࡜᪂つࢡࣝࢡ࣑ࣥㄏᑟయ࡟ࡼࡿไᚚ࡟ࡘ

66࠸࡚ࡢゎᯒ. ➨ 55 ᅇ᪥ᮏ࿧྾ჾᏛ఍ࠊᮾிࠊ4 ᭶ 2015 ᖺ11) ᆤ஭ㄔஓ, Ώ㑔Ꮫ, ஧⎼࿴⨾, ྜྷぢዉὠᏊ, ຍ⸨᫂ஂ, 㜰ཱྀ㞞ᘯ, ኱࿴ಟ,᷸⏣࿴ဢ,ࢳ࢙ࣥࣂ࣮ࢬࢪ࢙࣮࣒ࢬ, Ⳣ㔝⣧ኵ, ෆ⏣࿴ᖾ, ୰ᒣ⿱அ㸸Whole Exome Sequence ἲ࡟ࡼࡿ࢖ࢾ㑇ఏᛶ⚄⤒⑌ᝈࡢཎᅉ㑇ఏᏊྠᐃ. ➨ 11 ᅇ⋇་ෆ⛉࢔࢝ࢹ࣑࣮ࠊ
ᶓ὾ࠊ2 ᭶ 2014
ᖺ12) ཎ⏣ ᫴ࠊⓑ஭ ⚽἞ࠊ㉺ᮏ ▱኱ࠊ㜰ཱྀ 㞞ᘯ,ᕤ⸨ ẚ➼ᚿ㸸⿕ẟ࠾ࡼࡧၚᾮࡢ୧⪅࡬ࡢឤసࡀㄆࡵࡽࢀࡓࢪࣖࣥ࢞ࣜ࢔ࣥࣁ࣒ࢫࢱ࣮࡟ࡼࡿ࢔ࢼࣇ࢕ࣛ࢟ࢩ࣮ࡢ 1 ౛. ➨ 44 ᅇ᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍ࠊ௝ྎࠊ11 ᭶ 2024 ᖺ13) 㜰ཱྀ 㞞ᘯࠊⓑ஭ ⚽἞㸸ᐊෆ⎔ቃ࢔ࣞࣝࢤࣥ.➨ 23 ᅇ᪥ᮏ⮫ᗋ⎔ቃ་Ꮫ఍ࠊி㒔ࠊ6
᭶ 2014 ᖺ14) ▼஭ᓫྐ, ᪂಴㞝୍, ⓑ஭⚽἞, 㜰ཱྀ㞞ᘯ, Ჴඖ᠇୍, ᐊ஭ṇᚿ, 㛗℩㝯ⱥ, ᒣୗ┤⨾:IRF3 ࡢዲ㓟⌫ᛶẼ㐨⅖⑕⑓ែ࡟࠾ࡅࡿᙺ๭. ➨ 26 ᅇ᪥ᮏ࢔ࣞࣝࢠ࣮Ꮫ఍᫓Ꮨ⥲఍ࠊ5
᭶ 2014 ᖺ15) 㕥ᮌ㯞㔛Ꮚࠊ㯮ᓮ᫭ᙪࠊ㣕⏣ၨ㍜ࠊ㟷ᮌᗣኵࠊᯘ㔝῟ࠊ㎷ᮏඖࠊ㜰ཱྀ㞞ᘯ㸸≟ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡟࠾ࡅࡿ᪂つங㓟⳦ Lactobacillus crispatus KT-11 〇๣ࡢᢞ୚ヨ㦂. ➨㸯㸵ᅇ᪥ᮏ⋇་⓶⭵⛉Ꮫ఍Ꮫ⾡኱఍, ࡉ࠸ࡓࡲࠊ3 ᭶ 2014 ᖺ16) 㜰ཱྀ 㞞ᘯ, ὠஂ஭ ฼ᗈ, ኱㝮 ᑛྐ, ㎷ᮏ ඖ:≟ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࡢ࢔ࣞࣝࢤࣥࢆ
⏝࠸ࡓච␿⒪ἲ࡟ࡘ࠸࡚.➨㸯㸮ᅇ⋇་ෆ⛉࢔࢝ࢹ࣑࣮ࠊᶓ὾ࠊ2 ᭶ 2014 ᖺ17) ༳∾ಙ⾜ࠊᕝཎ஭᫴ᖹࠊ㜰ཱྀ㞞ᘯ㸸࢖ࢾࡢࣂ࢖࢜ࣂࣥࢡࣉࣟࢪ࢙ࢡࢺ࡟࠾ࡅࡿ࢖ࢾ⥳ෆ㞀ឤཷᛶ㑇ఏᏊࡢ◊✲.➨㸯㸮ᅇ⋇་⮫ᗋ㑇ఏ◊✲఍ࠊኳ❺ࠊ12 ᭶ 2013 ᖺ18) 㔝㷂῟ኵࠊ㕥ᮌ┤ஓࠊᡂ⏣Ὀ❶ࠊⓑ஭⚽἞ࠊ㜰ཱྀ㞞ᘯ㸸ᕷ㈍࣐ࢫࢡ࡟࠾ࡅࡿ✵Ẽởᰁ≀㉁ࡢ㝖ཤ≉ᛶ࡟ࡘ࠸࡚ ࡑࡢ 1 ࢞ࢫ≧≀㉁㸦࢔ࣥࣔࢽ࢔ࠊ㹔㹍㹁㸧.2013 ᖺᗘ࿧྾ಖㆤ࡟㛵ࡍࡿ◊✲Ⓨ⾲఍ࠊᮾிࠊ12 ᭶ 2013 ᖺ19) ⓑ஭⚽἞ࠊᡂ⏣Ὀ❶ࠊᯇᮏ⹒ୡࠊᰘ⏣ᙲࠊ㔝ᓮ῟ኵࠊ㜰ཱྀ㞞ᘯ㸸࣐ࢫࢡ╔⏝࡟ࡼࡿࢲࢽ࢔ࣞࣝࢤࣥ྾ධண㜵ຠᯝࡢ᳨ウ. ➨ 63 ᅇ᪥ᮏ࢔ࣞࣝࢠ࣮Ꮫ఍⛅ᏘᏛ⾡኱఍ࠊᮾிࠊ
11 ᭶ 2013 ᖺ20) ᓥ಴⚽຾ࠊᒸᒣኴ㑻ࠊቑ⏣೺୍ࠊὠஂ஭฼ᗈࠊ⸨ᮧṇேࠊ㜰ཱྀ㞞ᘯ㸸࢖ࢾࡢ⾑Ύ୰࢔ࣞࣝࢤࣥ≉␗ⓗ IgE ᢠయ㔞࡜࢔ࣞࣝࢠ࣮཯ᛂ㜈್⃰ᗘ࡜ࡢẚ㍑.➨㸯㸳㸴ᅇ᪥ᮏ⋇་Ꮫ఍ࠊᒱ㜧ࠊ9 ᭶ 2013 ᖺ21) 㜰ཱྀ 㞞ᘯ:ᐇ㦂ື≀࢔ࣞࣝࢠ࣮࡜⎔ቃᅉᏊ.➨ 50 ᅇ᪥ᮏᐇ㦂ື≀⎔ቃ◊✲఍,ᮾி,7 ᭶
27 ᪥ 2013 ᖺ22) 㜰ཱྀ㞞ᘯ: ≟ࡢࣂ࢖࢜ࣂࣥࢡࣉࣟࢪ࢙ࢡࢺ. ➨ 155 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊ ᮾிࠊ3 ᭶ 2013ᖺ23) ⚄ᓮ⨾⋹, ⓑ஭⚽἞, ⓙᕝᜨᏊ ᶫᮏ▱ᖾࠊ㜰ཱྀ㞞ᘯࠊ┾ቨ㑳, 㣤ᓥⱱᏊ:࠾ዲࡳ↝ࡁ⢊࡟⦾Ṫࡋࡓࢳࣜࢲࢽ㢮࡟ࡼࡿ༶᫬ᆺ࢔ࣞࣝࢠ࣮ࡢẕᏊ౛. ➨ 62 ᅇ᪥ᮏ࢔ࣞࣝࢠ࣮Ꮫ఍
⛅ᏘᏛ⾡኱఍. ኱㜰ࠊ11 ᭶ 2012 ᖺ6724) ⓑ஭⚽἞ࠊᡂ⏣Ὀ❶ࠊᯇᮏ⹒ୡࠊᰘ⏣ᙲࠊ㔝ᓮ῟ኵࠊ㜰ཱྀ㞞ᘯ: ࣐ࢫࢡ╔⏝࡟ࡼࡿࢲࢽ
࢔ࣞࣝࢤࣥ྾ධண㜵ຠᯝࡢ᳨ウ. ➨ 62 ᅇ᪥ᮏ࢔ࣞࣝࢠ࣮Ꮫ఍⛅ᏘᏛ⾡኱఍. ኱㜰,11 ᭶2012 ᖺ
25) ᓥ಴⚽຾ࠊ࿘⸨᫂⨾ࠊቑ⏣೺୍ࠊᮌෆ᫂⏨ࠊ㜰ཱྀ㞞ᘯ㸸ங㓟⳦ࢆ⏝࠸ࡓ㣗≀࢔ࣞࣝࢠ࣮ࡢ᪂つ἞⒪ἲࡢ㛤Ⓨ㸸࢖ࢾ࡟࠾ࡅࡿ⑌ᝈࣔࢹࣝ࡜ࡋ࡚ࡢ㣗≀࢔ࣞࣝࢠ࣮≟ࡢ᳨ウ.➨87 ᅇ㯞ᕸ⋇་Ꮫ఍ࠊ┦ᶍཎࠊ11 ᭶ 2012 ᖺ26) ᒸᮏ᠇᫂ࠊ▼୸ᾈ㟹ࠊᮌෆ ᫂⏨ࠊ⸨ᮧ ṇேࠊ㜰ཱྀ 㞞ᘯ㸸࢔ࢺࣆ࣮ᛶ⓶⭵⅖≟࡟࠾ࡅࡿ Malassezia pachydermatis ࡢឤస≧ἣ. ➨ 87 ᅇ㯞ᕸ⋇་Ꮫ఍ࠊ┦ᶍཎࠊ11
᭶ 2012 ᖺ27) ᐑᆅ୍ᶞࠊᮌෆ ᫂⏨ࠊ⸨ᮧ ṇேࠊᰩ⏣ ࿃㑻ࠊ㜰ཱྀ 㞞ᘯ㸸᪥ᮏ࡟࠾ࡅࡿ≟ΰྜ࣡ࢡࢳࣥ᥋✀ᚋ๪཯ᛂ࡟㛵ࡍࡿ኱つᶍ࡞␿Ꮫㄪᰝ. ➨ 87 ᅇ㯞ᕸ⋇་Ꮫ఍ࠊ┦ᶍཎࠊ11
᭶ 2012 ᖺ28) 㜰ཱྀ㞞ᘯ:≟ࡢ⑌ᝈゎᯒࡢࡓࡵࡢ㑇ఏᏊࣂࣥࢡ. ➨㸴ᅇᐙ␆㹂㹌㸿す㒓ࢩ࣏ࣥࢪ࣮࣒ࣗ,⚟ᓥࠊ10 ᭶ 2012 ᖺ29) 㜰ཱྀ㞞ᘯ:ᖏ㟁ᚤ⢏ᏊỈࠕࢼࣀ࢖࣮ࠖ࡟ࡼࡿ࣌ࢵࢺ࢔ࣞࣝࢤࣥ୙ά໬ࡢ᳨ウ.᪥ᮏ⮫ᗋ⋇་Ꮫࣇ࢛࣮࣒ࣛࠊᮾிࠊ9 ᭶ 2012 ᖺ30) ᇼᮏὈ௓ࠊ⋞ᩥᏹࠊᒾ㝃◊Ꮚࠊຍ⸨೺ኴ㑻ࠊஂᮎṇᬕࠊ㜰ཱྀ㞞ᘯࠊ᫂▼༤⮧ࠊఀ⸨ኖࠊ
Ἑᒸ⩏⿱ࠊ㕥ᮌ࿴⏨ࠊ๓⏣೺: ࢃࡀᅜࡢ့ஙື≀࡟࠾ࡅࡿ࢖ࣥࣇ࢚ࣝࣥࢨ࢘࢖ࣝࢫឤᰁ.
➨ 154 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊᒾᡭࠊ9 ᭶ 2012 ᖺ31) ᡂᮏ἞㸪ᯇᑿ⏤⣖Ꮚ㸪㜿㒊࿴✑ࠊ㜰ཱྀ㞞ᘯࠊ㛗℩㝯ⱥ㸪ᒣୗ┤⨾㸸ዲ୰⌫ᾐ₶ࢆక࠺ႍᜥࣔࢹࣝࢆ⏝࠸ࡓẼ㐨⅖⑕ࡢไᚚࡢゎᯒ.➨ 52 ᅇ᪥ᮏ࿧྾ჾᏛ఍, ⚄ᡞࠊ4 ᭶ 2012ᖺ32) 㜰ཱྀ㞞ᘯ 㸸≟ࡢࣂ࢖࢜ࣂࣥࢡࣉࣟࢪ࢙ࢡࢺࡢヨࡳ ᖹᡂ 23 ᖺᗘ᪥ᮏ⋇་ᖌ఍⋇་Ꮫ⾡Ꮫ఍,ᮐᖠࠊ2 ᭶ 2012 ᖺ33) ཎ⏣ ᫴ࠊⓑ஭⚽἞ࠊ㜰ཱྀ㞞ᘯ㸸࠾ዲࡳ↝ࡁ⢊ࠊࢱࢥ↝ࡁ⢊࡟ΰධࡋࡓࢲࢽ࡟ࡼࡿ࢔
ࣞࣝࢠ࣮཯ᛂࡢⓎ⑕ᶵᗎ࡟㛵ࡍࡿ⪃ᐹ ➨ 61 ᅇ᪥ᮏ࢔ࣞࣝࢠ࣮Ꮫ఍⥲఍ࠊᮾிࠊ11 ᭶
2011 ᖺ
34) ᓥ಴⚽຾ࠊ࿘⸨᫂⨾ࠊஂಖ⏣⩧ኴࠊᐑᆅ୍ᶞࠊ㧗℩᭷ຍ㔛ࠊᮌෆ᫂⏨ࠊ㜰ཱྀ㞞ᘯ:㑇ఏᛶࡀ␲ࢃࢀࡿ㣗≀࢔ࣞࣝࢠ࣮≟࡟࠾ࡅࡿᐙ⣔ࡢㄪᰝ. ➨ 152 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊሜࠊ9
᭶ 2011 ᖺ35) Fujimura T, Yonekura S, Horiguchi S, Sakaguchi M, Taniguchi M, Okamoto Y.㸸
Candidate for response monitoring or prognostic biomarkers in two-year sublingual
immunotherapy for Japanese cedar pollinosis. 22th World Allergy Congress,
Cancun, Mexico, Decmeber, 2011.
36) Tsukui T, Fukui M, Ohsumi T, Maeda S, Chimura N, Sakaguchi M, Tsujimoto, H.
Immunotherapy Using Pullulan-conjugated Recombinant House Dust Mite

68

$OOHUJHQ 'HU I -P). Asian Meeting of Animal Medicine SpecialtiesࠊBangkok,
Thailand, May, 2011.
37) ᡂᮏ἞ࠊᯇᑿ⏤⣖Ꮚࠊ㜿㒊࿴✑ࠊⓑ஭⚽἞ࠊ㜰ཱྀ㞞ᘯࠊ㛗℩㝯ⱥࠊᒣୗ┤⨾㸸ႍᜥ⑓ែ࡟࠾ࡅࡿẼ㐨ୖ⓶⣽⬊ࡢάᛶ໬࡜᪂つࢡࣝࢡ࣑ࣥㄏᑟయ࡟ࡼࡿไᚚ࡟ࡘ࠸࡚ࡢゎᯒ.
➨ 51 ᅇ᪥ᮏ࿧྾ჾᏛ఍ࠊᮾிࠊ4 ᭶ 2011 ᖺ

69

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Allergology International 64 (2015) 181e186

Contents lists available at ScienceDirect

Allergology International
journal homepage: http://www.elsevier.com/locate/alit

Original article

Japanese Society of Allergology task force report on standardization ofhouse dust mite allergen vaccines e Secondary publication*
Toshiro Takai a, b, Yoshitaka Okamoto a, c, Kimihiro Okubo a, d, Makoto Nagata a, e, f,Masahiro Sakaguchi a, g, Yuma Fukutomi a, h, Akemi Saito h, Hiroshi Yasueda a, h,
Keisuke Masuyama a, i, *aTask Force for House Dust Mite Allergen Standardization of the Committee for Allergens and Immunotherapy of the Japanese Society of Allergology, Tokyo,Japanb
Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japanc
Department of Otorhinolaryngology-Head and Neck Surgery, Graduate School of Medicine, Chiba University, Chiba, Japand
Department of Head and Neck Sensory Organ Science (Otolaryngology), Graduate School of Medicine, Nippon Medical School, Tokyo, Japane
Department of Respiratory Medicine, Saitama Medical University, Saitama, JapanfAllergy Center, Saitama Medical University, Saitama, Japang
Laboratory of Microbiology I, School of Veterinary Medicine, Azabu University, Kanagawa, Japanh
Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital, Kanagawa, Japani
Department of Otorhinolaryngology-Head and Neck Surgery, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi,
Yamanashi, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 13 November 2014
Received in revised form
21 December 2014
Accepted 6 January 2015
Available online 4 March 2015

Background: In the 1990s, the Japanese Society of Allergology (JSA) standardized Japanese cedar pollen
allergen vaccines. In the present study, the task force for house dust mite (HDM) allergen standardizationof the Committee for Allergens and Immunotherapy of JSA reports the standardization of HDM allergenvaccines in Japan.
Methods: In vivo allergenic potency was determined by intradermal testing of 51 Japanese adults withpositive serum speciﬁc IgE to HDM allergens. In vitro total IgE binding potency was analyzed bycompetition ELISA using a pooled serum, with sera obtained from 10 allergic patients. The amounts of
HDM group 1 (Der 1) and group 2 major allergens in eight HDM allergen extracts were measured bysandwich ELISAs. Correlation between the in vitro total IgE binding potency and major allergen levels wasanalyzed.
Results: We selected a JSA reference HDM extract and determined its in vivo allergenic potency. The
in vitro total IgE binding potency signiﬁcantly correlated with Der 1 content, group 2 allergen content,
and their combined amount, indicating that measurement of major allergen contents can be used as asurrogate in vitro assay.
Conclusions: The task force determined the in vivo allergenic potency (100,000 JAU/ml) and Der 1
content (38.5 mg/ml) of the JSA reference HDM extract, selected the measurement of Der 1 content as thesurrogate in vitro assay, and decided that manufacturers can label a HDM allergen extract as having a titerof 100,000 JAU/ml if it contains 22.2e66.7 mg/ml of Der 1.
Copyright © 2015, Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

Keywords:
House dust mite allergen standardization
Intradermal testingIn vivo allergenic potency
Major allergen content
Surrogate in vitro assay
Abbreviations:AU, allergy unit; BAU, bioequivalent allergy
unit; CBER, Center for Biologics Evaluation
and Research; Der 1, Der p 1 and Der f 1; Der
2, Der p 2 and Der f 2; DF, Dermatophagoides
farinae; DP, Dermatophagoides
pteronyssinus; FDA, Food and Drug
Administration; HDM, house dust mite;
JAU, Japanese allergy unit; JCP, Japanesecedar pollen; JSA, Japanese Society ofAllergology; UAS, universal allergenstandard*
This article is a secondary publication of “Japanese Society of Allergology task force report on standardization of house dust mite allergen vaccines” published in
Arerugi [Japanese Journal of Allergology] 2014; 63: 1229-40 (in Japanese).
* Corresponding author. Department of Otorhinolaryngology-Head and Neck Surgery, Interdisciplinary Graduate School of Medicine and Engineering, University of
Yamanashi, Yamanashi 409-3898, Japan.
E-mail address: mkeisuke@yamanashi.ac.jp (K. Masuyama).
Peer review under responsibility of Japanese Society of Allergology.

http://dx.doi.org/10.1016/j.alit.2015.01.005
1323-8930/Copyright © 2015, Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

77

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T. Takai et al. / Allergology International 64 (2015) 181e186

Introduction
Allergen-speciﬁc immunotherapy has been performed in general medical practice since it was ﬁrst described by Noon in 1911.1e7
Standardization of allergen vaccines/extracts used for therapy anddiagnosis is necessary because their qualities are variable
depending on production methods and manufactured lots.3e5,8e10
In the United States, allergen standardization is based on intradermal testing of allergic patients and the potencies of lots are
determined by appropriate surrogate in vitro assays, which are
based on inhibition of binding of IgE from pooled allergic sera tosolid phase reference allergen extracts, or measurement of speciﬁcallergen contents in the allergen vaccines.8 In the European Union,
products are standardized using manufacturers' in-house references and labeled in manufacturer-speciﬁc units.9 In Japan, theJapanese Society of Allergology (JSA) standardized Japanese cedarpollen (JCP) allergen vaccines.11,12 In vivo allergenic potency of the
JSA reference JCP extract was determined by intradermal testing
and measurement of the content of the major allergen Cry j 1 wasselected as the surrogate in vitro assay.
House dust mites (HDMs) are a major allergen source that provokes allergic rhinitis, asthma, conjunctivitis, and atopic dermatitis.13,14 However, standardization of HDM allergen vaccines/extracts
based on intradermal testing in Japanese subjects with positive serum
speciﬁc IgE to HDM allergens has not been performed. This report,
produced by the task force for HDM allergen standardization of theCommittee for Allergens and Immunotherapy of JSA, selected a JSA
reference HDM extract, determined its in vivo allergenic potency inJapanese allergy units (JAU) by intradermal testing of HDM-sensitized
Japanese adults, and determined a surrogate in vitro assay that issuitable for HDM allergen standardization in Japan.
Methods
HDM allergen extracts
Each HDM extract was prepared as a mixture of equivalent volumes of extracts from two mite species, Dermatophagoides pteronyssinus (DP) and Dermatophagoides farinae (DF). United States Foodand Drug Administration (FDA) reference extracts, E11-DP (10,000
AU/ml) and E10-DF (10,000 AU/ml), were acquired from the Laboratory of Immunobiochemistry, Division of Bacterial, Parasitic andAllergenic Products, Ofﬁce of Vaccines Research and Review, Center
for Biologics Evaluation and Research (CBER), FDA. Commercial extracts for subcutaneous immunotherapy (DP: lot#B3117094 and DF:
lot#F21G6279, 10,000 AU/ml) were purchased from Hollister-Stier(Spokane, WA, USA). Five extracts identiﬁed by alphabet letters
 AS (Hørsholm,(Extracts AeE) were obtained from ALK-Abello
Denmark) and Stallergenes SA (Antony, France). JSA chose an extract
other than those described above as the JSA reference HDM extract.
Measurement of group 1 allergens and in vitro relative IgE binding
potency testing took place in the laboratories of Sagamihara National
Hospital (Laboratory 1) and Azabu University (Laboratory 2). Measurement of the combined total content of group 2 allergens was
conducted only in Laboratory 1.
HDM-sensitized subjects
Inclusion criteria for the HDM-sensitized subjects enrolled in thisstudy were: Japanese adults between the age of 20 and 50 years who
were positive for DP or DF-speciﬁc IgE, showing 0.70 Ua/ml (class 2)
in ImmunoCAP assay (ThermoFisher Scientiﬁc, Uppsala, Sweden).
Subjects were excluded from the study if they had (a) skin disease on
the forearm that affects intradermal injection reactions; (b) used (1)external application of medication within one day of study initiation,(2) oral antihistamine, oral a- or b-adrenergic agonists, and topicalcorticosteroid or immunosuppressive medicine for external application to the injection site within one week of study initiation, (3)tricyclic antidepressants and phenothiazines with an antihistamine
effect within 2 weeks of study initiation, (4) non-selective b-adrenergic blockers within 3 weeks of study initiation, (5) systemic
immunosuppressive drugs within 30 days of study initiation, (6)speciﬁc antibodies within 90 days of study initiation; or if they were(c) pregnant or were possibly pregnancy on the study day; were
under (d) HDM-speciﬁc immunotherapy; or had (e) severe bronchialasthma; (f) anaphylaxis to adrenaline; (g) concomitant systemic
diseases such as cardiac, hepatic, renal, and hematologic disorders or
infection that could affect the study trial; or (h) were judged by theexaminers as being inappropriate for study enrollment.
Intradermal testing and collection of sera were conducted at the
Department of OtorhinolaryngologyeHead and Neck Surgery of
Chiba University Graduate School of Medicine, Department ofOtorhinolaryngology of Nippon Medical School, Graduate School of
Medicine, Department of Respiratory Medicine of Saitama Medical
University, and Department of OtorhinolaryngologyeHead andNeck Surgery of the University of Yamanashi Interdisciplinary
Graduate School of Medicine and Engineering. The ethical review
committee of each institution approved the protocol of this study.
Written informed consent was obtained from all patients before
study enrollment and anonymity was preserved using documents
and methods approved by the ethical review committees.
Intradermal testingFirst, we selected a candidate HDM extract as the JSA reference
HDM extract to be used for intradermal testing. The candidate
extract was diluted using the 0.005% Polysorbate 80-added control
solution for intradermal skin testing, “TORI-I”, which contained
0.9% (w/v) NaCl and 0.5% (w/v) phenol (Torii Pharmaceutical, Tokyo,
Japan). The dilution factors were from 37 to 319 (2187e1.162  109).
Control solution for intradermal skin testing, “TORI-I”, was used as
the negative control.
Intradermal testing was performed according to a previously
described method.11,12 The protocol for intradermal testing waslargely based on the FDA protocol except for criteria for determining the threshold concentration. Brieﬂy, 20 ml of each diluted
HDM extract was administered intradermally into the forearm from
low to high concentrations using 1-ml tuberculin syringes. Fifteen
minutes after the injection, the diameters of the wheal or erythema
were measured based on Ishizaki's criteria15e17 (i.e. positive reaction is deﬁned as presence of a wheal or erythematous skin reaction
with a diameter of more than 9 mm or 20 mm, respectively). The
threshold dilution factor was deﬁned as the maximum dilution
factor of diluted extract that can induce a positive reaction.
With respect to the determination of in vivo allergenic potency,
threshold values of all patients were represented as logarithms thathave 3 as the base of the threshold dilution factors, and then an
average value for them was calculated. Values, which were from 9
to 11, 11 to 13, and 13 to 15, were assigned the in vivo allergenic
potencies of 1000 JAU/ml, 10,000 JAU/ml, and 100,000 JAU/ml,
respectively, based on the plan used in the standardization of JCP
allergen vaccines.11,12 With this method, the same titer could be
assigned to extracts with, at the most, a 32-fold (9-fold) difference
in allergenic potencies.
Measurement of HDM group 1 major allergens
The group 1 allergens, Der p 1 and Der f 1, in the HDM extracts
were measured by a previously described method.18 Brieﬂy,

78

T. Takai et al. / Allergology International 64 (2015) 181e186

sandwich ELISAs for Der p 1 or Der f 1 were performed using
murine monoclonal antibodies and 92-Dp or 92-Df extract,18,19
respectively, as the standard antigen for ELISA. Previously, the 92Dp and 92-Df extracts were prepared from HDM bodies and thecontents of group 1 and group 2 allergens in 92-Dp and 92-Df were
determined.18,19 For validation of the measurement obtained bythis method, group 1 allergen content was also determined using
other ELISA kits purchased from Indoor Biotechnologies (Charlottesville, VA, USA) and Nichinichi Pharmaceutical (Mie, Japan).

Measurement of the total amount of HDM group 2 major allergens
The total amount of group 2 allergens (Der 2), Der p 2 and Der f
2, was measured using a previously described method.20,21 Brieﬂy,a sandwich ELISA for Der 2 was performed using rabbit polyclonal
antibodies and a mixture of 92-Dp and 92-Df extracts18,19 as thestandard antigen for ELISA.

Sera for in vitro relative IgE binding potency testing
A total of 20 ml of blood sample was taken from 10 randomly
selected patients out of 19 who had 17.5 Ua/ml (class 4) for DP- or
DF-speciﬁc IgE in the ImmunoCAP assay (ThermoFisher). Blood
samples were centrifuged and sera were separated and storedat 80  C. A pooled serum was prepared as a mixture of equivalent
volumes of the 10 sera.

183

Measurement of in vivo allergenic potency of the JSA reference HDM
extract
Fifty-two adults (24 men and 28 women) were enrolled in thisstudy. Their mean age (±SD) was 32.9 (±6.5) years. Of the 52 participants, 42 (80.7%) were diagnosed with persistent allergic
rhinitis and among them, 5 and 4 also had bronchial asthma andallergic rhinitis provoked by JCP, respectively. Another 4 participants had bronchial asthma and 1 had cough variant asthma. One
person was excluded from study analysis because the thresholdconcentration in intradermal testing could not be examined. Fig. 1
shows the frequency and distribution of threshold values of intradermal testing of the 51 subjects. The average (±SD) thresholdvalue was 13.22 (±1.43), which corresponded to 100,000 JAU/ml.
Major allergen contents in the JSA reference HDM extract
The concentrations of HDM group 1 and group 2 allergens in
HDM extracts designated as 92-Dp and 92-Df had been alreadydetermined.18,19 The group 1 and group 2 allergen contents in the
JSA reference HDM extract were measured by sandwich ELISAs
using the 92-Dp and/or 92-Df extract as the standard antigen for
ELISA (Table 1). Measurement of the group 1 allergens produced
similar results in the two laboratories. The concentrations of Der p
1, Der f 1, and total concentration of Der p 1 and Der f 1 (Der 1) in
the JSA reference HDM extract were (as the geometric means of thevalues obtained in the two laboratories) 25.6, 12.9, and 38.5 mg/ml,
respectively. The total concentration of Der p 2 and Der f 2 (Der 2)
measured in Laboratory 1 was 55.5 mg/ml.

In vitro relative IgE binding potency testing
Competition ELISA was used to evaluate the inhibition ofallergen-speciﬁc IgE binding as described previously.22 Brieﬂy, theplates were coated with the JSA reference HDM extract or anotherextract (Extract C). The pooled serum (dilution factor: 50) was
mixed with an equivalent volume of each serially diluted inhibitor
extract (ﬁnal serum dilution factor: 100), and after incubation for
30 min at room temperature, the mixtures were added to the wellsof the plates. The allergen-speciﬁc IgE binding to plate wells weredetected with enzyme-conjugated anti-human IgE and a ﬂuorogenic substrate. Relative potencies of extracts were calculated asratios of the dilution factors of the extracts that gave half the
maximum (50%) ﬂuorescence relative to that of the JSA reference
HDM extract.

Validation of measurement of Der p 1 and Der f 1
Other ELISA systems were used to validate the measurement of
Der p 1 and Der f 1 in this study (Table 2). Similar results wereobtained using two commercially available ELISA kits. Lower values(approximately 20% less) were obtained with the kit purchasedfrom Indoor Biotechnologies. The standard used in the kit isdesignated as an “universal” allergen standard (UAS), which wasdeveloped in the CREATE project funded through the European
Union.23,24 Regardless of whether the monoclonal antibodies werein the kit from Indoor Biotechnologies or developed in Laboratory 1,
use of UAS as the standard antigen for ELISA resulted in lower

Statistical analysis
Pearson correlation coefﬁcients for the association between
major allergen content and in vitro relative IgE binding potency
were calculated after logarithmic transformation. P < 0.05 wasregarded as statistically signiﬁcant.

Results
Selection of a HDM allergen extract as the JSA reference HDM extract
We selected one extract, which can be used for intradermal
testing and would be approved for allergen-speciﬁc immunotherapy in Japan, as a candidate for the JSA reference HDM extract.
The candidate extract showed appropriate values in terms of in vivo
allergenic potency and HDM group 1 and 2 major allergen contents,
as described below. Accordingly, we selected the candidate extract
as the JSA reference HDM extract.

79

Figure 1. Distribution of the threshold values that induced positive reactions in theintradermal testing of 51 Japanese adults with positive serum speciﬁc IgE to house dust
mite allergens. Prepared dilution factors of the Japanese Society of Allergology reference house dust mite extract were from 37 (2187) to 319 (1.162  109). A total of 20 ml ofeach diluted extract was administered intradermally into the forearm from low to highconcentrations. The diameters of the wheal or erythematous skin reaction were
measured 15 min after the injection. Threshold value: the exponent of the maximum
dilution factor that can induce a positive reaction in each subject.

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T. Takai et al. / Allergology International 64 (2015) 181e186

Table 1
House dust mite major allergen content in the Japanese Society of Allergology
reference house dust mite extract.

Der 2, and combined total of Der 1 and Der 2 (Der 1 þ Der 2) (Fig. 2).
Pearson correlation coefﬁcients were greater than 0.9 and were
statistically signiﬁcant.

Concentration (mg/ml)
Group 1 allergens
Institute
Laboratory 1
Laboratory 2
Geometric mean

Der p 1
28.2
23.2
25.6

Der f 1
14.1
11.8
12.9

Group 2 allergens
Der 1
42.3
35.0
38.5

Der 2
55.5
N.D.

Der p 1, Der f 1, and Der 2 were measured by sandwich ELISA in two institutes. Der 1,
combined total of Der p 1 and Der f 1; Der 2, combined total of Der p 2 and Der f 2;
Laboratory 1, Sagamihara National Hospital; Laboratory 2, Azabu University; N.D.,
not determined.

values compared with the use of 92-Dp or 92-Df. This suggests thatthe small discrepancy was caused by the difference in the deﬁned
concentrations of Der p 1 and Der f 1 between 92-Dp/92-Df and UAS
and was not due to the detection system, such as speciﬁcity of
monoclonal antibodies. The results indicated that the ELISA system
for Der p 1 and Der f 1 developed in Laboratory 1 was valid.
Major allergen contents in the HDM extracts tested
Major allergen contents in the seven HDM extracts other thanthe JSA reference HDM extract were measured (Table 3). Five extracts were from ALK or Stallergenes (Extract AeE) and two werefrom CBER/FDA and Hollister-Stier. Similar results were obtained in
the two laboratories (Tables 3, 4, concentration).
Correlation between the major allergen contents and in vitro
relative IgE binding potencies
Competition ELISA was used to evaluate the inhibition ofallergen-speciﬁc IgE binding to plates coated with the JSA reference
HDM extract or Extract C. A pooled serum was prepared as a
mixture of equivalent volumes of sera from 10 of 19 patients who
had 17.5 Ua/ml (class 4) for DP- or DF-speciﬁc IgE in the ImmunoCAP assay. The in vitro total IgE binding potencies of the HDM
extracts relative to the JSA reference HDM extract were determined(Tables 3, 4, in vitro relative potency). Similar results were obtainedusing JSA reference HDM extract-coated plates and Extract Ccoated plates. Similar results were obtained in the two laboratories.
Correlation between major allergen contents and the relative
IgE binding potency was analyzed. The relative IgE binding potency
correlated well with each of the concentrations of Der 1 (Figs. 2, 3),

Table 2Comparison of house dust mite group 1 allergen contents in the Japanese Society ofAllergology reference house dust mite extract determined by sandwich ELISAs using
different antibodies and standards.
Institute

Lab1

Lab2

Concentration (mg/ml)

ELISA
Antibodiesy

Standardz

Der p 1

Der f 1

Der 1

Lab1
Lab1
IndoorIndoorNichinichi
Lab1
Indoor

92-Dp/92-Df
UAS
92-Dp/92-Df
UAS
Nichinichi
92-Dp/92-Df
92-Dp/92-Df

28.2
22.1
27.4
22.3
24.3
23.2
22.1

14.1
9.69
14.6
11.7
13.6
11.8
14.0

42.3
31.8
42.0
34.0
37.9
35.0
36.1

Der p 1 and Der f 1 were measured by sandwich ELISA in two institutes.
Der 1, combined total of Der p 1 and Der f 1; Lab1, Sagamihara National Hospital;
Lab2, Azabu University; UAS, universal allergen standard; Indoor, Indoor Biotechnologies; Nichinichi, Nichinichi Pharmaceutical.
yCapture and detection antibodies used in ELISA.
z
Standards with deﬁned concentrations used in ELISA.

Discussion
In the 1990s, JSA standardized the JCP allergen vaccines/extracts.11,12 The JSA standard JCP extract showed an in vivo allergenic
potency of 10,000 JAU/ml. The unit JAU11,12 was determined byintradermal testing, similar to the bioequivalent allergy unit (BAU)
and the allergy unit (AU) deﬁned by FDA,8,25e27 but criteria for thereaction threshold are different between JAU and BAU/AU, and theinjection volume is 20 ml for JAU and 50 ml for BAU/AU. JSA selected
measurement of Cry j 1 content as the surrogate in vitro assay for
determining the potencies of other JCP extracts, with the concentration of 12.5 mg/ml of Cry j 1 corresponding to 10,000 JAU/ml. JSA
decided that manufacturers can label JCP extracts as having a titerof 10,000 JAU/ml if they contain 7.3e21 mg/ml of Cry j 1 i.e., within arange that is approximately three times the lowest value, thegeometric center of which is 12.5 mg/ml.11,12 In the present study,
the task force selected a JSA reference HDM extract, determined its
in vivo allergic potency in JAU using the same method for JCP
allergen standardization, and analyzed the correlation between thein vitro total potency determined by IgE binding inhibition ELISA
and the major allergen contents in eight HDM extracts.
Through intradermal testing of 51 Japanese HDM-sensitized
adults, the in vivo allergenic potency of the candidate for the JSA
reference HDM extract was determined as 100,000 JAU/ml. As thecandidate extract showed an appropriate in vivo allergenic potency(Fig. 1) and major allergen contents (Table 1), we decided to use it as
the JSA reference HDM extract. Surrogate in vitro assay to determine the potencies of extracts from different manufacturers andlots can be based on inhibition of binding of IgE from pooled allergic
sera to a reference allergen extract, or measurement of speciﬁcallergen contents in the allergen vaccines/extracts. In the UnitedStates, FDA adopted relative IgE binding potency determined by IgEbinding inhibition assay for HDM and mold allergen vaccines;
speciﬁc allergen contents for short ragweed pollen and cat allergenvaccines (Amb a 1 and Fel d 1, respectively); and enzymatic activity(hyaluronidase and phospholipases) for Hymenoptera venom
allergen vaccines.8 It has been reported that the major allergencontent correlates with the relative IgE binding potency.28e30
Among more than 20 groups of HDM allergens, group 1 and 2 allergens are considered the major allergens, although some reports
showed that other HDM allergens were also important, albeit lessso than the major allergens.13,14 We judged that measurement of
HDM major allergens is appropriate as a surrogate assay because
the relative IgE binding potency correlated well with each of the
major allergen contents (Figs. 2, 3).
The in vitro measurement of major allergen content has someadvantages as the surrogate assay for allergen standardization i.e., it
can determine absolute and not relative values, and does not needsera in which differing individual titers are seen. Species-speciﬁc
ELISAs using monoclonal antibodies to measure each HDM group
1 allergen, Der p 1 and Der f 1, with high accuracy are available
commercially (see the Methods section). However, those for eachgroup 2 allergen, Der p 2 and Der f 2, are not widely available, andthe total contents of Der p 2 and Der f 2 (Der 2) determined by ELISA
using polyclonal antibodies in the present study are approximate
values. Therefore, we selected measurement of the total content of
Der p 1 and Der f 1 (Der 1) as the surrogate in vitro assay. We
determined that the in vivo allergenic potency and Der 1 content of
the JSA reference HDM extract were 100,000 JAU/ml and 38.5 mg/
ml, respectively. Similar to the previous JCP allergen standardization,11,12 we decided that manufacturers can label an HDM allergen

80T. Takai et al. / Allergology International 64 (2015) 181e186

185

Table 3
House dust mite major allergen contents and in vitro total IgE binding potencies of the eight house dust mite extracts determined in Laboratory 1.
Concentration (mg/ml)y

SampleJSA
CBER/FDA
Hollister-Stier
Extract A
Extract B
Extract C
Extract D
Extract E

In vitro relative potencyz

Der p 1

Der f 1

Der 1

Der 2

Der 1 þ Der 2

JSA coating

Extract C coating

28.2
16.7
10.1
80.0
2.69
30.9
3.59
2.27

14.1
16.3
2.54
54.2
0.98
52.7
2.50
5.45

42.3
33.0
12.6
134
3.67
83.6
6.09
7.72

55.5
19.6
11.3
254
6.76
45.8
2.38
3.28

97.8
52.6
23.9
388
10.4
129
8.47
11.0

1.00
0.56
0.35
4.89
0.14
1.83
0.08
0.15

1.00
0.51
0.33
4.85
0.13
2.54
0.16
0.26

Der 1, combined total of Der p 1 and Der f 1; Der 2, combined total of Der p 2 and Der f 2; JSA, JSA reference house dust mite extract.
y
Der p 1, Der f 1, and Der 2 were measured by sandwich ELISA.
z
In vitro total IgE binding potency relative to the Japanese Society of Allergology (JSA) reference house dust mite extract was determined on the basis of the results of acompetition ELISA that measures the inhibition of allergen-speciﬁc IgE binding to plates coated with the JSA reference house dust mite extract (JSA coating) or Extract C(Extract C coating).

extract as having a titer of 100,000 JAU/ml if it contains
22.2e66.7 mg/ml of Der 1 i.e., within a range that is approximately
three times the lowest value, the geometric center of which is
38.5 mg/ml.
We validated the measurement of Der p 1 and Der f 1 (Table 2).
The amounts of group 1 and group 2 allergens in 92-Dp and 92-Df
extracts were determined previously in Laboratory 118, 19. The
measurement of the concentrations of puriﬁed group 1 and group 2
allergens previously used as the standard antigens for the ELISA
that determine the major allergen contents in 92-Dp and 92-Df
were based on absorbance at 280 nm. UAS is composed of eight
puriﬁed allergens (Der p 1, Der f 1, Der p 2, Fel d 1, Can f 1, Rat n 1,
Mus m 1, and Bla g 2) and the concentrations of the allergens were
determined by amino acid analysis.23,24 The difference in theoriginal methods for determining the concentrations of the standard antigens for ELISA might have caused the lower values(approximately 20% less) obtained by the Indoor Biotechnologies
kit compared with the ELISA developed in Laboratory 1 (Table 2).
In conclusion, the task force (1) selected the JSA reference HDM
extract and determined its in vivo allergenic potency via intradermal testing, (2) showed the correlation between in vitro IgE binding
potency and major allergen contents, and selected measurement of
Der 1 content as the surrogate in vitro assay for determining thepotencies of other HDM extracts by deﬁning 38.5 mg/ml of Der 1 as
100,000 JAU/ml. Furthermore, (3) the task force decided that

manufacturers can label a HDM allergen extract as having a titer of
100,000 JAU/ml if it contains 22.2e66.7 mg/ml of Der 1 i.e., within arange that is approximately three times the lowest value. Der 1
contents can be measured by appropriate methods such ascommercially available sandwich ELISAs (see the Methods section).
JSA is planning to store and distribute the JSA reference HDM
extract. The manufacturers of HDM allergen vaccines would ﬁnd

Table 4
House dust mite group 1 allergen contents and in vitro total IgE binding potencies ofthe eight house dust mite extracts determined in Laboratory 2.
SampleJSA
CBER/FDA
Hollister-Stier
Extract A
Extract B
Extract C
Extract D
Extract E

Concentration (mg/ml)

In vivo relative potency

Der p 1

Der f 1

Der 1

JSA coatingy

Extract C
coatingz

23.2
17.6
9.25
80.5
2.28
38.8
3.40
2.24

11.8
13.2
1.79
49.5
0.93
54.2
4.08
6.87

35.0
30.8
11.0
130
3.21
93.0
7.48
9.11

1.00
0.71
0.34
5.75
0.14
1.47
0.11
0.12

1.00
0.75
0.34
5.71
0.14
2.11
0.16
0.20

Der 1, combined total of Der p 1 and Der f 1; Der 2, combined total of Der p 2 and Der
f 2; JSA, JSA reference house dust mite extract.
y
Der p 1, Der f 1, and Der 2 were measured by sandwich ELISA.
z
In vitro total IgE binding potency relative to the Japanese Society of Allergology(JSA) reference house dust mite extract was determined on the basis of the results ofa competition ELISA that measures the inhibition of allergen-speciﬁc IgE binding toplates coated with the JSA reference house dust mite extract (JSA coating) or Extract
C (Extract C coating).

81

Figure 2. Correlation between house dust mite major allergen contents and in vitro
total IgE binding potencies of the eight house dust mite extracts determined in Laboratory 1. The values in Table 3 were used for the analysis. In vitro total IgE binding
potency relative to the Japanese Society of Allergology (JSA) reference house dust miteextract was determined based on the results of the competition ELISA that measured
the inhibition of allergen-speciﬁc IgE binding to plates coated with the JSA reference
house dust mite extract (A) or Extract C (B). r: Pearson correlation coefﬁcient. *p < 0.01.

186

T. Takai et al. / Allergology International 64 (2015) 181e186

Figure 3. Correlation between house dust mite group 1 allergen contents and in vitro
total IgE binding potencies of the eight house dust mite extracts determined in Laboratory 2. The values in Table 4 were used for the analysis. In vitro total allergenic
potency relative to the Japanese Society of Allergology (JSA) reference house dust miteextract was determined based on the results of the competition ELISA that measured
the inhibition of allergen-speciﬁc IgE binding to plates coated with the JSA reference
house dust mite extract (A) or Extract C (B). r: Pearson correlation coefﬁcient. *p < 0.01.

the HDM extract useful as the standard antigen for ELISAs thatmeasure the amounts of Der p 1 and Der f 1.
Acknowledgments
The authors thank CBER/FDA for supplying FDA reference HDM
 AS andextracts and FDA reference HDM-allergic serum; ALK-Abello
Stallergenes SA for supplying HDM allergen extracts; and Torii
Pharmaceutical Co., Ltd. and Shionogi & Co., Ltd. for their AS and Stallergenes SA,
communication with ALK-Abello
respectively.
This work was ﬁnancially supported by a Grant-in-Aid for Scientiﬁc Research from the Ministry of Heath, Labour, and Welfare ofJapan (Immunologic and Allergic Diseases Prevention and Treatment Research Enterprise, Immunology and Allergy Research Field,
H25-General-007) (Group Leader: Kimihiro Okubo) and the Japanese Society of Allergology.
Conﬂict of interest
YO received lecture fees from Torii, Shionogi, Kyowa Kirin, MSD, GlaxoSmithKline (GSK), and Kyorin, and research funding from the Japanese Rhinologic Society
and GSK. KO received lecture fees from Torii, GSK, MSD, Mitsubishi Tanabe, Ono, andKyowa Kirin, and research funding from GSK. MN received lecture fees from MSD
and AstraZeneca. KM received lecture fees from GSK, MSD, and Torii, and researchfunding from GSK.
Authors' contributions
TT: study design and interpretation of the in vitro study, and drafting andrevision of the manuscript; YO, KO, MN, and KM: study design, data acquisition,
analysis and interpretation of intradermal testing, and drafting and revision of themanuscript; MS, YF, and HY: study design, data acquisition, analysis and interpretation of the in vitro study, and drafting and revision of the manuscript; AS: dataacquisition and analysis of the in vitro study.

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VIROLOGICA SINICA 2015, 30 (3): 1-3
DOI 10.1007/s12250-015-3580-9

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Short Communication

Short Communication
to human influenza viruses in the sera, we performed a virus-neutralisation (VN) test (Itoh and others 2009) with a former seasonal H1N1
virus (A/Kawasaki/UTK4/09), seasonal H3N2 virus (A/Kawasaki/
UTK20/08), A(H1N1)pdm09 virus, and influenza B virus (B/Tokyo/
UT-E2/08), after receptor-destroying enzyme treatment and heatinactivation of the sera to remove non-specific inhibitors. The VN
antibody-positive sera (titres ≥10) were then further tested in an
immunoblot assay using the same virus antigens to confirm reaction
specificity.
In these assays, 14 sera were positive for VN antibody to A(H1N1)
pdm09 virus, representing 3.8 per cent positivity (Table 1). One serum
sample was also positive for VN antibody to the former seasonal H1N1
virus, but the reaction was weak, suggesting it may have been due tocross-reactivity. Eight other samples were seropositive to seasonal
H3N2 virus, representing 2.2 per cent positivity; however, these sera
did not react with dog H3N8 virus (A/canine/NE/52-14/06) or avian
H3N2 virus (A/duck/Mongolia/301/01), suggesting human-to-dog
transmission of the human virus. Notably, six sera were positive for
VN antibody to type B virus, representing 1.6 per cent positivity.
None of the sera was antibody-positive for both type A and B viruses.
Moreover, all samples were negative for VN antibody to highly pathogenic H5N1 avian virus (A/whooper swan/Mongolia/4/05; clade 2.2.).
None of the seropositive dogs showed any typical signs of acute respiratory illness according to the clinical records, suggesting that they
were asymptomatic or had very minor infections, as supported by the
low VN titres for the positive cases.
We conclude that dogs can be infected with human influenza
viruses, including type B virus. To our knowledge, there is only one
previous report of a possible natural influenza B virus infection of adog (Chang and others 1976), and even in this case, the infection was
not proven with established criteria. There has also been a report of
influenza C virus infection of dogs (Ohwada and others 1987). Thus,
domestic dogs may act as a vector for human influenza virus transmission within households, posing a potential public health concern.
Previous reports that dogs are susceptible to avian viruses, such as
H3N2 and H5N1, raise the possibility that dogs, like pigs, may act as
an intermediate host for the emergence of new, potentially pandemic
viruses. Continued surveillance of influenza viruses in dog populations will be important to achieve the “one health” concept for thiszoonotic disease.

Serological evidence
of infection of dogs
with human influenza
viruses in JapanT. Horimoto, F. Gen, S. Murakami,
K. Iwatsuki-Horimoto, K. Kato, H. Akashi,
M. Hisasue, M. Sakaguchi, Y. Kawaoka,
K. Maeda
HISTORICALLY, influenza virus has not been regarded as a major
pathogen of dogs. However, recent infections of racing and pet dogs
with H3N8 virus of equine origin in the USA after 2004 (Crawford
and others 2005) and retrospectively in the UK in 2002 (Daly andothers 2008), as well as with highly pathogenic H5N1 avian virus in
Thailand in 2004 (Songserm and others 2006), revealed that dogs are
susceptible to influenza A viruses. These infections caused respiratory
disease in the dogs and several proved fatal. Moreover, H3N2 virusof avian origin infected pet dogs in Korea in 2007 (Song and others2008) and China in 2010 (Li and others 2010), supporting the belief
that dogs should be included among the animals that are responsible for interspecies transmission of influenza A virus (Kim and others2013). Furthermore, there were reports in various parts of the world(Dundon and others 2010, Lin and others 2012) of dogs infected with
A(H1N1)pdm09 virus of swine origin, possibly due to contact withpeople infected with this H1N1 virus, suggesting human-to-dog transmission. Together, these reports suggest that dogs may play a role as
an intermediate host in which a mutant virus with pandemic potential could emerge. To partially address this possibility, we conducteda serological survey of human influenza virus infection in domestic
dogs in Japan.
A total of 366 serum samples were collected from mostly
indoor domestic dogs between January 2009 and February 2010 atanimal hospitals in the prefectures of Yamaguchi (162 samples) and
Kanagawa (204 samples), which are located in western and easternJapan, respectively. The dogs came to the hospitals with various
symptoms, and their serum samples were randomly collected for the
study with the pet owners’ permission. To detect antibodies specific

Veterinary Record (2013)
T. Horimoto, DVM, MSc, PhD,
F. Gen, MSc, PhD,
S. Murakami, MSc, PhD,
K. Kato, DVM, PhD,
H. Akashi, DVM, MSc, PhD,
Department of Veterinary Microbiology,
The University of Tokyo,
Tokyo 113-8657, JapanK. Iwatsuki-Horimoto, DVM, PhD,
Y. Kawaoka, DVM, MSc, PhD,
Division of Virology, Institute of Medical
Science, The University of Tokyo,
Tokyo 108-8639, Japan
M. Hisasue, DVM, PhD,
Department of Internal Medicine II,

Acknowledgements
We thank Sue Watson for editing the manuscript, Ryoko Yamao
for technical assistance, and veterinarians in Yamaguchi University
and Azabu University for collecting the samples. This work was
supported by grants-in-aid for Scientific Research (B), from the
Ministry of Education, Culture, Sports, Science, and Technology,
Japan.

Referencesdoi: 10.1136/vr.101929

CHANG, C. P., NEW, A. E., TAYLOR, J. F. & CHIANG, H. S. (1976) Influenza virus
isolations from dogs during a human epidemic in Taiwan. International Journal of Zoonoses
3, 61–64
CRAWFORD, P. C., DUBOVI, E. J., CASTLEMAN, W. L., STEPHENSON, I., GIBBS, E. P.,
CHEN, L., SMITH, C., HILL, R. C., FERRO, P., POMPEY, J., BRIGHT, R. A.,
MEDINA, M. J., JOHNSON, C. M., OLSEN, C. W., COX, N. J., KLIMOV, A. I.,
KATZ, J. M. & DONIS, R. O. (2005) Transmission of equine influenza virus to dogs.
Science 310, 482–485
DALY, J. M., BLUNDEN, A. S., MACRAE, S., MILLER, J., BOWMAN, S. J.,
KOLODZIEJEK, J., NOWOTNY, N. & SMITH, K. C. (2008) Transmission of influenza virus to English foxhounds. Emerging Infectious Diseases 14, 461–464
DUNDON, W. G., DE BENEDICTIS, P., VIALE, E. & CAPUA, I. (2010) Serologicalevidence of pandemic (H1N1) 2009 infection in dogs, Italy. Emerging Infectious Diseases
16, 2019–2021
ITOH, Y., SHINYA, K., KISO, M., WATANABE, T., SAKODA, Y., HATTA, M.,
MURAMOTO, Y., TAMURA, D., SAKAI-TAGAWA, Y., NODA, T., SAKABE,
S., IMAI, M., HATTA, Y., WATANABE, S., LI, C., YAMADA, S., FUJII,
K., MURAKAMI, S., IMAI, H., KAKUGAWA, S., ITO, M., TAKANO, R.,
IWATSUKI-HORIMOTO, K., SHIMOJIMA, M., HORIMOTO, T., GOTO, H.,
TAKAHASHI, K., MAKINO, A., ISHIGAKI, H., NAKAYAMA, M., OKAMATSU, M.,

M. Sakaguchi, DVM, MSc, PhD,
Department of Veterinary Microbiology I,
Azabu University, Kanagawa, JapanK. Maeda, DVM, PhD,
Department of Veterinary Microbiology,
Yamaguchi University, Yamaguchi,JapanE-mail for correspondence:
ahorimo@mail.ecc.u-tokyo.ac.jp
Provenance: not commissioned;
externally peer reviewed
Accepted November 24, 2013

January 25, 2014 | Veterinary Record

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Short Communication
TABLE 1: Dogs in the study that were seropositive to human influenza viruses
Prefecture

Number of samples Virus

Yamaguchi

162

Kanagawa

204

Number of positive
samples (%)
VN titres (number)A(H1N1)pdm09
H1N1
H3N2
B
A(H1N1)pdm09

4 (2.5)1 (0.6)
1 (0.6)4 (2.5)
10 (4.9)

10 (3), 20 (1*)
10 (1*)
40 (1)
10 (2), 20 (2)10 (4), 20 (5), 80 (1)

H1N1
H3N2
B

0 (0)
7 (3.4)2 (1.0)–10 (4), 40 (3)10 (1), 20 (1)Species (number)
Miniature dachshund (1), Chihuahua (1), toy poodle (1*), shih tzu (1)Toy poodle (1*)
Miniature dachshund (1)Mongrel (2), toy poodle (1), miniature dachshund (1)Miniature dachshund (3), papillon (2), toy poodle (1), mongrel (1), shiba (1),springer spaniel (1), long coat Chihuahua (1)–Miniature dachshund (3), toy poodle (2), Welsh corgi (1), Yorkshire terrier (1)Miniature dachshund (1), Maltese (1)*The same dog
VN, virus-neutralisation

OHWADA, K., KITAME, F., SUGAWARA, K., NISHIMURA, H., HONMA, M. &NAKAMURA, K. (1987) Distribution of the antibody to influenza C virus in dogs andpigs in Yamagata Prefecture, Japan. Microbiology & Immunology 31, 1173–1180
SONG, D., KANG, B., LEE, C., JUNG, K., HA, G., KANG, D., PARK, S., PARK, B., &OH, J. (2008) Transmission of avian influenza virus (H3N2) to dogs. Emerging Infectious
Diseases 14, 741–746
SONGSERM, T., AMONSIN, A., JAM-ON, R., SAE-HENG, N., PRIYOTHORN, N.,
PAYUNGPORN, S., THEAMBOONLERS, A., CHUTINIMITKUL, S.,
THANAWONGNUWECH, R., & POOYORAWAN, Y. (2006) Fatal avian influenza
A H5N1 in a dog. Emerging Infectious Diseases 12, 1744–1747

TAKAHASHI, K., WARSHAUER, D., SHULT, P. A., SAITO, R., SUZUKI, H.,
FURUTA, Y., YAMASHITA, M., MITAMURA, K., NAKANO, K., NAKAMURA,
M., BROCKMAN-SCHNEIDER, R., MITAMURA, H., YAMAZAKI, M.,
SUGAYA, N., SURESH, M., OZAWA, M., NEUMANN, G., GEM, J., KIDA, H.,
OGASAWARA, K., & KAWAOKA, Y. (2009) In vitro and in vivo characterization of
new swine-origin H1N1 influenza viruses. Nature 460, 1021–1025
KIM, H., SONG, D., MOON, H., YEOM, M., PARK, S., HONG, M., NA, W.,
WEBBY, R. J., WEBSTER, R. G., PARK, B., KIM, J.-K., & KANG, B. (2013) Interand intraspecies transmission of canine influenza virus (H3N2) in dogs, cats, and ferrets.
Influenza and Other Respiratory Viruses 7, 265–270
LI, S., SHI, Z., JIAO, P., ZHANG, G., ZHONG, Z., TIAN, W., LONG, L. P., CAI, Z.,
ZHU, X., LIAO, M., & WAN, X. F. (2010) Avian-origin H3N2 canine influenza A
viruses in Southern China. Infection Genetics and Evolution 10, 1286–1288
LIN, D., SUN, S., DU, L., MA, J., FAN, L., PU, J., SUN, Y., ZHAO, J., SUN, H., &LIU, J. (2012) Natural and experimental infection of dogs with pandemic H1N1/2009
influenza virus. Journal of General Virology 93, 119–123

Veterinary Record | January 25, 201487

J Clin Immunol (2013) 33:977–983
DOI 10.1007/s10875-013-9880-7

ORIGINAL RESEARCH

Analysis of Conformational and Sequential IgE Epitopes on the Major
Allergen Cry j 2 of Japanese Cedar (Cryptomeria japonica) Pollen
in Humans by Using Monoclonal Antibodies for Cry j 2
Kazuki Miyaji & Terumi Yurimoto & Akemi Saito &Hiroshi Yasueda & Yukari Takase & Hidekatsu Shimakura &Noriaki Okamoto & Akio Kiuchi & Saburo Saito &Masahiro Sakaguchi

Received: 18 November 2012 / Accepted: 25 February 2013 / Published online: 16 March 2013
# Springer Science+Business Media New York 2013pollinosis, and allergenic epitopes for mAbs and human IgE
were identified using ELISA with synthetic peptides. The
importance of the epitopes for human IgE was analyzed
using an inhibition ELISA.
Results Four independent epitopes (epitope #1, #2, #3, and #4)
were identified on Cry j 2 with the use of mAbs. Epitope #3
and #4, corresponding to peptides No. 25 and No. 33, respectively, were newly determined as epitopes for mAbs and human
IgE. Inhibition ELISA showed that not only epitope #2(sequential) but epitope #1 (conformational) may play an important role in the CJ pollinosis.
Conclusions Our results revealed 4 epitopes, including two
new ones, on Cry j 2. We also found that inhibition ELISA
with appropriate mAbs could be a viable method of evaluating the importance of the conformational and sequential
epitopes for human IgE. These results are beneficial for thedevelopment of safer and more efficient therapeutic strategies for treating CJ pollinosis.

Abstract
Purpose Japanese cedar (Cryptomeria japonica; CJ) pollinosis is a type I allergy induced by CJ pollen, and Cry j 2 is oneof the major allergens in this pollen. In a previous study, we
analyzed IgE epitopes on Cry j 2 in humans by using synthetic
peptides. The main purpose of this study was to identify B-cell
epitopes on Cry j 2 in patients with CJ pollinosis by using
monoclonal antibodies (mAbs) for Cry j 2.
Methods We used ELISA with mAbs for the epitope analysis. Sera samples were collected from 80 patients with CJ

Electronic supplementary material The online version of this article(doi:10.1007/s10875-013-9880-7) contains supplementary material,
which is available to authorized users.
K. Miyaji : T. Yurimoto : Y. Takase : H. Shimakura :N. Okamoto : A. Kiuchi : M. Sakaguchi (*)
Department of Veterinary Microbiology,
School of Veterinary Medicine, Azabu University,
1-17-71 Fuchinobe, Chuo-ku, Sagamihara,
Kanagawa 252-5201, Japane-mail: sakagum@azabu-u.ac.jp

Keywords Japanese cedar pollen . pollinosis . Cry j 2 .
IgE . epitope

A. Saito : H. Yasueda
Clinical Research Center for Allergy
and Rheumatology, National Sagamihara Hospital,
18-1 Sakuradai, Minami-ku, Sagamihara,
Kanagawa 252-0392, Japan

Introduction
Pollinosis is a typical type I allergy induced by pollens, andis defined as the appearance of respiratory symptoms suchas rhinoconjunctivitis. Japanese cedar (Cryptomeria japonica; CJ) pollinosis is one of the most common allergic
diseases in Japan [1]. In an epidemiological study conducted

S. Saito
Department of Molecular Immunology,
Institute of DNA Medicine, Jikei University
School of Medicine, 3-25-8 Nishi-Shinbashi,
Minato-ku, Tokyo 105-8461, Japan

88

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J Clin Immunol (2013) 33:977–983

ELISA Cross-Inhibition for Epitopes of Cry j 2 Recognized
by mAbsin the central Hokuriku area of Japan in May and June ofboth 2006 and 2007 [2], 36.7 % of the study participants(566 of the 1,540 subjects) had allergic rhinitis to CJ pollen.
Impaired performance and a voluntary ban on leaving home
for patients with CJ pollinosis comprise the tremendous
social impact and economic loss associated with this disease. Therefore, CJ pollinosis is one of the most serious
public health problems in Japan.
Yasueda et al. [3] purified Cry j 1 as a major allergen fromCJ pollen, and we isolated Cry j 2 as another major allergen [4].
Of 145 patients with CJ pollinosis tested for specific IgE levels,
134 (92 %) had specific IgE antibodies to both Cry j 1 and Cry j
2 [5]. Cry j 2 is completely different from Cry j 1 in terms of its
chemical and immunophysical properties, N-terminal aminoacid sequence, and its molecular mass of 37 kDa under nonreducing conditions [4]. Furthermore, we also recently isolated
Cry j 3 as a causative allergen of CJ pollinosis [6].
The identification and characterization of allergenic epitopes
are important for establishing safer and more efficient immunotherapy protocols [7, 8]. Previously, we used monoclonal
antibodies (mAbs) to characterize the allergenic epitopes of
Cry j 1 in CJ pollens, and identified five independent epitopes
on Cry j 1 [9]. In our preliminary study [10], in which epitopes
on Cry j 2 were analyzed using a synthetic peptide of Cry j 2,
we found that 124KWVNGREI131 is an important major linear
epitope. However, we had not classified IgE epitopes on Cry j 2
with mAbs for the allergen, and the significance of a previously
identified epitopes remains to be evaluated.
In the present study, we identified four independent epitopes on Cry j 2 by using mAbs for Cry j 2; two are new
epitopes. Analysis of a large number of sera samples
obtained from patients with CJ pollinosis has confirmed thatthey are also new epitopes for human IgE. We additionallypredicted whether amino acids that made synthetic peptides
recognizable by mAbs and human IgE could be exposed on
Cry j 2 by means of bioinformatics tools. Finally, fluorometric and inhibition ELISA assays were used to evaluate
the importance of conformational and sequential epitopes
recognized by mAbs and human IgE.

Epitopes recognized by the mAbs were grouped using the
ELISA cross-inhibition test. This test is based on the abilityof a mAb to inhibit the binding of a coexisting biotin-labeled
mAb to solid-phase Cry j 2. Briefly, Cry j 2 (0.5 μg/mL) was
adsorbed onto a microplate (F96 Maxisorp® NUNCImmuno™ Plate, ThermoFisher Scientific, Waltham, MA,
USA). Biotin-labeled mAbs (0.5 μg/mL) were then reactedwith the solid-phase antigen in the presence or absence of
unlabeled mAbs (5 μg/mL) for 1 h at room temperature. Next,
a streptavidin-peroxidase polymer (streptavidin-HRP, SigmaAldrich, St. Louis, MO, USA) was added to the wells. After a
1-h incubation period at room temperature, a substrate solution of o-phenylenediamine dihydrochloride was added. Afterthe enzyme reaction was quenched with 2 M H2SO4, theoptical density (OD) was measured using a multi-mode
microplate reader (Powerscan MX, DS Pharma Biomedical,
Osaka, Japan).
Patient Sera Samples
Informed consent was obtained from all subjects. The study
protocol was approved by the ethics committee at the Jikei
University School of Medicine. Sera samples from 80 patients with CJ pollinosis. The patents were selected on thebases of clinical symptoms of seasonal allergic rhinitis andpositive CAP (Phadia AB, Uppsala, Sweden) to CJ pollen.
For the negative control, sera samples were obtained from
25 non-allergic subjects who had previously been confirmed
to be negative for the CJ pollen allergen.
Colorimetric ELISA for Overlapping Synthetic Peptides
with mAbs
As previously described, the reaction of mAbs to Cry j 2
peptides was measured using a colorimetric ELISA [10].
Briefly, Cry j 2 protein (1 μg/mL) or synthetic peptides(10 μg/mL) were immobilized in the wells of a microplate(F96 MaxiSorp® NUNC-Immuno™ Plate) overnight at 4 °C.
The microplate was then washed with phosphate-bufferedsaline containing 0.05 % Tween 20 (PBST buffer) and incubated with biotin-labeled mAbs (0.1 μg/mL) for 1 h at room
temperature. The subsequent procedures were the same as
those for the cross-inhibition ELISA described above.

Methods
The Cry j 2 Antigen and Monoclonal Antibodies
Cry j 2 was purified as previously described [4]. A library of
peptides, consisting of 18, 20, 21, or 25 amino acids overlappingby 10 to 12, was commercially synthesized (Hokkaido System
Science Co., Ltd., Sapporo, Japan) [10, 11]. The eight mAbs
(S1, S2, S3, N26, T27, 24, 9E7, and J2A01) against Cry j 2 usedin our laboratory were categorized according to cross-inhibition
ELISA. These mAbs were kindly provided or generated byimmunizing mice with Cry j 2 [10, 12–14].

Fluorometric ELISA for Overlapping Synthetic Peptides
with Human IgE in Patient Sera
As previously described, specific IgE in the sera of patientswith pollinosis for Cry j 2 peptides was measured using
fluorometric ELISA [5]. Briefly, Cry j 2 protein (1 μg/mL)

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J Clin Immunol (2013) 33:977–983

979

or synthetic peptides (10 μg/mL) were immobilized in thewells of a microplate (FLUOTRAC™ 600, Greiner Bio-One
GmbH, Frickenhausen, Germany) overnight at 4 °C. The
microplate was then washed with PBST buffer and finally
incubated with diluted (1:10) sera samples for 3 h at room
temperature. The plates were washed, and anti-human IgE
antibodies conjugated to β-D-galactosidase (diluted 1:10;
Phadia AB) were added to each well. As the enzyme reaction
substrate, 0.2 mM 4-methylumbelliferyl-β-D-galactoside(Sigma-Aldrich) was added to the wells, and the plates wereincubated at 37 °C for 2 h. After quenching the reaction,
fluorescence units (FU) were measured on a multi-mode
microplate reader. Cutoff values were determined using serafrom pollinosis-negative patients as controls.

present study, the epitope on the Cry j 2 molecule related tomAbs S1, S2, and S3 was defined as epitope #1, mAbs N26,
T27, and 24 as epitope #2, mAb 9E7 as epitope #3, andmAb J2A01 as epitope #4 (Table I).
We examined the binding of these mAbs to synthetic
peptides of Cry j 2. Our observations were similar to thoseof a previous report [10]; we reconfirmed that mouse Cry j
2-specific mAb S1 did not react with any of the peptides,
and mAbs N26 and 24 reacted with peptide No. 13(121GQCKWVNGREICNDRDRPTA140). We found thatthe mAbs 9E7 and J2A01 reacted with peptides No. 25( 2 41 GRENSRAEVSYVHVNGAKFI 2 6 0 ) and No. 33(321TYKNIRGTSATAAAIQLKCS340), respectively. It wasdetermined that epitope # 2 is relevant to peptide No. 13,
epitope #3 to peptide No. 25, and epitope #4 to peptide No.
33. We found that epitope #3 and epitope #4 were new
epitopes recognized by mAbs.

Inhibition ELISA for the Conformational and Sequential
Epitopes with the Use of mAbs

Binding of IgE in Sera of Human Patients to Synthetic
Peptides of Cry j 2

To evaluate the importance of the conformational and sequential epitopes, inhibition ELISA was conducted using
mAbs as inhibitors [9]. Briefly, Cry j 2 protein (1 μg/mL)
was immobilized in the wells of a microplate overnight at
4 °C. Non-labeled mAbs S1 or N26 (20 μg/mL) werereacted with the immobilized protein for 1 h at room temperature. Plates were then washed with PBST buffer andsubsequently incubated with diluted (1:10) sera samples for
3 h at room temperature. Sera samples from 50 patients wereused for conducting the assay because of their sufficientvolume (>100 μL). Subsequent procedures were the sameas those of the fluorometric ELISA described above. The
inhibition ratio (%) was calculated as follows:

Figure 1 shows the reactivity of human IgE obtained from80 patients with anti-Cry j 2 IgE to synthetic peptides. The
cutoff values for Cry j 2 and peptides were obtained in the
maximal FU values of 25 negative controls, because thevalues were higher than mean value+3SD. We reconfirmedthat IgE from 61 patients (76 %) reacted with peptide No. 13(relevant to epitope #2). We also found that IgE from 11(14 %) patients reacted with peptide No. 25 (relevant toepitope #3), and IgE from 19 (24 %) patients reacted withpeptide No. 33 (relevant to epitope #4). In our previous
study, none of the samples obtained from 20 patients reactedwith these peptides [10].

FU in presence of inhibitor
1 100
FU in absence of inhibitor

Exposed Amino Acids of Sequential Epitopes on Cry j 2
The solvent-accessible amino acids of epitopes epitope#2, epitope #3, and epitope #4 were predicted using
NetSurfP (Table II). Core amino acids for epitope #2(124KWVNGREI131), which were identified using small
peptides of peptide No. 13 in the previous study [10], allseemed to be exposed on the Cry j 2 surface. The exposed
regions of epitope #3 were predicted to be 242R243E244N
245 246 247 248 250 256 258 260
S R A E S G K I, and those of epitope #4
to be 323Y324N326R328T330A331T332A. All computed results areshown in the Supplementary Table.

Bioinformatics Tool
The surface exposure of the Cry j 2 amino acids was predicted using NetSurfP (http://www.cbs.dtu.dk/services/
NetSurfP/) [15, 16].

Results
Epitope Specificity of Anti-Cry j 2 mAbs and Their Binding
to Synthetic Peptides of Cry j 2

Evaluating the Significance of the Epitopes, Epitope #1 and #2,
for Human IgEFour independent epitopes were identified on the Cry j 2
molecule by using the ELISA cross-inhibition test with
mAbs. An inhibition reaction was not observed with a
non-labeled mAb that recognized different epitopes. In the

We determined the inhibition rates of mAbs S1 (for epitope #1)
and N26 (for epitope #2) against human IgE for comparison of
their importance. Table III indicated FU value of human IgE to

90

980
Table I Classification of antiCry j 2 mAbs by inhibition
ELISA

J Clin Immunol (2013) 33:977–983

Unlabeled mAba

Biotin-labeled mAb toepitope #1

aInhibitor. +, >30 % inhibition.
Experiments were repeated atleast three times.

epitope #3

epitope #4

S1

S2

S3

N26

T27

24

9E7

J2A01

S1

+

+

+

−

−

−

−

−

S2

+

+

+

−

−

−

−

−

S3

+

+

+

−

−

−

−

−

N26

−

−

−

+

+

+

−

−

T27
24

−
−

−
−

−
−

+
+

+
+

+
+

−
−

−
−

9E7

−

−

−

−

−

−

+

−

J2A01

−

−

−

−

−

−

−

+

peptide No. 13 (relevant to epitope #2), and inhibition ratio ofmAbs to epitope # 1 and #2 against human IgE for each subject.
We found that their patterns were substantially different. Of the
10 sera samples that showed >50 % inhibition rates for mAb toepitope #1, eight sera samples had negative FU value for IgE toepitope #2 relative peptide.

amino acids play an important role in the binding between
antibody and epitope [18], and pentapeptides have beenreported as IgE epitopes in wheat gliadin [19]. All core aminoacids of epitope #2 determined in our previous study [10] werepredicted to be exposed, indicating that 124KWVNGREI131
was a completely sequential epitope. Epitope #3 was predicted
to contain seven consecutive amino acids, 242RENSRAE248,
exposed on Cry j 2, suggesting that these amino acids may
contribute as the epitope. Meanwhile, epitope #4 was notexpected to contain three or more consecutive amino acids.
Our result raises the possibility that the core determinants may
result from discontinuous amino acids within epitope #4.
Further study is needed to identify the core determinants of
those epitopes.
The mAb S1 did not react with any of the synthetic
peptides. This result coincides with that from our previous

Discussion

FU [log10]

The bioinformatics tool NetSurfP can predict solventaccessible regions on proteins. Using this tool, we predicted
exposed amino acids in Cry j 2 epitopes (epitope #2, #3,
and #4). A review article relevant to food allergies discussed
that the majority of IgE epitopes are eight amino acids orlonger [17]. However, it has been suggested that five to six

Fig. 1 Binding of human IgE
to Cry j 2 and synthetic
peptides. Related epitope
numbers are indicated withinparentheses below to thesynthetic peptide numbers.
Binding activity is expressed influorescence units (FU). The
dashed line represents cutoff
values, which were determinedas highest FU in negative
control. The cutoff values are
233.5, 156.0, and 185.5 fromthe left column. Sera samples
from 50 patients were used toprovide adequate amounts, andexperiments were repeated atleast three times

epitope #2

10

5

10

4

10

3

10

2

Pollinosis
Allergen+

Cry j 2

91+-

No. 13(epitope #2)+-

No. 25(epitope #3)+-

No. 33(epitope #4)

J Clin Immunol (2013) 33:977–983

981

Table II Prediction of solvent-accessible regions of Cry j 2 aminoacids using NetSurfP
EpitopePosition

Amino acids

epitope #2a

124–131

epitope #3

241–260

epitope #4

321–340KWVNGREI********GRENSRAEVSYVHVNGAKFI-*******-*-----*-*-*TYKNIRGTSATAAAIQLKCS--**-*-*--**--------

apeptide No. 13 (relevant to epitope #2), the importance ofa conformational epitope on Cry j 2 for human IgE had notbeen previously reported. Therefore, the responsiveness ofhuman IgE to epitope #1 (a conformational epitope) andepitope #2 (a sequential epitope) was compared by inhibition ELISA. The reaction of mAb with the epitope #1
inhibited the binding of human IgE to Cry j 2 in a different
manner in the reaction of mAb with epitope #2. Eighty
percent of the tested sera samples showing >50 % inhibition
rates for mAb to epitope #1 were negative for epitope #2.
Conformational epitopes on allergens are considered to play
an important role in initiating human IgE-mediated allergic
reactions [21–23]. Our results suggested that not only conformational but also sequential epitope may play an important role in the CJ pollinosis. Variations in responsiveness to
Cry j 2 epitopes could have been influenced by unknown
factors such as an individual’s genetic background and/or
clinical history. It may be worthwhile to analyze IgEbinding epitopes to understand the etiology of this allergy
and develop safer and more efficient therapeutic strategies
for treating CJ pollinosis.

Core sequence for epitope #2 was identified by Tamura et al. [10]Surface exposure of Cry j 2 amino acids was predicted by NetSurfP,and each amino acid was assigned to “Exposed (*)” or “Buried (-)”.
See also supplementary table

study, in which the mAb S1 also did not bind to any
synthetic peptides [10]. Along with decreasing reaction of
S1 to heat treated Cry j 2 [20], these findings indicate that
epitope #1 could be a conformational epitope. Although it
has been shown that a number of patient IgE reacted toTable III Inhibition rates ofmAbs binding to epitope #1 and#2 for evaluating the importanceof the conformational epitope(epitope #1) on Cry j 2 forhuman IgE

FU values of human IgE were
calculated based on the activity
to epitope #2 relative peptide
No. 13, and the cutoff value is233.5 (see also Fig. 1). The epitope #2 was chosen as a target
for comparison, since the affinityof human IgE to peptide No. 13
was higher than to other peptides. Sera samples from 50 patients were used to have enoughvolume (>100 μL) to conduct
inhibition ELISA. Inhibition rateover 50 % is shown in bold. The
experiments were repeated atleast three times.

Inhibition rate (%)
Subject

Inhibition rate (%)

FU valueepitope #1

epitope #2

Subject

FU value

1
2

0
0

27.1
46.3

0.0
21.2

26
27

3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

0
0
1
7
8
30
47
52
56
80
93
135
219
226
230
235

50.5
57.2
34.5
50.1
58.0
46.5
36.6
63.4
34.6
61.0
41.7
50.6
26.4
50.1
44.0
54.7

40.5
8.5
16.5
39.8
41.6
30.8
28.4
27.4
16.9
28.4
25.6
25.6
19.7
44.7
26.9
36.3

19
20
21

464
576
670

31.5
37.3
17.9

22
23
24
25

676
815
849
983

47.6
43.2
20.4
45.0

92

epitope #1

epitope #2

1038
1214

47.0
32.4

50.6
80.7

28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43

1229
1317
1719
1885
2061
2071
2207
2257
2303
2322
2371
2670
2953
3306
3857
4214

23.3
29.0
20.0
42.3
21.1
31.2
25.9
35.7
23.1
62.3
19.3
3.2
12.4
34.5
37.0
19.6

71.0
50.9
20.9
59.0
39.4
72.6
34.8
54.4
48.5
44.8
54.4
44.2
72.7
79.2
67.4
75.2

68.2
50.3
35.8

44
45
46

6490
6686
7944

29.1
22.7
9.4

54.5
59.6
57.1

28.9
41.5
42.5
44.6

47
48
49
50

9159
11372
13088
26236

24.6
12.2
6.4
7.5

71.7
65.6
35.7
22.2

982

J Clin Immunol (2013) 33:977–983

A previous study reported that no patients’ sera included
reactive IgE to the synthetic peptides No. 25 and No. 33[10]. Our present results show that these peptides wererecognized by the mAbs and human IgE (Fig. 1), suggestingthat the determinants are newly detected epitopes. The lackof detection in our previous analysis is probably because
those sites are minor epitopes and were thus incidentally notrecognized in the small sample size. In fact, we tested 80
patients with CJ pollinosis, and only 11 (14 %) patients for
peptide No. 25 (relevant to epitope #3) and 19 (24 %) patientsfor peptide No. 33 (relevant to epitope #4) were detected as
being epitope-specific IgE-positive at low FU compared to
peptide No. 13 (relevant to epitope #2). Moreover, differences
between the methodologies used in previous studies and ourstudy, especially with regard to the detection procedures, may
have contributed to the fact that those epitopes were notdetected by the other investigators.
Many studies have analyzed IgE epitopes of group 1 conifer
pollen allergens, such as Cry j 1 and Jun a 1 [9, 21, 24–26].
Using mAbs for Cry j 1, a major allergen of the CJ pollen, we
identified five independent epitopes on Cry j 1 in human patients[9]. Major mountain cedar allergen, Jun a 1, contains conformational and sequential IgE epitopes [26]. Other group 2 conifer
pollen allergens, such as Jun a 2 and Cha o 2 from cypress
pollen, have also been identified [27–29]. However, few studies
have analyzed IgE epitopes in group 2 conifer pollen allergens[10]. In our previous study, we identified important sequential
IgE epitopes of Cry j 2 by using synthetic peptides on thisallergen [10]. In the present study, we have identified two new
IgE epitopes by using mAbs for Cry j 2. These studies
concerning IgE epitopes on Cry j 2 will help in developing
new allergen vaccines against cedar and cypress pollinosis [30].

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RM, Yasueda H, et al. Isolation and characterization of native
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Watanabe M, et al. Epitope specificity of IgE antibodies to a major
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10. Tamura Y, Kawaguchi J, Serizawa N, Hirahara K, Shiraishi A, Nigi
H, et al. Analysis of sequential immunoglobulin E-binding epitopeof Japanese cedar pollen allergen (Cry j 2) in humans, monkeys
and mice. Clin Exp Allergy. 2003;33:211–7.
11. Namba M, Kurose M, Torigoe K, Hino K, Taniguchi Y, Fukuda S,
et al. Molecular cloning of the second major allergen, Cry j II, fromJapanese cedar pollen. FEBS Lett. 1994;353:124–8.
12. Taniai M, Kayano T, Takakura R, Yamamoto S, Usui M, Ando S,
et al. Epitopes on Cry j I and Cry j II for the human IgE antibodies
cross-reactive between Cupressus sempervirens and Cryptomeria
japonica pollen. Mol Immunol. 1993;30:183–9.
13. Homma R, Uesato N, Miyahara A, Kimura H, Hantani Y, Ito G, etal. Production of a monoclonal antibody against Cry j 2. Arerugi.
1995;44:461–6 (in Japanese with English abstract).
14. Yasueda H, Saito A, Sakaguchi M, Ide T, Saito S, Tniguchi Y, et al.
Identification and characterization of a group 2 conifer pollen allergenfrom Chamaecyparis obtusa, a homologue of Cry j 2 from Cryptomeria japonica. Clin Exp Allergy. 2000;30:546–50.
15. Petersen B, Petersen TN, Andersen P, Nielsen M, Lundegaard C. A
generic method for assignment of reliability scores applied tosolvent accessibility predictions. BMC Struct Biol. 2009;9:51.
16. Ingale AG. Antigenic epitopes prediction and MHC binder of aparalytic insecticidal toxin (ITX-1) of Tegenaria agrestis (hobospider). Open Access Bioinforma. 2010;2:97–103.
17. Bannon GA, Ogawa T. Evaluation of available IgE-binding epitope dataand its utility in bioinformatics. Mol Nutr Food Res. 2006;50:638–44.
18. Laver WG, Air GM, Webster RG, Smith-Gill SJ. Epitopes on protein
antigens: misconceptions and realities. Cell. 1990;61:553–6.
19. Tanabe S. IgE-binding abilities of pentapeptides, QQPFP and
PQQPF, in wheat gliadin. J Nutr Sci Vitaminol. 2004;50:367–70.
20. Sawatani M, Yasueda H, Akiyama K, Shida T, Taniguchi Y, Usui M,
et al. Immunological and physicochemical properties of Cry j II, theConclusion
Four epitopes, including two new epitopes, were identified on
Cry j 2 by using mAbs, and IgE from patients with pollinosis
also reacted to the epitopes. The bioinformatics tool NetSurfP
could predict the candidate amino acids that accounted for thecore determinants in one of the two new epitopes. Our study
also demonstrated that inhibition ELISA using mAbs is a
viable method to evaluate the importance of the conformational and sequential epitopes for human IgE. These results areuseful for the development of safer and more efficient therapeutic strategies for treating CJ pollinosis.

Acknowledgments We are grateful to Dr. Reiko Homma (Torii Pharmaceutical Co., Ltd., Tokyo, Japan) for kindly giving us monoclonal
antibodies. This research was partially supported by the Promotion and
Mutual Aid Corporation for Private Schools of Japan, Grant-in-Aid forMatching Fund Subsidy for Private University, and by a Project Grant
awarded by the Azabu University Research Services Division.

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EW, Togawa A, et al. Structural basis for epitope sharing between group 1 allergens of cedar pollen. Mol Immunol.
2006;43:509–18.
27. Mori T, Yokoyama M, Komiyama N, Okano M, Kino K. Purification, identification, and cDNA cloning of Cha o 2, the second
major allergen of Japanese cypress pollen. Biochem Biophys Res
Commun. 1999;263:166–71.
28. Yasueda H, Saito A, Sakaguchi M, Ide T, Saito S, Taniguchi Y, etal. Identification and characterization of a group 2 conifer pollen
allergen from Chamaecyparis obtusa, a homologue of Cry j 2 from
Cryptomeria japonica. Clin Exp Allergy. 2000;30:546–50.
29. Yokoyama M, Miyahara M, Shimizu K, Kino K, Tsunoo H.
Purification, identification, and cDNA cloning of Jun a 2, thesecond major allergen of mountain cedar pollen. Biochem Biophys
Res Commun. 2000;275:195–202.
30. Sakaguchi M, Hirahara K, Fujimura T, Toda M. Approaches toimmunotherapies for Japanese cedar pollinosis. Auris Nasus Larynx.
2011;38:431–8.

second major allergen of Japanese cedar pollen (Cryptomeria japonica). Arerugi. 1993;42:738–47 (in Japanese with English abstract).
21. Varshney S, Goldblum RM, Kearney C, Watanabe M, Midoro-Horiuti
T. Major mountain cedar allergen, Jun a 1, contains conformational aswell as linear IgE epitopes. Mol Immunol. 2007;44:2781–5.
22. Padavattan S, Flicker S, Schirmer T, Madritsch C, Randow S,
Reese G, et al. High-affinity IgE recognition of a conformational
epitope of the major respiratory allergen Phl p 2 as revealed by X-ray
crystallography. J Immunol. 2009;182:2141–51.
23. Tiwari R, Negi S, Braun B. Validation of a phage display andcomputational algorithm by mapping a conformational epitope ofBla g 2. Int Arch Allergy Immunol. 2011;157:323–30.
24. Tamura Y, Sasaki R, Inouye S, Kawaguchi J, Serizawa N, Toda M,
et al. Identification of a sequential B-cell epitope on major allergen(Cry j 1) of Japanese cedar (Cryptomeria japonica) pollen in mice.
Int Arch Allergy Immunol. 2000;123:228–35.
25. Midoro-Horiuti T, Mathura V, Schein CH, Braun W, Yu S, Watanabe M,
et al. Major linear IgE epitopes of mountain cedar pollen allergen Jun a 1
map to the pectate lyase catalytic site. Mol Immunol. 2003;40:555–62.

94Veterinary Immunology and Immunopathology 149 (2012) 132–135

Contents lists available at SciVerse ScienceDirect

Veterinary Immunology and Immunopathology
journal homepage: www.elsevier.com/locate/vetimm

Short communication

IgE reactivity to a Cry j 3, an allergen of Japanese cedar (Cryptomeria
japonica) pollen in dogs with canine atopic dermatitis
Shota Kubota a , Kazuki Miyaji a , Yusaku Shimo a , Hidekatsu Shimakura a , Yukari Takase a ,Noriaki Okamoto a , Akio Kiuchi a , Masato Fujimura b , Takashi Fujimura c ,Douglas J. DeBoer d , Toshihiro Tsukui e , Masahiro Sakaguchi a,∗a
Department of Veterinary Microbiology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201,
Japanb
Fujimura Animal Hospital, 5-10-26 Aomatanihigashi, Minou, Osaka 652-0022, Japanc
Research Center for Allergy and Immunology, RIEKN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japand
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USAe
Nippon Zenyaku Kogyo Co., Ltd, 1-1 Tairanoue Sasagawa, Asaka-machi, Koriyama, Fukushima 963-0196, Japana r t i c l e

i n f oArticle history:
Received 17 April 2012
Received in revised form 24 May 2012
Accepted 6 June 2012
Keywords:
AllergenCanine atopic dermatitis
IgE
Japanese cedar pollena b s t r a c t
The present study investigated IgE reactivity to a new Cryptomeria japonica pollen allergen(Cry j 3) in dogs with atopic dermatitis by using a ﬂuorometric ELISA. Serum samples from15 dogs that showed IgE sensitivity to crude C. japonica pollen allergen by ELISA were tested
for speciﬁc IgE to each allergen, individually. All 15 dogs had anti-Cry j 1 IgE, 6 (40%) had
anti-Cry j 2 IgE, and 11 (73%) had anti-Cry j 3 IgE. Further, we found that these anti-Cry j
3 IgE reacted to Cry j 3 with immunoblotting analysis. These ﬁndings indicate that Cry j 3
may be a major allergen in dogs.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction

C. japonica pollen allergen (Masuda et al., 2000a). Theseﬁndings suggested that C. japonica pollen allergen might
be associated with the pathogenesis of CAD.
Two major allergens have been previously isolated fromC. japonica pollen and characterized as Cry j 1 (Yasueda
et al., 1983) and Cry j 2 (Sakaguchi et al., 1990). Cry j 1
was identiﬁed as a 41−46 kDa allergen with pectate lyase
enzyme activity (Yasueda et al., 1983; Taniguchi et al.,1995), while Cry j 2 was identiﬁed as a 45 kDa allergen withpolymethylgalacturonase enzyme activity (Sakaguchi et al.,
1990; Ohtsuki et al., 1995). We previously showed thathumans (Sakaguchi et al., 1990; Hashimoto et al., 1995),
Japanese monkeys (Macaca fuscata) (Sakaguchi et al., 1992;
Hashimoto et al., 1994), and dogs (Masuda et al., 2000b) had
speciﬁc IgE to Cry j 1 and Cry j 2.
Of the 100 human patients with C. japonica pollinosis, 27
serum samples contained IgE against the recently isolated
Cry j 3, suggesting that Cry j 3 is an important allergen in some patients (Fujimura et al., 2007). Further, weJapanese cedar (Cryptomeria japonica) pollinosis is
one of the most common immediate-type allergic diseases in Japan (Yasueda et al., 1983), which has been
also reported in dogs (Sasaki et al., 1995) as well as
in humans. The dogs sensitive to C. japonica pollen
showed clinical symptoms of canine atopic dermatitis(CAD) and positive immune reactions to the pollen allergenin Prausnitz–Küstner test (Rupa et al., 2008). CAD is a common dermatitis in dogs and deﬁned as an inﬂammatory
and pruritic skin disease with IgE antibodies, most commonly directed against environmental allergens (Halliwell,2006). Our epidemiological study in Japan reported thatapproximately 20% of atopic dogs had speciﬁc IgE to crude∗ Corresponding author. Tel.: +81 42 850 2481; fax: +81 42 850 2481.
E-mail address: sakagum@azabu-u.ac.jp (M. Sakaguchi).
0165-2427/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetimm.2012.06.007

95

S. Kubota et al. / Veterinary Immunology and Immunopathology 149 (2012) 132–135

found that Cry j 3 is 27 kDa protein and has relatively highhomology with thaumatin-like protein categorized in thepathogenesis-related-5 (PR-5) family protein which may
act as causative allergens of oral allergy syndrome (OAS)in human patients (Fujimura et al., 2007). OAS is a condition characterized by IgE-mediated immediate allergic
symptoms restricted to the oral mucosa, which may involve
itching, and vascular edema of the lips, tongue, and pharynx
with a sudden onset (Kondo and Urisu, 2009). OAS symptoms occur in association with IgE cross-reactivity arisingfrom structural homologies between pollen allergens andfood proteins. A typical manifestation of OAS would be theoral mucosal symptoms that appear when a patient withbirch pollen allergy has eaten some foods associated withthe family Rosaceae such as an apple, cherry, or peach. However, to date, there have been no studies to investigate theallergic responses to Cry j 3 in dogs.
In the present study, we investigated IgE reactivity to
Cry j 3 in atopic dogs sensitive to crude C. japonica pollen
allergen by a ﬂuorometric ELISA.

133

anti-mouse IgG1 (Zymed Laboratories, San Francisco, CA,
USA) at room temperature for 1 h. After washing, the plate
was incubated with ␤-d-galactosidase-conjugated streptavidin (Zymed Laboratories) at room temperature for 1 h.
After ﬁnal washing, the plate was incubated with 0.1 mM
4-methylumbelliferyl-␤-d-galactopyranoside (Sigma, St.
Louis, MO, USA) at 37 ◦ C for 2 h. The enzyme reaction was
stopped with 0.1 M glycine–NaOH (pH10.2), and the ﬂuorescence intensity was read as ﬂuorescence units (FU) on a
microplate ﬂuorescence reader (Fluoroskan; Flow Laboratories, McLean, VA, USA). The ﬂuorometric ELISA for speciﬁc
IgE in each sample was repeated more than three times.
The cut-off value was determined as the average + 3SD FU
in serum samples of 19 dogs as negative control.
2.4. Detection of allergen-speciﬁc IgE by immunoblotting
analysis
Cry j 1 and Cry j 3 were electrophoresed by SDSPAGE and then electrotransferred to PVDF membrane (GEHealthcare, Piscataway, NJ, USA). The three sera showing higher FU values of anti-Cry j 1 or anti-Cry j 3 IgE
had been respectively pooled for immunoblotting analysis. After blocking with PBS containing 5% skimmed milk,
the membrane was incubated with pooled sera diluted
1:10 in PBS containing 0.1% Tween-20 and 5% non-fat
dried milk for 2 h. After washing, horseradish peroxidase(HRP)-conjugated goat anti-dog IgE (0.1 ␮g/ml) (Bethyl
Laboratories, Montgomery, TX, USA) was added on themembrane and incubated for 2 h. After washing, the membrane was incubated with ECL reagent (GE Healthcare) for
5 min and exposed to a radiograph ﬁlm for 5–30 s.

2. Materials and methods
2.1. Crude and major C. japonica pollen allergens
Crude C. japonica pollen allergen was prepared as
described previously (Yasueda et al., 1983). Cry j 1 and Cry
j 2 as major allergens were puriﬁed as described previously(Sakaguchi et al., 1990). Cry j 3 was puriﬁed as described
previously (Fujimura et al., 2007).
2.2. Sera
CAD was diagnosed based on Willemse’s criteria(Willemse, 1986) and Prélaud’s major criteria (Prélaud
et al., 1998). The sera were obtained from 100 dogs withAD visiting to Fujimura Animal Hospital owed by one of
the authors (M.F.) (Osaka, Japan). The sera used as negative controls in the pollen-speciﬁc IgE assay were obtained
from 19 dogs that had been kept indoors as experimental laboratory animals and had never been exposed to C.
japonica pollen. They had shown no signs of AD and demonstrated no speciﬁc reaction to crude C. japonica allergen byintradermal skin testing (IDST).

3. Results and discussion
Speciﬁc IgE to crude C. japonica pollen allergen was measured in the sera of the 100 dogs with AD by ELISA (Fig. 1).
The cut-off value calculated from negative control samples
was 55 FU. Of the 100 dogs with AD, 15 had IgE sensitivity
to crude C. japonica pollen allergen similar to the results ofour previous epidemiological study (Masuda et al., 2000a).
Next, IgE reactivity to Cry j 1 and Cry j 2 was examined inthese 15 dogs sensitive with crude C. japonica pollen allergen by ELISA (Fig. 2A and B). The cut-off values of speciﬁc
IgE levels to Cry j 1 and Cry j 2 calculated from the negative
control samples were found to be 114 and 96 FU, respectively. Of the 15 serum samples which included speciﬁc IgE
to crude C. japonica pollen allergen, all had speciﬁc IgE to
Cry j 1, and 6 (40%) had speciﬁc IgE to Cry j 2. In humans,
more than 90% of the patients with C. japonica pollinosis
had speciﬁc IgE to Cry j 1 and Cry j 2 (Hashimoto et al.,1995). Further, in wild Japanese monkeys, we found that of
the 45 monkeys sensitive to crude C. japonica pollen allergen, 23 (51%) had speciﬁc IgE to both Cry j 1 and Cry j 2, 21(47%) had speciﬁc IgE only to Cry j 1, and 1 (2%) had speciﬁc
IgE only to Cry j 2 (Hashimoto et al., 1994). In humans andmonkeys, Cry j 1 and Cry j 2 are major pollen allergens.
In the present study, however, 40% of the dog sera that
had speciﬁc IgE to crude pollen had anti-Cry j 2 IgE. This
result was similar to that of our previous study (Masuda

2.3. Fluorometric ELISA for speciﬁc IgE to crude andmajor C. japonica pollen allergens
Speciﬁc IgE to crude and major C. japonica pollen allergens were measured by a ﬂuorometric ELISA. A microplate(NUNC-Immuno Plate Maxisorp F96; NalgeNunc International, Roskilde, Denmark) was coated with crude C.
japonica pollen allergen (4 ␮g/ml) or the puriﬁed allergens, Cry j 1, Cry j 2, or Cry j 3 allergens (1 ␮g/ml),
at 4 ◦ C overnight. After washing, the plate was incubated with diluted sera (1:10) at room temperature for
3 h. The plate was then washed and incubated witha mouse monoclonal anti-dog IgE antibody (0.5 ␮g/ml)(DeBoer et al., 1993) was then added. The plate wasincubated at 4 ◦ C overnight. After washing, the plate
was then incubated with biotinylated rat monoclonal

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S. Kubota et al. / Veterinary Immunology and Immunopathology 149 (2012) 132–135

134

(A)

Specific IgE to Cry j 1 (FU)Specific IgE to crude pollen allergen (FU)

2000

1500

1000

500

1500

1200

900

600

300

0

(B)

Pollinosis

Control

Pollinosis

Control

Pollinosis

Control

500

0Control

Specific IgE to Cry j 2 (FU)Pollinosis

Fig. 1. IgE reactivity to crude C. japonica pollen allergen in dogs. The data
were obtained by using diluted sera (1:10). The dashed line shows the
cut-off value. The cut-off value calculated from negative control samples
was 55 FU.

et al., 2000b). These ﬁndings indicate that Cry j 1 is a major
allergen in dogs as well as humans and Japanese monkeys.
IgE reactivity to Cry j 3 was examined in these 15 dogs
sensitive to crude pollen allergen (Fig. 2C). The cut-off values of speciﬁc IgE to Cry j 3 calculated from negative control
samples were 126 FU. Of the 15 sera which contained speciﬁc IgE to crude C. japonica pollen allergen, 11 (73%) had
speciﬁc IgE to Cry j 3. Of 11 sera samples, ﬁve also reactedwith Cry j 1, and six with both Cry j 1 and 2. The IgE-binding
frequency of Cry j 3 in the human patients with C. japonica pollinosis was estimated as 27% (Fujimura et al., 2007).
More than 70% of CAD with speciﬁc IgE to crude C. japonica
pollen allergen had anti-Cry j 3 IgE. These ﬁndings suggestthat Cry j 3 may be a more important allergen in dogs thanin humans.
To further evaluate anti-Cry j 3 IgE in dogs reactivity
with Cry j 3, we conducted immunoblotting analysis. Cry j
1 and Cry j 3 were electrophoresed by SDS-PAGE and thenelectrotransferred to PVDF membrane. The pooled serumof 3 dogs with anti-Cry j 1 IgE was added to the membrane,
and Cry j 1 was detected with anti-Cry j 1 IgE (Fig. 3, lane1). Similarly, the pooled serum of 3 dogs with anti-Cry j 3
IgE was added to the membrane, and Cry j 3 was detectedwith anti-Cry j 3 IgE (Fig. 3, lane 3). In contrast, the pooled
serum of 3 dogs as negative controls with no anti-Cry j 1
and Cry j 3 IgE was added to the membrane, and Cry j 1

400

300

200

100

0

Specific IgE to Cry j 3 (FU)(C) 1000

800

600

400

200

0
Fig. 2. IgE reactivity to (A) Cry j 1, (B) Cry j 2, and (C) Cry j 3 in atopic dogs
sensitive to crude C. japonica pollen allergen. The data were obtained byusing diluted sera (1:10). The dashed line shows the cut-off value. The
cut-off values calculated from negative control samples were 114, 96 and
121 FU, respectively.

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S. Kubota et al. / Veterinary Immunology and Immunopathology 149 (2012) 132–135

135

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Inouye, S., 1995. Hypersensitivity to Japanese cedar (Cryptomeria
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Taniguchi, Y., Ono, A., Sawatani, M., Nanba, M., Kohno, K., Usui, M., Kurimoto, T., Matuhasi, T., 1995. Cry j I, a major allergen of Japanese cedarpollen, has pectate lyase enzyme activity. Allergy 50, 90–93.
Willemse, T., 1986. Atopic skin disease: a review and a reconsideration ofdiagnostic criteria. J. Small Anim. Pract. 27, 771–778.
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Fig. 3. Immunoblotting analysis of Cry j 1 and Cry j 3. Cry j 1 (lanes 1
and 2) or Cry j 3 (lanes 3 and 4) was electrophoresed by SDS-PAGE andthen electrotransferred to PVDF membrane. Lane 1: pooled serum of 3
dogs with anti-Cry j 1 IgE (FU values: 1410, 1090, and 925), lanes 2 and 4:
pooled serum of 3 dogs as negative controls with no anti-Cry j 1 and Cry
j 3, lane 3: pooled serum of 3 dogs with anti-Cry j 3 IgE (FU values: 812,
507, and 412).

and Cry j 3 were not detected (Fig. 3, lanes 2 and 4). These
results reconﬁrmed that sera of dogs with anti-Cry j 3 IgEreacted to Cry j 3 with immunoblotting analysis.
We found that Cry j 3 has signiﬁcant homology with thethaumatin-like protein categorized in PR-5 family protein
which might act as a causative allergen in OAS (Fujimura
et al., 2007). Cross-reactivity between allergens from C.
japonica pollen and tomato has been reported in dogs
and humans (Fujimura et al., 2002; Kondo et al., 2002).
PR-5 protein, which was considered as one of the importantpollen allergens responsible for pollinosis and OAS, havebeen identiﬁed as Jun a 3 from mountain cedar (Juniperus
ashei), Jun v 3 from eastern red cedar (Juniperus virginiana), and Cup a 3 from Arizona cypress (Cupressus arizonica)(Midoro-Horiuti et al., 2000, 2001; Cortegano et al., 2004).
Fujimura et al. (2007) revealed that Cry j 3 has signiﬁcanthomology with the thaumatin-like protein and suggested
that Cry j 3 might act as a causative allergen for OAS in
human patients. Unfortunately, we could not evaluate thepresence of OAS in the dogs with IgE to Cry j 3. The present
results, however, suggest that Cry j 3 is an important allergen in dogs and might play an important role in the etiology
of OAS in dogs.
In conclusion, we found that many dogs with speciﬁc IgE
to crude C. japonica pollen had anti-Cry j 3 IgE. Our results
indicate that Cry j 3 can be considered as one of the major
allergens in CAD. Further studies are needed to reveal therole of cross-reactivity to other allergens in the etiology of
OAS in CAD.
Conﬂicts of interest
The authors declare no conﬂicts of interest.
Acknowledgments
This research was partially supported by Grant-in-Aid
Matching Fund Subsidy for Private University and by aproject grant awarded by the Azabu University ResearchServices Division.

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Cellular Immunology 275 (2012) 24–32

Contents lists available at SciVerse ScienceDirect

Cellular Immunology
journal homepage: www.elsevier.com/locate/ycimm

The role of CCR7 in allergic airway inﬂammation induced by house dust
mite exposure
Masaki Kawakami a,b, Osamu Narumoto a,b, Yukiko Matsuo a, Kazuhide Horiguchi c, Satomi Horiguchi c,
Naohide Yamashita d, Masahiro Sakaguchi e, Martin Lipp f, Takahide Nagase b, Naomi Yamashita a,⇑a

Department of Pharmacotherapy, Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan
Department of Pulmonary Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan
Department of Anatomy, Faculty of Medical Science, University of Fukui, Fukui, Japand
Department of Advanced Medical Science, Institute of Medical Science, University of Tokyo, Japane
Department of Veterinary Microbiology, School of Veterinary Medicine, Azabu University, Kanagawa, Japanf
Tumorgenetics and Immunogenetics, Max-Delbrück-Center for Molecular Medicine, Berlin, Germanybca r t i c l e

i n f oArticle history:
Received 13 December 2011
Accepted 29 March 2012
Available online 5 April 2012
Keywords:
AsthmaAirway hyperresponsiveness
CCR7
CCL21
House dust mite
Airway inﬂammationa b s t r a c t
House dust mite (HDM), the most common allergen, activate both the IgE-associated and innate immune
responses. To clarify the process of sensitization, we investigated the role of the CCL21, CCL19, and CCR7
axis in a mouse model of HDM-induced allergic asthma. HDM inhalation without systemic immunization
resulted in a HDM-speciﬁc IgE response. CCR7-knockout (CCR7KO) mice exhibited greater airway inﬂammation and IgE responses compared to wild-type mice. We examined FoxP3 expression in these mice toclarify the contribution of regulatory cells to the responses. FoxP3 expression was higher in the lungs but
not in the lymph nodes of CCR7KO mice compared to wild-type mice. In CCR7KO mice, FoxP3-positive
cells were found in lung, but we observed higher release of IL-13, IL-5, TGF-b, IL-17, and HMGB1 in bronchoalveolar lavage ﬂuid. We demonstrate here that immuno-regulation through CCR7 expression in T
cells plays a role in HDM-speciﬁc sensitization in the airway.
 2012 Elsevier Inc. All rights reserved.

1. Introduction
The inhalation of allergens induces airway inﬂammation, which
is a prominent feature of asthma [1–4]. Dendritic cells are antigenpresenting cells that initiate several immune responses [5]. After
dendritic cells take up allergens, they enter afferent lymphatic vessels through a process promoted by CCL21 [6,7]. Antigen-laden
antigen-presenting cells then induce activation, expansion, anddifferentiation of antigen-speciﬁc T cells within the draining lymph
nodes. We previously showed that paucity of lymph node T cells(plt) mice, which are deﬁcient in CCL21ser and CCL19, resulted in
reduced but still substantial airway inﬂammation, suggesting thatallergen sensitization and airway inﬂammation can be induced
without secondary lymph nodes [8]. In contrast, CCL21 andAbbreviations: HDM, house dust mite; CCR7KO, CCR7-knockout; LPS, lipopolysaccharide; BALF, bronchoalveolar lavage ﬂuid; HE, haematoxylin-eosin; PAS,
periodic acid-Schiff; iBALT, inducible bronchus-associated lymphoid tissue; HMGB,
high-mobility group box; Treg, regulatory T; ACh, acetylcholine; Raw, airway
resistance.
⇑ Corresponding author. Address: Department of Pharmacotherapy, ResearchInstitute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi
Nishitokyo-shi, Tokyo 202-8585, Japan. Fax: +81 424 68 8647.
E-mail address: naoyama@musashino-u.ac.jp (N. Yamashita).

CCL19 play a critical role in the resolution phase of airway inﬂammation, which is similar to their role in the mucosal immune system [8–10].
In animal asthmatic models, systemic immunization using
allergens formulated with alum is used to induce IgE. In plt mice
we used ovalbumin in alum to clarify the role of CCL21 [8]. Administration of house dust mite (HDM) by inhalation was recently
shown to induce airway inﬂammation without systemic immunization [11,12]. In many countries, HDM is one of the most common
allergens triggering asthma [13–15]. HDM allergens have proteinase activity, which is critical for sensitization [16,17]. HDM allergens react with toll-like receptor 4 (TLR4), which is a receptor for
lipopolysaccharide (LPS) [18,19]. In addition, Derf 2, a major
HDM allergen, possesses structural homology to myeloid differentiation factor (MD) 2, the LPS-binding component of the TLR4 signaling complex [20,21]. Taken together, these ﬁndings indicatethat allergen sensitization involves complex interactions between
antigen-speciﬁc and innate immune responses that have not yet
been clariﬁed.
CCR7 is the receptor for CCL19 and CCL21, and CCR7-knockout(CCR7KO) mice possess similar impairment of the secondary
lymph nodes as plt mice [22]. The impairment of contact
sensitivity in CCR7KO mice is greater than that in plt mice [22].

0008-8749/$ - see front matter  2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.cellimm.2012.03.009

99M. Kawakami et al. / Cellular Immunology 275 (2012) 24–32

25

Furthermore, CCR7 is important in maintaining immune responses
and immune tolerance [23]. CCR7 is also essential to migrate andexhibit regulatory function of regulatory T (Treg) cells [24–26].
To clarify the process of allergen sensitization and the role of
CCR7 expression in this process, we investigated the ability of
CCR7KO mice to exhibit HDM-speciﬁc airway inﬂammation. We
found that airway inﬂammation was signiﬁcantly augmented andlymphocyte accumulation was prominent in CCR7KO mice.
Although FoxP3-positive cells were present in the lung, they did
not function effectively in CCR7KO mice. This ﬁnding suggests thatallergen sensitization is controlled by the expression of CCR7. The
targeted control of CCR7 expression has potential as an immunotherapy for asthma.

fuged at 1500 rpm for 10 min at 4 C. Pellets were dissolved in 1 ml
PBS and the number of cells counted. Cytospin preparations wereobtained by spinning at 640 rpm for 2 min, stained with Diff Quik
(International Reagents Corporation, Osaka, Japan), and then examined by microscopy.
The content of cytokine in the BALF was measured using the
ELISA kits; IL-13, IL-5 and TGF-b by Quantising kit (R&D System,
Inc. (Minneapolis, MN), IL-35 by speciﬁc ELISA kit (Cusabio Biotec,
P.R., China). HMGB1 was measured by using an HMGB1 ELISA Kit II(Shino-test Co., Tokyo, Japan), which speciﬁcally detects HMGB1[28].

2. Methods

The assessment of airway responsiveness was undertaken 24 hafter the last mite administration, as previously reported [27].
Brieﬂy, the anesthetized mice were tracheostomized and injectedwith pancuronium bromide. The animals were connected to a Harvard ventilator with 0.25 ml tidal volume and a respiratory frequency of 150/min. Next, they were placed in the whole-bodyplethysmograph (Buxco Electronics) to measure airway resistance(Raw). To assess airway responsiveness, increasing dose of acetylcholine (ACh) (3.12–25 mg/mL) was administered by nebulization.
Data are expressed as [Raw after inhalation of ACh/Raw before
inhalation)  100 (%)].

2.1. Animals
C57BL/6. mice were purchased from SLC (Shizuoka, Japan).
CCR7/ C57BL/6 mice were established as previously reported[22] and bred in the animal facilities of Musashino University
School of Medicine under speciﬁc pathogen-free conditions underapproval of Institutional Animal experiments Committee ofMusashino University. Care and use of the animals followed theguidelines of the ‘‘Principles of Laboratory Animal Care’’ formulated by the National Society for Medical Research. To genotype
the pups, tail DNA was extracted by using a DNA Isolation Kit(GENTRA, Minneapolis, MN, USA) and examined by PCR analysis
to detect the wild-type and knockout alleles. The sense and antisense primers to amplify the 600-bp CCR7 wild-type allele were
50 -CCCCGGGCAATGTCCTGA-30 and 50 -CGTGTCCTCGCCGCTGTT-30 ,respectively. The sense and antisense alleles to amplify the 400bp knockout allele were 50 -CTCTCGTGGGATCATTGTTTTTCT-30 and
50 -GTCTCCGCCTCCATGCTTCACC-30 , respectively.
2.2. Treatment
Mice were treated by intranasal administration with 40 lg of
HDM extract dissolved in saline once per day for 7–10 days. The
HDM extract was kindly provided by ITEA Inc., Tokyo, Japan. Micetreated with saline were used as controls.
2.3. Measurements of serum and HDM-speciﬁc IgE levels
For total IgE detection, total IgE ELISA kits (Morinaga Institute of
Biological Science, Yokohama, Japan) were used following the
manufacturer’s instructions. For mite-speciﬁc IgE detection, plates
were coated with 20 lg/ml HDM extract in 50 mM carbonate buffer (pH 9.0). After blocking the coated plates with SuperBlock(Pierce, Rockford, In.), samples were diluted 20-fold and added tothe plates. Antibodies were detected with rat anti-mouse IgE speciﬁc monoclonal antibody (YAMASA, Chiba, Japan), followed by theaddition of Streptavidin-HRP (Dako, Corp., Glodtrup, Denmark).
The color was developed with tetramethylbenzidine + substrate(BioFx Labo., MD, USA) and the reaction stopped by addition of
1 N H2SO4. Optical density at 450 nm (OD450) was measured using
a microplate reader. Antibody titers were calculated by comparison
with internal standards.
2.4. Bronchoalveolar lavage ﬂuid (BALF) cell analysis
BALF cell analysis was performed 24 h after the last HDM
administration, as previously reported [27]. BALF was obtainedby incubating and repeatedly washing the lung with 1 ml of saline
until the recovered volume was 2 ml. The isolated BALF was centri-

100

2.5. Assessment of airway responsiveness2.6. Histological examination
Mice were perfused transcardially with PBS and then 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4). The lungs
were removed and then immersed in 4% paraformaldehyde for
2 h at 4 C. After washing with PBS, the specimens were soaked
overnight in 30% sucrose in PBS, embedded in optimal cutting temperature (OCT) compound (Miles, Elkhart, IN, USA), and thenquickly frozen. Cryostat sections were cut at a thickness of
10 lm by using a Leica CM3050 cryostat (Leica Microsystems,
Wetzlar, Germany), and thaw-mounted on poly-L-lysine-coatedglass slides (Matsunami Glass, Osaka, Japan). The samples wereanalyzed by Haematoxylin-eosin (HE) and periodic acid-Schiff(PAS) staining.
For immunohistological analysis, lung sections were preincubated overnight with anti-FoxP3 (1:1000; eBioscience, San Diego,CA) and then incubated with Alexa Fluor 488-coupled donkey
anti-IgG (Molecular Probes, Eugene, OR, USA). Rat anti-mouse
CD4, CD8 and CD3 antibodies (BD Pharmigen) were used for CD4,CD8and CD3 positive cell detection. Goat anti-mouse CD4 antibody(R&D Systems) was used for double staining experiments. Sections
were counterstained with 40 ,60 -diamidino-2-phenylindole (DAPI;
Roche Diagnostics, GmbH, Mannheim, Germany) and mountedwith Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA). Control tissues were prepared by omitting primary antibodies from the incubation solutions; no speciﬁc immunoreactivity
was observed. Immunoﬂuorescence images were examined with a
Leica TCS-SP2 confocal microscope (Leica Microsystems) with excitation wavelengths of 350, 488, and 543 nm. Images were collected
and analyzed in Leica Confocal Software (Leica Microsystems).
2.7. CD4+CD25+ and dendritic cell isolation
Lung was homogenized in 1% penicillin/streptomycin/
RPMI1640 using cell strainers with 70 lm pores (BD Falcon, NJ,
USA). Specimens were then centrifuged at 1500 rpm for 10 min at
4 C, and the cell pellets were washed twice. Lung cells were puriﬁed using NycoPrep (AXIS-SHIELD plc, Dundee, Scotland) followed
by separation using CD4+CD25+ regulatory T cell isolation kits
(Miltenyi Biotech GmbH, Nordrhein-Westfalen, Germany). In the

26

M. Kawakami et al. / Cellular Immunology 275 (2012) 24–32

Fig. 1. Airway inﬂammation and airway response. (A) IgE and HDM-speciﬁc IgE synthesis. Each bar represents the mean ± SEM of values obtained from 10–12 different mice.
Control indicated mice treated with saline. HDM indicated mice treated with house dust mite (HDM).⁄p < 0.05. (B) BALF cell analysis BALF cells were placed on glass slides
using Cytospin. Slides were then stained with Diff Quik, and cell differentiation was assessed microscopically. Control indicated mice treated with saline. HDM indicated micetreated with HDM. The Y axis shows the number of cells in total BALF (2 mL). Each bar indicates mean ± SEM of 5–7 different mice. ⁄⁄p < 0.01. ⁄p < 0.05, CCR7KO comparedwith wild type mice. (C) Airway response to acetylcholine. The indicated concentrations of ACh were inhaled for 3 min. Airway resistance (Raw) was measured using wholebody plethysmographs as described in the Methods. ACh-evoked changes in Raw are expressed as a percentage of the Raw observed before ACh inhalation (100%). The data
shown are means ± SEM for six mice.⁄p < 0.05, HDM-treated vs control mice.

101

M. Kawakami et al. / Cellular Immunology 275 (2012) 24–32

27

Fig. 3. Confocal micrographs. CD3 (green) and CD4 (red) (A) or CD8 (red) (B) were
stained using the speciﬁc antibodies. DNA was counterstained with DAPI (blue).
original magniﬁcation, 400. (For interpretation of the references to color in thisﬁgure legend, the reader is referred to the web version of this article.)mAb-coated microbeads (Miltenyi Biotech). Bead-bound cells werethen isolated using magnetic separation columns. The purities of
the CD11c+ cell populations were 90% as shown in Fig. 4.
For ﬂow cytometric analysis, anti-mouse CD16/CD32(Fc block),CD11c, CD40, CD80, CD86, CD83 antibodies (BD Pharmigen) wereused. Data were collected using FACSAria (B D Biosciences).
2.8. Examination of mRNA expression using real-time PCR

Fig. 2. Histological analysis. Histological analysis was performed with HE and PAS
staining. (A) Wild type mice and (B) CCR7KO mice. Control indicated mice treatedwith saline. HDM indicated mice treated with house dust mite (HDM). Figures are
representative data from histological examination of ﬁve mice.

ﬁrst step, CD4+ cells were separated from all non-CD4 cells, and in
the second step CD25+ cells were positively selected by using mAbcoated microbeads and magnetic separation columns. The purity of
the resultant CD4+CD25+ cell populations was 95% (data notshown). After 2 days of culture, the supernatant was harvested.
For dendritic cells isolation, the lung cells were puriﬁed using
NycoPrep (AXIS-SHIELD), followed by incubation with anti-CD11c

102

Total RNA was extracted with ISOGEN (Nippon Gene Co., Ltd.,Tokyo, Japan) by a modiﬁed acid–guanidium thiocyanate–phenol–chloroform method. RNA purity was established by means ofa NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies,
Wilmington, DE). The RNA had an OD260/280 ratio of 1.8 indicating high purity. After measuring the amount of total RNA, cDNA
synthesis was performed with a cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, California, USA). The levels of mRNA were
examined by real-time PCR using a Light Cycler-Fast Start DNA
Master Syber Green I kit (Roche Diagnostics, Mannheim, Germany).
In this system, double-stranded DNA labeled with Syber Green I is
detected by real-time PCR. Quantiﬁcation was performed bycomparison to a standard curve generated from a serial dilutionof speciﬁc PCR products. mRNA levels were standardized according
to b-actin mRNA levels and were expressed as ratios of control values. The primers used were as follows: bactin, 50 -CCTGTATGCCTCTGGTCGTA-30 (sense) and 50 -CCATCTCCTGCTCGAAGTCT-30 (antisense) to amplify a 260 bp product; FoxP3, 50 -TCTTCGAGGAGCCAGAAGAG-30 and 50 -TACTGGTGGCTACGATGCAG-3 to amplify a
240 bp product.
2.9. Statistics
For comparisons of multiple parameters, we used multiple analysis of variance (MANOVA). The level of statistical signiﬁcance wasset at p < 0.05. Data were expressed as the mean ± SEM.

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Fig. 4. Flow cytometric analysis of dendritic cells. Dendritic cells positively separated from lung were analyzed for activation marker and co-stimulatory molecules.

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M. Kawakami et al. / Cellular Immunology 275 (2012) 24–32
Table 1
Characteristics of CD11c positive dendritic cells in the lung.
Percentage of positive cells(%)

Wild type

CCR7KOMHC class II positive
CD40
CD83
CD80
CD86

43.8 (8.9)
12.6 (4.9)
4.7 (2.3)
15.4 (6.5)
7.3 (4.0)

42.1(13.9)
11.5 (8,1)
5.8 (3.3)
16.4 (11.4)
17.1 (14.1)

NS
NS
NS
NS
NS

NS: not statistically signiﬁcant. The number is mean (SE), n = 3–4.

Table 2
Characteristics of CD4+CD25+ cells in the lung.

Number of cells (105)
IL-10 production (pg/ml)
IL-35 production (pg/ml)

Wild type

CCR7KO

3.4 (1.3)
1453 (134)190 (23)

11.9 (3.4)
1714 (6)256 (24)

p < 0.001
NS
NS

Number of cells was number of CD4+CD25+ cells separated from one lung. Cytokine
production was from 1  104 cells. NS: not statistically signiﬁcant. The number isMean (SE), n = 3–4.

3. Results
3.1. Total and HDM-speciﬁc IgE synthesis after HDM exposure
Total and HDM-speciﬁc IgE levels were measured in serum by
ELISA to assess the allergic sensitization induced by inhalation of
HDM. Inhaled HDM induced IgE synthesis (p < 0.01 compared tocontrol) and that IgE synthesis was signiﬁcantly augmented in
CCR7KO mice compared to the wild type (p < 0.01) (Fig. 1A).
HDM-speciﬁc IgE was also elevated in CCR7KO mice treated with
HDM. These results indicate that continuous HDM inhalation induced systemic allergic sensitization in both the wild type and
CCR7KO mice and that the level of sensitization was higher in
CCR7KO mice.

3.2. Role of CCR7 in HDM-induced airway inﬂammation

Fig. 5. FoxP3 expression. (A) Confocal micrographs. FoxP3 (green) was stainedusing the speciﬁc antibodies. DNA was counterstained with DAPI (blue). (B) Numberof FoxP3 positive cells in lung sections examined by confocal microscopy. (C)Number of FoxP3 positive cells in lymph node. (D) FoxP3 mRNA expression in thelung. ⁄p < 0.05, ⁄⁄p < 0.01. (For interpretation of the references to color in this ﬁgurelegend, the reader is referred to the web version of this article.)

104

We analyzed the inﬁltration of inﬂammatory cells into the BALF
to examine the role of CCR7 in HDM-induced airway inﬂammation(Fig. 1B). Signiﬁcant inﬁltration of eosinophils and macrophages
was observed in mice exposed to HDM compared to controls a(Fig. 1B, p < 0.01). The number of eosinophils, neutrophils, lymphocytes and macrophages were signiﬁcantly higher in CCR7KO micethan in the wild type (p < 0.01 for eosinophils, p < 0.05 for neutrophils, lymphocytes and macrophages).
Airway responsiveness was also analyzed. CCR7KO mice exhibited signiﬁcant higher response compared with untreated mice at
12.5 and 25 mg/ml ACh. Wild type mice showed signiﬁcant response at 25 mg/ml ACh (Fig. 1C).
Histological analysis by HE staining revealed that HDM exposure caused more severe airway inﬂammation in CCR7KO micethan in the wild type (Fig. 2). As shown by PAS staining, there weresigniﬁcantly more mucus-producing cells in the airways of
CCR7KO mice than in the wild type (Fig. 2). These data indicate that
HDM induced severe airway inﬂammation in CCR7KO mice. We
also observed prominent lymphoid aggregates in the bronchiolesof CCR7KO mice (Fig. 2). Immuno-histochemical examination
revealed that these aggregates were composed predominantly of
CD4+CD3+ cells and contained a few CD4 single positive cells
and few CD8+ cells (Fig. 3).

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M. Kawakami et al. / Cellular Immunology 275 (2012) 24–32

Dendritic cells play a key role for cell differentiation and antigen
presentation [5]. When dendritic cell immobilize antigen, theirphenotype changes to mature type and express CCR7 to migrate
secondary lymph node [23,29,30]. We examined the mature status
of dendritic cells in the lung by ﬂow cytometric analysis. As shownin the Fig. 4, dendritic cells with wild type and those from CCR7KO
mice expressed some amount of MHC class II, CD40, CD80, CD83
and CD86 molecules. The amount of expression varied but there
were no signiﬁcant difference between dendritic cells with wild
type and those from CCR7KO mice (Table 1). These data suggested
that dendritic cells acquired some activation without CCR7
expression.

CCR7 expression is critical for T cells to exit peripheral tissues[31,32]. FoxP3 positive T cells, which are known to suppress airway
inﬂammation, express CCR7 and are activated by pulmonary
dendritic cells within secondary lymph nodes [26,33]. We next
examined FoxP3 expression in the lung. The results from immuno-histochemical analyses using Foxp3 antibodies are shown in
Fig. 5A. In lung sections, we observed signiﬁcantly more FoxP3positive cells in CCR7KO mice than in the wild type (Fig. 5B). In
the lymph node, however, there were signiﬁcantly more FoxP3positive cells in the wild type than in CCR7KO mice (Fig. 5C). FoxP3mRNA expression was also signiﬁcantly higher in CCR7KO micethan in the wild type in the lung (Fig. 5D).

Fig. 6. Analysis of cytokine production in the BALF. BALF supernatants were analyzed for cytokine production by cytokine-speciﬁc ELISA. Each bar indicates mean ± SEM of 5–
8 different mice. ⁄p < 0.05, ⁄⁄p < 0.01.

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Fig. 7. HMGB1 production in the BALF. BALF supernatants were analyzed for
HMGB1 production by speciﬁc ELISA. Each bar indicates mean ± SEM of four
different mice. ⁄⁄p < 0.01.

To further examine the Treg cell function in the lung, we separated Treg cell using CD4+CD25+ cell selection kits. The number of
CD4+CD25+ cells was signiﬁcantly increased in CCR7KO mice compared to wild type mice (Table 2). To examine the regulatory function of CD4+CD25+ cells, we examined the synthesis of IL-10 and
IL-35, which is reported the one of the key cytokine of regulatory
function [34,35]. When we examined the cytokine production,
CD4+CD25+ cells produced equal amount of IL-10 and IL-35 in wild
type and CCR7KO mice (Table 2).
3.3. Role of CCR7 in HDM-induced cytokine production
We examined cytokine production in BALF in CCR7KO and wildtype mice to explore the type of airway inﬂammation induced by
HDM in the absence of CCR7. Levels of the Th2 cytokines, IL-13
and IL-5, were signiﬁcantly higher in CCR7KO mice than in the wild
type (Fig. 6A and B). By comparison, levels of the Th1 cytokine, IFNc, were below the detection limit (9.4 pg/ml, n = 6 for each group),suggesting that the inﬂammation in CCR7KO mice is Th2-dominant
allergic inﬂammation. We also analyzed the expression levels of
the regulatory cytokines TGF-b and IL-17 [36] and found that bothwere expressed at higher levels in CCR7KO mice than in the wild
type (Fig. 6C and D). The formation of lymphocyte aggregates in
the CCR7KO mice is consistent with the increased expression of
IL-17 in these mice compared to the wild type and with the roleof IL-17 in the development of inducible bronchus-associated lymphoid tissue (iBALT) [37].
Next, we examined HMGB1 production in the BALF of CCR7KO
and wild-type mice. HMGB1 is a critical mediator of the innate immune response to inﬂammation and infection [38–40]. HMGB1 isalso a regulator of Treg cells [41,42]. We found that HMGB1 production is signiﬁcantly higher in CCR7KO mice than in the wild
type, suggesting that innate immune response is augmented andTreg cell function is depressed in CCR7KO mice (p < 0.01, Fig. 7).

31

CD4+ cells, but also contained a few CD8+ cells. We also observed
FoxP3-positive cells. Although there was an increased number of
FoxP3-positive cells in the lung of CCR7KO mice compared towild-type mice, the levels of Th2 cytokines as well as IL-17 andTGF-b were also increased. In addition, HMGB1 production was
also increased in CCR7KO mice. These results suggest that CCR7deﬁcient FoxP3-positive cells, which down-regulate allergic airway
inﬂammation [43,44], did not function efﬁciently in CCR7KO mice.
Intra-tracheal continuous HDM exposure to mice induces theasthmatic phenotype [11,12,45]. In the current study, we also
found that HDM exposure by inhalation induced IgE responses.
These responses were increased in CCR7KO mice, which was consistent with the increase in systemic immunization seen in
CCR7KO and plt mice [8,23,46]. CCR7 is a receptor of CCL21 andCCL19 that plays a critical role in the development of secondary
lymph nodes [22,47]. CCR7, which is expressed by dendritic cells
and T cells, plays a critical role in the movement of lymphocyte
and dendritic cells from peripheral tissue into afferent lymphatics[32,47]. Disruption of this process in CCR7KO mice causes lymphocytes to accumulate in the tissue of these mice [32] and is the reason why HDM exposure caused lymphoid aggregates to form intheir bronchioles.
Another reason for T cell accumulation in CCR7KO mice may berelated to an impairment of Treg cells in these mice [33]. CCR7KO
mice accumulate BALT from day 5 after birth [33]. Treg cells suppress effector T cell function by homing to lymphoid organs, whichis conﬁrmed by the fact that Treg cells from wild-type mice work
perfectly in CCR7KO mice [33]. Consistent with the previous reportthat CCR7-deﬁcient T reg cells work normally in vitro [10], wefound that Treg cells, present in the BALT, produced IL-10 and IL35 in vitro. We also found that dendritic cells in the lung of
CCR7KO mice expressed activation makers such as MHC class II
and co-stimulatory molecules. It is possible that the structure of
BALT may not be sufﬁcient for FoxP3 positive cell to possess suppressive function with CCR7 deﬁcient condition. In the case of
inﬂuenza infection, iBALT, which is essential for local immunity
to inﬂuenza, is not efﬁciently formed in the absence of CCL21
and CCL19 [48]. Recently, Ishimaru et al. have reported that CCR7
negative FoxP3-positive cells have defects in the c-Jun and FoxP3
interaction [24]. Thus, it is also possible that FoxP3-positive cells
in CCR7KO mice possess defects in local migration to exhibit regulatory function in iBALT. To conﬁrm the Treg function of CCR7KO
mice in allergen sensitization, we should precede the experimentsby transfusion of Treg cells in vivo and precise functional analysis
in vitro, which is continuing subject to be solved.
HDM is known to accelerate not only antigen-speciﬁc immunity
but also innate immunity through the TLRs [19,20]. TLR, TLR2, andTLR4 are thought to be required to induce allergic inﬂammation[19,20]. A recent ﬁnding of homology between Derf2 and MD2,
the relation to TLR4 has been stressed, because MD2 is criticalfor signal transduction through TLR4 [20]. HMGB1 was producedin CCR7KO mice as an accelerated innate immune response. Beyond its role in maintaining chromosomal integrity, HMGB1 is secreted as a cytokine and is considered an alarmin [40].
In conclusion, we provide results here to highlight CCR7 expression as a promising target for regulation of HDM-induced innate
immunity and antigen responses. Further studies are needed to develop the targeted control of CCR7 expression as immunotherapy
for asthma.

4. Discussion

Acknowledgments

In the present study, we showed that continuous HDM exposure induced allergic airway inﬂammation, which was augmented
in CCR7KO mice. The prominent lymphoid aggregates observed in
the bronchioles of CCR7 KO mice were predominantly composed ofThis study was supported in part by grants-in-aid for ScientiﬁcResearch from the Ministry of Education, Culture, Sports, Scienceand Technology, Japan; a grant for Allergic Disease and Intractable
Diseases from the Ministry of Health, Labor and Welfare, Japan;

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and a grant to the Respiratory Failure Research Group from Healthand Labor Sciences, Japan.
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Experimental and Molecular Pathology 93 (2012) 18–25

Contents lists available at SciVerse ScienceDirect

Experimental and Molecular Pathology
journal homepage: www.elsevier.com/locate/yexmp

Suppressive effects of a pyrazole derivative of curcumin on airway inﬂammation
and remodeling
Osamu Narumoto a, b, Yukiko Matsuo a, Masahiro Sakaguchi c, Shunsuke Shoji d, Naohide Yamashita e,
David Schubert f, Kazuho Abe g, Kazuhide Horiguchi h, Takahide Nagase b, Naomi Yamashita a,⁎a

Department of Pharmacotherapy, Research Institute of Pharmaceutical Sciences Musashino University, Tokyo, Japan
Department of Pulmonary Medicine, Faculty of Medicine University of Tokyo, Tokyo, Japanc
Department of Veterinary Microbiology, School of Veterinary Medicine, Azabu University, Kanagawa, Japand
Division of Clinical Research, National Hospital Organization Tokyo National Hospital, Tokyo, Japane
Department of Advanced Medical Science, Institute of Medical Science University of Tokyo, Tokyo, Japanf
The Salk Institute, Laboratories for Cellular Neurobiology, La Jolla, CA, USAg
Department of Pharmacology, Research Institute of Pharmaceutical Sciences Musashino University, Tokyo, Japanh
Department of Anatomy, Faculty of Medical Science, University of Fukui, Fukui, Japanba r t i c l e

i n f oArticle history:
Received 27 December 2011
and in revised form 22 March 2012
Available online 19 April 2012
Keywords:
AsthmaCurcumin derivative
Airway remodelingEMT

a b s t r a c t
To advance the control of airway epithelial cell function and asthma, we investigated the effects of a new curcumin derivative, CNB001, which possesses improved pharmacological properties. Normal human bronchial
epithelial (NHBE) cells were stimulated with synthetic double-stranded RNA, Poly(I:C). CNB001 signiﬁcantly
suppressed IL-6, TNF-α, and GM-CSF production by NHBE cells, and did so more effectively than did curcumin
or dexamethasone (DEX). CNB001 signiﬁcantly inhibited the decrease of E-cadherin mRNA expression andincrease of vimentin mRNA expression observed in NHBE cells induced by a combination of TGF-β1 andTNF-α, which are markers of airway remodeling. In NHBE cells stimulated by TGF-β1, CNB001 signiﬁcantly
downregulated the level of active serine peptidase inhibitor clade E member (SERPINE) 1, which is also
reported to be related to airway remodeling. Whereas DEX alone signiﬁcantly increased the active SERPINE1
level, the combination of DEX and CNB001 signiﬁcantly suppressed active SERPINE1. In addition, CNB001 signiﬁcantly suppressed neutrophil inﬁltration, IL-6, TNF-α, IL-13 and active SERPINE1 production in bronchoalveolar lavage ﬂuid of the murine asthma model, which was not observed in the case of DEX. In conclusion, thecurcumin derivative, CNB001, is a promising candidate to treat asthma associated with neutrophilic airway
inﬂammation and remodeling.
© 2012 Elsevier Inc. All rights reserved.

Introduction
Although various effector cells reside within asthmatic airways,
airway epithelial cells actively involved in airway inﬂammation andremodeling, by producing inﬂammatory cytokines and inﬂuencing
other cells (Holgate, 2010; Holgate et al., 2000; Lee et al., 2009). Airway epithelial cells are the ﬁrst cells that encounter various stimulants in the airway and produce cytokines such as IL-6 and TNF-α,Abbreviations: NHBE cells, normal human bronchial epithelial cells; SERPINE1, serine peptidase inhibitor clade E member 1; EMT, epithelial–mesenchymal transitions;
ECM, extracellular matrix; TNF, tumor necrosis factor; DEX, dexamethasone; Poly(I:C), polyinosinic:polycytidylic acid; GM-CSF, granulocyte-macrophage colony stimulating factor; BALF, bronchoalveolar lavage ﬂuid; TLR3, Toll-like receptor 3; MAPK,
mitogen-activated protein kinases; CUR, curcumin; OPM, OVA + Poly(I:C) + house
dust mite.
⁎ Corresponding author at: Department of Pharmacotherapy, Research Institute ofPharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi Nishitokyo-shi,
Tokyo 202-8585, Japan. Fax: + 81 424 68 8647.
E-mail address: naoyama@musashino-u.ac.jp (N. Yamashita).

which are closely related to the pathophysiology of asthma (Ather
et al., 2011; Takizawa et al., 1996; Vieira et al., 2011; Yadav et al.,2011). Viral infection of the respiratory tract is the most frequent factor involved in asthmatic exacerbations and may trigger neutrophilic
inﬂammation in asthmatic airways (Fahy et al., 1995; Wark et al.,2002).
Airway remodeling, which is known to induce irreversible airﬂow limitation, as in the case of chronic obstructive pulmonary disease is another problem to be solved (Gizycki et al., 1997). Airway
remodeling is characterized by structural changes within the airway
wall, including smooth muscle hypertrophy, basement membranethickening, submucosal ﬁbrosis, mucus cell metaplasia, epithelial
shedding, and angiogenesis (Gizycki et al., 1997). Among the various factors involved in the pathogenesis of remodeling, we focusedon serine peptidase inhibitor clade E member 1 (SERPINE1, also
known as plasminogen activator inhibitor-1, PAI-1) and the processof epithelial–mesenchymal transition (EMT) in epithelial cells(Hackett et al., 2009; Kowal et al., 2008; Kucharewicz et al., 2003;
Kuramoto et al., 2009; Miyamoto et al., 2011). SERPINE1 regulates

0014-4800/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexmp.2012.04.011

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extracellular matrix (ECM) proteolysis (Kowal et al., 2008; Kucharewicz
et al., 2003), and the attenuation of SERPINE1 has been reported to
exhibit therapeutic properties for airway remodeling (Kuramoto et al.,2009; Miyamoto et al., 2011). Although EMT is a complex process
involving various cells, the airway epithelial cells are one of the main
targets related to airway remodeling (Hackett et al., 2009; Holgate etal., 2000; Lee et al., 2009).
Here, we explored the effects of a new curcumin derivative on
epithelial cell function. Curcumin is a polyphenol derived from turmeric (Curcuma longa), a plant that has been reported to possess anticancer, antioxidant, and anti-inﬂammatory properties (Aggarwal
and Harikumar, 2009; Fang et al., 2005; Ray et al., 2003; Santel etal., 2008; Weber et al., 2005; Wong et al., 2010). Although curcuminpossesses good pharmacological properties, the utility of curcumin
as a potential therapeutic drug is limited because of its poor bioavailability (Anand et al., 2007). Curcumin blocks multiple sites oftransforming growth factor beta (TGF-β) signaling (Gaedeke et al.,2004), which plays a key role in the production of SERPINE1(Dennler et al., 1998) and induction of EMT (Zavadil and Bottinger,
2005). Therefore, we hypothesized that a new curcumin derivative
might control not only airway inﬂammation but also EMT in asthmatic conditions that currently cannot be satisfactorily controlled
by any other drugs, including steroids.
We used a curcumin derivative called CNB001, which is more stable
and shows stronger cell-protecting effects than curcumin (Liu et al.,2008; Maher et al., 2010). We ﬁrst compared the anti-inﬂammatory
effect of CNB001 with that of dexamethasone (DEX) and curcumin. We
then evaluated the effects of CNB001 on pathways that are thought tobe involved in remodeling.
Materials and methods

19of CNB001, curcumin, and DEX (10 mmol/L) were prepared in ethanoland diluted to the appropriate concentration with culture medium.
CNB001, curcumin, or DEX was added to the medium at the concentrations described in each experiment. One hour later, Poly(I:C) was
added at a concentration of 10 μmol/L. Cells and supernatants werecollected 18 h after treatment of Poly:IC. Preliminary experiments
showed that CNB001 was not toxic at the dose of 10 μmol/L. A
trypan-blue exclusion test of viability showed no signiﬁcant difference between the control and the CNB001 group (trypan-blue positive cells, 4.6% ± 0.6%, 3.2% ± 1.7%, respectively, P > 0.05 NS, n = 6).
Initial control experiments included solvent (ethanol) at doses of up
to 1 μL/mL, which did not signiﬁcantly affect cell viability or production of cytokines.
To induce EMT, cells were grown on 10-cm dishes until 80% to100% conﬂuency. They were then incubated with TGF-β1 (5 ng/mL)with or without TNF-α (10 ng/mL).
Real-time PCR and immunoassay
Total RNA was extracted by using Trizol Reagent (Invitrogen Corporation, Carlsbad, CA, USA) and then reverse transcribed by using a
cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). A
Light Cycler-Fast Start DNA Master SYBR Green I kit (Roche Diagnostics, Mannheim, Germany) was used for real-time PCR. Primers arelisted in Table 1.
Interleukin-6 (IL-6), granulocyte-macrophage colony stimulating
factor (GM-CSF), and TNF-α levels were measured by using a Quantikine Immunoassay kit (R&D systems, Minneapolis, MN, USA). Active
human SERPINE1 (PAI-1) was measured by using a speciﬁc ELISA
Quantifying kit (Molecular Innovations, MI, USA).

Chemicals

Animals

Curcumin, dexamethasone (DEX), polyinosinic:polycystidylic acid
(Poly(I:C)), human recombinant tumor necrosis factor (TNF)-α, andhuman TGF-β1 were purchased from Sigma-Aldrich (St. Louis, MO,
USA). House dust mite allergen (HDM) derived from Dermatophagoides farinae (Derf) was provided by Dr. Shirai (Itea Inc., Tokyo,
Japan), and contained 8 μg/mg major allergen Derf1. Ovalbumin(OVA) was purchased from Sigma (St. Louis, MO, USA).

Speciﬁc-pathogen-free female BALB/c mice (8 weeks old) werepurchased from SLC (Shizuoka, Japan). The animals were housed ina speciﬁc pathogen-free environment and allowed free access tofood and water. All experimental protocols were approved by theMusashino University Animal Care Committee and were in accordance with the NIH Guide for the care and use of laboratory animals.
Mice were initially intraperitoneally immunized with 10 μg OVA and2 mg aluminum hydroxide gel on days 0 and 14, as previously
reported (Matsuo et al., 2009; Yamashita et al., 2006). From day 16
onwards, the animals were treated every other day for 2 weeks as follows. Mice were intraperitoneally injected with 25 mg/kg CNB001,
25 mg/kg curcumin, 1 mg/kg DEX, or solvent alone. CNB001 and curcumin were dissolved in 100% ethanol (40 μL for 1 mg) and then diluted 10 fold with 1% (v/v) Tween80/Saline (Sigma). DEX wasdissolved in 100% ethanol to a concentration of 0.1% w/v and then diluted 10 fold with 1% Tween80/Saline. Thirty minutes after intraperitoneal injection, the mice were subjected to inhalation of OVA,
followed by intranasal administration of Poly(I:C) (500 μg/kg) andHDM (8 μg/kg) dissolved in 80 μL of saline. Saline alone was used
for control mice. Inhalation of OVA (20 mg/mL) was conducted at a

CNB001
CNB001 was synthesized as a hybrid molecule between cyclohexyl
bisphenol A and curcumin, both of which have a proven neuroprotective effect (Liu et al., 2008). The purity of CNB001 was more than 98%.
CNB001 is reported to be superior to curcumin in neuroprotective effects in cell-culture assays for trophic factor withdrawal, oxidative
stress, and glucose starvation, as well as amyloid-induced toxicity(Liu et al., 2008).
Cell culture and treatment
Normal human bronchial epithelial (NHBE) cells were purchasedfrom Lonza (Walkersville, MD, USA). The cells were cultured in bronchial epithelial growth medium (BEGM) supplemented with BulletKit(Lonza Walkersville, Inc. MD, USA) on collagen type I-coated dishes at
37 °C in a 5% CO2/95% air incubator. BulletKit contains human recombinant epidermal growth factor, hydrocortisone, transferrin, insulin,
bovine pituitary extract, retinoic acid, triiodothyronine, gentamicin,
and amphotericin (concentrations not stated). Cells were used onpassage 3 for experiments pertaining to EMT, and on passages 4 to
6 for other experiments.
After the NHBE cells reached 80% to 100% conﬂuency, the cells
were incubated overnight in BEGM without BulletKit. Stock solutions

109

Table 1
Primers used for real-time PCR.
Targetgene

Primer sequences

Amplicon size (bp)

GAPDH

Forward 5′-TGCACCACCAACTGCTTAGC-3′
Reverse 5′-GGCATGGACTGTGGTCATGAG-3′
Forward 5′-TGAAGGTGACAGAGCCTCTGGAT3′
Reverse 5′-TGGGTGAATTCGGGCTTGTT-3′
Forward 5′-CCTTGAACGCAAAGTGGAATC-3′
Reverse 5′-CTGAGTTTCCGAATAGCCTG-3′

87

E-cadherin

Vimentin

151

106

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O. Narumoto et al. / Experimental and Molecular Pathology 93 (2012) 18–25

A
IL-6 (pg/mL)

ﬂow rate of 0.3 mL/min for 10 min by using a micromist nebulizer(Devilbiss, Somerset, PA, USA).
Twenty-four hours after the last treatment, the mice were sacriﬁced and bronchoalveolar lavage ﬂuid (BALF) was obtained by instilling and aspirating 3 times with 1 mL aliquots of PBS (recovery>85%). BALF was centrifuged at 540 g for 10 min (at 4 °C) and the supernatants were stored at −80 °C for ELISA. The cell pellets were
resuspended in 1 mL PBS. After total BALF cell counts were obtained,
the resuspended cells were adhered onto glass slides at 640 rpm for
2 min by using a Shandon Cytospin 4 (Thermo Electron Corp., Waltham,
MA, USA). The BALF cells on glass slides were stained with Diff-Quik(International Reagents Corp, Osaka, Japan) and were differentiallycounted at 200 cells per slide (one slide per animal).
For histological examination, mice were perfused transcardiallywith PBS and then 4% paraformaldehyde in 100 mM phosphate buffer(pH 7.4). The lungs were removed and then immersed in 4% paraformaldehyde. After washing with PBS, the specimens were soaked
overnight in 30% sucrose in PBS, embedded in optimal cutting temperature (OCT) compound (Miles, Elkhart, IN, USA), and then quickly
frozen. Cryostat sections were cut and thaw-mounted on poly-L-lysinecoated glass slides (Matsunami Glass, Osaka, Japan). The samples wereanalyzed by haematoxylin–eosin (HE) staining.
Concentrations of IL-6, TNF-α, IL-13, and active murine SERPINE1(PAI-1) in BALF were measured by using a speciﬁc ELISA Quantifyingkit (R&D systems, MN, USA and Molecular Innovations, MI, USA, respectively) according to the manufacturers' instructions.

1800
1500
1200
900
600
300
0

*

1500

*

*

*
*

IL-6 (pg/mL)

B
*

1200
900
600
300
0

C
IL-6 (pg/mL)

Statistical analysis
The signiﬁcance of the difference between groups was assessed byusing the Wilcoxon/Kruskal–Wallis nonparametric test as implemented in JMP8.0.2 (SAS Institute, Cary, NC, USA). The test was conducted
as two sided. P values less than 0.05 were considered to be signiﬁcant.
The data were expressed as mean ± SEM.

2000
1600
1200
800
400
0

*

*

*

Results
Comparing the effects of CNB001, curcumin, and DEX on
Poly(I:C)-induced expression of inﬂammatory cytokines
Poly(I:C), a synthetic analog of viral double-stranded RNA and a
Toll-like receptor (TLR) 3 ligand, acts as a potent proinﬂammatory
stimulus for lung epithelial cells by inducing the secretion of cytokines, which are important for allergic airway inﬂammation (Bachar
et al., 2004; Groskreutz et al., 2006). As shown in Fig. 1A, and laterin Fig. 2, stimulation of NHBE cells with 10 μg/mL Poly(I:C) caused asigniﬁcant increase in expression of inﬂammatory cytokines, IL-6,
GM-CSF, and TNF-α. We ﬁrst tested whether CNB001, DEX, or curcumin could suppress the expression of inﬂammatory cytokines. Pretreatment with either CNB001 or curcumin for 1 h signiﬁcantly
blocked the expression of IL-6 in a dose-dependent manner (Figs.
1A, B). DEX signiﬁcantly inhibited the expression of IL-6 in a dosedependent manner up to a concentration of 0.1 μmol/L; however,higher concentrations of DEX did not further reduce the expressionof IL-6 (Fig. 1C). The maximum suppression was 94% at the dose of
10 μmol/L for CNB001, 88% at the dose of 10 μmol/L for curcumin,
and 73% at the dose of 0.1 μmol/L for DEX. In all further experiments,
we used CNB001 and curcumin at a concentration of 10 μmol/L, andDEX at a concentration of 0.1 μmol/L.
We next compared the anti-inﬂammatory effects of CNB001 withthose of DEX and curcumin. CNB001 inhibited Poly(I:C)-induced production of IL-6, GM-CSF, and TNF-α to a signiﬁcantly greater extent
than did DEX or curcumin (Fig. 2). Addition of CNB001 to DEX suppressed inﬂammatory cytokines signiﬁcantly more effectively thandid DEX alone; however, the levels of inﬂammatory cytokines observed were almost the same as those obtained with CNB001 alone.

Fig. 1. Dose-dependent effects of CNB001, dexamethasone, and curcumin on Poly(I:C)induced expression of IL-6. NHBE cells were treated with CNB001 (panel A), curcumin(panel B), or dexamethasone (DEX) (panel C) at the concentrations indicated for 1 hprior to stimulation by Poly(I:C). Cell supernatants and cells were harvested 18 h
after the addition of Poly(I:C). Deﬁnition of abbreviations: CNB1, 1 μmol/L CNB001;
CNB2.5, 2.5 μmol/L CNB001; CNB5, 5 μmol/L CNB001; CNB10, 10 μmol/L CNB001;
CUR1, 1 μmol/L curcumin; CUR2.5, 2.5 μmol/L curcumin; CUR5, 5 μmol/L curcumin;
CUR10, 10 μmol/L curcumin; DEX0.01, 0.01 μmol/L DEX; DEX0.1, 0.1 μmol/L DEX;
DEX1, 1 μmol/L DEX; DEX10, 10 μmol/L DEX. Data represent means ± SD, *P b 0.05,
n = 3 for each group. Similar experiments were conducted 3 times, and one representative experiment is shown.

CNB001 reduces EMT induced by TGF-β1 plus TNF-α
Next, we examined the effect of CNB001 on EMT, which is related
to airway remodeling. TGF-β1 is the best-known inducer of EMT in
airway epithelial cells, and induction of EMT is enhanced by combining TGF-β1 with TNF-α (Doerner and Zuraw, 2009; Takahashi et al.,2010). To examine the suppressive effects of CNB001, we used
TGF-β1 and TNF-α together as stimulants to induce EMT in NHBE
cells. We examined the expression of E-cadherin and vimentin,
which are markers of EMT (Zavadil and Bottinger, 2005). After 72 h,
the cells were harvested, and the expression levels of E-cadherin
and vimentin were analyzed by performing real-time PCR. Costimulation by TGF-β1 and TNF-α signiﬁcantly decreased the expression of E-cadherin and signiﬁcantly increased the expression ofvimentin (Fig. 3). Treatment with CNB001 signiﬁcantly suppressed
the decrease in E-cadherin expression and the increase of vimentin

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O. Narumoto et al. / Experimental and Molecular Pathology 93 (2012) 18–25

*

A

*
*

900
600

E-Cadherin/
GAPDHmRNA

1200

*

*

300
0

B
GM-CSF(pg/mL)

1200

*

*

*

*

600
300
0

TNF-α(pg/mL)

C

90
60

*

*

0.06
0.04
0.02
0

B

*

900

*

0.08

vimentin/
GAPDHmRNA

IL-6 (pg/mL)

A

21

*

*

0.6
0.4
0.2
0

Fig.3. Effects of CNB001 and dexamethasone on the expression of E-cadherin andvimentin. EMT was induced in NHBE cells by 72-h treatment with TGF-β1 (2 ng/mL)
plus TNF-α (4 ng/mL). The cells were either treated with TGF-β1 plus TNF-α, or TGFβ1 plus TNF-α in combination with CNB001 (10 μmol/L) or dexamethasone (DEX;
0.1 μmol/L). At the end of the induction period, the E-cadherin (panel A) and vimentin(panel B) mRNA expression levels in the cells were measured and normalized to levelsof GAPDH mRNA. Data represent means ± SD, *P b 0.05, n = 3 for each group. Similar
experiments were conducted 3 times, and one representative experiment is shown.

*

*

0.8

*

*

30
0

the production of active SERPINE1. When CNB001 was added to theDEX, the induction of active SERPINE1 was signiﬁcantly and greatly suppressed (Fig. 4).

expression, suggesting that CNB001 efﬁciently suppresses EMT. In
contrast, DEX caused no signiﬁcant change either in E-cadherin or
vimentin expression.
CNB001 inhibits active SERPINE1 synthesis
We next evaluated active SERPINE1 (PAI-1), which plays an important role in airway remodeling by regulating ECM proteolysis. SERPINE1
acts directly through plasmin formation and indirectly throughplasmin-mediated activation of matrix metalloproteinases (Kowal etal., 2008; Kucharewicz et al., 2003). For the stimulant, we used TGFβ1 (2 ng/mL), which is an inducer of SERPINE1 and is related to airway
remodeling (Dennler et al., 1998; Kowal et al., 2008; Miyamoto et al.,2011). Both CNB001 and curcumin suppressed TGF-β1-induced active
SERPINE1 (Fig. 4). CNB001 showed a signiﬁcantly stronger effect thanthat of curcumin. In contrast to CNB001, DEX signiﬁcantly augmented

111

Comparing the in vivo effects of CNB001, DEX, and curcumin by using
a murine model of asthma
We next evaluated the effect of CNB001 on the prevention of airway inﬂammation in vivo. An OVA-speciﬁc IgE induced mousemodel has been commonly used to examine allergic inﬂammation,
which can be efﬁciently suppressed by treatment of the model with
DEX alone (Brusselle et al., 1995; Chi et al., 2011; Wills-Karp et al.,1998). Intra-tracheal administration of HDM, which is a common allergen in humans, is recently shown to be a new asthmatic model

Active SERPINE1(U/mL)

Fig. 2. Anti-inﬂammatory effects of CNB001, curcumin, and dexamethasone. NHBE cells
were treated with CNB001, curcumin, or dexamethasone (DEX) for 1 h prior to stimulation by Poly(I:C). Control cells were not treated or stimulated. Eighteen hours after
the addition of Poly(I:C), cell supernatants and cells were harvested. Protein expression of IL-6 (panel A), GM-CSF (panel B), and TNF-α (panel C) was compared between
cell supernatants obtained from control cells, Poly(I:C)-stimulated cells, and Poly(I:C)stimulated cells treated with CNB001, curcumin, DEX, or CNB plus DEX. Deﬁnition ofabbreviations: CNB, 10 μmol/L CNB001; DEX, 0.1 μmol/L DEX; CUR, 10 μmol/L curcumin. Data represent means ± SD, *P b 0.05, n = 3 for each group. Similar experiments
were conducted 3 times, and one representative experiment is shown.

*

80
60

*

*

*
*

40
20
0

Fig. 4. Effect of CNB001 and dexamethasone on levels of active human SERPINE1. NHBE
cells were treated with CNB001 (10 μmol/L) or dexamethasone (DEX; 0.1 μmol/L) for
1 h prior to stimulation by TGF-β1 (2 ng/mL). *P b 0.05, n = 3 for each group. Similar experiments were conducted 3 times, and one representative experiment is shown.

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O. Narumoto et al. / Experimental and Molecular Pathology 93 (2012) 18–25

without systemic immunization (Fattouh et al., 2005; Gough et al.,
2003; Phipps et al., 2009; Trompette et al., 2009). In addition, combining Poly(I:C) stimulation with the OVA-speciﬁc IgE induced
model represents exacerbation of asthma induced by respiratory
viral infections and induced neutrophil accumulation in the BALF
(Kim et al., 2007; Takayama et al., 2011). To examine the effects ofvarious treatments on the control of severe airway inﬂammation,
we used the mouse model exposed to OVA plus HDM plus Poly(I:C).
In these mice, the total cell counts in BALF, especially neutrophil
counts, were signiﬁcantly suppressed by CNB001 treatment (Figs.
5A, B), but not by curcumin treatment. In addition, adding CNB001
to DEX signiﬁcantly increased the suppressive effects comparedwith those of DEX alone. Eosinophil counts were not signiﬁcantly
suppressed by CNB001 alone, but were signiﬁcantly suppressed when
CNB001 was used in combination with DEX (Fig. 5C). Histological

Discussion

Fig. 5. In vivo effect of CNB001, curcumin, and dexamethasone on blood cell counts inbronchoalveolar lavage ﬂuid of a murine asthmatic model. OVA-sensitized BALB/cmice were intraperitoneally injected with 25 mg/kg CNB001 (CNB), 25 mg/kg curcumin (CUR), 1 mg/kg dexamethasone (DEX), or solvent alone. Thirty minutes after,
the mice were intranasally administered house dust mite and Poly(I:C), and OVAimmunized by inhalation of ovalbumin (OVA) (indicated as OPM in the ﬁgure). Control
mice were treated with saline alone. Total cell numbers (A), neutrophil (B) and eosinophil numbers (C) in bronchoalveolar lavage ﬂuid are shown. Data represent means± SD, *P b 0.05, n = 7–8 for each group.

We studied the effects of a new curcumin derivative, CNB001, as atherapeutic agent for airway inﬂammation and remodeling. By using
epithelial cells stimulated by Poly(I:C) in vitro, we showed that
CNB001 suppressed the cytokines IL-6, TNF-α, and GM-CSF, which
are produced by epithelial cells during the inﬂammation and play important roles in the pathogenesis of asthma (Doherty and Croft, 2011;
Morjaria et al., 2011; Saha et al., 2009). Moreover, CNB001 suppressed these cytokines more effectively than did DEX or curcumin.
Our in vivo study of a mouse model of asthma showed that CNB001
suppressed IL-6, TNF-α, and IL-13 production, and reduced the number of inﬂammatory cells, especially neutrophils, in BALF. Furthermore, CNB001 prevented EMT in vitro, and suppressed the activityof SERPINE1, a protein that is associated with airway remodelingboth in vitro and in vivo. In addition, combining CNB001 with DEX
suppressed the activity of SERPINE1, which was augmented by DEX
alone. Our results showed the promising effects of CNB001, in combination with DEX, on airway inﬂammation and remodeling.
The anti-inﬂammatory effects of curcumin are mediated throughinhibition of the activation of NF-κB and p38MAPK (mitogen-activated protein kinases) (Camacho-Barquero et al., 2007; Jang et al., 2007;
Song et al., 2010; Sugimoto et al., 2002; Venugopal et al., 2007). Curcumin inhibits the phosphorylation of I-κB (inhibitor of NF-κB) andprevents translocation of NF-κB into the nucleus (Oh et al., 2011;
Song et al., 2010). NF-κB is activated by Poly(I:C), which we usedhere for stimulation. NF-κB plays a central role in the production of
inﬂammatory cytokines such as TNF-α, IL-6, and IL-8 (Bruewer etal., 2003; Wong and Tergaonkar, 2009). In our in vitro and in vivo
study, CNB001 suppressed the synthesis of inﬂammatory cytokines
to a signiﬁcantly greater extent than did curcumin. By using the invivo model, we also showed signiﬁcant inhibition of IL-13, which isa critical cytokine for asthma pathogenesis (Wills-Karp et al., 1998).
Airway remodeling, which is a cause of decreased respiratory
function in asthma and cannot be effectively treated by standard
therapies, has become one of the major targets of therapies for asthma. In airway remodeling, epithelial cells produce inﬂammatory cytokines that induce smooth muscle cell hyperplasia and an increase inactivated ﬁbroblasts and myoﬁbroblasts that deposit excessive extracellular matrix (Skold, 2010; Westergren-Thorsson et al., 2010). Recent evidence suggests that epithelial cells play an active role ininitiating this process (Hackett et al., 2009; Holgate et al., 2000;
Willis et al., 2006). Because there is no generally accepted methods
to deﬁne airway remodeling yet, the evidence of reduced EMT is acceptable to explain the prevent airway remodeling, we examinedthe effect of CNB001 on EMT. Loss of E-cadherin deﬁnes epithelial dedifferentiation, and an increase in vimentin represents differentiation
to mesenchymal cells, which represents EMT (Willis et al., 2006;
Zavadil and Bottinger, 2005). EMT can be induced by TGF-β1 and enhanced by TNF-α, IL-1β, and ﬁbronectins (Dennler et al., 1998;
Takahashi et al., 2010). Here, we conﬁrmed the loss of E-cadherin expression and the increase in vimentin expression in epithelial cells

analysis also demonstrated that CNB001 suppressed airway inﬂammation beneath airway epithelial cells (Fig. 6C).
We then measured cytokine production in the BALF of the mousemodel exposed to OVA plus HDM plus Poly(I:C). CNB001 signiﬁcantly
suppressed IL-6, TNF-α, and IL-13 expression in these mice (Figs. 7A–C).
We chose IL-6 and TNF-α in order to compare effects of CNB001 in vivo
with that in vitro. We also examined IL-13 to analyze the effects of
CNB001 on Th2 allergic inﬂammation in vivo. Furthermore, the concentration of active murine SERPINE1 in BALF was signiﬁcantly suppressed by
CNB001, and signiﬁcantly enhanced by DEX (Fig. 7D). Addition of
CNB001 plus DEX tended to suppress the level of active SERPINE1 whencompared with that observed after addition of DEX alone, though the difference was not statistically signiﬁcant (Fig. 7D).

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A

B

C

D

E

F

23

Fig. 6. Histological analysis by hematoxylin and eosin staining. OVA-sensitized BALB/c mice, treated as described in the legend of Fig. 5, were subjected to histological examination.
The mice were perfused transcardially with PBS and then 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4). A, control; B, OVA + Poly(I:C) + house dust mite (OPM);
C, OPM plus CNB001; D; OPM plus curcumin; E; OPM plus dexamethasone (DEX), F; OPM plus CNB001 plus DEX. → eosinophils ➨ neutrophils (arrows indicated some of typicalcells, original magniﬁcation × 1000).

treated with TGF-β1 and TNF-α in vitro, suggesting the occurrenceof EMT.
We also focused on the production of active SERPINE1, which isreported to play an essential role in tissue remodeling of lung, kidney,and cardiovascular systems after inﬂammation (Ha et al., 2009; Huang
et al., 2008; Kuramoto et al., 2009; Shetty et al., 2008; Sisson et al.,2009). Kuramoto et al. reported increased expression of SERPINE1 in

BALF of murine asthma models compared with that of healthy mice
(Kuramoto et al., 2009). In addition, they showed that inhalation of
urokinase-type plasminogen activator reduces the level of active
SERPINE1 and suppresses airway remodeling in these mice
(Kuramoto et al., 2009). Miyamoto et al. report that airway remodeling is suppressed in Serpine1-deﬁcient mice compared to wildtype mice, and following intra-airway administration of Serpine1

B

C

D
*

Active SERPINE1(ng/mL)

A

1

*

0.8
0.6

*

*

0.4
0.2
0

Fig. 7. In vivo effect of CNB001, curcumin, and dexamethasone on cytokine synthesis in a murine asthma model. IL-6 (A), TNF-α (B), IL-13 (C), and active murine SERPINE1(D) levels were measured in bronchoalveolar lavage ﬂuid obtained from OVA-sensitized BALB/c mice that were treated as described in the legend of Fig. 5. OPM indicates OVA+ Poly(I:C) + house dust mite. Data represent means ± SD, *P b 0.05, n = 5–6 for each group.

113

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O. Narumoto et al. / Experimental and Molecular Pathology 93 (2012) 18–25

siRNA in wild-type mice (Miyamoto et al., 2011). Although DEX possesses strong anti-inﬂammatory effects, DEX and its receptor complex promote the expression of Serpine1 by affecting the promoter
region of the Serpine1 gene (Kimura et al., 2009; Wickert et al.,2007; Yamamoto et al., 2004). This may be one of the reasons that
glucocorticoids cannot prevent airway remodeling (Kuramoto etal., 2009). Here, we observed that DEX increased the level of active
SERPINE1 both in vivo and in vitro, but, when combined with
CNB001, it suppressed the level of active SERPINE1. These results
suggest that a combination of glucocorticoids and CNB001 might
prevent increased expression of SERPINE1, and thereby suppress
airway remodeling.
Our results suggest that a new curcumin derivative, CNB001, could
potentially be used for controlling the function of airway epithelial
cells by reducing inﬂammation and airway remodeling in asthma.
We focused on epithelial cells in this study, and further studies areneeded to determine the effects of this derivative on other cell
types involved in EMT and airway remodeling.

Conﬂict of interest statement
No conﬂicts of interests are declared by the authors.

Acknowledgments
This study was supported in part by Grants-in-Aid for Scientiﬁc Research from the Ministry of Education, Science, Sports and Culture,
Japan; a grant for Allergic Disease and Intractable Diseases from
the Ministry of Health, Labor and Welfare; a grant from the Respiratory
Failure Research Group from the Health and Labor Sciences; andYamazaki Spice Promotion Foundation. We also thank Michiko Tachibana for her excellent technical assistance.
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Veterinary Immunology and Immunopathology 145 (2012) 447–452

Contents lists available at SciVerse ScienceDirect

Veterinary Immunology and Immunopathology
journal homepage: www.elsevier.com/locate/vetimm

Research paper

Large-scale survey of adverse reactions to canine non-rabies combined
vaccines in Japan
Kazuki Miyaji a , Aki Suzuki a , Hidekatsu Shimakura a , Yukari Takase a , Akio Kiuchi a ,Masato Fujimura b , Goro Kurita c , Hajime Tsujimoto d , Masahiro Sakaguchi a,∗a

Department of Veterinary Microbiology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara,
Kanagawa 252-5201, Japanb
Fujimura Animal Hospital, 5-10-26, Aomatanihigashi, Minou, Osaka 652-0022, Japanc
Kurita Animal Hospital, Furukawa, Furukawa, Ibaraki 306-0016, Japand
Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo
113-8657, Japana r t i c l e

i n f oArticle history:
Received 1 December 2011
Received in revised form
27 December 2011
Accepted 29 December 2011
Keywords:
Canine non-rabies combined vaccine
Adverse event
Anaphylaxisa b s t r a c t
Canine non-rabies combined vaccines are widely used to protect animals from infectious
agents, and also play an important role in public health. We performed a large-scale survey
to investigate vaccine-associated adverse events (VAAEs), including anaphylaxis, in Japanby distributing questionnaires on VAAEs to veterinary hospitals from April 1, 2006 throughMay 31, 2007. Valid responses were obtained for 57,300 vaccinated dogs at 573 animal
hospitals; we obtained VAAEs information for last 100 vaccinated dogs in each veterinary
hospital. We found that of the 57,300, 359 dogs showed VAAEs. Of the 359 dogs, death wasobserved in 1, anaphylaxis in 41, dermatological signs in 244, gastrointestinal signs in 160,
and other signs in 106. Onset of VAAEs was mostly observed within 12 h after vaccination(n = 299, 83.3%). In this study, anaphylaxis events occurred within 60 min after vaccination,
and about half of these events occurred within 5 min (n = 19, 46.3%). Furthermore, whereanaphylaxis was reported, additional information to support the diagnosis was obtainedby reinvestigation. Our resurvey of dogs with anaphylaxis yielded responses on 31 dogs;
27 of these demonstrated collapse (87.1%), 24 demonstrated cyanosis (77.4%), and bothsigns occurred in 22 (71.0%). Higher rates of animal VAAEs, anaphylaxis, and death were
found in Japan than in other countries. Further investigations, including survey studies,
will be necessary to elucidate the interaction between death and vaccination and the risk
factors for VAAEs, and thus develop safer vaccines. Moreover, it may also be necessary tocontinually update the data of VAAEs.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction

protecting animals from infectious agents and plays animportant role in public health. No vaccine, however, iscompletely effective or without adverse reactions, andvaccine-associated adverse events (VAAEs) do occur, albeit
infrequently, after vaccinations. Epidemiological surveys in
the United Kingdom (Gaskell et al., 2002) and the UnitedStates (Moore et al., 2005) identiﬁed the rates for VAAEs as
0.093 and 38.2/10,000 vaccinated dogs, respectively.
VAAEs can include anaphylaxis, which is an acute multisystem, severe type I hypersensitivity reaction (Sakaguchi

Canine non-rabies combined vaccines, containing
zoonotic pathogen leptospira, are widely used in small
animal veterinary medicine. Vaccination is aimed at∗ Corresponding author. Tel.: +81 42 754 7111x705;
fax: +81 42 754 7661.
E-mail address: sakagum@azabu-u.ac.jp (M. Sakaguchi).
0165-2427/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2011.12.023

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K. Miyaji et al. / Veterinary Immunology and Immunopathology 145 (2012) 447–452

Fig. 1. The questionnaire form used for the investigation of vaccine-associated adverse events (VAAEs). The questionnaire was distributed by the Japan
Small Animal Veterinary Association to veterinary hospitals in Japan from April 1, 2006 through May 31, 2007.

hospitals in Japan from April 1, 2006 through May 31, 2007.
The questionnaires not only recorded standard information: date of birth, breed, sex and neuter status, weight,
and date of vaccination, but also included important factors
associated with adverse reactions, such as type of vaccination, signs, and time of their onset since vaccination (Fig. 1).
Practicing veterinarians diagnosed adverse reactions andclassiﬁed them into 5 groups according to clinical signs:
death, anaphylaxis, dermatological signs (swelling of face,pruritus, urticaria, ﬂush, and erythema), gastro-intestinal
signs (vomiting and diarrhea), and other signs (including hypodynamia and anorexia). In cases of anaphylaxis,
additional information was obtained by reinvestigation
to support the diagnoses. To deﬁne the population, each
responding veterinarian also stated the number of affectedcases per last 100 vaccinated dogs.

et al., 1995; Roth, 1999) occasionally observed in humans
and dogs after vaccination, and which sometimes causes
death (Brooks, 1991; Sakaguchi et al., 2000). According
to large epidemiological studies on canine VAAEs, theincidences of anaphylaxis were 0.018/10,000 vaccinateddogs in the United Kingdom (Gaskell et al., 2002) and
0.65/10,000 vaccinated dogs in the United States (Moore
et al., 2005). Based on a survey of a small number (35)of Japanese veterinary hospitals, the anaphylaxis rate inJapan appeared to be somewhat higher, at 0.17% (6/3477
vaccinated dogs) (Fujimura, 2006). However, to date, nolarge-scale investigation had been carried out on VAAEs
associated with canine non-rabies combined vaccine inJapan. Moreover, although incidences of anaphylaxis were
provided, none of the previous studies (Gaskell et al., 2002;
Moore et al., 2005; Fujimura, 2006) considered importantdetails, such as concurrent symptomatic states.
Here, we report the results of a large-scale survey of
VAAEs in Japan, based on diagnosis made by experienced
veterinarians in their practices. Our results reveal useful
information on VAAEs, including critical detail on anaphylaxis and death caused by canine non-rabies combined
vaccines. It is useful to record vaccine reactions routinely,
and report VAAEs to speciﬁc agents such as local governments in veterinary medicine as well as human medicine.

2.2. Vaccines
The following non-rabies combined vaccines for dogs
are commonly used in Japan: combined live vaccines composed of canine parvovirus, canine distemper virus, canine
adenovirus type 2, and canine parainﬂuenza virus (group1); adding live or inactivated coronavirus to group 1 (group2); adding inactivated leptospira to group 1 (group 3);
adding inactivated coronavirus and leptospira to group 1(group 4); adding live coronavirus and inactivated leptospira to group 1 (group 5). Duramune MX5 (Kyoritsu
Seiyaku Corporation, Tokyo, Japan; Fort Dodge Animal
Health, Iowa, USA), Eurican 5 (Merial Animal Health,Lyon, France), and Nobivac DHPPi (Intervet, Boxmeer,
Netherlands) were classiﬁed as group 1; Canine-6II (Kyoto
Biken Laboratories, Kyoto, Japan), Duramune DX6, and Vanguard Plus 5/CV (Pﬁzer Animal Health, New York, USA)2. Materials and methods
2.1. Experimental design and questionnaire for study ofcanine vaccine associated adverse events
The questionnaires about adverse reactions to canine
non-rabies combined vaccines were distributed by theJapan Small Animal Veterinary Association to veterinary117

K. Miyaji et al. / Veterinary Immunology and Immunopathology 145 (2012) 447–452

449

Table 1
Number and incidence of clinical signs of vaccine-associated adverse
events (VAAEs) in 57,300 vaccinated dogs.
Clinical signs

Numbers of VAAEsa(per 10,000 dogs)

Anaphylaxis
Death
Dermatological signs
Gastrointestinal signs
Others41 (7.2)
1 (0.2)244 (42.6)
160 (27.9)
106 (18.5)

aTotal number was over 359 because multiple signs were present insome dogs.

were classiﬁed as group 2; Canine-8, Eurican 7, and Nobivac DHPPi+L as group 3; Duramune DX8 and Vanguard Plus
5/CV-L as group 4; and Canine-9 (II) as group 5.

Fig. 2. Numbers of dogs with vaccine-associated adverse events (VAAEs),
grouped by time of onset of signs. A total of 359 dogs exhibited VAAEs,
including one death. Signs manifested within 12 h of vaccination in 299
cases.

2.3. Statistical analyses
events (n = 299, 83.3%) were observed within 12 h after vaccination (Fig. 2).

Sex and neuter status were analyzed as categorical
data. Continuous variables of age and weight were converted to categorical variables because nonlinear trends
were detected in the model-ﬁtting process. Dogs weregrouped on the basis of age at date of vaccination as
follows: 2–9 months, 9 months to 1.5 years, 1.5–2.5 years,
2.5–3.5 years, 3.5–5.5 years, 5.5–8.5 years, and >8.5 years.
Weight was converted from continuous to categorical dataof 0–5 kg, 5–10 kg, and > 10 kg. The P values for anaphylaxis
in total VAAEs were evaluated using a multivariate unconditional logistic regression model. The variants included
sex and neuter status, weight, age, and vaccine group.
The model was assessed for signiﬁcance by use of the leCessie–van Houwelingen test. A value of P < 0.05 was considered signiﬁcant. Statistical analysis was performed withR version 2.11.1 (www.r-project.org/).

3.2. Anaphylaxis and deathMore detailed information about vaccine-associatedanaphylaxis is provided in Tables 2 and 3, as well as
Fig. 3. Miniature Dachshunds (n = 13) accounted for approximately 30% of the anaphylaxis cases. Anaphylaxis occurredin four Miniature Schnauzers out of a population of theeight with VAAEs. Of 41 dogs diagnosed with anaphylaxis,
17 (41.5%) were sexually intact males, 18 (43.9%) wereintact females, 3 (7.3%) were castrated males, and 3 (7.3%)
were spayed females. According to age and weight, thegreatest frequency of anaphylaxis was recorded amongdogs aged 2–9 months (n = 20, 48.8%) and among dogs
weighting less than 5 kg (n = 27, 65.9%), respectively. A
multivariate logistic regression model, including sex andneuter status, weight, and age satisﬁed requirements for
goodness of ﬁt (P = 0.12). In the ﬁnal model, there was no
signiﬁcant relationship between anaphylaxis and the factors of sex and neuter status (P = 0.58), weight (P = 0.15), age(P = 0.24), or vaccine group (P = 0.96). All cases of anaphylaxis occurred within 60 min after vaccinations, and about
half occurred within 5 min (n = 19, 46.3%) (Fig. 3).

3. Results
3.1. Total adverse reactions
Valid responses were obtained for 57,300 vaccinateddogs, from 573 animal hospitals; of these, 359 dogs
were diagnosed with VAAE (62.7/10,000 vaccinated dogs).
Unfortunately, the detailed information regarding dogs
with no adverse events could not be obtained in the present
study. Of the 359 dogs, anaphylaxis was observed in 41,
dermatological signs in 244, gastrointestinal signs in 160,
and other signs in 106 (Table 1). A single death (0.2/10,000
vaccinated dogs) was reported within a few days after vaccination.
In decreasing order of frequency, 181 (50.4%) Miniature
Dachshunds, 37 (10.3%) Chihuahuas, 18 (5.0%) Mixedbreeds, and 17 (4.7%) Toy Poodles were reported to have
VAAEs in the present survey; other breeds were involved
less frequently. The population with VAAEs included 145(40.4%) sexually intact males, 156 (43.5%) sexually intact
females, 24 (6.7%) castrated males, 24 (6.7%) spayedfemales, and 10 (2.8%) unknown. Almost half these dogs
(n = 164, 45.7%) were between the ages of 2 and 9 months.
Dogs in the weight category of 0–5 kg had the highest frequency of adverse reactions (n = 249, 69.4%). Most adverse

Fig. 3. Numbers of dogs with anaphylaxis, grouped by time of onset. A
total of 41 dogs exhibited anaphylaxis signs; anaphylaxis set in within
60 min of vaccination, and within 5 min for 19 cases.

118

K. Miyaji et al. / Veterinary Immunology and Immunopathology 145 (2012) 447–452

450

Table 2
Data obtained for 41 cases, where anaphylaxis followed vaccination.
BreedaSexb

Age (years)

Weight (kg)

Onset time (min)Diagnostic reasonc

BeagleC.K.C. Spaniel
Chihuahua
J.R. Terrier
M. Dachshund

M
F
F
M
F
F
F
F
F
M
S
M
C
S
M
C
M
M
M
F
F
M
M
F
M
M
M
F
M
F
F
F
M
F
F
F
S
M
M
F
C

3.4
0.2
0.2
1.8
0.2
0.2
0.2
0.2
0.3
0.4
1.4
2.1
3.3
3.5
4.7
5.3
6.8
0.2
0.4
2.2
2.4
0.2
4.2
7.1
0.3
2.6
0.3
3.0
0.2
NA
9.6
0.2
0.3
0.3
0.2
0.2
1.4
1.4
13.3
0.2
1.3

8.0
2.2
0.8
4.4
1.7
1.7
2.4
2.6
1.5
1.7
4.9
4.4
5.6
6.8
5.6
4.4
4.6
0.6
3.7
6.2
9.5
2.0
11.0
3.7
16.1
5.9
1.0
7.2
10.5
9.0
11.5
1.6
1.9
2.2
1.3
2.4
2.5
2.6
3.8
1.7
14.8

10
1
3
2
5
10
5
10
5
30–40
15
30
30
40
<5
30
15
1
15
10
10
10
<10
15
11
1
15
5
10
5
5
<5
1
60
<5
<5
5–10
5
5
1
60

ND
Collapse, Cyanosis, Hyperpnea
ND
Collapse, Cyanosis
Collapse, Cyanosis, Hyperpnea
Collapse, Cyanosis
Collapse, Cyanosis
Collapse, Cyanosis
ND
Collapse, Cyanosis
ND
Cyanosis
Collapse, Cyanosis, Hypothermia
Collapse, Cyanosis
Collapse, Cyanosis
Collapse, Cyanosis
ND
Collapse, Hyperpnea
Collapse, Cyanosis
ND
Collapse, Cyanosis, Hypothermia, Dyspnea
Collapse, Cyanosis
ND
ND
Collapse, Cyanosis
ND
Collapse
Collapse, Cyanosis
Collapse, Cyanosis
Collapse, Cyanosis
Collapse, Cyanosis
ND
Collapse, Hyperpnea
Cyanosis, Hyperpnea
Collapse
Collapse, Cyanosis
Collapse, Cyanosis, Hyperpnea
Collapse, Cyanosis
ND
Collapse
ND

M. Schnauzer

Mix

Newfoundland
Pekingese
Pomeranian
Pug
Shiba
S. Sheepdog
Shih Tzu

Toy Poodle

Welsh Corgi

ND: no data.
aC.K.C. Spaniel: Cavalier King Charles Spaniel, J.R. Terrier: Jack Russell Terrier, M. Dachshund; Miniature Dachshund, M. Schnauzer: Miniature Schnauzer,
S. Sheepdog: Shetland Sheepdog.
b
M: sexually intact male, F: sexually intact female, C: castrated male, S: spayed female.
c
Diagnostic reasons were obtained from reinvestigation.

When we attempted to resurvey the 41 dogs that experienced anaphylaxis, responses were received for 31 of thesedogs (recovery ratio = 75.6%). As shown in Table 3, collapse
and cyanosis were observed in almost all 31 of the dogs,
with 27 (87.1%) demonstrating collapse, 24 (77.4%) demonstrating cyanosis, and 22 (71.0%) showing both signs. A
single death (Chihuahua, intact female, 2 months, 0.9 kg),

occurring 38 h after vaccination, was reported in our questionnaire.
4. Discussion
The risks involved in vaccination of dogs were highlighted by previous large epidemiological studies (Gaskell
et al., 2002; Moore et al., 2005); however, surveys on
VAAEs had only been performed on a small scale in Japan(Ohmori et al., 2002, 2005a; Fujimura, 2006). Here, we
present the results of a large-scale investigation of VAAEs
at Japanese veterinary hospitals. We found a VAAE rate of
62.7/10,000 vaccinated dogs, which is much higher than
the rates reported in the United Kingdom (0.093/10,000
vaccinated dogs) (Gaskell et al., 2002) and the United States(38.2/10,000 vaccinated dogs) (Moore et al., 2005). Previous reports suggested that small breed dogs (<10 kg),
especially Dachshunds, were more prone to VAAEs than

Table 3
Number of signs exhibited by anaphylactic dogs (n = 31).
Anaphylactic signsna%Collapse
CyanosisHyperpnea
Hypothermia
Dyspnea

27
24
6
2
1

87.1
77.4
19.4
6.5
3.2

a
Total number was over 31 because multiple signs were present insome dogs.

119

K. Miyaji et al. / Veterinary Immunology and Immunopathology 145 (2012) 447–452

larger dogs (Gaskell et al., 2002; Moore et al., 2005; Moore
and Hogenesch, 2010). According to the Japan Kennel Club(http://www.jkc.or.jp), Miniature Dachshund, Chihuahua,
and Toy Poodle are the most popular breeds in this country, and account for just over half of the total registrationsin the Club (50.3%). This popular breed bias and the proposed breed susceptibility may contribute to higher VAAEs
rate in Japan than in other countries.
Factors known to cause vaccine reactions include theprimary vaccine agent or antigen, adjuvants, preservatives, stabilizers, and residues from tissue culture used in
vaccine production (Hogenesch et al., 1999; Roth, 1999;
Georgitis and Fasano, 2001). Our previous study reported
large amounts of bovine serum albumin (BSA) and bovine
IgG contents in canine vaccines (Ohmori et al., 2005b). Furthermore, we found IgE reactivity against fetal calf serum(FCS) components in dogs with allergic reactions after vaccination, suggesting that most of these reactions might
be caused by FCS components derived from the culture
media used to produce vaccines (Ohmori et al., 2005b,
2007). Many vaccines that are commonly used in Japan areimported from abroad, although a few are made in Japan.
Multilateral studies are necessary to clarify the relationshipbetween VAAEs and vaccine by-products, and to ascertain
whether certain vaccines are more likely to give rise to
VAAEs.
Among the many clinical signs of adverse reactions tovaccines, anaphylaxis is the most dramatic (Roth, 1999).
In the present investigation, the incidence of anaphylaxis (7.2/10,000 vaccinated dogs) in dogs was remarkably
higher than those reported in previous studies (Gaskell
et al., 2002; Moore et al., 2005; Moore and Hogenesch,2010). The dogs showing anaphylaxis in the present study
included many small breeds such as Miniature Dachshundand Miniature Schnauzer. This implies that a breed predisposition may play a key role in anaphylaxis after
vaccination of dogs, just as in total VAAEs, and that genetic
factors may be involved in these clinical signs.
Moore et al. (2005) have indicated that the risk of VAAEs
was signiﬁcantly increased for small and neutered dogs,
highest for dogs approximately 1–3 years old, and leastfor dogs ≥6 years of age. Unfortunately, as the information regarding dogs with no adverse events could not be
obtained in the present study, the odds ratios of VAAEs
could not be estimated; however, we did estimate the Pvalues for anaphylaxis according to adverse reactions after
vaccine administrations using a logistic regression model.
Additionally, we were not able to analyze the relationship between breed and anaphylaxis due to small sample
numbers for many breeds. However, no signiﬁcant differences were observed in the anaphylaxis risk by sex andneuter status, weight, and age; thus, our data seem toshow the same tendency of potential risk factors for VAAEs
in the results of Moore et al. (2005). The results of thepresent study suggest that various factors such as weight
and age could predict the potential risk of adverse reactions, including anaphylaxis after vaccination, which may
allow practicing veterinarians to allay the anxiety of at leasta few owners regarding vaccination.
As only a single death was reported in this investigation, a direct association between death and vaccine

451

administration was not clear; however, as an incidence of
0.02/10,000 vaccinated dogs was reported in the UnitedState (Moore et al., 2005), the mortality rate also appeared
to be increased in Japan. This case did not show anaphylactic signs, although other clinical sign was observed. Further
extensive surveys are needed to uncover the predictive factors and clinical signs that precede such outcomes, in orderto prevent such unfortunate events. Moreover, to clarify
the interaction between death and vaccination, comparison with randomized unvaccinated dogs may be effective.
5. Conclusions
We present data regarding VAAEs, and in particular, on
anaphylaxis, based on a large-scale study in Japanese veterinary hospitals. Higher rates of VAAEs, anaphylaxis, anddeath were observed in Japan compared to other countries.
Further international studies aimed at obtaining similar
information will be necessary to elucidate the risk factors, including breed predisposition, the quality of vaccine
products, and prior sensitization with variable allergens,
for VAAEs, with a view to developing safer vaccines. In
addition, as our study was not current, it may be also benecessary to continually update the data of VAAEs.
Conﬂict of interest statement
None of the authors of this paper has a ﬁnancial or personal relationship with other people or organizations that
could inappropriately inﬂuence or bias the content of thepaper.
Acknowledgments
The authors would like to thank all staff of participating veterinary hospitals for their kind assistance in
responding to the questionnaires. We also thank the Japan
Small Animal Veterinary Association for its support. We
thank Dr. Sakae Inouye in Otsuma Women’s University
for critical review of this manuscript. This research waspartially supported by the Promotion and Mutual Aid
Corporation for Private Schools of Japan, Grant-in-Aid forMatching Fund Subsidy for Private University, and by a
Project Grant awarded by the Azabu University ResearchServices Division.
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FULL PAPER

Internal Medicine

Toshihiro TSUKUI1) , Masahiro SAKAGUCHI 2)*, Keigo KURATA 3), Sadatoshi MAEDA4), Keitaro OHMORI5),
Kenichi MASUDA6), Hajime TSUJIMOTO7) and Shigehiro IWABUCHI1)
1)Nippon Zenyaku Kogyo Co., Ltd., 1–1 Tairanoue Sasagawa, Asaka-machi, Koriyama, Fukushima 963– 0196, Japan2)Department of Veterinary Microbiology, School of Veterinary Medicine, Azabu University, 1–17–71 Fuchinobe, Chuo-ku,

Sagamihara, Kanagawa 252–5201, Japan
3)Institute of Tokyo Environmental Allergy, 2–2–4 Yushima, Bunkyo-ku, Tokyo 113– 0034, Japan4)Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1–1 Yanagido, Gifu, 501–1193, Japan
5)Laboratory of Veterinary Molecular Pathology and Therapeutics, Division of Animal Life Science, Graduate School, Institute ofSymbiotic Science and Technology, Tokyo University of Agriculture and Technology, 3–5–8 Saiwai-cho, Fuchu, Tokyo 183–8509, Japan
6)Animal Allergy Clinical Laboratories, Inc., 5–4–30 Nishihashimoto, Midori-ku, Sagamihara Kanagawa 252–0131, Japan
7)Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1–1–1

Yayoi, Bunkyo-ku, Tok yo 113–8657, Japan(Received 22 November 2010/Accepted 26 January 2012/Published online in J-STAGE 9 February 2012)
Escherichia coli

ABSTRACT.

Dermatophagoides farinae

KEY WORDS:

doi: 10.1292/jvms.10-0520; J. Vet. Med. Sci. 74(7): 851–856, 2012

in vitro
data describing the assays and comparing the available tests.
-

to be an immediate type hypersensitivity disease caused bydogs might represent an improvement in reagents available
In vitro-

techniques.

*C ORRESPONDENCE TO: S AKAGUCHI
252–5201, Japan.

Dogs naturally sensitized with mite allergen: All dogs

122

852

T. TSUKUI ET AL.

CA, U.S.A.).
: Microtiter-D. farinae
ng/ml

:
μ

-

Dermatophagoides farinae allergen

D. farinae allergen

above.
Heat inactivation of IgE in dog sera: To assess the heat

: A cDNA clone

D. farinae-

cially D. farinae
D. farinae

Escherichia
coli competent cells (Top10: Invitrogen Corp, Carlsbad, CA,-analysisμg/ml).

μg/ml) (Asahi

E.colil

μg/ml)
5–30 sec.
-

et al.
:
μg/ml

ng/ml

μg/ml

Statistical analysis
t-test; P-μg/ml(2 μg/ml): The E. coli

U.S.A.) in 0.1 M sodium citrate (pH 4.5). The optical density

123

853

MEASUREMENT FOR CANINE IGE

E. colito canine IgE: The results presented in Fig. 2 demonstrate the-: To evaluate the utilIgE to D. farinae
D. farinae
D. farinae
negative dogs.
Heat inactivation of IgE in dog sera

E. coli-E. colito 100 μl

allergens. The results presented in Fig. 4 document that all
dog sera contained D. farinae

ng/ml. The
sity.

124

-

854

T. TSUKUI ET AL.

-

DISCUSSION
D. farinae-

E. coli
et al.
IgE in western blotting-

D. farinae
D. farinae
D. farinae

125

855

MEASUREMENT FOR CANINE IGE

reactivity at concentrations as little as 3 ng, but reactivity
n

-

-

-

-

remains to be determined, it is logical to assume that its

represent a good adjunct to currently available monoclonal

μg/ml
μg/ml).

and immunoblotting detection reagents.
--

E. coli lysate. At---

University.
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Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 157–161

Contents lists available at ScienceDirect

Comparative Immunology, Microbiology
and Infectious Diseases
journal homepage: www.elsevier.com/locate/cimid

Induction of Th1 immune responses to Japanese cedar pollen allergen(Cry j 1) in mice immunized with Cry j 1 conjugated with CpG
oligodeoxynucleotide
Y. Kaburaki a , T. Fujimura b , K. Kurata c , K. Masuda d , M. Toda e , H. Yasueda f , K. Chida a ,S. Kawarai g , M. Sakaguchi g,∗a
Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku,
Tokyo 113-8657, Japanb
Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japanc
Institute of Tokyo Environmental Allergy, 2-2-4 Yushima, Bunkyo-ku, Tokyo 113-0034, Japand
Animal Allergy Clinical Laboratories, 5-4-21 Nishihashimoto, Sagamihara, Kanagawa 229-1131, Japane
Experimental Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Street 59, Langen 63225, GermanyfClinical Research Center for Allergy and Rheumatology, National Sagamihara Hospital, 18-1 Sakuradai, Sagamihara, Kanagawa 228-8522, Japang
Department of Veterinary Microbiology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 229-8501, Japana r t i c l e

i n f oArticle history:
Received 22 April 2010
Accepted 22 June 2010
Keywords:
Allergens
CpG
PollenIgEa b s t r a c t
We determined whether a major Japanese cedar pollen allergen (Cry j 1) conjugated withCpG oligodeoxynucleotide would enhance allergen-speciﬁc Th1 responses in mice. Cry j 1
conjugated with CpG (Cry j 1–CpG) induced IL-12 in the spleen cells of naïve mice. Cry j
1–CpG immunization of BALB/c mice suppressed anti-Cry j 1 IgE response and enhanced
anti-Cry j 1 IgG2a to subsequent Cry j 1 and alum adjuvant injection. CD4+ T cells isolated
from the spleens in mice immunized with Cry j 1–CpG produced higher IFN-␥ levels than
did CD4+ T cells obtained from mice as negative controls. Our results suggested that Cry j
1–CpG immunization can induce Cry j 1-speciﬁc Th1 immune responses, thereby inhibiting
IgE response to the pollen allergen.
© 2010 Elsevier Ltd. All rights reserved.

1. Introduction

and Cry j 2 [5]. Recently, we isolated Cry j 3, which may
play crucial roles in the cross-reactivity with oral allergy
syndrome [6].
Bacterial DNA and synthetic oligodeoxynucleotides
containing CpG are potent adjuvants of Th1 responses characterized by production of IFN-␥ and IgG2a [7–9]. Tokunaga
and his colleagues had found that DNA extracted from
Mycobacterium bovis activated NK cells to induce interferon and anti-tumor activities [10,11]. Several short DNA
sequences containing CpG motif were determined by thesequencing of mycobacterium DNA.
The administration of allergen conjugated to CpG is an
attractive approach to the prevention and treatment ofallergic diseases [9]. In a clinical study, ragweed pollen
allergen (Amb a 1) conjugated with CpG vaccine showedclinical efﬁcacy in the treatment of ragweed allergic rhinitis

Japanese cedar (Cryptomeria japonica; CJ) pollinosis is atypical type I allergy induced by CJ pollen and one of themost common allergic diseases in Japan. In areas where
there are CJ trees, approximately 10% of the population suffers from CJ pollinosis [1]. Two major allergens, Cry j 1 [2]and Cry j 2 [3] have been isolated from CJ pollen. More than
90% of 145 CJ pollinosis patients had speciﬁc IgE antibodies to both Cry j 1 and Cry j 2 allergens: the remainder had
speciﬁc IgE to either one or the other [4]. The patients with
CJ pollinosis had high levels of T-cell reactivity to Cry j 1∗ Corresponding author. Tel.: +81 42 850 2481; fax: +81 42 850 2481.
E-mail address: sakagum@azabu-u.ac.jp (M. Sakaguchi).
0147-9571/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cimid.2010.06.005

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Y. Kaburaki et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 157–161

2.5. Immunization[12]. Moreover, allergens conjugated with CpG reduced thebinding activity of speciﬁc IgE to native allergens and also
reduced allergenesis of the allergens [9,13]. Recently, it wasreported that Cry j 2 T-cell epitope peptides conjugated to
CpG vaccine have no allergencity [14].
In this study, we investigated whether Cry j 1–CpG
immunization suppressed the development of Cry j
1-speciﬁc IgE production in mice. Furthermore, we investigated the effect of Cry j 1–CpG on Th1 and Th2 cytokine
production in Cry j 1-speciﬁc CD4+ T cells.

Ten BALB/c mice in each group were subcutaneously
immunized with Cry j 1 (5 ␮g)–CpG, Cry j 1 (5 ␮g)–mCpG,
or Cry j 1 (5 ␮g) in PBS three times weekly. All mice wereintraperitoneally immunized with 5 ␮g of Cry j 1 and 2 mgof aluminum hydroxide (alum)(Pierce, Rockford, IL, USA)mixture at 3 and 6 weeks. Blood samples were collected bytail bleeding at weeks 1, 2, 4, 5, and 7 weeks, and serum
samples were stored at −30 ◦ C. Spleens for assays of IFN-␥,
IL-4, and IL-5 were collected at 7 weeks.

2. Materials and methods

2.6. Measurement of mouse anti-Cry j 1 IgG1, IgG2a , IgE

2.1. Mice

IgG1 and IgG2a antibody responses to Cry j 1 wereassayed by ELISA as described previously [20]. The IgE antibody response to Cry j 1 was analyzed by ﬂuorometric ELISA
as described previously [20].

Female BALB/c mice (7 weeks old) were obtained fromJapan SLC Inc (Shizuoka, Japan). They were housed under
speciﬁc pathogen-free conditions.

2.7. ELISA inhibition with human anti-Cry j 1 IgE
2.2. Antigens
The ability of Cry j 1–CpG to bind human Cry j 1-speciﬁc
IgE was measured by using ﬂuorometric ELISA inhibition
as described previously [21].

Cry j 1 is a basic glycoprotein isolated from CJ pollen
by afﬁnity chromatography using monoclonal antibodies(mAb) speciﬁc to Cry j 1 [15,16].

2.8. Cytokine assays in CD4+ T cells from spleens of
immunized mice

2.3. Conjugation of CpG oligodeoxynucleotides to Cry j 1

CD4+ T cells were puriﬁed from the spleens of mice by
MACS [22]. Puriﬁed CD4+ T cells (2 × 105 ) were cultured
with irradiated syngenic splenocytes (3 × 105 ) as antigenpresenting cells in 96-well plates for 3 days in the presenceor absence of Cry j 1 (10 ␮g/ml). The culture supernatant
was collected 3 days after cultivation and kept at −30 ◦ C
until use. IFN-␥, IL-4, and IL-5 levels were determined by
ELISA using anti-IFN-␥ mAb, anti-IL-4 mAb, and anti-IL-5
mAb (BD Biosciences) [20].

The phosphorothioated CpG, consisting of 22 bases containing two CpG motifs 5 -TGACTGTGAACGTTCGAGATGA3 (immunostimulatory sequence: ISS 1018) [17,18] wasproduced by Invitrogen Corp. (Carlsbad, CA, USA). Asa negative control, phosphorothioated oligodeoxynucleotide consisting of 22 bases containing the mutated5 -TGACTGTGAAGGTTAGAGATGA-3 (mCpG) was used inthis study. Each of these CpGs had a molecular mass ofapproximately 7000. The 5 -phosphorothioate CpG was
coupled to amino groups on Cry j 1. Cry j 1 was modiﬁed toincorporate thiol moieties by reacting to it with excess of
succinimidyl-S-acetyl thiol acetate (Pierce Biotechnology,
Rockford, IL, USA), as described previously [19]. Endotoxin
levels were measured by Bio-Whittaker, QCL-1000 (Cambrex Bio Science Walkersville, Walkersville, MD, USA). The
endotoxin levels of Cry j 1–CpG and Cry j 1–mCpG were<0.03 endotoxin unit/␮g.

2.9. Statistics
The Kruskal–Wallis test was applied to compare data
among the four groups. If the difference was signiﬁcant, post hoc analysis using the Mann–Whitney U-test
with Bonferroni correction in antibody levels and theTurkey–Kramer test in cytokine levels were performed toanalyze the difference between the ODN-Cry j 1 groupand the other group. Statistical signiﬁcance was deﬁned as
p < 0.05.

2.4. In vitro assay for IL-12 p40 assays in the naive
spleen cells

3. Results

Mice were sacriﬁced by means of anesthesia and theirspleens were removed. Spleen cells (2 × 105 ) were added
to wells in a total volume of 100 ␮L and stimulated withCry j 1–CpG, CpG, and mCpG. Cultures were incubated for0, 6, 11, 24, 35, and 48 h at 37 ◦ C in 5% CO2 . Supernatants
were harvested after each incubation period and frozen at−30 ◦ C before being analyzed. The levels of IL-12 p40 in theculture supernatant were determined by sandwich ELISA(BD Biosciences, Franklin Lakes, NJ, USA).

3.1. IL-12 p40 from naïve spleen cells
We investigated whether Cry j 1–CpG induced IL-12
p40 in the spleens of naïve mice. After spleen cells were
cultivated for 36 h, signiﬁcant IL-12 p40 production wasobserved in the cells stimulated with CpG-Cry j 1 and CpG
as compared with mCpG as a negative control (Fig. 1).

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Y. Kaburaki et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 157–161

Fig. 1. IL-12 p40 production by Cry j 1–CpG or CpG stimulation in
the spleens of naive mice. mCpG (1 ␮g/ml: 142 pmol), CpG (1 ␮g/ml:
142 pmol), Cry j 1–CpG (1 ␮g/ml: 142 pmol). *p < 0.05 for Cry j 1–CpG
compared with mCpG.

159

Fig. 3. Inhibition of anti-Cry j 1 IgE production by Cry j 1–CpG inoculation.
BALB/c mice were immunized with 5 ␮g each of Cry j 1, Cry j 1–CpG, Cry
j 1–mCpG, or PBS. **p < 0.01 for the group immunized with Cry j 1–CpG
compared with other groups.

3.2. Human anti-Cry j 1 IgE binding to Cry j 1–CpG
To determine whether the conjugation of CpG to Cry j
1 diminished the ability of Cry j 1 to react with anti-Cry j
1 IgE in pooled serum from patients with CJ pollinosis, theability of Cry j 1–CpG to bind anti-Cry j 1 IgE was tested by
the ELISA inhibition. Cry j 1–CpG reacted less well with antiCry j 1 IgE than did Cry j 1. An approximate 50-fold higher
concentration of Cry j 1–CpG was necessary to achieve 50%
inhibition compared with Cry j 1 (Fig. 2).

IgE production to a secondary immunization of Cry j 1 andalum.
3.4. Anti-Cry j 1 IgG1 and IgG2a in mice immunized withCry j 1–CpG
The effects of initial immunization of Cry j 1–CpG on the
production of anti-Cry j 1 IgG1 production were examined.
After ﬁrst immunization of Cry j 1 and alum at 3 weeks, antiCry j 1 IgG1 production in groups immunized with Cry j 1
and Cry j 1–mCpG was enhanced (Fig. 4A). After secondary
immunization of Cry j 1 and alum at 7 weeks, anti-Cry j 1
IgG1 production showed no signiﬁcant difference among
any of the groups.
The effects of initial immunization of Cry j 1–CpG on the
production of anti-Cry j 1 IgG2a production were examined.
At 1 and 2 weeks, anti-Cry j 1 IgG2a levels showed no differences among any of the groups. After immunization of
Cry j 1 and alum at 7 weeks, Cry j 1–CpG immunization
developed signiﬁcantly higher levels of anti-Cry j 1 IgG2acompared with groups immunized with PBS, Cry j 1, or Cry
j 1–mCpG (p < 0.01) (Fig. 4B).

3.3. Anti-Cry j 1 IgE in mice immunized with Cry j 1–CpG
We investigated whether initial immunization of Cry
j 1–CpG suppressed anti-Cry j 1 IgE development after
immunization of Cry j 1 and alum. In groups immunizedwith Cry j 1 or Cry j 1–mCpG, anti-Cry j 1 IgE production
was induced by boosting with Cry j 1 and alum at 4 and 6
weeks. Anti-Cry j 1 IgE production was signiﬁcantly inhibited in mice immunized with Cry j 1–CpG up to 7 weeks(Fig. 3). Cry j 1–CpG immunization suppressed anti-Cry j 1

3.5. Cytokine production in Cry j 1-speciﬁc CD4+ T cells
cultures
The levels of IFN-␥, IL-4, and IL-5 in response to Cry j 1
were measured in CD4+ T cells obtained 2 weeks after thesecond boosting of Cry j 1 in alum. CD4+ T cells from mice
immunized with Cry j 1–CpG secreted signiﬁcantly higher
levels of IFN-␥ than did CD4+ T cells obtained from mice
immunized with PBS, Cry j 1, or Cry j 1–mCpG (p < 0.05)(Fig. 5A).
Cry j 1-speciﬁc IL-5 levels in mice immunized with Cry
j 1–CpG showed signiﬁcantly lower than those in mice
immunized with Cry j 1–mCpG, but showed no signiﬁcantdifferences among other groups (Fig. 5B). Cry j 1-speciﬁc
IL-4 levels in mice immunized with Cry j 1–CpG showed nosigniﬁcant differences among other groups (Fig. 5C).

Fig. 2. Inhibition of human anti-Cry j 1 IgE from CJ allergic patients by Cry
j 1 or Cry j 1–CpG.

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Y. Kaburaki et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 157–161

Fig. 4. Anti-Cry j 1 IgG1 and IG2a production by Cry j 1–CpG inoculation.
BALB/c mice immunized with 5 ␮g each of either Cry j 1, Cry j 1–CpG, Cry j
1–mCpG, or PBS. (A) IgG1 and (B) IgG2a . **p < 0.01 for the group immunizedwith Cry j 1–CpG compared with other groups.

4. Discussion
It has been reported that IFN-␥ induction is an IgM to
IgG2a switch factor, which accounts for the association of
IgG2a with a Th1 response [23]. Cry j 1–CpG immunization signiﬁcantly induced the production of IFN-␥ fromCry j 1-speciﬁc CD4+ T cells. These observations are similar
to those from the study of Iho et al. [24]. Those investigators showed that CpG oligodeoxynucleotide containing
AACGTT induced the production of IFN-␥ and the expansionof antigen-speciﬁc human peripheral blood mononuclear
cells. In our study, we used CpG oligodeoxynucleotide
containing AACGTT. This CpG might be a Th1 cell differentiation inducer and promote IFN-␥ production.
In this study, inoculation with Cry j 1–CpG reduced
anti-Cry j 1 IgE antibody and enhanced IgG2a responses
after immunization with alum-precipitated Cry j 1. Cry
j 1–CpG induced the allergen-speciﬁc Th1-type immune
response, resulting in the inhibition of the IgE response to
the allergen. Tight et al. reported that in mice primed forTh2 response, injection with Amb a 1 (a major ragweed
allergen)-conjugated CpG suppressed speciﬁc Amb a 1 IgE[9]. Also, Suzuki et al. reported that anti-Cry j 2 IgE levels
were lower in mice immunized with Cry j 2 T-cell peptide conjugated with CpG [14]. These ﬁndings indicated theFig. 5. The effect of Cry j 1–CpG on cytokine production in Cry j 1-speciﬁc
CD4+ T-cell cultures. CD4+ T cells from mice immunized with Cry j 1, Cry j
1–CpG, Cry j 1–mCpG, or PBS were stimulated with 10 ␮g/ml of Cry j 1 for
3 days. (A) IFN-␥, (B) IL-5 and (C) IL-4. *p < 0.05 for the group immunizedwith Cry j 1–CpG compared with other groups.

potential for a CpG-conjugated allergen or T-cell peptide
vaccine to serve as a tool for the prevention and treatment
of CJ pollinosis.
In the present study, IL-4 levels in mice immunized withCry j 1–CpG were no different from those in any otherimmunized mice. IL-5 levels in mice immunized with Cry
j 1–CpG showed signiﬁcantly lower than those in mice
immunized with Cry j 1–mCpG, but showed no signiﬁcantdifferences among other groups. Similarly, our previous
study showed that the inoculation of DNA vaccine encoding the CD4+ T-cell epitope in Cry j 2 (a major CJ allergen)131

Y. Kaburaki et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 157–161

inhibited the allergen-speciﬁc IgE response to subsequent
allergen and alum injection, but did not inhibit allergenspeciﬁc IL-4 and IL-5 production [22]. The alum can induce
strong antigen-speciﬁc Th2 response and Th2-associatedIL-4 production [25,26]. The subsequent allergen and alum
injection in the present study might induce too strong Th2
responses to suppress Th2-cytokines in mice.
CpG oligodeoxynucleotide is a potent adjuvant of Th1
response, so it is hoped that it will be useful in the development of immunotherapies for allergic diseases and cancer.
Marshall et al. demonstrated that ISS 1018-conjugated
Amb a 1 allergen would induce IFN-␥ production in peripheral blood mononuclear cells from human patients with
ragweed allergy [18]. We used the same single strand synthetic ISS 1018-conjugated Cry j 1. Our data suggested thatCpG played a role in switching on Th1 immunity to a protein antigen through the upregulation of IgG2a and IFN-␥.
Furthermore, Cry j 1–CpG induced Th1 cell differentiationand suppressed anti-Cry j 1 IgE. This immunization may be
useful as an immunotherapy for CJ pollinosis.

161

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Acknowledgments
This work was supported by a Grant-in Aid for ScientiﬁcResearch from the Ministry of Health, Labor, and Welfareof Japan, by the Promotion and Mutual Aid Corporation
for Private Schools of Japan, by a Grant-in-Aid for Matching Fund Subsidy for Private University, and by a project
grant awarded by the Azabu University Research Services
Division.
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et al. Conjugation of immunostimulatory DNA to the short ragweed132Author's personal copy
Clinical Immunology (2011) 139, 65–74

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Clinical Immunology
www.elsevier.com/locate/yclim

Increase of regulatory T cells and the ratio of specificIgE to total IgE are candidates for response monitoringor prognostic biomarkers in 2-year sublingual
immunotherapy (SLIT) for Japanese cedar pollinosis
Takashi Fujimura a,1 , Syuji Yonekura a , Shigetoshi Horiguchi a ,Yuriko Taniguchi b , Akemi Saito c , Hiroshi Yasueda c , Ayako Inamine a ,Toshinori Nakayama b , Toshitada Takemori d , Masaru Taniguchi d ,Masahiro Sakaguchi e , Yoshitaka Okamoto a,⁎a

Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan
Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japanc
Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital, Kanagawa, Japand
Research Center for Allergy and Immunology, Yokohama Institute, RIKEN (The Institute of Physical and Chemical Research),
Kanagawa, Japane
Department of Veterinary Microbiology, School of Veterinary Medicine, Azabu University, Kanagawa, Japanb

Received 24 September 2010; accepted with revision 31 December 2010
KEYWORDS
Allergic rhinitis;
Biomarker;
Immunotherapy;
Japanese cedar pollinosis;
Regulatory T cell;
Sublingual immunotherapy

Abstract The aims of this study were to examine the therapeutic effects of sublingual
immunotherapy (SLIT) and to identify potential biomarkers that would predict the therapeutic
response in a randomized, double-blind, placebo-controlled clinical trial. The trial was carriedout over two pollinosis seasons in 2007 and 2008. Carry-over therapeutic effects were analyzed in2009. SLIT significantly ameliorated the symptoms of pollinosis during the 2008 and 2009 pollen
seasons. Cry j 1-specific cytokine production in a subgroup of patients with mild disease in the
SLIT group was significantly attenuated. The ratio of specific IgE to total IgE before treatment
correlated with the symptom-medication score in the SLIT group in 2008. Patients with increased

Abbreviations: DBPC, double-blind, placebo-controlled; ELISA, enzyme-linked immunosorbent assay; ELISPOT, enzyme-linked immunospot
assay; iTreg, induced regulatory T cells; ITT analysis, intention-to-treat analysis; JAU, Japanese allergy unit; N.S., not significant; OT analysis,on-treatment analysis; PBMCs, peripheral blood mononuclear cells; RAST, radioallergosorbent test; SLIT, sublingual immunotherapy; SMS,
symptom-medication score; Treg, regulatory T cells; QOL, quality-of-life.
⁎ Corresponding author. Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Chiba University, 1-8-1,
Inohara, Chuo-ku, Chiba 260-8670, Japan. Fax: +81 43 227 3442.
E-mail address: yokamoto@faculty.chiba-u.jp (Y. Okamoto).
1
Present address: Research Center for Allergy and Immunology, Yokohama Institute, RIKEN (The Institute of Physical and Chemical
Research), Kanagawa, Japan.
1521-6616/$ - see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.clim.2010.12.022

133

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66

T. Fujimura et al.
Cry j 1-iTreg in the SLIT group had significantly improved QOL and QOL-symptom scores. In
summary, the specific IgE to total IgE ratio and upregulation of Cry j 1-iTreg are candidates forbiomarker of the clinical response to SLIT.
© 2011 Elsevier Inc. All rights reserved.

1. Introduction
Japanese cedar (Cryptomeria japonica) pollinosis is acommon allergy in Japan, with a prevalence estimated to
be 26.5% in a nationwide survey conducted in 2008 [1].
A 2000 Japanese allergy unit (JAU) sample of standardized
extract from Japanese cedar pollen is the only available
allergen for subcutaneous and sublingual immunotherapy(SLIT) against pollinosis in Japan. The 2000 JAU extractcontains 1.5 to 4.2 μg of the major allergen, Cry j 1 [2]. The
common monthly cumulative dose for SLIT is 8000 JAU, whichcontains approximately 10 μg of Cry j 1. This maintenancedose is 200-fold higher than that used in traditional
subcutaneous immunotherapy using 0.2 ml of a 200 JAU/ml
extract, which contains approximately 50 ng of Cry j 1.
Despite using a low dose of the major allergen compared withthat in European trials, positive effects on pollinosis havebeen shown in randomized double-blind, placebo-controlled(DBPC) studies, in which SLIT significantly ameliorated thesymptom score, symptom-medication score (SMS), andquality-of-life (QOL) score [3,4].
SLIT induces Cry j 1-specific IgG4 production and attenuates
the seasonal increase in the number of Th2 cells specific toepitopes from Cry j 1 and Cry j 2 [3]. Involvement of antigenspecific Tr1 cells or regulatory T cells (Treg) in the therapeuticmechanism has also been suggested [5,6]. We previously foundthat SLIT increased the levels of Cry j 1-specific induced Treg
cells (Cry j 1-iTreg; IL10+Foxp3+ cells in CD25+CD4+ leukocytes)
and that the increase in Cry j 1-iTreg after the pollen season
may serve as a response monitoring biomarker that correlateswith a positive therapeutic effect based on the QOL-symptomscore and distinguishes responders from non-responders after
SLIT [6].
In this report, we examined the reproducibility of thepositive therapeutic effects and safety of SLIT and upregulation of iTregs as a response monitoring biomarker, with thegoal of confirming our previous results in a larger randomized
DBPC study. Therefore, the safety and clinical effect of SLIT
for Japanese cedar pollinosis were used as the primary
endpoint, and carry-over effects, immunological changes,
and biomarkers for a positive clinical effect induced by SLIT
were secondary endpoints.

2. Materials and methods
2.1. Study population
The study was conducted as a randomized, DBPC, parallelgroup, single center trial in subjects with Japanese cedar
pollinosis. This study was performed for two pollen seasons
between September 2006 and May 2008, with follow-up in
the pollen season in 2009. We recruited 130 participants in

134

September 2006. Diagnosis of Japanese cedar pollinosis was
based on clinical history and the presence of IgE specific toJapanese cedar pollen of at least class 2 (CAP-RAST method,
Phadia, Tokyo, Japan). Participants with a history of
immunotherapy or a diagnosis of asthma, or those who
were pregnant, were excluded from the study. Patients who
suffered seasonal or chronic rhinitis that required medical
treatment were also excluded.
A total of 103 patients were eligible for the study, and all
had moderate or severe symptoms in the previous pollen
season [7]. We anticipated that some participants in the SLIT
group would drop out from the study due to side effects andwe planned to evaluate the risk of mild or severe side effects
due to the vaccination. Therefore, we randomly divided thepatients into treatment (SLIT) and placebo groups with aratio of 6:4 according to the table of random numbers
prepared by the Department of Pharmacy at Chiba University
Hospital (Fig. 1). The sample size was determined based on aprevious study [3]. Briefly, we planned to have 50 patients ineach group with anticipation of dropout. We set 1.0 as amagnitude for the difference of average SMS between thatfrom the SLIT and placebo groups and 1.5 as a standard
deviation according to the result of previous study. Therefore, when the power was set to 0.8 and the α-error to 0.05,
the number of required cases was 35 in each group. A person
who was not directly involved in the study was responsible
for group allocation. To prevent leakage of information, theallocation table was kept by this person and a member of the
ethics committee who was also not directly involved in the
study, until accessed with the key after completion of thestudy. The protocol was approved by the Ethics Committee of
Chiba University, and written informed consent was obtained
from each patient prior to participation in the study.

2.2. Clinical protocols
The SLIT group included 58 patients who received standardized Japanese cedar pollen extract (Torii Pharmaceutical Co.
Ltd., Tokyo, Japan) [8], and the placebo group included 45
patients who received an inactive placebo. The protocol
consisted of treatment with graded courses of the extract in
50% glycerol, followed by maintenance therapy [6]. Briefly,
the extract was graded in three strengths: 20, 200, and 2000
JAU/ml. Patients received increasing doses with each vial,
beginning with 0.2 ml from the 20 JAU/ml vial and increasing
by 0.2 ml a day for 5 days per week. The vaccine was taken
sublingually, kept in place for 2 min without a retention
reagent, and then spit out. The procedure was repeated until
the maximum dose (1.0 ml of 2000 JAU/ml) was reached.
The maintenance dose was 1.0 ml of 2000 JAU/ml given oncea week until the end of May 2008. The patients in the placebogroup received inactive 50% glycerol in saline. All participants were allowed to take symptom-reducing drugs asneeded.

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Biomarkers for the clinical effects of SLIT

67

Assessed for eligibility(N=130)Excluded (N=27)
Not meeting inclusion criteria (N=10)
Refused to participate (N=2)
Other reasons (N=15)

Enrollment
Randomized

Active group
Allocated intervention(N=58)<ITT analysis>Placebo group
Allocated intervention(N=45)

Analyzed (N=51)<OT analysis>

Analyzed (N=37)

Double-blind in 2006 to 2008
Broke blind to physicians and researchers
Analyzed (N=36)<Follow-up>
Single-blind in 2009

Analyzed (N=27)

Figure 1 Flow diagram for groups and individuals in the phases of the randomized trial. Fifteen participants from the SLIT (N = 7) andplacebo (N = 8) groups were lost to follow-up due to reasons such as moving house and transfer. The double-blind status was maintained
until completion of analysis of all clinical and immunological parameters (December 2008). Follow-up analysis in 2009 was undertaken
in a single-blind manner.

2.3. Clinical symptoms and safety measurements
The patients completed a pollinosis diary to record theirnasal symptoms and use of symptom-reducing drugs in the2007, 2008, and 2009 pollen seasons. The total amounts ofpollen scattered from Japanese cedar and Japanese cypress(Chamaecyparis obtusa) in Chiba prefecture were 2777,
6596, and 5486 grains/cm2 during the 2007, 2008, and 2009pollen seasons, respectively, based on measurements with aDurham pollen sampler. The duration and amount of
scattered Japanese cedar pollen differed greatly among
these years, but the daily amount of scattered pollen
typically followed a wide-based bell-shaped curve over thewhole pollen season from the middle of January or earlyFebruary to the middle or end of May. The duration of thepeak pollen season was relatively constant in the 3 years,
and therefore, we analyzed the SMS during the peak period.
The peak pollen season was defined as the period from thefirst day that the pollen count was ≥20 grains/cm2/day for 3
consecutive days until the last day that the pollen count was≥ 20 grains/cm2/day before a period in which the pollencount was b 20 grains/cm2/day for 7 consecutive days.
The daily SMS was calculated as described previously [3].
Briefly, daily episodes of sneezing and nose blowing were rated
as 0–4: none, 0; 1–5 episodes, 1; 6–10 episodes, 2; 11–20
episodes, 3; N 20 episodes, 4. Daily medication was recorded
based on drug types and duration of usage using the following
guidelines: antihistamines, mast cell stabilizers, and vasoconstrictors, 1; topical ocular or nasal steroids, 2. Patients with anaverage daily SMS in the peak pollen season of ≤4 were

judged to have mild symptoms based on guidelines for allergic
rhinitis [7].
In the middle of the 2007 and 2008 pollen seasons, theparticipants completed the Japanese Allergic Rhinitis QOLStandard Questionnaire No.1 (JRQLQ No.1) for assessment of
QOL-symptom and total QOL scores [9]. These scores were
calculated as previously described [4,6]. The total QOLsymptom score was calculated as the sum of each componentscore: none, 0; mild, 1; moderate, 2; severe, 3; and very
severe, 4. Nasal and ocular symptoms covered by thequestionnaire included runny nose, sneezing, nasal congestion, itchy nose, itchy eyes, and watery eyes. Adverse eventswere graded using Common Terminology Criteria for Adverse
Events (CTCAE) v.3.0 [10]. Briefly, adverse events were
graded as mild, grade 1; moderate, grade 2; severe, grade 3;
life threatening, grade 4; death, grade 5 according to acategory for allergy/immunology in the CTCAE v.3.0 scoring
system.

2.4. Blood samples
Peripheral blood was obtained from each patient before
treatment (September to October 2006) and before and after
the pollen seasons in 2007 (December 2006 to January 2007,
and May to June 2007, respectively) and 2008 (November toDecember 2007, and May 2008, respectively). Peripheral
blood mononuclear cells (PBMCs) were isolated, frozen, andstored in liquid nitrogen [6]. However, the PBMCs isolated
before treatment, and before and after the 2007 pollen
season were damaged during storage and we were unable to135Author's personal copy
68

T. Fujimura et al.

analyze their immunological responses. Therefore, immunological data were obtained only from PBMCs collected before
and after the 2008 pollen season.

2.5. Total and antigen-specific immunoglobulin titer
The Cry j 1-specific IgE and IgG4 titers in plasma were
measured by ELISA [3,11]. Total IgE and specific IgE titers forJapanese cedar, orchard grass, mugwort, and house dust
mites were evaluated by the CAP-RAST method (Phadia).

2.6. Flow cytometric analysis
The levels of Cry j 1-iTreg were analyzed by flow cytometry[6]. Briefly, PBMCs were cultured with or without Cry j 1 for
3 days, followed by a culture with 10 ng/ml phorbol 12myristate 13-acetate, 1 μM ionomycin, and 2 μM monensin
for 6 h. The PBMCs were stained with PE-Cy7-anti-CD4
antibody, APC-anti-IL10 antibody (BD Biosciences, San
Diego, CA, USA), PE-anti-CD25, and FITC-anti-Foxp3 (clone:
PCH101) using a Foxp3 staining buffer set (eBioscience, San
Diego, CA, USA).

2.7. Analysis of the number of IL4-producing cells
and the concentration of cytokines
The number of IL4-producing cells stimulated with Cry j 1
was determined by enzyme-linked immunospot (ELISPOT)
assay, and the concentrations of IL2, IL5, and IL13 in theculture supernatant were measured using a BD™ Cytometricbead assay (CBA) Flex system (BD Biosciences) [6]. Briefly, a
96-well sterile filter plate (Millipore, Billerica, MA, USA) was
coated with monoclonal antibody to human IL4 (Mabtech AB,
Nacka Strand, Sweden). The plate was pre-incubated with
AIM-V medium at 37 °C for 1 h. The medium was discarded,
and then PBMCs (3 × 105 cells/well) were cultured with fresh
medium alone or with 10 μg/ml Cry j 1 for 17 h at 37 °C in
AIM-V medium containing 5% human AB serum (SigmaAldrich, St. Louis, MO, USA). The plates were then incubatedwith a biotinylated monoclonal antibody to human IL4 for
2 h, and then with streptavidin-conjugated alkaline phosphatase for 1 h at room temperature. After washing with
PBS, the plates were incubated with BCIP/NBTPLUS (Mabtech)for 5 min at 37 °C. For the CBA, isolated PBMCs were cultured
at 2.5 × 106 cells/ml with or without 5 μg/ml Cry j 1 for 3 days
at 37 °C in AIM-V medium containing 5% human AB serum(Sigma-Aldrich). After centrifugation at 300×g for 10 min, thesupernatant was divided into aliquots and stored at − 20 °C
until the cytokine assay was performed.

2.8. Data representation

2.9. Statistical analysis
Two-group comparisons were performed using a Wilcoxon t-test
or Mann–Whitney U-test to determine the significance ofdifferences, or using an unpaired t-test as indicated. P-values
b 0.05 were considered to be significant.

3. Results
3.1. Clinical effects and adverse events
A total of 103 patients were included in the overall analysis ofefficacy for the 2007 and 2008 pollen seasons. These patientswere randomly divided into the SLIT (N = 58) and placebo(N = 45) groups at a ratio of 6:4. Diaries and QOL questionnaires
for 88 patients were available at the end of the DBPC study.
The overall randomized population was considered to be the
ITT population. The SMS in the SLIT group did not differ
significantly from that in the placebo group in ITT analysis after
2-year SLIT (P = N.S.; Student t-test, data not shown).
The final sample size included 88 subjects for OT analysis(SLIT; N = 51, placebo; N = 37, ratio 4:3). The demographic
characteristics of the OT population before treatment are
shown in Table 1. The SMS in the SLIT group did not differ
significantly from that in the placebo group in the 2007
peak pollen season (February 19 to March 31, P = N.S.;
Student t-test). However, the average SMS in the 2008 peakpollen season (February 29 to April 1) was significantly
ameliorated in the SLIT group compared with the placebogroup (4.2 vs. 5.3, P = 0.02; Student t-test). The percentagesof subjects with mild symptoms (SMS ≤ 4) were 55% and 28% in
the SLIT and placebo groups, respectively, in the peak pollen

Table 1study.
Group

SLIT

PlaceboNumberSex (M/F)
Mean age
Range
Total IgE [IU/ml]
Range
Specific IgE c
Range
Class [mean]
Range
Other allergies d (%)
Orchard grass
MugwortHouse dust mite

51
17/34
44.4
16–73
198
6.8–148027
0.8–100
3.5
2–6

37
8/29
42.3
19–70
258
8.6–2090
29
1.5–100
3.8
2–6

16 (31%)5 (10%)
24 (47%)

11 (30%)3 (8%)
13 (35%)a

The full analysis set (N = 103) was used for the intention-totreat (ITT) analysis and per protocol populations (N = 88) wereused for on-treatment (OT) analysis (Fig. 1). Cry j 1-specificcytokine production is shown as the difference between cells
stimulated with Cry j 1 and controls stimulated with medium
only. Changes after the 2008 pollen season are shown as
differences between pre- and post-pollen season values.

136

Clinical data of participants at the start of the
P-value
N.S.a
N.S.b
N.S.b
N.S.b
N.S.b

N.S.e
b 0.05 f
N.S.e

Yates2 × 2 Chi-squared test.
Student t-test.
cSpecific IgE to Japanese cedar pollen; CAP-RAST raw value[kAU/L], mean.
dNumber of subjects with specific IgE of at least CAP-RAST
class 2.
e
2 × 2 Chi-squared test.
fFisher exact probability.
bAuthor's personal copy
Biomarkers for the clinical effects of SLIT

69

season (Fig. 2A). QOL-symptom and total QOL scores werealso significantly ameliorated in the SLIT group compared tothose in the placebo group in the middle of the 2008 pollen
season (Fig. 2B).
There were no severe adverse events that required apatient to withdraw from the study; however, some subjects
reported adverse events of mild discomfort: six of grade 2(oral pruritus: 2; gingivostomatitis: 2; asthma: 1; rash innasal cavity: 1) in the SLIT group (6/51; 11.8%); and one ofgrade 1 (bitter taste) in the placebo group (1/37; 2.7%).

3.2. Immunoglobulin production
There were no significant differences in Cry j 1-specific IgE
and IgG4 production between patients in the SLIT andplacebo groups before treatment, or before and after thepollen seasons. The SLIT group was divided into subgroups
based on the SMS in the 2008 peak pollen season: a mild
subgroup with SMS ≤ 4 (classified as responders; N = 28) and asevere subgroup with SMS N4 (non-responders; N = 23). IgE

14

SMS in 2008 peak pollen season

SMS in 2007 peak pollen season

(A)

12
10
8
6
4
2
0

Act.

*

14
12
10
8

2
0

Act.

Total QOL score

QOL-symptom score

80

25
20
15
10

Plc.

**

60

40

20

5
0

0
Act.

Plc.

IL2, IL5, and IL13 levels were analyzed in the culture
supernatant. The number of IL4-producing cells was measured by ELISPOT because IL4 was undetectable in the
supernatant. There were no significant differences between
the SLIT and placebo groups in the production of eachcytokine following stimulation with Cry j 1 (Fig. 3A). IL5 wassignificantly increased after the pollen season in all groups(P b 0.05; Wilcoxon t-test), and the IL2 and IL13 levels andthe number of IL4-producing cells were significantly
increased after the pollen season in the SLIT and placebogroups and in the severe subgroup (P b 0.05; Wilcoxon t-test).
Patients in the mild subgroup (responder to SLIT) did notshow significant increase of IL2 and IL13 or of IL4-producing
cells after the pollen season (P = N.S.; Wilcoxon t-test). The
increases in the number of IL4-producing cells and IL5 level
after the pollen season in the mild subgroup were significantly
less than those in the severe subgroup (non-responders) andthe placebo group. The increase of IL13 in the mild subgroupwas significantly less than that in the severe subgroup andshowed a tendency to be attenuated compared with theplacebo group (P = N.S.; Mann–Whitney U-test). The increaseof IL2 in the mild subgroup was significantly less than that in
the placebo group (P b 0.05) and showed a tendency to beattenuated compared with the severe subgroup (P = 0.053;
Mann–Whitney U-test, Fig. 3B).

3.4. Prognostic biomarkers for clinical effects

4

Plc.

*

3.3. Cry j 1-specific cytokine production

6

(B)
30

and IgG4 production in patients in the mild subgroup wereboth similar to those in patients in the severe subgroup and in
the placebo group at various time points (data not shown).

Act.

Plc.

Figure 2 Clinical scores after 2-year SLIT. (A) Average daily
symptom-medication scores (SMS) in the SLIT (Act.; N = 51) andplacebo (Plc.; N = 37) groups in the 2007 and 2008 peak pollen
seasons. (B) QOL-symptom and total QOL scores from the QOL
questionnaire were plotted for the SLIT (Act.; N = 51) and placebo(Plc.; N = 37) groups in the middle of the 2008 pollen season. Eachdiamond shows a value for an individual. Two-group comparisonswere performed using an unpaired Student t-test. *P b 0.05,**P b 0.01.

The average ratio of Japanese cedar pollen-specific IgE tototal IgE (sIgE/tIgE ratio) in all patients in the study was
0.193 before treatment. The SLIT group was divided into
subgroups with a sIgE/tIgE ratio ≤ 0.19 (low, N = 28) andN 0.19 (high, N = 23) before treatment. Similar subgroups
were established in the placebo group. The SMS in the 2008
peak pollen season for the low subgroup was significantly
improved compared to that in the high subgroup in the SLIT
group (P = 0.02; Mann–Whitney U-test); however, in theplacebo group, the low and high subgroups had comparable
SMSs (P = N.S.; Mann–Whitney U-test, Fig. 4A). Furthermore,
the SMS was correlated with the sIgE/tIgE ratio in the SLIT
group (Rs = 0.39, P b 0.01; Spearman correlation analysis), but
not in the placebo group (Rs = 0.08, P = N.S.; Spearman
correlation analysis, Fig. 4B).

3.5. Upregulation of Cry j 1-iTreg levels as a
response monitoring biomarker
A population of IL10+Foxp3+ cells in CD25+CD4+ leukocytes
was evaluated as a potential marker for iTreg after
stimulation with Cry j 1 or medium only before andafter the pollen season in 2008. Neither the changes in Cry
j 1-iTreg levels after stimulation with and without Cry j 1 nor
the upregulation of Cry j 1-iTreg from pre- to post-pollen
season differed significantly different between the groups(data not shown).

137

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T. Fujimura et al.

We previously reported that upregulation of Cry j 1-iTreg
is a candidate biomarker that may distinguish SLIT responders from non-responders based on QOL-symptom scores [6].
Therefore, we divided the SLIT group into subgroups basedon an increase (N = 24) or decrease (N = 27) in Cry j 1-iTreg
levels from before to after the pollen season in 2008. QOLsymptom and total QOL scores in the increased iTreg
subgroup significantly improved compared with those in
the placebo group. In contrast, the scores in the decreased
iTreg subgroup were similar to those in the placebo group(Fig. 4C).

3.6. Carry-over effects in the year after treatment
A total of 63 patients completed a pollinosis-symptom diaryduring the 2009 pollen season; 1 year after the 2-year SLIT

treatment (Fig. 1). All participants remained blinded to theirtreatment with SLIT or a placebo. The SMS in the peak pollen
season in 2009 (February 15 to March 6) in the SLIT group(N = 36) was significantly attenuated compared to theplacebo group (N = 27, P = 0.03). The average SMSs for the
SLIT and placebo groups were 3.5 and 4.5, respectively, inthe peak pollen season (Fig. 5).

4. Discussion
The primary endpoint of this randomized DBPC trial was thetherapeutic effect evaluated in ITT analysis. No significant
positive effect was observed between the SLIT and placebogroups after exchanging the perceived improvement ofpatients who dropped out with each median score from thecounter group. In OT analysis, the SMS in the SLIT group was

(A)
**
**

IL4-producing cells****

IL5 production [pg/ml]

250

200

150

100

**

*

**
**

1500

1000

500

50

0

0
A M S P
pre-pollen season

A M S P
post-pollen season

A M S P
pre-pollen season

**
**

**

A M S P
post-pollen season

**
600

*

IL2 production [pg/ml]

2000

IL13 production [pg/ml]

2000

**

1500

1000

500

**

**

500
400
300
200
100

0

0
A M S
P
pre-pollen season

A M S P
pre-pollen season

A M S
Ppost-pollen season

A M S P
post-pollen season

Figure 3 Cytokine production from PBMCs. (A) Number of Cry j 1-specific IL4-producing cells and Cry j 1-specific cytokine levels in
the SLIT group (A; N = 51), the mild subgroup of the SLIT group (M; N = 28), the severe subgroup of the SLIT group (S; N = 23), and theplacebo group (P; N = 37) at before and after the 2008 pollen season. Comparisons with a significant difference are indicated as * and **;otherwise, comparisons are not significantly different (P = N.S.). (B) Increases in the number of Cry j 1-specific IL4-producing cells and
Cry j 1-specific cytokine levels occurred from before to after the 2008 pollen season in the SLIT group (Act.; N = 51), the mild subgroupof the SLIT group (Mild; N = 28), the severe subgroup of the SLIT group (Sev.; N = 23), and the placebo group (Plc.; N = 37). Each diamondshows the value for an individual. Two-group comparison was performed using a Mann–Whitney U-test. *P b 0.05, **P b 0.01.

138Author's personal copy
Biomarkers for the clinical effects of SLIT

71

(B)
2000

*

Concentrationof IL5 [pg/ml]

IL4-producing cells

200

**

150

100

50

0

-50

*
*

1500

1000

500

0

-500
Act.

Mild

Act.

Plc.

Mild

Concentrationof IL2 [pg/ml]

1000
800
600
400
200
0
-200
-400

Sev.

Plc.

*

500

*

1200

Concentrationof IL13 [pg/ml]

Sev.

400
300
200
100
0
-100
-200
-300

Act.

Mild

Sev.

Plc.

Act.

Mild

Sev.

Plc.

Figure 3 (continued).

significantly ameliorated compared to the placebo group in
2008. The percentage of mild subjects (SMS ≤ 4) in the SLIT
group was 28% higher than that in the placebo group (SLIT,
55%; placebo, 27%), and the SMS was reduced by approximately 21% in the SLIT group compared with the placebogroup (SLIT, 4.2; placebo, 5.3). This percentage of mild
subjects differ significantly between the SLIT and placebogroups (P = 0.009; 2 × 2 Chi-squared test). These effects
following 2-year treatment were comparable to those in atrial of 1-year daily treatment using grass pollen tablets [12].
The low dose of the extract (about 1/40th of that used inEurope) may be one reason for the poor clinical outcome in
the first year [13]. An extract of concentration N 2000 JAU is
not available for clinical use in Japan, and the clinicaleffects, safety, and optimum schedule for administration ofan extract with a much higher allergen concentration remain
unclear.
Positive clinical therapeutic effects were not obtained
following 1-year treatment in our study, even in OT analysis(data not shown). In contrast, two previous reports demonstrated positive therapeutic effects after 1-year SLIT for
Japanese cedar pollinosis [3,4]. However, in these studies,
the annual pollen count (1154 grains/cm2/season) [3] wasless than in our study, and daily SMS was significantly
attenuated on only 4 days in the pollen season [4]. The
severity of SMS is affected by the amount of Japanese cedarpollen in the total and peak pollen season. Natural resolution
and tolerance are not usually induced by natural exposure to
Japanese cedar pollen, regardless of the amount of pollen[14].
Whether there are detectable alterations in peripheral
T-cell responses after specific immunotherapy is still under
debate [15–18]. The Cry j 1-specific cytokine profile from the
SLIT group did not differ significantly from that in the placebogroup. However, the increases in IL2, IL4, IL5, and IL13
production in the mild subgroup in the SLIT group weresignificantly attenuated (or showed a tendency to beattenuated) compared to the severe subgroup and the placebo139

Author's personal copy
72

T. Fujimura et al.

Figure 4 Biomarkers for positive therapeutic effects following SLIT. (A) SMSs in the 2008 peak pollen season for patients with low(low; N = 28) and high (high; N = 23) sIgE/tIgE ratios in the SLIT group (Act.), and for those with low (N = 25) and high (N = 12) sIgE/tIgE
ratios in the placebo group (Plc.). *P b 0.05. (B) Correlation between SMSs in the 2008 peak pollen season and sIgE/tIgE ratios before
treatment in the SLIT (Act.; N = 51) and placebo (Plc.; N = 37) groups. Statistical data were obtained with Spearman correlation
analysis. (C) QOL-symptom and total QOL scores from the QOL questionnaire plotted for a subgroup with increased Cry j 1-iTreg in the
SLIT group (Inc.; N = 24), a subgroup with decreased Cry j 1-iTreg in the SLIT group (Dec.; N = 27), and the placebo group (Plc.; N = 37) inthe middle of the 2008 pollen season. Each diamond shows the value for an individual. *P b 0.05, **P b 0.01.

group (Fig. 3B). The SMS in all patients in the study correlatedwith the seasonal increases in IL4 (R = 0.35, P b 0.01), IL5(R = 0.35, P b 0.01), and IL13 (R = 0.36, P b 0.01). The discrepancy in our current results and the results of previous studies
with regard to downregulation of cytokine production from
PBMCs may depend on the extent of the therapeutic effects
achieved in each clinical trial.
Cry j 1-specific IgE production was not changed bytreatment, even in the mild subgroup, as also found in our
preliminary study [6]. We speculate that more time isrequired for changing antibody production following thechanges of antigen-specific T cell profiles, because thealteration of T cell profiles strongly influences subsequentclass switch recombination of B cells and antibody produc-

140

tion. Another possibility is that the dose for SLIT used in thisstudy was not high enough to alter the antibody profiles.
The sIgE/tIgE ratio has been found to be significantly higher
in responders than in non-responders following 4-year immunotherapy [19]. In our trial, this ratio did not differ significantly
between responders and non-responders (P = N.S.; Mann–
Whitney U-test). However, subjects with a low sIgE/tIgE ratio
before treatment were more likely to be responders to 2-year
SLIT, and the ratio correlated with the SMS only in patientstreated with SLIT (Fig. 4A, B). This suggests that SLIT was more
effective in patients with a low sIgE/tIgE ratio than in thosewith a high sIgE/tIgE ratio. The range of total IgE levels for theparticipants were relatively wide (6.8–2090 IU/ml in all
patients); however, the change of the total IgE for eachAuthor's personal copy
Biomarkers for the clinical effects of SLIT

Figure 5 Carry-over effects following 2-year treatment with
SLIT. SMSs in the 2009 peak pollen season were plotted for the
SLIT (Act.; N = 36) and placebo (Plc.; N = 27) groups. Eachdiamond shows the value for an individual. Two-group comparisons were performed using an unpaired t-test.

individuals after 2-year treatment was not significantly
different compared to before treatment (1.5 ± 1.0 times
higher, P = N.S.; paired t-test). Therefore, the wide range of
total IgE levels was due to the variability on the allergic status
for individuals, but not on method for measurement. The
serum IgE level may affect the surface IgE level on effector
cells such as mast cells and basophils, and Tregs can downregulate activation of mast cells and eosinophils [20,21]. We
speculate that effector cells with a low specific IgE level areless likely to be activated by antigen crosslinking or are more
susceptible to downregulation by Tregs than those with a highspecific IgE level. It is also possible that the symptoms ofpatients with a low sIgE/tIgE ratio may be more readilyattenuated by suboptimal potentiation of iTreg induced by
SLIT.
We previously reported that an increased count of Cry j
1-iTregs was a candidate biomarker that could be used to
distinguish between responders and non-responders to SLIT, as
evaluated by the QOL-symptom score. In this report, thesubgroup with increased Cry j 1-iTregs showed significant
amelioration of the QOL-symptom and total QOL scorescompared to the placebo group, while the subgroup withdecreased Cry j 1-iTregs did not show this response (Fig. 4C).
However, there was no significant difference in Cry j 1-specificcytokine production from PBMCs among patients with increased
iTregs and decreased iTregs, and those in the placebo group(data not shown). Foxp3-expressing CD25+CD3+ cells and IL10expressing CD3+ cells, which are induced in the nasal mucosa
after subcutaneous immunotherapy, have been linked to theclinical efficacy and suppression of seasonal inflammation[22]. Immunotherapy using an Amb a 1-immunostimulatory
oligodeoxynucleotide conjugate also induced CD4+CD25+ T cells
and IL10-producing cells in the nasal mucosa after the pollen
season [23]. These data suggest that iTregs may downregulate
effector cells at local sites of inflammation to suppress clinicalsymptoms. Induction of iTregs in the nasal mucosa andfunctional analysis of these cells may be necessary to determine
the regulatory mechanisms affected by SLIT. Mucosal biopsy in73
the peak pollen season is useful for evaluation of local inductionof iTregs and downregulation of effector cells. However, nasal
biopsy in the pollen season significantly influences the daily SMS
in the peak pollen season. Mucosal biopsy outside the pollen
season after exposure using an artificial pollen chamber may be
a powerful tool for evaluation of local regulatory mechanisms
induced by SLIT [24]. Upregulation of iTregs in nasal mucosa
may be difficult to determine since the evaluation may be
painful for patients. However, upregulation of iTregs inperipheral blood is simple to analyze and may be a usefulbiomarker because an increase of peripheral Cry j 1-iTregs iscorrelated with QOL and QOL-symptom scores in the pollen
season, as discussed here and elsewhere [6].
Cry j 1-specific IgG4 production was not induced by SLIT inthis study to the same extent as that in our previous study[6]. A clinical trial showing that daily 2500 SQ-T (14 μg Phl p 5
per 4 weeks) tablets failed to induce IgG production supports
our current results [13]. A change in the immunoglobulin
profile may require a higher allergen dose or longer durationof exposure. However, our study suggests that detectable
quantitative changes in IgG4 are not essential for theamelioration of clinical symptoms.
In summary, we suggest that the sIgE/tIgE ratio andupregulation of iTregs may be considered as prognostic andresponse monitoring biomarkers, respectively, for SLIT.
However, further investigation of induction of iTregs at
local inflammatory sites and downregulation of inflammatory cells is needed. Furthermore, validation studies withlarger sample size would be required before either biomarkers should be applied widely in the clinical management ofpollinosis patients. Development of a more effective vaccine
and better protocols may reveal more significant differencesin the Cry j 1-specific cytokine profiles and iTreg induction,and these results may increase our understanding of the rolesof iTregs or Tr1 in the therapeutic mechanisms underlying
the efficacy of SLIT.

Acknowledgments
We sincerely thank Drs. Takashi Saito, Yasuyuki Ishii, Masato
Kubo, Tsuneyasu Kaisho, and Hisahiro Yoshida (RIKEN,
Kanagawa, Japan) for their helpful comments and fruitful
discussions. This work was partially supported by a grantfrom the Ministry of Health, Labour and Welfare in Japan, inpart by the Global COE program (Global Center for
Education and Research in Immune System Regulation andTreatment), MEXT, Japan, and in part by the Promotion and
Mutual Aid Corporation for Private Schools of Japan, Grantin-Aid for Matching Fund Subsidy for Private University,
Japan.

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Author's personal copy
Veterinary Immunology and Immunopathology 139 (2011) 99–106

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology
journal homepage: www.elsevier.com/locate/vetimm

Research paper

Establishment of a quantitative ELISA for the measurement ofallergen-speciﬁc IgE in dogs using anti-IgE antibody cross-reactive tomouse and dog IgETaro Okayama a , Yukiko Matsuno a , Nobutaka Yasuda a , Toshihiro Tsukui b ,Yasuyuki Suzuta b , Masanori Koyanagi b , Masahiro Sakaguchi c , Yasuyuki Ishii d ,Thierry Olivry e , Kenichi Masuda a,∗aAnimal Allergy Clinical Laboratories Inc., SIC-2 #301 5-4-30 Nishihashimoto, Midori-ku, Sagamihara, Kanagawa 229-1131, Japan
Zenoaq Nippon Zenyaku Kogyo Co., Ltd., Sasagawa Tairanoue 1-1, Asaka-machi, Koriyama, Fukushima 963-0196, Japanc
Department of Veterinary Microbiology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuou-ku, Sagamihara,
Kanagawa 229-8501, JapandLaboratory for Vaccine Design, RIKEN Research Center for Allergy and Immunology (RCAI), Suehiro-chou 1-7-22, Tsurumi-ku, Yokohama,
Kanagawa 230-0045, Japane
Department of Clinical Sciences and Center for Comparative Medicine and Translational Research, College of Veterinary Medicine,North Carolina State University, Raleigh, NC, USAba r t i c l e

i n f oArticle history:
Received 3 March 2010
Received in revised form 2 August 2010
Accepted 5 September 2010
Keywords:
Dogs
IgE
Canine atopic dermatitis
ELISA
Allergya b s t r a c t
As IgE plays a pivotal role in type I hypersensitivity-mediated allergic diseases, it is valuable
to measure absolute quantity of serum antigen-speciﬁc IgE for clinical and research purposes. Here we describe a novel ELISA system that enables quantiﬁcation of antigen-speciﬁc
IgE in ng/ml in dogs. A newly developed monoclonal antibody (CRE-DM) was shown to recognize canine and mouse IgE equally in a dose dependent manner, but it did not recognize
canine IgG. The reactivity of CRE-DM to canine IgE was also conﬁrmed by an inhibition
ELISA using canine IgE as an inhibitor and the maximum inhibition rate was 91.3%. In orderto know whether canine IgE speciﬁc to an allergen could be quantitatively measured with
an ELISA using CRE-DM, we established a quantitative ELISA that could measure canine IgE
recognizing Cry j 1, one of the major allergens of Japanese cedar pollen. In this ELISA, astandard curve was created by using concentration-predetermined Cry j 1-speciﬁc monoclonal mouse IgE. According to the standard curve, the concentration of Cry j 1-speciﬁc IgEin dogs that were experimentally sensitized to Japanese cedar pollen could be calculated
and determined in ng/ml. The speciﬁcity of the Cry j 1-speciﬁc IgE ELISA using CRE-DM wasalso conﬁrmed by inhibition ELISA using canine IgE as an inhibitor and the inhibition rate
was 97.0%. Reproducibility of the ELISA in three independent assays was determined usinggroups of pooled canine sera whose Cry j 1-IgE titers ranged from 155.9 to 888.2 ng/ml.
Intra- and inter-assay reproducibility was determined with coefﬁcient of variation ranging
between 3.1–5.2% and 2.2–8.0%, respectively. These results demonstrated that the ELISA
utilizing CRE-DM was a speciﬁc, reliable and robust new laboratory test that could quantify
absolute amount of antigen-speciﬁc IgE in canine serum. The ELISA will serve as a useful
tool in the clinics to evaluate the change of serum IgE titers during anti-allergic treatments
as well as during seasonal ﬂuctuation of allergen exposure.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction∗ Corresponding author. Tel.: +81 042 770 9437; fax: +81 042 770 9438.

IgE plays a pivotal role in provoking IgE-mediated allergic reaction and inducing various allergic diseases such

E-mail address: masuda@aacl.co.jp (K. Masuda).
0165-2427/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2010.09.002

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the amount of IgE in the tested serum. The intensity of signals is further converted into units, scores or grades that aredeﬁned by each laboratory and reported to the clinicians.
Because the reference reagents and the data processing
platforms used in the tests differ for each laboratory, it
is difﬁcult to compare results among the different laboratories. Also the results must be regarded as qualitative orsemi-quantitative because the results are not given in an
absolute quantiﬁcation such as in ng/ml but in comparative
scales. Moreover, there have been continuous debates until
now on how to interpret and link the serum IgE test resultswith clinical ﬁndings (DeBoer and Hillier, 2001b; Zur et al.,2002). For these reasons, a truly quantitative serum IgE test
has been sought.
In basic immunological research utilizing mice, there
are a number of reports that utilize ELISAs which are able
to quantify allergen-speciﬁc mouse IgE in ␮g/ml. In these
ELISAs, anti-mouse IgE antibodies are used for the detection of IgE and pre-quantiﬁed allergen-speciﬁc monoclonal
mouse IgEs are used to create standard curves. Then, theconcentrations of allergen-speciﬁc IgE in mouse sera arecalculated according to the standard curves. For instance,
a quantitative ELISA system for ovalbumin (OVA)-speciﬁcmouse IgE was established with a monoclonal mouse IgE
recognizing OVA and used to detect the serum titer of OVAspeciﬁc IgE at ␮g/ml in mice (Hamada et al., 2003). As indogs, monoclonal canine IgE speciﬁc to ﬁlarial antigen has
been developed and reported (Gebhard et al., 1995). However, the IgE described in the literature can only be used
as a standard reference to measure the worm-speciﬁc IgE,
which seems to have little use in allergy testing. In orderto establish an ELISA for the measurement of allergen speciﬁc IgE in dogs by such systems, it is required to establish
monoclonal canine IgE for each allergen to be tested, which
seems to be difﬁcult to achieve.
To overcome these issues, we attempted to establish a
novel ELISA system that could measure the absolute quantity of antigen-speciﬁc serum IgE in dogs by using a newly
developed anti-IgE antibody that cross reacts equally to
IgEs of dog and mouse (CRE-DM). A monoclonal mouse IgE
speciﬁc to Cry j 1, one of the major allergens of Japanesecedar pollen, was used as a reference standard to quantify
Cry j 1-speciﬁc IgE in canine sera. This is the ﬁrst report todemonstrate the method of an absolute quantiﬁcation ofallergen-speciﬁc IgE in ng/ml scale in canine sera.

as canine atopic dermatitis (AD) and cutaneous adverse
food reactions in dogs. The relationship between IgE andallergic diseases has been extensively investigated in dogs.
Allergen-speciﬁc IgE has been known to initiate degranulation of the basophils in the skin of experimental ﬂea allergy
model dogs (Halliwell and Schemmer, 1987). It was also
shown that the basophils of the peripheral blood of dogs
with AD (Masuda et al., 2000) and food allergy (Ishida et al.,2003) are initiated to release histamines through antigenIgE mediated stimulation. The serum titer of Japanese cedarpollen was reported to ﬂuctuate in parallel to the manifestation of allergy-related clinical signs and the pollinationseason (Masuda et al., 2002). An oral exposure to food antigens in dogs with food allergy induced clinical signs, whichwere accompanied by the increase of allergen-speciﬁc IgE
in the sera (Jackson et al., 2003). Because of the accumulating evidence suggesting the involvement of IgE in thepathogenesis of IgE-mediated allergic diseases in dogs, it
is becoming quite important to detect allergen-speciﬁc
IgE in clinical cases of canine AD and food allergy for
diagnosis and therapeutic purposes (DeBoer and Hillier,
2001a).
Two major test methods have been used in dogs todetect allergen-speciﬁc IgE; intradermal test (IDT) andserum antigen-speciﬁc IgE test (serum IgE test). IDT is
the in vivo test to see whether wheals are formed at thesites of intradermal injection of allergens. When a positive immediate reaction to an allergen solution is detectedin IDT, it can be concluded that the patient has an IgEmediated hypersensitivity to that allergen, thus supporting
the diagnosis of IgE-mediated allergic disease. IDT has been
popularly used in veterinary practice (Hillier and DeBoer,
2001), but however, there are some limitations for its clinical use. Because of the nature of this test as a qualitative
method, the test results are only obtained as negative or
positive. Therefore, it is difﬁcult for clinicians to understand the magnitude of sensitization and to monitor thetherapeutic efﬁcacies using IDT results. It is also reported
that IDT is prone to false negative results because the sufﬁcient allergen concentrations that give the proper test
results differ from allergen to allergen and from patient topatient (Hensel et al., 2004). Moreover, IDT is also reported
to exhibit false positive reactions due to sub-clinical hypersensitivity (Pastorello, 1993; Codner and Tinker, 1995). In
addition, because IDT requires particular procedures suchas intradermal injections, antigen preparation and animals
to be immobilized during the test, IDT is inconvenient for
veterinary practitioners who are not specialized in allergic
diseases to perform as a routine clinical test.
Serum IgE tests have been developed during the last
three decades in expectation to overcome the limitationsof IDT. Serum IgE tests are the methods to detect allergenspeciﬁc IgE in canine sera. Currently in most commercial
tests available in the veterinary ﬁeld, various forms of
ELISAs are utilized as assay systems. For the detection ofcanine IgE, anti-IgE antibodies or recombinant extracellular segments of the human high afﬁnity IgE receptor(rhFc␧RI␣) are used (DeBoer and Hillier, 2001b). The
amount of the detecting reagent bound to IgE is measured
by detecting the signals developed by enzyme-mediatedmethods. Thus the generated signals are deemed to reﬂect

2. Materials and methods
2.1. Preparation of anti-IgE mAb cross-reactive to canine
and mouse IgESix female rats (Wistar/ST) were all immunized with20 ␮g of mouse IgE (BD Bioscienses, San Jose, CA, USA) andcanine IgE (Bethyl Laboratories, Montgomery, TX, USA) two
times with a 2-week interval. After 4 weeks, the spleencells were obtained and fused with a mouse myeloma cellline, X63Ag8.653. A hybridoma cell clone that produced an
antibody reacting with canine and mouse IgE in the culture supernatant was selected by direct ELISAs using mouse
IgE or canine IgE as antigens. The cell clone was expanded
and injected intraperitoneously into rats (Wistar/ST) and144

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101

To obtain serum of various titers of IgE to Cry j 1, four
Maltese-beagle atopic (MBA) dogs were sensitized withCry j 1 with a combination of intraperitoneal and epicutaneous antigen stimulations. Dogs ﬁrst received two
intraperitoneal injections of 200 ␮g of a mixture of Cry
j 1 and 2 (ratio: 5:2). Dogs were further sensitized withadditional epicutaneous applications of 200 ␮g of crude
Japanese cedar pollen in mineral oil, with or without tape
stripping, on a weekly to monthly basis depending on theirallergen-speciﬁc IgE serum levels. The serum samples werecollected at several time points before and after the sensitization procedure. Successful sensitization was conﬁrmedby the positive reactions to Cry j 1 in IDT, which showed
wheal formation comparable to that of the histamine positive control. The sera were used for the quantiﬁcationof serum Cry j 1-speciﬁc IgE pre- and post-sensitizationand for determining the reproducibility of the Cry j 1speciﬁc IgE ELISA utilizing CRE-DM. All aspects of thestudy were approved beforehand by North Carolina State
University Institutional Animal Care and Use Committee(IACUC).

ascites was obtained 6 months after the injection. From
the ascites, rat IgG was puriﬁed with ammonium sulfate
precipitation and biotinylated with a kit (EZ-Link, Pierce
Biotechnology, Rockford, IL, USA). The IgG concentration
was determined with a spectrophotometer at 280 nm. Thisanti-IgE antibody was supposed to be cross-reactive to IgEs
of dog and mouse, thus the antibody was named CRE-DM.
All aspects of this experiment were approved by Animal
Care and Use Committee of Zenoaq Nippon Zenyaku Kogyo
Co., Ltd. (Zenoaq, Fukushima, Japan).
2.2. Evaluation of the reactivity and the speciﬁcity of
CRE-DM
Reactivity of the CRE-DM to canine IgE, canine IgG andmouse IgE were evaluated by ELISA using biotinylated CREDM as a detecting antibody. The microwells were coatedwith various concentrations ranging from 1 to 100 ng/mlof canine IgE (Bethyl Laboratories), canine IgG (Rockland
Immunochemicals, Inc. Gilbertsville, PA, USA), or mouse IgE(BD Bioscienses). The wells were blocked with 1% gelatin
in PBS for 2 h at room temperature. After washing threetimes with PBST, 100 ␮l of 0.5 ␮g/ml biotinylated CREDM diluted in dilution buffer (1% gelatin in PBST) was
added to each well. After 2-h incubation at room temperature, the wells were washed three times with PBST
and 100 ␮l of 0.05 U/ml Streptavidin-beta-Gal conjugate(Roche Diagnostics GmbH, Penzberg Germany) in dilution
buffer was added to each well and incubated for 2 h at room
temperature. The wells were then washed three times
with PBST and 100 ␮l of 0.1 mM 4-methylumbelliferyl ␤d-galactopyranoside (4MU, Sigma–Aldrich, Saint Louis, MS,
USA) in reaction buffer (0.1% gelatin, 0.1 M NaCl and 1 mM
MgCl2 in phosphate buffer, pH 7.4) was added. Following 1-h incubation at room temperature, 100 ␮l of stop
solution (0.25 M Na2 CO3 ) was added and detected withmicroplate reader Gemini XPS (Molecular Devices, Sunnyvale, CA, USA). The data were analyzed using Softmax Pro(Molecular Devices). In the inhibition ELISA assay, whichaims to qualify the speciﬁcity of the reaction, biotinylated
CRE-DM was pre-incubated overnight at 4 ◦ C with various
amounts of canine IgE, and the assays were performed similarly as described above.

2.4. Determination of the optimal dilution rate for the
dog serum sample
To address an issue that IgGs in the serum that share
reactive antigens with IgEs could possibly inﬂuence thereactions between antigens and IgEs in the ELISA, it wasconsidered essential to use the appropriate serum dilution rate to measure antigen-speciﬁc IgE in the serum. To
determine the optimal dilution rate for serum samples,high Cry j 1-IgE titer serum described above was diluted
to various dilution rates ranging from 10 to 100,000 andused in the ELISA. Because the dog was intensely immunized so as to produce large amounts of antibodies, thesample was considered to contain large amounts of IgGs
speciﬁc for Cry j 1 as well, which should well exceed
the amount of IgGs that could possibly be produced in
the spontaneous sensitization to the allergen. From thesereasons we used this serum as a representative sample.
A serum of healthy beagle, obtained from Zenoaq, wasdiluted simultaneously and used in the assay to serve asa blank control for each dilution rates. Microwells were
coated with 100 ␮l of 1 ␮g/ml Cry j 1 (Hayashibara Biochemical Labs., Inc., Okayama, Japan). Wells were blockedwith 1% gelatin PBS for 2 h at room temperature and thenwashed three times with PBST. Then the diluted samples
were applied to wells and incubated overnight at 4 ◦ C.
After washing three times with PBST, 100 ␮l of biotinylated CRE-DM in dilution buffer was added to each well.
The rest of the procedure was carried out as describedabove.

2.3. Serum samples from experimentally sensitized dogs
To obtain high titer serum used for the measurement
and inhibition experiments in Cry j 1-speciﬁc IgE ELISA,
a dog was experimentally sensitized to Japanese cedarpollen. The sensitization procedure was carried out aspreviously described (Yamashita et al., 2000). A dog wasmaintained and the experiment was carried out at Zenoaq.
Brieﬂy, a healthy beagle was subcutaneously injected twice
at 2-week interval with 100 ␮g of Japanese cedar pollen
antigen in 3 ml of saline-emulsiﬁed alum. Two weeks after
the second injection, production of IgE to Japanese cedarpollen antigen was conﬁrmed by IDT (data not shown),
and serum sample was collected. All aspects of this experiment were approved by Animal Care and Use Committeeof Zenoaq.

2.5. Quantiﬁcation of Cry j 1-speciﬁc IgE in canine serum
To determine whether canine IgE speciﬁc to an allergen could be quantitatively measured with an ELISA using
CRE-DM and Cry j 1-speciﬁc monoclonal mouse IgE, weperformed a quantitative ELISA speciﬁc for canine IgE to
Cry j 1. Microwells were coated with 100 ␮l of 1 ␮g/ml
Cry j 1 (Hayashibara Biochemical Labs., Inc.). Wells were145

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blocked with 1% gelatin PBS for 2 h at room temperatureand then washed three times with PBST. Sera from clinical cases were diluted 1:1000 in dilution buffer and 100 ␮lwere applied to microwells. To create a standard curve,anti-Cry j 1 monoclonal mouse IgE was obtained by a standard method to be used as a standard reference. Brieﬂy,
BALB/c mice were immunized twice in a week interval with
5 ␮g of Cry j 1 and 2 mg of alum per mouse. Three days after
the last immunization, the mice were sacriﬁced and theirsplenic cells were fused with P3U1 myeloma cells at 3:1
ratio with polyethylene glycol 4000 (Wako Pure Chemical
Industries, Osaka, Japan). The fused cells were seeded in
96 well ﬂat bottom plate at the concentration of 6 × 104
cells per well (splenic cells) and cultured in HAT selection
medium (complete RPMI with 10% FCS containing 0.1 mM
hypoxanthine, 0.016 mM thymidine 0.4 nM aminopterin)
for 10 days. The culture supernatants of growing cells werescreened for the presence of mouse IgE with the Cry j 1-IgE
ELISA using anti-mouse IgE monoclonal antibody (Yamasa
Corporation, Chiba, Japan). Finally, a positive clone wasestablished as a hybridoma secreting IgE speciﬁc to Cry j
1.
Obtained anti-Cry j 1 monoclonal mouse IgE was quantiﬁed with mouse IgE EIA kit (Yamasa Corporation) anddiluted to concentrations ranging 0.156–10 ng/ml and100 ␮l of each solution was applied to wells of ELISA plates.
After incubating overnight at 4 ◦ C, wells were washed and100 ␮l of biotinylated CRE-DM diluted in dilution buffer at
0.5 ␮g/ml was added to each well. The rest of the procedure
was carried out as described above. To further investigate
whether CRE-DM could speciﬁcally detect IgE in canine
serum, CRE-DM was incubated beforehand with canine
IgE as an inhibitor for overnight at 4 ◦ C (pre-treated CREDM). Inhibition ELISA was performed similarly as describedabove but replacing CRE-DM with the pre-treated CREDM.

Fig. 1. Reactivity of CRE-DM against canine IgE, canine IgG and mouse
IgE. Reactivity of the CRE-DM was evaluated in ELISA by applying the
CRE-DM to the wells coated with canine IgE, canine IgG or mouse IgE.
Resulting signals are plotted as ﬂuorescence unit. Open diamonds, squares
and triangles indicate signals from mouse IgE, canine IgE and canine IgG,
respectively. The result is the representative of three independent experiments which gave similar results.

3. Results
3.1. Reactivity and speciﬁcity of CRE-DM
The reactivity of CRE-DM to IgE was ﬁrst determinedby the direct ELISA. It was shown that the CRE-DM reacted
equally to canine IgE and mouse IgE in a dose dependent
manner (Fig. 1). However, the CRE-DM did not react tocanine IgG. The reactivity was further analyzed by an inhibition ELISA using canine IgE as an inhibitor. When CRE-DM
was pre-incubated with various amounts of canine IgE, thereaction was obviously reduced. The inhibition rate ranged
from 83.0% to 91.3% with the inhibitor/CRE-DM ratio varying from 0.5 to 10. The maximum reduction was obtained
when the ratio was 10 (Fig. 2). Taken together, the CREDM was considered to react speciﬁcally to canine IgE andmouse IgE, but not to canine IgG.

2.6. Evaluation of the reproducibility of the ELISA
utilizing CRE-DM
The reproducibility of the ELISA utilizing CRE-DM wasevaluated by comparing results among the wells in threedifferent plates within an assay (intra-assay) and among
the wells of three independent assays which were performed on different days (inter-assay). Canine sera withvarious titers were prepared from the Cry j 1-sensitized
MBA dogs as described above and the concentrations of
Cry j 1-speciﬁc IgE in each serum were determined beforehand with the ELISA using CRE-DM. According to theconcentrations, sera were divided into three groups, high,intermediate and low. Three serum samples were chosenfrom each group and pooled. On each plate the sera ofhigh, intermediate and low groups were applied to wells.
The ﬁrst assay was carried out with three plates in parallel and the data were used for intra-assay evaluation. The
second and third assays were carried out with one plateeach. For inter-assay evaluation, data from three plates,
each from three independent assays, were collected andanalyzed. Coefﬁcient of variation (CV) was calculated as(SD/mean) × 100 (%) for each sera group.

3.2. Determination of the optimal dilution rate for the
dog serum sample
To determine the serum dilution rate that is optimal forthe quantiﬁcation of Cry j 1-speciﬁc IgE in the dog serum,
the serum with high IgE titer was diluted into indicateddilution rates and the ELISA was performed (Fig. 3). As theresult, the ﬂuorescence units and the dilution rates showed
good correlation at the dilution rates between 100,000 and1000 but plateaued at the dilution rates lower than 300.
This was considered to indicate that the reactions between
the Cry j 1-speciﬁc IgEs and the antigens were inhibited at
the dilution rates <300 possibly due to the inﬂuence of the
Cry j 1-speciﬁc IgGs in the serum that shared antigens with
IgEs. From these results, we concluded that the dilution
rate of 1000 was optimal in the measurement of allergenspeciﬁc IgE in dog sera.

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103

Inhibition=97.0%
14000

11951.6

12000

IgE conc. (ng/mL)

10000
8000
6000
4000
2000

85.1
0

Fig. 2. Inhibition ELISA using canine IgE as an inhibitor. The CRE-DM was
pre-incubated prior to ELISA with canine IgE to evaluate the speciﬁcity of
the reaction. Resulting signals are plotted as ﬂuorescence units with open
diamonds. Percentages of the reductions are shown in the table. The result
is the representative of three independent experiments that gave similar
results.

Inhibitor

To test whether allergen-speciﬁc IgE in canine serum
could be measured with an ELISA using CRE-DM, Cry j
1-speciﬁc IgE was measured in serum from the dog experimentally sensitized to Japanese cedar pollen. A standard
curve in the ELISA was created by using the concentrationpredetermined monoclonal mouse IgE speciﬁc to Cry j 1. As
a result, Cry j 1-speciﬁc IgE in the serum could be measured
and the concentration was determined as 11951.6 ng/ml.
Furthermore, in order to conﬁrm the speciﬁcity of this
ELISA, an inhibition ELISA was performed with canine IgE
as an inhibitor. When canine IgE was used as an inhibitor,
the concentration of Cry j 1-speciﬁc IgE in the serum was

reduced to 85.1 ng/ml, which indicated that the inhibition
rate was 97.0% (Fig. 4). These data indicated that the reactivity of CRE-DM to canine IgE was maintained in the ELISA
for canine serum as samples and allergen-speciﬁc IgE concentration in dog serum could be measured. To further
conﬁrm that serum antigen-speciﬁc IgE could be measured
in other dog serum samples with different sensitization statuses, we measured the concentrations of Cry j 1-speciﬁc
IgE in MBA dogs during pre- and post-sensitization withCry j 1 (Table 1). The results clearly demonstrated that
Cry j 1-speciﬁc IgE concentrations increased from nearly
undetectable levels before the sensitization to high concentrations such as 544.8–1852.5 ng/ml after the sensitization.
From these results, it was concluded that the ELISA utilizing
CRE-DM could be used to measure allergen-speciﬁc IgE in
the serum of dogs hypersensitive to Japanese cedar major
allergens.

100000

Fluorescence units(+)

Fig. 4. Cry j 1-speciﬁc ELISA utilizing CRE-DM in canine serum samples.
The absolute concentration of Cry j 1-speciﬁc IgE in canine sera wasanalyzed. Measurement of Cry j 1-speciﬁc IgE in a dog experimentallysensitized to Japanese cedar pollen. Also, the speciﬁcity of the ELISA wasevaluated by pre incubating CRE-DM with canine IgE as an inhibitor. Inhibition rates are indicated in the ﬁgure. The result is the representative of
two independent experiments that gave similar results.

3.3. Quantiﬁcation of Cry j 1-speciﬁc IgE in canine serum
samples

10000

3.4. Reproducibility of the Cry j 1-speciﬁc IgE ELISA
utilizing CRE-DM
Using sera from MBA dogs experimentally sensitized to
Cry j 1, the reliability of the Cry j 1-speciﬁc IgE ELISA wasdetermined by evaluating the reproducibility of the values in pooled sera with the high, intermediate, and low

1000

100
100000

(-)

10000

1000

100

10

Table 1
Cry j 1-speciﬁc IgE concentration in MBA dogs sensitized with Cry j 1.

1

Dilution factor

Dog no.

Fig. 3. Determination of the optimal dilution rate for the dog serum sample. The dog serum with high Cry j 1-speciﬁc IgE was diluted at various
dilution rates as indicated. The resulting signals from each dilution rates
are indicated as ﬂuorescence units and plotted with solid diamonds. The
result is the representative of two independent experiments that gave
similar results.

1
2
3
4
a

147

Pre-sensitization (ng/ml)
15.6
naa
0
0

Post-sensitization (ng/ml)
1852.51513.4613.1
544.8

Not applicable due to a failure in sample collection.

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T. Okayama et al. / Veterinary Immunology and Immunopathology 139 (2011) 99–106

104

Table 2
Reproducibility of the Cry j 1-speciﬁc IgE ELISA utilizing CRE-DM.
Comparison

GroupsIntra-assay

Low
IntermediateHigh

Data1 (ng/ml)
155.9
413.4
841.3

Data2 (ng/ml)
172.2
391.2
888.2

Data3 (ng/ml)
168.7
388.5
841.9

Mean (ng/ml)
165.6
397.7
857.1

CV (%)
5.2
3.4
3.1

Inter-assay

Low
Intermediate
High

172.2
391.2
888.2

156.0
411.6
887.4

180.0
456.5
854.2

169.4
419.8
876.6

7.2
8.0
2.2

so far is against ﬁlarial antigen (Gebhard et al., 1995).
Alternatively, the pooled serum of clinical cases sensitized
to an allergen such as Japanese cedar pollen has beenused as a reference in a comparative measurement ELISA
whose test results were expressed at units/ml (Masuda
et al., 2000). However, in a comparative measurement
ELISA, the results must be considered as semi-quantitative
and, as a drawback of this method, results cannot becompared among different allergens since the referencesmust differ from assay to assay. In contrast, allergenspeciﬁc mouse IgE measurement has been implemented for
different allergens by establishing pre-quantiﬁed allergenspeciﬁc monoclonal mouse IgE as reference standards
for each allergen (Hamada et al., 2003). Therefore, theresults could be expressed in, for example, ␮g/ml, whichenables the comparison of the amount of allergen-speciﬁc
IgE among different allergens. With this information, weconceived the objective that we could measure canine
allergen-speciﬁc IgE by using allergen speciﬁc mouse IgE
as a reference standard if we had an antibody that could
cross-react to both canine and mouse IgE. Therefore, it wasa great advantage to obtain an anti-IgE antibody, CRE-DM,
which was proven to recognize both canine and mouse
IgE in a dose dependent manner. In fact, we could quantify Cry j 1-speciﬁc IgE in canine sera at ng/ml by using
Cry j 1-speciﬁc monoclonal mouse IgE as a reference standard in this study. The established method in this study
can be applied to other allergen-speciﬁc IgE ELISAs by creating pre-quantiﬁed allergen-speciﬁc mouse IgE to various
allergens. For this purpose, we are currently working on to
expand the test system to measure allergen-speciﬁc canine
IgE to various allergens by establishing reference standards
using mouse sera sensitized with different allergens. With
such a multi-allergen quantiﬁcation system, it will become
possible to compare the exact amount of allergen-speciﬁc
IgEs among the different allergens in dogs. Since serum IgElevels have been shown to increase and decrease in parallel
to allergen exposure (Masuda et al., 2002; Horiguchi et al.,2008), it will become possible to estimate the exposure levels of each allergen at the tested time point by comparing
the serum IgE levels among allergens.
We conclude that the results obtained from the anti-Cry
j 1 IgE ELISA were reliable since the dispersion of the results
obtained by this assay had low CV (3.1–5.2% and 2.2–8.0%
in intra- and inter-assay comparisons, respectively). These
CV values are low compared to those in a previous report(Lee et al., 2009), which was considered to demonstrate
the reliability of this assay. With such a low inter-assay
variance among the results, this ELISA could be an appropriate test to monitor the possible seasonal ﬂuctuation ofserum allergen-speciﬁc IgE in dogs, since the amounts ofconcentrations of Cry j 1-speciﬁc IgE in three independent experiments. As the results, the intra-assay CV wasfound to be 5.3%, 3.4% and 3.1% in the pooled sera of low,intermediate and high IgE groups, respectively. Likewise,
the inter-assay CV was 7.2%, 8.0% and 2.2% in the pooledsera of low, intermediate and high IgE groups, respectively(Table 2). These results indicated that the values obtainedwith the Cry j 1-speciﬁc IgE ELISA in this study were highly
reproducible and reliable.
4. Discussion
In order to measure an absolute concentration ofallergen-speciﬁc IgE in canine sera, it is necessary to use
a monoclonal antibody that is highly speciﬁc to canine
IgE. Since there are an excess amount of IgG which share
reactive antigen with IgE in the serum (Hill et al., 1995),
and that IgE and IgG have high similarities (Patel et al.,1995), IgE measurement will be inﬂuenced if the anti-IgE
antibody used in the measurement can cross-react to IgGeven at a low level. Therefore, an anti-IgE antibody usedin the detection of IgE must be highly speciﬁc to IgE andrequired to be conﬁrmed by proper assays so that the antibody does not react to IgG. We ﬁrst examined the reactivityof the anti-IgE antibody, CRE-DM, especially focusing on
the reactivity to canine IgE and IgG. In our study, it was
shown that CRE-DM reacted speciﬁcally to puriﬁed canine
IgE and mouse IgE in a dose dependent manner, but not tocanine IgG, which was further conﬁrmed by the inhibition
ELISA. The speciﬁcity of CRE-DM was also conﬁrmed in the
ELISA for canine antigen-speciﬁc IgE using the serum of anexperimentally sensitized dog. Unlike in the other previous
studies that evaluated the reactivity of anti-IgE antibody or
rhFc␧RI␣ to canine IgE only by heating the serum at 56 ◦ C(Stedman et al., 2001; Lee et al., 2009), we could obtain
direct evidence that veriﬁed the reactivity of CRE-DM by
the combination of ELISA and inhibition ELISA experiments.
Also, in a currently ongoing study, we could determine thelinear epitope sequence that is recognized by CRE-DM. CREDM was found to recognize an epitope located in the CH3
regions of dog and mouse IgEs that showed high amino acidsequence similarity between both species (Tsukui T., et al.,article in preparation). Furthermore, the identical sequence
was not found in the amino acid sequence of dog IgG.
According to these results, it was concluded that the ELISA
using CRE-DM was highly speciﬁc for the measurement ofcanine IgE.
To measure an allergen-speciﬁc IgE by a quantitative
ELISA, pre-quantiﬁed allergen-speciﬁc IgE is required as
the reference standard for each allergen to be tested. However, the only puriﬁed canine allergen-speciﬁc IgE reported

148

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T. Okayama et al. / Veterinary Immunology and Immunopathology 139 (2011) 99–106

105

To summarize, we have established a novel ELISA system for the quantitative measurement of allergen-speciﬁc
IgE in ng/ml in dogs. As we could demonstrate the reliability of the ELISA utilizing CRE-DM, we are aiming at
the implementation of CRE-DM in multi-allergen IgE test,
which might possibly become a standard allergy test. Since
the concept of the quantitative measurement of serum
allergen-speciﬁc IgE by ELISA utilizing CRE-DM can be further applied not only to dogs but also to other animal
species including humans, the ELISA has a potential to be
expanded to IgE testing in various species. In the future,comparison of the results on a same scale from medical, veterinary and experimental allergy model studies
should enhance the understanding of the pathophysiological mechanism of IgE-mediated allergic diseases.

serum allergen-speciﬁc IgE in dogs were reported to ﬂuctuate depending on seasonal allergen exposure (Masuda
et al., 2002; Horiguchi et al., 2008). By monitoring the
ﬂuctuations of IgE to seasonal environmental allergens,
it is possible to understand which environmental allergen might be associated with the manifestation of clinicalsigns in each patient at certain time points. This should
aid clinicians to deﬁne more accurate therapeutic strategies by determining the speciﬁc target allergens at eachtime point. Another possible application of this ELISA isthe evaluation of treatment effect of anti-allergic drugs,
allergen avoidance and allergen immunotherapy. Efﬁcaciesof the therapies can be evaluated by comparing allergenspeciﬁc IgE concentrations before and after treatment (vanHalteren et al., 1997; Takagi et al., 2005), which will serve
as a valuable tool to evaluate the proof of concept ofnovel therapies under investigation. The other advantage
of a quantitative system is the possible introduction of astandardization system by using pre-quantiﬁed reference
standard as a universal calibrator. It will be beneﬁcial for
veterinarians and pet owners if the test results become able
to be compared among different tests and the consistencyof the test results are validated by a proper standardization
program (DeBoer and Hillier, 2001b). Thus, the quantitative measurement of canine antigen-speciﬁc IgE utilizing
this ELISA could be beneﬁcial in many aspects of veterinary
allergy.
A relationship between the concentration of serum
antigen-speciﬁc IgE and the onset of in vivo allergic reaction has not yet been elucidated in dogs. In general, thetest results in antigen-speciﬁc IgE ELISA have been compared with those from IDT, since IDT was long considered
as a gold standard of the diagnosis of allergy (Mueller etal., 1999; Foster et al., 2003). However, in dogs, since IDT
has been shown to exhibit false-positive reactions due tosub-clinical hypersensitivity (Pastorello, 1993; Codner andTinker, 1995) and false negative results due to the insufﬁcient antigen concentrations that are required to provoke invivo reactions (Hensel et al., 2004), it is difﬁcult to consider
IDT as the deﬁnitive allergy test in dogs (DeBoer and Hillier,
2001b). Because it is advantageous to measure an absolute quantity of serum allergen-speciﬁc IgE by this ELISA,
we are currently attempting to evaluate the threshold of
IgE concentrations that are associated with in vivo allergic
reactions instead of comparing the results between ELISA
and IDT. In a currently on-going study, we have shown thata concentration of 100–200 ng/ml of antigen-speciﬁc IgEin dog serum is sufﬁcient to provoke in vivo allergic reaction by Prausnitz–Kustner test using experimental dogs
(unpublished data). Similar attempts have been made in
human medicine to predict the outcome of food provocation test in human patients with food allergy by deﬁning
the threshold of serum allergen-speciﬁc IgE titers thatelicit allergic reactions (Sampson and Ho, 1997; Sampson,
2001; Komata et al., 2009). However, because the levels ofallergen-speciﬁc IgE are usually measured in speciﬁc units(kUA /L) in human studies, it is not possible to extrapolate
the results to dogs. By further accumulating data in dogs,
we consider that this ELISA could be useful to determinea threshold concentration of serum IgE that elicit in vivo
allergic reaction or clinical symptoms of allergy.

Conﬂict of interest statement
This study was funded by Animal Allergy Clinical Laboratories, Inc., Kanagawa, Japan. Dr. Masuda is CEO andstock-holder of this company.
Acknowledgement
We would thank Dr. Takashi Saito (Research Center ofAllergy and Immunology, RIKEN) for providing monoclonal
mouse IgE speciﬁc to Cry j 1.
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⥳ෆ㞀ࡢデ᩿㸸㯞ᕸ኱Ꮫ㝃ᒓື≀⑓㝔ࢆ᮶㝔ࡋࡓࢩࣂ࢖ࢾ  ༉ࠊࢩ࣮ࢬ࣮ ༉ࢆᑐ㇟࡜ࡋࡓࠋ୧║࡟࣋ࣀ࢟ࢩ࣮ࣝࢆⅬ║ࡋ࡚ᒁ㒊㯞㓉ࢆ᪋ࡋࠊ║ᅽࢆࢺࣀ࡛࣌ࣥ

ᐃࡋࡓࠋ║ᅽ PP+J ௨ୖࡢಶయࢆ⥳ෆ㞀ࠊPP+J ᮍ‶ࡢಶయࢆ೺ᖖ࡜ุᐃࡋࡓࠋࢩࣂ࢖ࢾ 151༉࡜ࢩ࣮ࢬ࣮ ༉ࡀ೺ᖖࠊࢩࣂ࢖ࢾ  ༉࡜ࢩ࣮ࢬ࣮ ༉ࡀ⥳ෆ㞀࡜デ᩿ࡉࢀࡓࠋ㸲ṓ௨ୗࡢ࢖ࢾࡣ㝖እࡋࡓࠋ
'1$ ࡢ⢭〇㸸㢁㟼⬦ࡼࡾ⾑ᾮࢆ᥇ྲྀࡋࠊ'1$ ඲⾑࢟ࢵࢺ࣭ࢫࣆࣥἲ㸦)8-,),/0 ♫㸧࡛'1$ ࢆ⢭〇ࡋࡓࠋ'1$ ࡢ⃰ᗘ࡜⣧ᗘࢆ *HQH4XDQW3UR㸦*( ࣊ࣝࢫࢧ࢖࢚ࣥࢫ♫㸧࡛ᐃࡋࡓࠋ
'1$ ሷᇶ㓄ิࡢỴᐃ㸸65%' 㑇ఏᏊࡢ 613㸦UVUVUV
UVUV㸧ࠊ(/29/ 㑇ఏᏊࡢ 613㸦UVUVUV
UVUV㸧ྜࢃࡏ࡚  ࠿ᡤࢆゎᯒࡋࡓࠋ$'076 㑇ఏᏊ࡟ࡘ࠸࡚ࡣ
*O\$UJ *!$ ࢆゎᯒࡋࡓࠋ3&5 ࣉࣛ࢖࣐࣮ࢆ⾲㸯࡟♧ࡍࠋ3&5 ⏘≀ࢆ ࢔࣮࢞ࣟࢫࢤ࡛ࣝ㟁ẼὋືࡋࠊࢩࣥࢢࣝࣂࣥࢻࢆษࡾฟࡋ࡚Υ࡛෾⤖ࡋࡓࠋࢧࣥࣉࣝ
ࢆ⁐ゎࡋࠊ4LDJHQ'\H([ࢫࣆࣥ࢟ࢵࢺ㸦4,$*(1 ♫㸧࡛ '1$ ࢆ⢭〇ࡋࡓࠋ+LJK'\H
0L[㸦ࣛ࢖ࣇࢸࢡࣀࣟࢪ࣮ࢬ♫㸧ࢆῧຍࡋࢧ࢖ࢡࣝࢩ࣮ࢣࣥࢩࣥࢢࢆᐇ᪋ࡋࡓࠋࢧࣥࣉ
ࣝࢆࢩ࣮ࢣࣥࢩࣥࢢࢳ࣮ࣗࣈ࡟⛣ࡋࠊ$%,'1$$QDO\]HU㸦ࣛ࢖ࣇࢸࢡࣀࣟࢪ࣮ࢬ♫㸧࡛ሷᇶ㓄ิࢆゎᯒࡋࡓࠋ
⤫ィゎᯒ㸸⥳ෆ㞀⩌࡜೺ᖖ⩌ࡢ 613 ࢆ +DUG\:HLQEHUJ ᖹ⾮᳨࡛ᐃࡋࡓࠋ⥳ෆ㞀⩌࡜೺ᖖ⩌ࡢ࢔ࣞࣝ㢖ᗘࢆȮ⮬஌᳨ᐃ࡛ホ౯ࡋࡓࠋ࣓ࢱゎᯒࡢࡓࡵࡢ 3 ್࡜࢜ࢵࢬẚࢆ
0DQWHO+DHQV]HO ἲ᳨࡛ᐃࡋࡓࠋ/' ⤫ィ࡟ࡣ +DSORYLHZ ࣉࣟࢢ࣒ࣛࢆ౑⏝ࡋࡓࠋࣟࢪࢫࢸࢵࢡᅇᖐศᯒ࡟ࡣ 3/,1. ࢆ౑⏝ࡋࡓࠋ⤖ᯝ࠾ࡼࡧ⪃ᐹ⥳ෆ㞀⩌࡜೺ᖖ⩌ࡢᖹᆒᖺ㱋ࡣࢩࣂ࢖ࢾ࡛ࡣ 8.5s2.9 ṓ࡜ 10.0s3.0 ṓࠊࢩ࣮ࢬ࣮࡛
ࡣ 9.2s2.0 ṓ࡜ 10.1s2.5 ṓ࡛࠶ࡗࡓࠋ
ࢩࣂ࢖ࢾ 98 ༉࡜ࢩ࣮ࢬ࣮67 ༉ࡢ SRBD1ࠊELOVL5ࠊ
ADAMTS10 㑇ఏᏊࡢከᆺࢆࢲ࢖ࣞࢡࢺࢩ࣮ࢣࣥࢩࣥࢢἲ࡛ゎᯒࡋࡓ㸦⾲㸯㸧ࠋ ⾲㸰ࡣ SRBD1 㑇ఏᏊࡢ 5 ࠿ᡤࡢ SNP ࡢゎᯒ⤖ᯝࢆ♧ࡍࠋࢩࣂ࢖ࢾ࡛ࡣ rs22018513
࡛᭱ࡶᙉ࠸┦㛵㸦P=0.00039㸧ࢆ♧ࡋࠊG ࢔ࣞࣝࡣ⥳ෆ㞀ࣜࢫࢡࡀ 3.03 ಸ࡟࡞ࡿࡇ࡜ࡀ
ุ᫂ࡋࡓࠋrs8655283 ࡢ T ࢔ࣞࣝࡀ 2.20 ಸ㸦P=0.016㸧࡜ rs22018514 ࡢ G ࢔ࣞࣝࡀ 2.56
ಸ㸦P=0.0037㸧ࣜࢫࢡࡀ㧗ࡃ࡞ࡿࡇ࡜ࡀุ᫂ࡋࡓࠋࢩ࣮ࢬ࣮࡛ࡣ rs9172407 ࡢ G ࢔ࣞࣝࡀ 5.25 ಸ㸦P=0.0014㸧ࣜࢫࢡࡀ㧗ࡃ࡞ࡿࡇ࡜ࡀุ᫂ࡋࡓࡀࠊࢩࣂ࢖ࢾ࡛ࡣ᭷ពᕪ࡜࡞ࡽ
࡞࠿ࡗࡓࠋࢩ࣮ࢬ࣮࡛ࡣ rs8655283 ࡢ T ࢔ࣞࣝࠊrs22018514 ࡢ G ࢔ࣞࣝࠊrs22019922 ࡢ
A ࢔࡛ࣞࣝ࢜ࢵࢬẚࡀࡑࢀࡒࢀ 2.43ࠊ2.43ࠊ2.49 ಸ㧗ࡃ࡞ࡗࡓࡀ᭷ពᕪ࡟ࡣ⮳ࡽ࡞࠿ࡗࡓࠋࢩࣂ࢖ࢾ࡛ࡣ rs8655283ࠊ
ᅗ㸯ࡣ SRBD1 㑇ఏᏊࡢ 5 ࠿ᡤࡢ SNP ࡢ LD ࣈࣟࢵࢡࢆ♧ࡍࠋ
rs22018514ࠊrs22018513 ࡢ D’್ࡀ 0.78 ௨ୖ࡜᭷ព࡞┦㛵ࢆ♧ࡋࡓࠋࢩ࣮ࢬ࣮࡛ࡣ
rs22019922 ࡜ rs22018513 ࡢ D’್ࡣ 0.68 ௨ୖ࡜᭷ព࡞┦㛵ࢆ♧ࡋࡓࠋࢩࣂ࢖ࢾ⥳ෆ㞀Ⓨ⑕࡬ࡢ rs8655283ࠊrs22018514ࠊrs22018513 ࡢᙳ㡪ࢆゎ᫂ࡍࡿࡓࡵ
ࣟࢪࢫࢸ࢕ࢵࢡᅇᖐศᯒࢆ⾜ࡗࡓ⤖ᯝࢆ⾲㸱࡟♧ࡍࠋrs22018513 ࡢ㛵୚ࡀ᭱ࡶ㧗࠸ࡇ࡜

152

ࡀุ᫂ࡋࡓࠋ ⾲㸲ࡣ ELOVL5 ࡜ ADAMTS10 㑇ఏᏊࡢゎᯒ⤖ᯝ࡛࠶ࡿࠋELOVL5 㑇ఏᏊࡢ rs9194033
࡜ rs22202438 ࡣࢩࣂ࢖ࢾࠊ
ࢩ࣮ࢬ࣮࡜ࡶ࡟ከᆺ࡛࠶ࡗࡓࡀ⥳ෆ㞀࡜ࡢ┦㛵ࡣ࡞࠿ࡗࡓࠋELOVL5 ࡜ ADAMTS10 㑇ఏᏊࡢࡑࡢ௚ࡢ SNP ࡛ࡣከᆺࡀᏑᅾࡋ࡞࠿ࡗࡓࠋ
ࡇࡢ◊✲ࡢ┠ⓗࡣ SRBD1ࠊELOVL5ࠊADAMTS10 㑇ఏᏊࡢ඲ 11 ࠿ᡤࡢ SNP ࡢࢩࣂ࢖ࢾ࣭ࢩ࣮ࢬ࣮⥳ෆ㞀Ⓨ⑕࡬ࡢ㛵୚ࢆゎᯒࡍࡿࡇ࡜࡛࠶ࡗࡓࠋࡑࡢ⤖ᯝࠊࢩࣂ࢖ࢾ࡛ࡣ
SRBD1 㑇ఏᏊࡢ࢚ࢡࢯࣥ 4 ࡟Ꮡᅾࡍࡿ rs22018513 ࡀࠊࢩ࣮ࢬ࣮࡛ࡣ࢖ࣥࢺࣟࣥ 1 ࡟Ꮡ
ᅾࡍࡿ rs9172407 ࡀ⥳ෆ㞀Ⓨ⑕࡜᭷ព࡞┦㛵ᛶࢆ♧ࡋࠊ⥳ෆ㞀ឤཷᛶೃ⿵㑇ఏᏊ࡜⪃࠼
ࡽࢀࡓࠋBLAST ゎᯒ࡛ࡣ࢖ࢾ SRBD1 㑇ఏᏊࡣࣄࢺ SRBD1 㑇ఏᏊ࡜ 90%ࡢ┦ྠᛶࢆ♧ࡋࡓࠋ࢖ࢾ SRBD1 㑇ఏᏊࡢ rs22018513 ࡜ rs9172407 ࡟┦ᙜࡍࡿ㓄ิࡣࣄࢺ SRBD1 㑇ఏᏊࡢ࢚ࢡࢯࣥ 4 ࡜࢖ࣥࢺࣟࣥ 1 ࡟Ꮡᅾࡍࡿࡀ SNP ࡜ࡋ࡚ࡣሗ࿌ࡉࢀ࡚࠸࡞࠸ࠋࣄࢺ
NTG ឤཷᛶೃ⿵㑇ఏᏊ࡛࠶ࡿ SRBD1 㑇ఏᏊࡢ rs32137871 ࡣ࢖ࣥࢺࣟࣥ 17 ࡟఩⨨ࡍࡿࡀࠊ࢚ࢡࢯࣥ 4 ࡜࢖ࣥࢺࣟࣥ 1 ࡜ࡢ㐃㙐ࡀ㠀ᖖ࡟ᙉ࠸ࠋࣄࢺ SRBD1 㑇ఏᏊࡢࡉࡽ࡞ࡿゎᯒࡀᚲせ࡛࠶ࡿࠋ
SRBD1 㑇ఏᏊࡣ᭱ึ࡟኱⭠⳦ࡢ S1 RNA ⤖ྜᛶࢻ࣓࢖ࣥ࡜ࡋ࡚Ⓨぢࡉࢀࠊࣄࢺ࡛ࡣ
DNA ⤖ྜᛶࢱࣥࣃࢡ㉁ࡢ୍㒊ศ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋࣄࢺ࡜࢖ࢾࡢ SRBD1 㑇ఏᏊࡢᶵ⬟ࡣ୙࡛᫂࠶ࡿࠋ࢖ࢾ SRBD1 ࡣ➨ 10 ᰁⰍయ࡟㏫㓄ิ࡜ࡋ࡚Ꮡᅾࡍࡿ඲㛗 186kb ࡢ㑇ఏᏊ
࡛࠶ࡾࠊ3kb ࡢ࢚ࢡࢯࣥࢆྵࡴࠋ࣐࢘ࢫ࡛ࡣ⥙⭷⚄⤒⠇⣽⬊࡬ࡢᒁᅾࡀ▱ࡽࢀ࡚࠸ࡿࡀ
࢖ࢾ࡛ࡢሗ࿌ࡣ࡞࠸ࠋ௒ᅇⓎぢࡋࡓ rs22018513 ࡜ rs9172407 ࡢኚ␗ࡣ࢚ࢡࢯࣥྠ⩏ⓗ⨨᥮࡜࢖ࣥࢺࣟࣥኚ␗
࡛࠶ࡾࠊ㑇ఏᏊࡢⓎ⌧࡟㛵㐃ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡓࠋࣄࢺ࡛ࡢ◊✲ࡢࡼ࠺࡟ mRNA ࣞ࣋ࣝࡢゎᯒࡀᚲせ࡛࠶ࡿࠋSRBD1 ࡣ㛫᥋ⓗ࡟ࡣ⣽⬊ቑṪࠊࢱࣥࣃࢡ㉁ྜᡂࠊ࢔࣏ࢺ࣮
ࢩࢫㄏᑟࠊ࣓࣍࢜ࢫࢱࢩࢫ࡟㛵୚ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠾ࡾࠊSRBD1 ࡢάᛶ໬ࡣ⥙⭷⚄⤒⠇⣽⬊ࡢ⣽⬊Ṛ࠿ࡽ⥳ෆ㞀ࢆច㉳ࡍࡿ࡜⪃࠼ࡽࢀࡓࠋELOVL5 ࡣ㛗㙐୙㣬࿴⬡⫫㓟ࡢ⏕ྜᡂ࡟㛵୚ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ᪥ᮏ⥳ෆ㞀Ꮫ఍◊✲ࢢ࣮ࣝࣉࡣࣄࢺ ELOVL5 㑇ఏᏊࡀ NTG ឤཷᛶ㑇ఏᏊࡢೃ⿵࡛࠶ࡿࡇ࡜ࢆሗ࿌
ࡋࡓࡀࠊࡇࡢ◊✲࡛ࡣ࢖ࢾ⥳ෆ㞀࡜ࡢ┦㛵ᛶࡣ࡞࠿ࡗࡓࠋࡇࢀࡣࣄࢺ㸦NTG㸧࡜࢖ࢾ㸦POAG㸧ࡢ⥳ෆ㞀ࢱ࢖ࣉࡢ┦㐪࡟ࡼࡿ࡜⪃࠼ࡽࢀࡿࠋ୍᪉ࠊSRBD1 㑇ఏᏊࡣࣄࢺ࡜࢖
ࢾࡢ⥳ෆ㞀࡟ඹ㏻ࡋ࡚࠸ࡓࠋࡼࡗ࡚࢖ࢾ⥳ෆ㞀Ⓨ⑕࡟࠾ࡅࡿ SRBD1 㑇ఏᏊࡢᶵ⬟ࢆゎ᫂ࡍࢀࡤࣄࢺ⥳ෆ㞀ࡢⓎ⑕࣓࢝ࢽࢬ࣒ゎ᫂࡟ࡘ࡞ࡀࡿྍ⬟ᛶࡀ࠶ࡾࠊ࢖ࢾࡀዲ㐺࡞ື≀ࣔࢹࣝ࡟࡞ࡿ࡜ᛮࢃࢀࡿࠋ ຍ⸨ࡽࡢሗ࿌࡟ࡼࡿ࡜ࣅ࣮ࢢࣝ≟ࡣࢩࣂ࢖ࢾࠊࢩ࣮ࢬ࣮ࠊ࢔࣓ࣜ࢝ࣥࢥࢵ࣮࢝ࢫࣃ
ࢽ࢚ࣝ࡟⥆ࡃ 4 ␒┠ࡢ⥳ෆ㞀ዲⓎ≟✀࡛࠶ࡿࠋKuchtey ࡽࡣ᭱㏆ࠊࣅ࣮ࢢࣝ≟ POAG 㛵㐃㑇ఏᏊ࡜ࡋ࡚ ADAMTS10 ࡢ Gly661Arg ኚ␗ࢆሗ࿌ࡋࡓࠋᡃࠎࡶࡇࡢ SNP ࢆࢩࣂ࢖ࢾ
࡜ࢩ࣮ࢬ᳨࣮࡛ᰝࡋࡓࡀࠊ
ኚ␗ࢆ☜ㄆ࡛ࡁࡎ≟✀࡟౫Ꮡࡋࡓ SNP ࡜⪃࠼ࡽࢀࡓࠋ
Kuchteyࡽࡣ᭱㏆ࡉࡽ࡟ࡇࡢ SNP ࡀࣅ࣮ࢢࣝ≟≉␗ⓗ࡛࠶ࡾࠊ௚ࡢ≟✀࡛ࡣᏑᅾࡋ࡞࠸ࡇ࡜ࢆ
ሗ࿌ࡋࡓࠋ࢖ࢾ㑇ఏᏊࢹ࣮ࢱ࣮࣋ࢫ࡛ࡣ ADAMTS10 㑇ఏᏊ࡟ 16 ࠿ᡤ SNP ࡀᏑᅾࡍࡿ

153

ࡇ࡜ࡀࢃ࠿ࡗ࡚࠾ࡾࠊࡇࢀࡽ SNP ࡢ⥳ෆ㞀࡜ࡢ┦㛵ᛶࢆゎᯒࡍࡿᚲせࡀ࠶ࡿࠋ ࢖ࢾ⥳ෆ㞀Ⓨ⑕ࢆண᝿ࡍࡿ᭷ຊ࡞㑇ఏᏊ᳨ᰝ᪉ἲࡣࡲࡔ࡞࠸ࠋᡃࠎࡀ௒ᅇࢩࣂ࢖ࢾ
࡜ࢩ࣮ࢬ࣮࡛Ⓨぢࡋࡓ SNP ࡣ⥳ෆ㞀㑇ఏᏊ᳨ᰝᑐ㇟࡜ࡋ࡚᭷ຊ࡜⪃࠼ࡽࢀࡓࠋ
ࡋ࠿ࡋࠊ࡝ࡢࡼ࠺࡞ᶵᵓ࡛࢖ࢾ⥳ෆ㞀ࡀ㉳ࡁࡿ࠿ࡢ◊✲ࡣ኱ࡁ࡞ㄢ㢟࡛࠶ࡿࠋࣄࢺ࡜࢖ࢾ࡛ࡢ
SRBD1 㑇ఏᏊࡢᶵ⬟ࡢࡉࡽ࡞ࡿ◊✲ࡣ⥳ෆ㞀ࡢⓎ⑕ࣜࢫࢡࡢண

ࠊண㜵་Ꮫࠊ἞⒪᪉ἲࡢᶞ❧࡟኱ࡁࡃ㈉⊩ࡍࡿ࡜ᮇᚅࡉࢀࡿࠋࡲࡓࠊSRBD1 㑇ఏᏊ᳨ᰝ࡟ࡼࡾࠊ࢖ࢾࡢ⥳ෆ㞀ࢆ᪩ᮇ࡟ṇ☜࡟デ᩿ࡍࡿࡇ࡜ࡀྍ⬟࡜࡞ࡿࠋࡍ࡛࡟⥳ෆ㞀ࢆⓎ⑕ࡋࡓಶయ࡛ࡣ☜ᐃデ᩿ࡀྍ⬟࡜࡞ࡾࠊ✚ᴟⓗ࡞἞⒪ࢆ᪩ᮇ࡟㛤ጞࡍࡿࡇ࡜࡛ኻ᫂ࡢ༴ᶵࢆᅇ㑊࡛ࡁࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࠋ⾲㸯 ⥳ෆ㞀㑇ఏᏊゎᯒ࡟⏝࠸ࡓࣉࣛ࢖࣐࣮ࡢሷᇶ㓄ิ⾲㸰 SRBD1 㑇ఏᏊゎᯒ⤖ᯝ

154

ᅗ㸯 SRBD1 㑇ఏᏊࡢ LD ࣈࣟࢵࢡ㸦A ࡣࢩࣂ࢖ࢾࠊB ࡣࢩ࣮ࢬ࣮ࡢ⤖ᯝࢆ♧ࡍ㸧⾲㸱 ࢩࣂ࢖ࢾ SRBD1 㑇ఏᏊࡢࣟࢪࢫࢸࢵ࢕ࢡᅇᖐศᯒ

⾲㸲 ELOVL5 㑇ఏᏊ࡜ ADAMTS10 㑇ఏᏊࡢゎᯒ⤖ᯝ155㸰㸬≟ࡢⓑෆ㞀ࡢ㑇ఏᏊゎᯒ༳∾ಙ⾜ࠊᕝཎ஭᫴ᖹ㸦㯞ᕸ኱Ꮫ㝃ᒓື≀⑓㝔㸧㜰ཱྀ㞞ᘯࠊᒸᮏ᠇᫂ࠊ㧗℩᭷ຍ㔛㸦㯞ᕸ኱Ꮫᚤ⏕≀Ꮫ➨㸯㸧◊✲せ᪨࢔࣓࣭ࣜ࢝ࣥࢥࢵ࣮࣭࢝ࢫࣃࢽ࢚ࣝࡣⱝᖺᛶⓑෆ㞀࡟࠾ࡅࡿཎᅉ㑇ఏᏊࡢ㑇ఏᏊゎᯒࢆ⾜ࡗࡓࠋ௨๓࠿ࡽ࣎ࢫࢺ࣭ࣥࢸࣜ࢔࡞࡝࡛ሗ࿌ࡢ࠶ࡗࡓ㑇ఏᏊࡢኚ␗ࡣㄆࡵࡽࢀ࡞࠿ࡗ
ࡓࠋⱝᖺᛶⓑෆ㞀࢔࣓࣭ࣜ࢝ࣥࢥࢵ࣮࣭࢝ࢫࣃࢽ࢚ࣝࢆ DNA ᥇ྲྀ㡰఩࡟ A ࡜ B ࡢ 2ࡘࡢ㞟ᅋ࡟ศࡅ࡚ゎᯒࢆ࠾ࡇ࡞ࡗࡓࠋA 㞟ᅋ࡟࠾࠸࡚ࡣ⥙⨶ⓗ࡞ SNP ࢔࣮ࣞࢆ⾜࠸ࠊ15 ␒ᰁⰍయୖࡢ rs22428454(࢚ࣥࢻࢭࣜࣥཷᐜయࢱ࢖ࣉ A 㑇ఏᏊ)࡜ 31 ␒ᰁⰍయୖࡢ
㑇ఏᏊ㠀ࢥ࣮ࢻ㡿ᇦ rs23685575㸦㑇ఏᏊ㠀ࢥ࣮ࢻ㡿ᇦ㸧ࡀⓎ⑕࡜ࡢ㛵㐃ࢆ♧၀ࡍࡿ⤖ᯝࡀᚓࡽࢀࡓࠋࡋ࠿ࡋࠊB 㞟ᅋ࡟࠾࠸࡚ࡣࡇࡢ⤖ᯝࡢ෌⌧ᛶࡀᚓࡽࢀ࡞࠿ࡗࡓࠋ
◊✲┠ⓗ࢔࣓࣭ࣜ࢝ࣥࢥࢵ࣮࣭࢝ࢫࣃࢽ࢚ࣝࡣⱝᖺᛶⓑෆ㞀ࡢዲⓎ≟✀࡜▱ࡽࢀ࡚࠸ࡿࠋⱝᖺᛶⓑෆ㞀ࡣ⏕ᚋ 3 ṓࡃࡽ࠸ࡲ࡛࡟Ỉᬗయࡢΰ⃮ࢆ♧ࡋࠊどຊࡀኻࢃࢀࡿ⑌ᝈ࡛ᖖᰁⰍయᛶ
ຎᛶ㑇ఏ࡜⪃࠼ࡽࢀ࡚࠸ࡿࡀཎᅉ㑇ఏᏊࡣ୙࡛᫂࠶ࡿࠋᮏ◊✲ࡣ࢔࣓࣭ࣜ࢝ࣥࢥࢵ࢝
࣮࣭ࢫࣃࢽ࢚ࣝ࡟࠾ࡅࡿⱝᖺᛶⓑෆ㞀ࡢཎᅉ㑇ఏᏊࢆ᳨ウࡍࡿࡓࡵ࡟ࡇࡢ≟ࡢⱝᖺᛶⓑෆ㞀ࡢ DNA ࢆ཰㞟ࡋࠊ⥙⨶ⓗ࡞ SNP ࢔࣮ࣞࢆ⾜࠸ࠊࡑࡢ㑇ఏᏊゎᯒࢆ⾜ࡗࡓࠋᮦᩱ࡜᪉ἲ㯞ᕸ኱Ꮫ㝃ᒓື≀⑓㝔࡟⤂௓ࡉࢀࡓ࢔࣓࣭ࣜ࢝ࣥࢥࢵ࣮࣭࢝ࢫࣃࢽ࢚ࣝ≟࡛ⱝᖺᛶⓑෆ㞀࡜デ᩿ࡉࢀࡓ౛ࢆ㝧ᛶ౛ࠊⱝᖺᛶⓑෆ㞀ࢆⓎ⑕ࡋ࡚࠸࡞࠸౛ࢆ㝜ᛶ౛࡜ࡋࡓࠋ᭱ึ࡟
᥇ྲྀࡉࢀࡓ㝧ᛶ౛ 32 ౛ࠊ㝜ᛶ౛ 8 ౛ࢆ A 㞟ᅋ࡜ࡋࡓࠋࡲࡓࠊA 㞟ᅋ࡜ࡣู࡟཰㞟ࡉࢀࡓ㝧ᛶ౛ 17 ౛ࠊ㝜ᛶ౛ 16 ౛ࢆ B 㞟ᅋ࡜ࡋࡓࠋࡇࢀࡽࡢ≟࠿ࡽ DNA ࢆ⢭〇ࡋࠊA 㞟ᅋ࡟࠾࠸࡚⥙⨶ⓗ࡞ゎᯒ࡜ࡋ࡚ SNP ࢔࣮ࣞ㸦࢖࣑ࣝࢼ♫㸧ࢆ⾜ࡗࡓࠋࡑࡢ SNP ゎᯒࡢ⤖ᯝ࠿ࡽᚓࡽࢀࡓᡤぢ࠿ࡽⱝᖺᛶⓑෆ㞀࡜ࡢ㛵㐃ࡀ␲ࢃࢀࡿ SNP ࢆྵࡴ࢚ࣥࢻࢭࣜ
ࣥཷᐜయࢱ࢖ࣉ A 㑇ఏᏊࡢ࢚ࢡࢯࣥ㒊ศࡢ඲㑇ఏᏊࡢࢩ࣮ࢡ࢚ࣥࢫࢆ A 㞟ᅋࡢ୰࠿ࡽ
㝧ᛶ౛ 21 ౛ࠊ㝜ᛶ౛ 5 ౛ࢆ㑅ࢇ࡛ㄪ࡭ࡓࠋࡲࡓࠊSNP ゎᯒࡢ⤖ᯝ࠿ࡽᚓࡽࢀࡓ㛵㐃ࡍࡿ㑇ఏᏊࡢ SNP ࡟ࡘ࠸ู࡚ࡢ㞟ᅋ࡛࠶ࡿ B 㞟ᅋ࡟࠾࠸࡚㑇ఏᏊゎᯒࢆ⾜࠸ࠊ⥙⨶ⓗ࡞
SNP ࢔࣮ࣞࡢ⤖ᯝࡢ☜ㄆࢆ⾜ࡗࡓࠋ⤖ᯝ࠾ࡼࡧ⪃ᐹࡇࢀࡲ࡛࡟㑇ఏᛶⓑෆ㞀ࡢཎᅉ㑇ఏᏊ࡜ࡋ࡚ࡣࠊ࣎ࢫࢺ࣭ࣥࢸࣜ࢔ࠊ࣮࢜ࢫࢺࣛࣜ࢔࣭ࣥ
ࢩ࢙ࣃ࣮ࢻࠊࢫࢱࢵࣇ࢛࣮ࢻࢩ࣮࣭ࣕࣈ࣭ࣝࢸࣜ࢔࡟࠾࠸࡚ࠊHeat shock transcription

156

factor 4 㑇ఏᏊࡢኚ␗ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡇࢀࡣࡇࡢ㑇ఏᏊࡢ࢚ࢡࢯࣥ㸷࡟࠶ࡿ C ࡀ୍ሷᇶḞኻࡋ࡚ࠊࡑࡢ⤖ᯝࠊࣇ࣮࣒ࣞࢩࣇࢺࡀ࠾ࡇࡾࠊ೵Ṇࢥࢻࣥࡀฟ⌧ࡍࡿ࡜࠸࠺ࡶ
ࡢ࡛࠶ࡗࡓࠋᮏ◊✲࡟࠾࠸࡚ A 㞟ᅋࡢⱝᖺᛶⓑෆ㞀ࡢ㸰㸳౛࡟ࡘ࠸࡚ྠࡌኚ␗ࡀᏑᅾ
ࡍࡿ࠿ㄪ࡭ࡓࠋᮏ◊✲࡟࠾࠸࡚㸰㸳౛ࡍ࡭࡚ C 㑇ఏᏊࡢḞᦆࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡑ
ࡢࡇ࡜࠿ࡽ࢔࣓࣭ࣜ࢝ࣥࢥࢵ࣮࣭࢝ࢫࣃࢽ࢚ࣝ≟ࡢⱝᖺᛶⓑෆ㞀ࡣࡇࢀࡲ࡛ࡢሗ࿌࡜ࡣ
␗࡞ࡿ㑇ఏᏊኚ␗࡛㉳ࡇࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡓࠋ
࢔࣓࣭ࣜ࢝ࣥࢥࢵ࣮࣭࢝ࢫࣃࢽ࢚ࣝ≟࡛ⱝᖺᛶⓑෆ㞀࡜デ᩿ࡉࢀࡓ౛ࢆ㝧ᛶ౛ࠊⱝᖺᛶⓑෆ㞀ࢆⓎ⑕ࡋ࡚࠸࡞࠸౛ࢆ㝜ᛶᑐ↷࡜ࡋࡓࠋ᭱ึ࡟᥇ྲྀࡉࢀࡓ㝧ᛶ౛ 32 ౛ࠊ㝜ᛶ౛
8 ౛ࢆ⥙⨶ⓗ࡞ SNP ࢔࣮ࣞࢆ⾜࠸ࠊ15 ␒ᰁⰍయୖࡢ rs22428454(࢚ࣥࢻࢭࣜࣥཷᐜయ
ࢱ࢖ࣉ A 㑇ఏᏊ)࡜ 31 ␒ᰁⰍయୖࡢ㑇ఏᏊ㠀ࢥ࣮ࢻ㡿ᇦ rs23685575㸦㑇ఏᏊ㠀ࢥ࣮ࢻ㡿ᇦ㸧ࡀⓎ⑕࡜ࡢ㛵㐃ࢆ♧၀ࡍࡿ⤖ᯝࡀᚓࡽࢀࡓ㸦ᅗ 1ࠊ⾲㸯㸧ࠋ ࢚ࣥࢻࢭࣜࣥཷᐜయࢱ࢖ࣉ A 㑇ఏᏊࡣ㸵ࡘࡢ࢚ࢡࢯࣥ࠿ࡽ࡞ࡾࠊrs22428454 ࡣ࢚ࢡࢯࣥ㸯࡜࢚ࢡࢯࣥ㸰ࡢ㛫ࡢ࢖ࣥࢺࣟࣥ㡿ᇦ࡟Ꮡᅾࡍࡿ㸦ᅗ㸰㸧ࠋ࢚ࣥࢻࢭࣜࣥཷᐜయ࡟
ࣜ࢞ࣥࢻࡀ⤖ྜࡍࡿ࡜ࠊ⣽⬊ෆࡸ⣽⬊⭷ࡢ Ca2㸩ࢳࣕࢿࣝࡀ㛤ᨺࡋࠊ⣽⬊ෆ࡟ Ca2㸩ࡀ⵳✚ࡍࡿࠋⓑෆ㞀Ỉᬗయෆ࡛ࡣ Ca ⃰ᗘୖ᪼ࡀࡳࡽࢀࡿ࡜ࠊ࢝ࣝࣃ࢖ࣥ㸦Ca ࡟ࡼࡗ࡚άᛶࡍࡿࢱࣥࣃࢡ㉁ศゎ㓝⣲㸧࡟ࡼࡿࢡࣜࢫࢱࣜࣥࡢศゎࡀஹ㐍ࡋ࡚Ỉᬗయࡢⓑ⃮໬ࡀ㉳ࡇࡿ࡜ྍ⬟ᛶࡀ࠶ࡿࠋCa ⃰ᗘ࡟㛵ࢃࡿ࢚ࣥࢻࢭࣜࣥཷᐜయࡢ␗ᖖࡣࠊⓑෆ㞀Ⓨ⑕࡟㛵୚ࡍࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋࡇࢀࡽࡢࡇ࡜࠿ࡽ࢔࣓࣭ࣜ࢝ࣥࢥࢵ࣮࣭࢝ࢫࣃࢽ࢚ࣝ
ࡢⓑෆ㞀≟㝧ᛶ౛ 21 ౛ࠊ㝜ᛶ౛ 5 ౛ࡢ࢚ࣥࢻࢭࣜࣥཷᐜయࢱ࢖ࣉ A 㑇ఏᏊࡢ඲㑇ఏᏊ㓄ิࡢゎᯒࢆ⾜ࡗࡓࠋࡋ࠿ࡋࠊ࢚ࢡࢯࣥ㒊ศ࡟࠾࠸࡚௚ࡢኚ␗ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋࡇ
ࡢ࢚ࣥࢻࢭࣜࣥཷᐜయࢱ࢖ࣉ A 㑇ఏᏊࡢ࢚ࢡࢯࣥ㒊ศࡢ㑇ఏᏊኚ␗ࡀⓑෆ㞀Ⓨ⑕࡟㛵୚ࡍࡿྍ⬟ᛶࡣప࠸࡜⪃࠼ࡽࢀࡓࠋ

157

ᘙᲫ ႉϋᨦཚư᭗᫁ࡇƴᛐǊǒǕƨ 502%*4$250270#&,)%$10(*1./
TU'
TU'
TU'
TU'
TU'
TU'

158

◊✲Ⓨ⾲
㸯.ㄽᩥⓎ⾲1)༳∾ಙ⾜ࠊᕷᕝ㝧୍ᮁࠊᕝཎ஭᫴ᖹࠊⴠྜ⚽἞㸬㯞ᕸ኱Ꮫ║⛉࡟᮶㝔ࡋࡓ⥳ෆ㞀⑕౛ࡢ㝶క⑕ࡢศ㢮ࠊ᪥ᮏ⋇་ᖌ఍㞧ㄅࠊ68ࠊ55-58ࠊ2015㸬

2)

Kanemaki N, Tchedre K T, Imayasu M, Kawarai S, Sakaguchi M, Yoshino A, Itoh N,
Meguro A, Mizuki N.: Dogs and humans share a common susceptibility gene
SRBD1 for glaucoma risk. PLoS One, 8, 9, e74372, 2013.

2. Ꮫ఍Ⓨ⾲
1)

Ota N, Kanemaki N, Tsujita H, Kobayashi Y, Abe M, Takimoto Y, Imayasu M,
Meguro A, Mizuki N. Associations of single-nucleotide polymorphisms of SRBD1
gene 64 with glaucoma in Shiba-inu dog: A meta-analysis in six Japaneseveterinary hospitals. ACVO Annual meeting, Coeur d̓Alene, ID, USA, Oct, 2015.

2)

Imayasu M, Kanemaki N, Meguro A, Mizuki N. A novel single nucleotide
polymerism asocial woth glaucoma in Shiba-inu and Shih-Tzu dogs, ARVO Annual
Meeting, Orlando, FL, USA, May, 2014.

3)

Kanemaki N, Tchedre KT, Imayasu M, Meguro A, Mizuki N. Identification of S1
RNA binding domain-1 SRBD1 as a major gene determing glaucoma in dogs. ARVO
Annual Meeting, Seattle, WA, USA, May, 2013.

159

原

著

麻布大学眼科に来院した緑内障症例の随伴症の分類
印牧信行 1）

市川陽一朗 2）

川原井晋平 1）

1）麻布大学附属動物病院（〒 252‑5201
2）千葉県

落合秀治 3）†

相模原市中央区淵野辺 1‑17‑71）

開業（いちかわ動物病院：〒 270‑0011

3）麻布大学生物科学総合研究所（〒 252‑5201

松戸市根木内 168‑1）

相模原市中央区淵野辺 1‑17‑71）（2013 年 1 月 30 日受付・2014 年 9 月 22 日受理）

要約

麻布大学眼科に来院した犬の緑内障症例の発生状況と随伴症について調べた．1994 年 4 月から 2011 年 12 月までに麻布大学附属動物病院眼科に来院した犬 2,981 頭を対象とした．柴犬の緑内障罹患率は，来院数の上位 21 犬種のうちで，最も高かった（84 頭 /196 頭＝42.9％）．柴犬の緑内障眼 121 眼において，角膜混濁 57 眼（47.1％），眼球拡張 102 眼（84.3％），網膜視神経乳頭の萎縮及び陥凹 66 眼（54.5％），水晶体脱臼 50 眼（41.3％）が認められた．眼球拡張，視神経乳頭萎縮，水晶体脱臼を示す緑内障眼は高眼圧値を示した．水晶体脱臼を示す緑内障眼の 90％は水晶体亜脱臼であった．─キーワード：臨床徴候，緑内障，柴犬．
日獣会誌

68，55 〜 58（2015）

緑内障は眼圧上昇に起因して視覚障害をもたらす疾患る症例では主原因あるいは症候が重度であった原因の疾で，犬種依存性の高い眼科疾患の一つであることが知ら患を診断名として集計した．緑内障症例は臨床症候と眼れる［1‑3］．その罹患犬種には，アメリカンコッカー

圧値 25mmHg 以上を示したものを緑内障と診断した．

スパニエル，ビーグル，バゼットハウンド，チャウチャ

眼 圧 測 定 は 眼 圧 計（TONO-PEN XL, Reicher t,ウ，シャーペイ，ボストンテリア，ワイヤーフォックス

U.S.A.）を用いて，午前 9 時から午後 5 時までの外来診

テリア，柴犬などがある［4, 5］．

療時間帯で計測した．また，今回の調査では初診来院時緑内障の臨床徴候は病期によって変化し，急性期にはに眼圧値が記録されていなかった症例で臨床症候，病歴

赤目，角膜浮腫，突発的な視覚喪失がみられ，次いで慢から診断された緑内障を症候性緑内障とした．これらの性期には急性期の症候に加えて，網膜視神経乳頭の陥凹

症例はすべて，カルテの記載データ及び記録写真の所見または萎縮，眼球拡張や水晶体脱臼がみられる［1］．

から判断した．随伴所見は角膜混濁，眼球拡張，眼底像

緑内障好発犬種では原発緑内障と続発緑内障が混在しの視神経乳頭所見，水晶体脱臼について調べた．角膜混て発症することがある．また，原発緑内障は犬種間で，濁の程度は，肉眼では認めがたく細隙灯顕微鏡で識別可

発症年齢，性差及び発症病態は異なる［2］．そのため，能な軽度の混濁を軽度，虹彩が透視できる中等度の混犬種間における緑内障の発生状況は，日常診療で遭遇す

濁を中等度，また虹彩や瞳孔が見えない重度の混濁を重る臨床所見を知る重要情報と考えられる．

度とした．さらに，眼球拡張は肉眼的に明らかな症例とそこで，今回，麻布大学附属動物病院眼科に来院した

し，また水晶体脱臼は完全脱臼と亜脱臼のものに分類

緑内障の発生状況と緑内障における随伴所見について調し，脱臼した位置について記録した．視神経乳頭所見は，査した．

乳頭の萎縮，陥凹と正常所見に分類とした．
統計処理：来院頻度及び症例の頻度はフィッシャーの材 料 及 び 方 法

直接確率検定または分割表分析で行った．また随伴所見における眼圧値の有意差検定は Welch の t 検定並びに1994 年 4 月から 2011 年 12 月までに麻布大学附属動Schef f 多重比較法で行った．

物病院眼科に来院した 2,981 頭を対象とした．眼疾患症
例の集計は重複集計を避けるため，複数の診断名を有す† 連絡責任者：落合秀治（麻布大学生物科学総合研究所）〒 252‑5201 相模原市中央区淵野辺 1‑17‑71☎ 042‑754‑7111 FAX 042‑754‑9930
E-mail : ochiaih@azabu-u.ac.jp

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麻布大学眼科に来院した緑内障症例の随伴症の分類
犬種（緑内障発生率）
シーズー （9.0％）
ミニチュアダックスフンド （3.3％）
柴犬（42.9％）
アメリカンコッカースパニエル （7.0％）
トイプードル （2.2％）
キャバリアキングチャールズスパニエル （2.8％）
ゴールデンレトリーバー （7.6％）
マルチーズ （6.9％）
ヨークシャーテリア （1.1％）
シェットランドシープドッグ （2.5％）
ミニチュアシュナウザー （0％）
ラブラドールレトリーバー （7.7％）
チワワ （5.6％）
パグ （3.0％）
ポメラニアン （0％）
パピヨン （8.5％）
ウェルシュコーギー （0％）
ビーグル（11.9％）
ボーダーコリー，コリー種 （0％）
シベリアンハスキー（13.2％）
ウエストハイランドホワイトテリア （0％）
その他の 75 犬種
交雑種

＊

＊

0

100

200

300

P＜0.01
非緑内障症例
緑内障症例

400500（総症例数：2,981）

外来症例数
図1

眼底の疾患（5）2.6％

犬種間における緑内障発生状況表1

その他（11）5.5％

N

眼瞼疾患（9）4.6％

分類

角結膜疾患（9）4.6％
原発水晶体
脱臼（10）
5.1％

計

罹患眼数

初診来院
年齢

性

頭
片側性 片側性 両側性雄 雌（眼） （右眼）（左眼）（両眼）

緑内障
53
症例
（73）
症候性
20
緑内障
（26）
症例
11
混合
＊
（22）
症例

緑内障（84）
42.9％

前部ぶどう
膜疾患（21）
10.7％

図2

柴犬緑内障における患側眼，性及び年齢

84
（121）

（平均
±標準偏差）

20

13

40

21 32

7.4±2.38

7

7

12

8 12

7.8±2.78

─

─

22

5

6

7.5±1.75

27

20

74

34 50

7.5±2.39

白内障（47）24.0％＊：1 眼は緑内障で，他眼が症候性緑内障眼である症例

柴犬の眼科疾患の発生頻度

障と他の眼科疾患との来院状況（図 2）をみると，緑内
障が 42.9％で第 1 位，次いで，白内障が 24.0％，前房出血を含む前部ぶどう膜疾患 10.7％，原発水晶体脱臼

成5.1％，角結膜疾患 4.6％，眼瞼疾患 4.6％，眼底の疾患

績2.6％，その他 5.5％が認められた．

犬種間における緑内障発生状況を調べた（図 1）．そ次いで柴犬緑内障における患眼の左右差，性差並びにの結果，緑内障の来院頻度は来院上位 21 犬種のうち，初診来院年齢を調べた（表 1）．調査対象とした緑内障

柴 犬 42.9％， シ ベ リ ア ン ハ ス キ ー 13.2％， ビ ー グ ル

は緑内障症例 53 頭 73 眼，症候性緑内障症例 20 頭 26 眼，

11.9％，シーズー 9.0％，パピヨン 8.5％の順にみられ，その混合症例 11 頭 22 眼の計 84 頭 121 眼であった．緑

柴犬の緑内障罹患率は他の上位 20 犬種との間で有意に内障症例と症候性緑内障症例において，性差，患眼の左高かった（P＜0.01）．また，柴犬 196 頭において緑内

右差はみられなかった．また，初診来院年齢は全体とし

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印牧信行

市川陽一朗

川原井晋平

他

表 2 柴犬の緑内障眼及び症候性緑内障眼における随伴所見

背

側

12

緑内障眼（N＝84）
所

見

緑内障眼及び
症候性緑内障眼（N＝121）

10

背内側

背外側

8

眼圧値
（mmHg）

N

（％）

角膜混濁
の程度

重度
中等度
軽度
なし

20 23.8 42.2±7.63
13 15.5 40.9±10.14
2
2.4 46.5±2.12
49 58.3 40.2±8.52

37
18
2
64

30.6
14.9
1.6
52.9

眼球拡張

ある
なし

68 81.0 43.6±6.91a
16 19.0 29.5±3.80a

102
19

84.3
15.7

網膜視
神乳頭
所見

萎縮
陥凹
正常
判定＊
不能

38 45.2 44.7±6.92a
8
9.5 39.6±10.06b
13 15.5 30.2±3.85a，b，c

56
10
14

46.3
8.2
11.6

41

33.9

腹

5

4.1

緑内障眼（N＝32）

45
71

37.2
58.7

緑内障眼＋症候性緑内障眼（N＝45）

N （％）

水晶体
脱臼

完全
脱臼
亜脱臼
なし

25 29.8 41.1±7.253c

3.6 43.7±8.33
a32 38.1 43.8±8.21
49 58.3 38.8±8.24a

6
4
2

内

側

外

0

腹内側

図3

a‑c：2 群及び多重比較検定において，同一文字の間で有意差（P＜0.05）を認める．
＊透光体の混濁により観察されなかった．

側

腹外側
側

柴犬緑内障における水晶体亜脱臼の脱臼変位方向とに依ると考えられた．
種々の犬種で報告された原発緑内障の発症年齢は 4 〜
8 歳の間で，およそ 6 歳である［4］．今回の柴犬の成績て 7.5±2.39 歳であった．
柴犬の緑内障眼及び症候性緑内障眼における随伴所見

では初診来院年齢は平均 7.5 歳であった．これは本調査

は表 2 に示した．全患眼 121 眼のうち，角膜混濁は患症例がすべて紹介症例であったことにより高齢であった

眼の 45.5％が混濁明瞭な中等度及び重度を示したが，と考えられた．また，性差では雌での発症が多いとの報

残りのうち 52.9％は透明角膜を示した．眼球拡張は患

告［4］もあるが犬種により異なり，柴犬では性差は認眼の 84.3％を示した．視神経乳頭の萎縮・陥凹所見はめられなかった．

患眼の 54.5％で認められたが，角膜・前房・水晶体・

柴犬緑内障の随伴所見では，角膜混濁は調査緑内障眼

硝子体といった透光体の混濁によって乳頭観察が不能での約 4 割が中等度及び重度の混濁で，隅角が可視できなあった症例眼は 33.9％を占めた．また随伴所見と眼圧い程度の混濁を示した．また，眼球拡張並びに網膜視神

値との関係では，眼球拡張，視神経乳頭陥凹・萎縮，水経萎縮を示した緑内障眼は平均眼圧値が 40mmHg 以

晶体亜脱臼を示す緑内障眼は高眼圧値を示した（表 2）．

上を示し，Gelatt ら［2］の眼圧値分類による重度緑内水晶体脱臼は患眼 121 眼のうち，水晶体脱臼を示さ障に相当した．緑内障は非可逆的病態を示す疾患であるない眼球が 71 眼（58.7％）で，水晶体脱臼眼が 50 眼ことから，眼球拡張並びに網膜視神経萎縮を示す疾患眼（41.3％）であった（表 2）．また水晶体脱臼眼は完全脱では眼圧測定は不可欠であることが認められた．

臼 5 眼（4.1％）， 亜 脱 臼 45 眼（37.2％） が 認 め ら れ，

緑内障でみられる水晶体脱臼は，今回の調査眼の約 6

亜脱臼の発生頻度は水晶体脱臼眼の 9 割を占めた．その割で観察されなかったが，水晶体脱臼を示す患眼の 9 割

水晶体亜脱臼は腹内側から背側方向（腹内側，内側，背（45/50 眼）が水晶体亜脱臼を示し，またその脱臼方向

内側，背側）に変位したものが 45 眼中の 35 眼（77.8％）は背・内側方向であることを示した．近年，水晶体脱臼でみられた（図 3）．

の関連遺伝子が原発緑内障の素因に関わることが報告さ

考れ，緑内障と水晶体脱臼の関連が指摘されるようになっ察た［6, 7］．今回の緑内障眼における水晶体脱臼は眼球

日 本 国 内 に お い て， 緑 内 障 の 犬 種 発 生 率 は， 柴 犬拡張に関連するものと推察されるが，水晶体小帯断裂の33％，シーズー 16.5％，交雑種犬 7.9％，アメリカンコッ

原因が眼圧上昇と眼球拡張だけに起因するかは，さらな

カースパニエル 6.3％であると Kato ら［5］によって報る検討が必要であると考えられた．

告されている．今回の柴犬の罹患率 42.9％は Kato らの成績の整理にご協力いただいた小松紘之氏に感謝の意を表す
る．

報告よりも高く，眼科診療症例に限定されて集計したこ
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引

用

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son TM, Gelatt KN, Haines JL, Kuchtey R W : Map-

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4th ed, 767‑786, Blackwell Publishing, Oxford (2007)

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［ 7 ］ Morales J, Al-Sharif L, Khalil DS, Shinwari JM, Bavi P,

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Al Tassan N : Homozygous mutations in ADAMTS10［ 4 ］ Gelatt KN, MacKay EO : Prevalence of the breedrelated glaucoma in pure-breed dogs in Nor th Ameri-

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tis, glaucoma, spherophakia, and shor t stature, Am J
Hum Genet, 85, 558‑568 (2009)

H : Incidence of canine glaucoma with goniodyspla-

Classification of Clinical Findings of Glaucoma in Dogs Presenting at the Ophthalmic
Depar tment of Azabu Veterinar y Teaching Hospital
Nobuyuki KANEMAKI 1 ) , Yoichiro ICHIKAWA2) , Shinpei KAWARAI 1 ) and Hidehar u OCHIAI 3 )†1) Veterinar y Teaching Hospital of Azabu University, 1‑17‑71 Fuchinobe, Chuo-ku, Sagamihara, 252‑5201, Japan2) Ichikawa Animal Hospital, 168‑1 Negiuchi, Matusdo, 270‑0011, Japan
3) Research Institute of Biosciences, Azabu University, 1‑17‑71 Fuchinobe, Chuo-ku, Sagamihara, 252‑5201, JapanSUMMARY
We sur veyed incidences and clinical findings of glaucoma according to breed in 2,981 dogs presented to theophthalmic depar tment at the Veterinar y Teaching Hospital of Azabu University from April 1994 to December
2011. Among the 21 major breeds presented, Shiba Inu dogs displayed the highest rate of glaucoma, at 42.9%(84/196). Of 121 eyes of glaucomatous Shiba Inu dogs, there were 57 eyes with cor neal opacity (47.1%), 102eyes with enlargement of the eyeball (84.3%), 66 eyes with atrophy and cuppings of the retinal optic disc (54.5%)
and 50 eyes with lens luxation (41.3%). High intraocular pressure was presented in glaucomatous Shiba Inu
dogs with either enlargement of the eyeball, atrophy of the retinal optic disc or lens luxation. Subluxation of
the lens accounted for 90% in the glaucomatous eyeballs with lens luxation.
─ Key words : Clinical signs, Glaucoma, Shiba Inu.
† Correspondence to : Hideharu OCHIAI (Research Institute of Biosciences, Azabu University)
1‑17‑71 Fuchinobe, Chuo-ku, Sagamihara, 252‑5201, JapanTEL 042‑754‑7111 FAX 042‑754‑9930 E-mail : ochiaih@azabu-u.ac.jp

J. Jpn. Vet. Med. Assoc., 68, 55 〜 58 (2015)

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Dogs and Humans Share a Common Susceptibility Gene
SRBD1 for Glaucoma Risk
Nobuyuki Kanemaki1, Kissaou T. Tchedre2, Masaki Imayasu2, Shinpei Kawarai3, Masahiro Sakaguchi3,
Atsushi Yoshino4, Norihiko Itoh4, Akira Meguro4, Nobuhisa Mizuki4*
1 Veterinary Teaching Hospital, Azabu University, Sagamihara, Kanagawa, Japan, 2 Central R&D Laboratory, Menicon Co., Ltd., Kasugai, Aichi, Japan, 3 Department of
Veterinary Microbiology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan, 4 Department of Ophthalmology, Yokohama City University
School of Medicine, Yokohama, Kanagawa, Japan

Abstract
Glaucoma is a degenerative optic neuropathy that is associated with elevated intraocular pressure. Primary open angle
glaucoma is the most common type of glaucoma in canines, and its highest incidence among dog breeds has beenreported in Shiba-Inus, followed by Shih-Tzus. These breeds are known to have an abnormal iridocorneal angle anddysplastic prectinate ligament. However, the hereditary and genetic backgrounds of these dogs have not yet been clarified.
In this study, we investigated the association between polymorphisms of the glaucoma candidate genes, SRBD1, ELOVL5,
and ADAMTS10, and glaucoma in Shiba-Inus and Shih-Tzus. We analyzed 11 polymorphisms in these three genes using
direct DNA sequencing. Three SRBD1 SNPs, rs8655283, rs22018514 and rs22018513 were significantly associated withglaucoma in Shiba-Inus, while rs22018513, a synonymous SNP in exon 4, showed the strongest association (P = 0.00039,
OR = 3.03). Conditional analysis revealed that rs22018513 could account for most of the association of these SNPs withglaucoma in Shiba-Inus. In Shih-Tzus, only rs9172407 in the SRBD1 intron 1 was significantly associated with glaucoma(P = 0.0014, OR = 5.25). There were no significant associations between the ELOVL5 or ADAMTS10 polymorphisms andglaucoma in Shiba-Inus and Shih-Tzus. The results showed that SRBD1 polymorphisms play an important role in glaucoma
pathology in both Shiba-Inus and Shih-Tzus. SRBD1 polymorphisms have also been associated with normal- and hightension glaucomas in humans. Therefore, SRBD1 may be a common susceptibility gene for glaucoma in humans and dogs.
We anticipate that the nucleotide sequencing data from this study can be used in genetic testing to determine for the firsttime, the genetic status and susceptibility of glaucoma in dogs, with high precision. Moreover, canine glaucoma resulting
from SRBD1 polymorphisms could be a useful animal model to study human glaucoma.
Citation: Kanemaki N, Tchedre KT, Imayasu M, Kawarai S, Sakaguchi M, et al. (2013) Dogs and Humans Share a Common Susceptibility Gene SRBD1 for Glaucoma
Risk. PLoS ONE 8(9): e74372. doi:10.1371/journal.pone.0074372
Editor: Reiner Albert Veitia, Institut Jacques Monod, France
Received December 17, 2012; Accepted August 6, 2013; Published September 9, 2013
Copyright: ß 2013 Kanemaki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities. The funders had no role instudy design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Two of the authors, KTT and MI are employed by the Menicon Co., Ltd. This does not alter the authors’ adherence to all the PLOS ONE
policies on sharing data and materials.
* E-mail: mizunobu@med.yokohama-cu.ac.jp

population [2]. The study identified two new susceptibility genes
for NTG, SRBD1 and ELOVL5, with strong statistical significance.
Similarly, Mabuchi et al. also reported the association of an
SRBD1 polymorphism with Japanese POAG patients, including
late-onset NTG and high tension glaucoma [10].
Canine primary glaucoma has been investigated since almost 50
years ago [11], and high incidences have been reported in Beagles[12–14], Welsh Springer Spaniels [15], and other breeds [16,17].
Recent study reported the Gly661Arg variant in ADAMTS10 as
the candidate disease-causing variant for POAG Beagles [12].
Kato et al. investigated the incidence of canine POAG, andreported that Shiba-Inus exhibited the highest incidence ofglaucoma among 29 breeds, followed by Shih-Tzus [18]. They
also reported that an abnormal iridocorneal angle and dysplastic
prectinate ligament were associated with a high incidence ofglaucoma in Shiba-Inus and Shih-Tzus. However, the hereditaryand genetic backgrounds of glaucoma in these dogs have not yet
been clarified.
In this study, to verify recent genetic findings, we investigated
the association between glaucoma in Shiba-Inu and Shih-Tzu

Introduction
Glaucoma is a degenerative optic neuropathy comprising agroup of eye disorders, including visual field defects, progressive
loss of retinal ganglion cells, and degeneration of optic nerveaxons, and is frequently associated with elevated intraocularpressure (IOP) [1]. Glaucoma is classified into three types: primary
open angle glaucoma (POAG), primary closed angle glaucoma(PCAG), and primary congenital glaucoma (PCG) [1]. POAG is
the most common type of glaucoma, and is usually associated withhigh IOP. Japanese populations, however, have a substantiallyhigher incidence of normal tension glaucoma (NTG), a form ofglaucoma in which optic nerve damage occurs even though the
IOP is not elevated [2,3].
It is well known that glaucoma is genetically heterogeneous andmany genes, such as CYP1B1, MYOC, OPTN, and OPTC, arelinked to POAG and PCG in humans and/or dogs [4–9].
Recently, the Normal Tension Glaucoma Genetic Study Group of
the Japan Glaucoma Society performed a genome-wide association study with NTG patients and controls in a Japanese
PLOS ONE | www.plosone.org

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SRBD1 and Canine Glaucoma

dogs and polymorphisms of glaucoma candidate genes, SRBD1,ELOVL5 and ADAMTS10, using direct sequencing.

observed throughout the region from rs22019922 to rs22018513(D’ $0.68) (Figure 1B). rs8655283 and rs22018514 were in almost
complete LD in both Shiba-Inus and Shih-Tzus (r2 = 0.73 and
0.74, respectively). rs9172407 was not linked with any of the otherfour SNPs in either breed.
To elucidate the effect of rs8655283, rs22018514 andrs22018513 on the disease susceptibility in Shiba-Inus, we
performed conditional logistic regression analysis. Conditioningby rs22018513 eliminated the significant association of rs8655283
and rs22018514, while the association of rs22018513 remained
significant after conditioning by rs8655283 or rs22018514(Table 3). These results suggest that rs22018513 could accountfor most of the association of these SNPs with glaucoma in ShibaInus.
Table 4 shows the results of association analysis for thepolymorphisms of ELOVL5 and ADAMTS10. rs9194033 andrs22202438 of ELOVL5 were polymorphic in Shiba-Inus and ShihTzus, while three ELOVL5 polymorphisms (rs22226301,
rs8643563 and rs22194174) and the ADAMTS10 Gly661Arg
variant were monomorphic. The G allele of rs9194033 and the G
allele of rs22202438 had a 1.45- and a 1.39-fold increased risk ofglaucoma in Shiba-Inus, respectively. Conversely, these alleles
were decreased in cases compared to controls in Shih-Tzus(OR = 0.57 and 0.66, respectively). The differences in the allelicfrequencies of rs9194033 or rs22202438 between cases andcontrols did not reach statistical significance for either breed.

Results
The average ages of glaucoma cases and controls were 8.562.9
and 10.063.0 years old, respectively, in Shiba-Inu dogs. Those in
Shih-Tzu dogs were 9.262.0 and 10.162.5 years old, respectively.
We genotyped 11 polymorphisms in SRBD1, ELOVL5, and
ADAMTS10 in 98 Shiba-Inu and 67 Shih-Tzu dogs using thedirect DNA sequencing method (Table 1).
Table 2 shows the details of five single nucleotide polymorphisms (SNPs) in SRBD1, including their genomic locations andallele frequencies in Shiba-Inus and Shih-Tzus. In Shiba-Inus, themost statistically significant association was observed for
rs22018513 (P = 0.00039); the G allele of rs22018513 had a
3.03-fold (95% CI = 1.62–5.65) increased risk of glaucoma, with afrequency of 78.6% in cases vs. 54.8% in controls. Significant
associations were also observed for rs8655283 and rs22018514 in
Shiba-Inus; the frequencies of the T allele of rs8655283 and the G
allele of rs22018514 were significantly greater among glaucoma
cases than among controls (rs8655283, 37.5% vs. 21.4%,
P = 0.016, OR = 2.20, 95% CI = 1.15–4.20; rs22018514, 41.1%
vs. 21.4%, P = 0.0037, OR = 2.56, 95% CI = 1.34–4.86). In ShihTzus, we observed a significant association for rs9172407(P = 0.0014) and the G allele of rs9172407 had a 5.25-fold (95%CI = 1.76–15.63) increased risk of glaucoma (25.9% in cases vs.
6.3% in controls), whereas the frequency of this G allele was only
moderately, but not significantly, increased in cases compared tocontrols in Shiba-Inus (8.9% vs. 6.0%, OR = 1.55, 95%
CI = 0.51–4.71). rs22018513, which had the strongest association
with glaucoma in Shiba-Inus, did not show a significant
association in Shih-Tzus (P = 0.868, OR = 1.13, 95% CI = 0.26–
4.95). Two other SNPs (rs8655283 and rs22018514), which wereassociated with glaucoma in Shiba-Inus, as well as rs22019922,
also did not achieve statistically significant associations withglaucoma in Shih-Tzus. However, the odds-ratios of these variantsin Shih-Tzus were suggestive of an association, with the T allele ofrs8655283, the G allele of rs22018514, and the A allele ofrs22019922, each having a 2.43 or 2.49-fold increased risk ofglaucoma.
Figure 1 shows the strength of linkage disequilibrium (LD) for
the five SNPs of SRBD1 in Shiba-Inus and Shih-Tzus. Strong LD
was observed between rs8655283, rs22018514 and rs22018513 inShiba-Inus (D’ $0.78) (Figure 1A). In Shih-Tzus, strong LD was

Discussion
The aim of the present study was to assess the potential
associations of polymorphisms in the candidate genes SRBD1,ELOVL5, and ADAMTS10, with the development of canine
glaucoma. To this end, we genotyped 11 polymorphisms of thesegenes in two breeds of dogs with cases of glaucoma or without, as
controls. Here we report that the SRBD1 polymorphisms exhibited
significant association with canine glaucoma, while the ELOVL5
and ADAMTS10 polymorphisms that were examined in this study
were not associated with canine glaucoma. In Shiba-Inus, thestrongest association with glaucoma in SRBD1 was observed at
rs22018513, which is a synonymous SNP in exon 4. Two other
SNPs, rs8655283 and rs22018514, were also significantly associated with glaucoma; however, these significant associations were
calculated only secondarily from a strong LD with rs22018513. In
Shih-Tzus, however, rs22018513 was not associated with glaucoma. Only rs9172407 in intron 1 of SRBD1 showed a statistically

Table 1. Primer pairs for PCR of glaucoma-related genes.

Gene
SRBD1

ELOVL5ADAMTS10

Allele59-39 Forward

59-39 Reverse

rs22019922

TGTTGGTGTTGTCAGCAAGTTCACACTCTTTCTCTCACTCTCTC

rs8655283

TTAGGATGAAACCATGGAAC

TTGGCGATTTATTGAACTAAC

rs22018513, rs22018514

GCTATTGCTGATGTTGATTTG

TGCAGTGCTGGCCCTGTTGGA

rs9172407

GTGAACCTGAAATGGCAAA

TTAACTAGCTTCCTTGCTTCC

rs22226301

AGTTGTGCTGCTTACATTAGGAGCAAGGCAAGATGTGTTC

rs9194033

AGTGATGCTGCTATGGGATGGCTCAGGTCATGGGATCAAG

rs22202438

CATGCTGAACATCTGGTGGT

GCTGGTCTGGATGATTGTCAACCACCAGAGGACACGGATA

rs8643563

AATTGTATGGCTGGGACCAA

rs22194174

AATGCTTATCCTACCCAATCTCAGGCTCTATGCTCAGTG

Gly661Arg (56097365 G.A)

CACAGAGCAAGCAGGGAGT

GGGTGGAAGTGGGAGTG

doi:10.1371/journal.pone.0074372.t001

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Table 2. Association analysis for five polymorphisms in the SRBD1 gene region for Shiba-Inu and Shih-Tzu dog breeds.

SNP IDrs22019922

rs8655283

rs22018514

rs22018513

rs9172407

Position
Chr. (CanFam2.0) Allele SNP Type

10

10

10

10

10

50924623

50989281

51,049,600

51,049,604

51062753

A/C

C/T

C/G

A/G

A/G

IntronIntronNon-synonymous

Synonymous

IntronRisk
Allele Breed

A

T

G

G

G

Risk Allele
Frequency (%)

N

P

OR(95% CI)

7.1

0.65

1.27 (0.44–3.66)

91.3

0.25

2.49(0.50–12.49)

0.40

1.59 (0.66–3.80)

Cases

Controls Cases

Controls

Shiba-Inu

56

42

8.9

Shih-Tzu

27

40

96.3

Overall

83

82

Shiba-Inu

56

42

37.5

21.4

0.016

2.20 (1.15–4.20)

Shih-Tzu

27

40

92.6

83.8

0.13

2.43 (0.75–7.89)

Overall

83

82

0.00682.25 (1.28–3.97)

Shiba-Inu

56

42

41.1

21.4

0.0037

2.56 (1.34–4.86)

Shih-Tzu

27

40

92.6

83.8

0.13

2.43 (0.75–7.89)

Overall

83

82

Shiba-Inu

56

42

78.6

Shih-Tzu

27

40

94.4

Overall

83

82

Shiba-Inu

56

42

8.9

Shih-Tzu

27

40

25.9

Overall

83

82

0.00182.52 (1.43–4.44)54.8

0.00039

3.03 (1.62–5.65)

93.8

0.87

1.13 (0.26–4.95)

0.00152.59 (1.46–4.61)

6.0

0.44

1.55 (0.51–4.71)

6.3

0.00145.25 (1.76–15.63)

0.00742.90 (1.34–6.26)

OR, odds ratio; CI, confidence interval.
Overall P values and ORs for meta-analysis were calculated using the Mantel-Haenzel method.
doi:10.1371/journal.pone.0074372.t002

Figure 1. Linkage disequilibrium (LD) plot of five SNPs of the SRBD1 gene. A) LD structure in Shiba-Inus. B) LD structure in Shih-Tzus. The D’value and r2 value (in parentheses) corresponding to each SNP pair are expressed as a percentage and shown within the respective square. The color
scheme is based on D’ and LOD score values: bright red (LOD $2 and D’ = 1); shades of pink/red (LOD $2 and D’ ,1); blue (LOD ,2 and D’ = 1); white(LOD ,2 and D’ ,1).
doi:10.1371/journal.pone.0074372.g001

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Table 3. Conditional logistic regression analysis of rs8655283, rs22018514 and rs22018513 in the SRBD1 gene for Shiba-Inus.
P**

SNP ID
Risk Allele

Model

P*

Covariates
rs8655283

rs22018514

rs22018513

rs8655283

T

Additive

0.021

–

0.92

0.15

rs22018514

G

Additive

0.0066

0.13

–

0.10

rs22018513

G

Additive

0.000250.00100.0021–*P values for each SNP under the recessive, additive, or dominant model that provided the best fit by logistic regression analysis. The lowest P value was selected as thebest fit model. The indicated model showed the lowest P value for each SNP.
**P values adjusted for each SNP under the indicated model by conditional logistic regression analysis.
doi:10.1371/journal.pone.0074372.t003

SRBD1 region, suggesting that rs3213787 is strongly linked withpolymorphisms in exon 4 and intron 1. Further investigation will
be necessary to confirm the association of polymorphisms in theexon 4 and intron 1 regions of SRBD1 in human glaucoma
patients.
SRBD1 was initially identified as an S1 RNA-binding domain in
Escherichia coli [19]. Many proteins contain S1 RNA-binding
domains, such as the bacterial exonuclease polynucleotide
phosphorylase, eukaryotic translation initiation factor 2 alpha,and even a human DNA-binding protein [20,21]. The function of
SRBD1 in humans and dogs remains unknown. Canine SRBD1 islocated on the reverse strand of chromosome 10, and isapproximately 186 kb, including approximately 3 kb of the exon

significant association in Shih-Tzus, but not in Shiba-Inus. These
results suggest that rs22018513 and rs9172407, or a respective
neighboring polymorphism, may be a causative factor forglaucoma in Shiba-Inus and Shih-Tzus, respectively.
BLAST analysis (http://blast.ncbi.nlm.nih.gov/) reveals thatthe canine SRBD1 amino acid sequence shares a high degree ofsimilarity with its human homolog, with 90% identity. In humans,
the nucleotide sequences corresponding to rs22018513 andrs9172407 are located within exon 4 and intron 1 of SRBD1,
respectively. However, these sequences have never been reported
as polymorphic in humans. While the human NTG-associated
SNP, rs3213787, is located in intron 17 of SRBD1 [2], theHapMap database shows strong LD across the entire human

Table 4. Association analysis for six polymorphisms in the ELOVL5 and ADAMTS10 gene regions for Shiba-Inu and Shih-Tzu dog
breeds.

SNP ID

Position
Chr. (CanFam2.0) Gene

Allele SNP Type

Risk
Allele*Breed

Risk Allele
Frequency (%)

N

Cases Controls Cases
rs22226301 12

rs9194033 12

rs22202438 12

rs8643563 12

rs22194174 12

Gly661Arg 2020733716

20739417

20743516

20744701

20749077

56097365

ELOVL5ELOVL5ELOVL5ELOVL5ELOVL5

C/T

A/G

A/G

(–)/T

A/C

ADAMTS10 A/G

3’UTR

IntronSynonymous

T

G

G

Frameshift coding T

IntronNon-synonymousAA(56097365 G.A)

P

OR (95% CI)

Controls

Shiba-Inu 56

42

0.0

0.0

–

–

Shih-Tzu

27

40

0.0

0.0

–

–

Overall

83

82

Shiba-Inu 56

42

33.9

Shih-Tzu

27

40

55.6

Overall

83

82

Shiba-Inu 56

42

35.7

Shih-Tzu

27

40

59.3

Overall

83

82

Shiba-Inu 56

42

0.0

Shih-Tzu

27

40

0.0

Overall

83

82

Shiba-Inu 56

42

0.0

Shih-Tzu

27

40

0.0

Overall

83

82

Shiba-Inu 56

42

0.0

0.0

–

–

Shih-Tzu

27

40

0.0

0.0

–

–

Overall

83

82

–

–

–

–

26.2

0.25

1.45 (0.78–2.70)

68.8

0.12

0.57 (0.28–1.16)

0.99

0.97 (0.61–1.54)

28.6

0.29

1.39 (0.75–2.56)

68.8

0.26

0.66 (0.32–1.36)

0.98

1.02 (0.64–1.62)

0.0

–

–

0.0

–

–

–

–

0.0

–

–

0.0

–

–

–

–

OR, odds ratio; CI, confidence interval.
Overall P values and ORs for meta-analysis were calculated using the Mantel-Haenzel method.
*Risk allele is for Shiba-Inu dogs.
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the canine ADAMTS10 gene region. Since the dog genome
information is still incomplete, it is predicted that more evenpolymorphisms exist in the canine ADAMTS10 gene region.
Therefore, it is necessary to perform a comprehensive genetic
analysis of the region and clarify whether ADAMTS10 is acandidate gene for glaucoma not only in Beagles, but also in otherbreeds.
There are no genetic tests currently available to assist inglaucoma diagnosis, identification of people at risk, initiation oftreatment, and timing of surgical intervention. We performed thepresent SNP analysis of candidate genes in Shiba-Inu and ShihTzu dog breeds for the possibility to help develop diagnostic,
genetic analyses for glaucoma risk factors. However, the mechanism by which these genes contribute to the development ofglaucoma remains to be determined. Future studies are expected
to examine the roles of SRBD1 in humans and dogs, in an effort todetermine whether genetic testing might not only help predictwhether someone will develop glaucoma, but may also, perhaps,
be a valuable prognostic factor for the clinical course of thedisease, and/or predictive factor for its treatment. Despite new
and improving diagnostic and therapeutic options for glaucoma,
blindness resulting from glaucoma remains a major public healthproblem. These future experiments will help to optimize glaucoma
treatment.

region (http://asia.ensembl.org/Canis_familiaris/Transcript/Exons?
db = core;g = ENSCAFG00000002554;r = 10:47923593-47924593;
t = ENSCAFT00000049409). SRBD1 transcript is reportedly expressed in the retinal ganglion cell and neuroblast layers in neonatal
mouse tissue [2], but its localization in dogs remains unknown.
The present study found that a synonymous SNP (rs22018513)
and intronic SNP (rs9172407) in SRBD1 were associated withglaucoma in Shiba-Inus and Shih-Tzus, respectively. Synonymousand intronic polymorphisms can significantly affect gene expression by various mechanisms and lead to the development ofdisease [22–24]. We did not compare SRBD1 mRNA levels innormal and affected dogs; however, a similar experiment has been
conducted in humans [2]. Results from that study showed asignificant correlation between increased SRBD1 expression and
the NTG-associated risk allele of intronic SNP. These results
suggest that rs22018513 and rs9172407 in canine SRBD1 could
cause enhanced SRBD1 expression. Reports show that SRBD1 isindirectly involved in cell growth, general protein synthesis,
induction of apoptosis, and maintaining homeostasis [2]. Therefore, we hypothesize that enhanced expression, leading toincreased activity of SRBD1, which could induce apoptosis, could
result in retinal ganglion cell death during the development ofglaucoma.
ELOVL5 is a fatty acid condensing enzyme involved in thebiosynthesis of long-chain polyunsaturated fatty acids [25], and is
one of the candidate genes for retinitis pigmentosa [26]. The
Normal Tension Glaucoma Genetics Study Group of the Japan
Glaucoma Society reported ELOVL5 as a new susceptibility genefor human NTG [2]. However, the present study did not show any
significant association of ELOVL5 polymorphisms with canine
glaucoma. The difference between our results and those reportedby the study group may be due to the different forms of glaucoma
that were studied (human NTG without IOP elevation, and canine
glaucoma with IOP elevation, respectively). In contrast, SRBD1
polymorphisms were associated with canine glaucoma and humanglaucoma independent of IOP, suggesting that SRBD1 polymorphisms may affect a common disease condition in canine andhuman glaucoma. Therefore, detection of any common phenotypes in these glaucoma studies [2,10] is important because it will
help to clarify how SRBD1 affects the development of glaucoma.
Moreover, canine glaucoma resulting from SRBD1 polymorphisms
could be used as an excellent genetic animal model for humanglaucoma and contribute significantly to the development of novel
diagnostic and therapeutic options for glaucoma.
Kato, et al., reported that the Beagle breed has the fourthhighest incidence of canine glaucoma, after Shiba-Inu, Shih-Tzu,
and American Cocker Spaniel breeds [18]. Kuchtey et al. recently
reported that the Gly661Arg variant (56097365 G.A) of
ADAMTS10 in Beagles with POAG is a candidate, predictive
gene allele for canine POAG [12]. We did not observe anassociation between this 56097365 G.A variant and glaucoma in
Shiba-Inus or Shih-Tzus, possibly because of breed-specific allelicdifferences between Beagles and Shiba-Inus or Shih-Tzus. Morerecently, Kuchtey, et al., reported that the Gly661Arg variant was
not found in any of the other dog breeds analyzed, (Shiba-Inu,
Shih-Tzu, American Cocker Spaniels, Chihuahua, Australian
Cattle Dog, Jack Russell Terrier, Jindo, Siberian Husky, andYorkshire Terrier), suggesting that this allele is Beagle-specific, andthat other genes may be associated with glaucoma in other breeds[27]. However, ADAMTS10 may be still a candidate gene forglaucoma in dogs, including Shiba-Inu and Shih-Tzu, because
other ADAMTS10 variants have yet to be investigated for theirassociation with canine glaucoma. The Ensembl database (http://
asia.ensembl.org/index.html) shows 16 genetic polymorphisms in
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Materials and Methods
Ethics Statement
This study was performed as part of research approved by the
Ethical Committee of Azabu University (Permit Number: 1104082). Informed written consent was obtained from each dog owner.
All procedures in this study were conducted in accordance with
the Guide for the Care and Use of Laboratory Animals of Azabu
University.

Diagnosis of Glaucoma
98 Japanese Shiba-Inu dogs and 67 Shih-Tzu dogs wererecruited from the Veterinary Teaching Hospital at Azabu
University. All dogs received complete ophthalmologic examinations using a hand-held slit-lamp biomicroscope (SL-14; Kowa,
Tokyo, Japan), indirect ophthalmoscopy, and tonometry. After theapplication of topical anesthesia (oxybuprocaine hydrochloride,
Santen, Osaka, Japan), IOP was measured by tonometry using the
Tono-Pen XL (Mentor O&O Inc., Norwell, MA). 42 Shiba-Inus
and 40 Shih-Tzus were diagnosed as normal (,25 mmHg IOP).
56 Shiba-Inus and 27 Shih-Tzus had elevated IOP (.25 mmHg)
in at least one eye, and were diagnosed with glaucoma. Since
glaucoma is a late-onset disorder, we did not recruit dogs younger
than four years in the control group, in an attempt to exclude
potential glaucomatous dogs.

DNA Preparation
Genomic DNA from glaucomatous and normal dogs wascollected from peripheral blood and purified using a DNA whole
blood spin kit (Fuji Film, Tokyo, Japan). The purity andconcentration of DNA were examined using GeneQuant Pro(GE Healthcare, Cambridge, UK).

Determination of DNA Sequences
In the SRBD1 and ELOVL5 gene regions, a total of ten
polymorphisms were selected to cover the entire gene regions(rs22019922, rs8655283, rs22018514, rs22018513 and rs9172407
in SRBD1; rs22226301, rs9194033, rs22202438, rs8643563 andrs22194174 in ELOVL5) (Table 2,4). In the ADAMTS10 gene
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SRBD1 and Canine Glaucoma

between glaucomatous and normal dogs were assessed using the x2
test, and P,0.05 was considered statistically significant. Overall, P
values and odds ratios (ORs) for meta-analysis were calculated by
the Mantel-Haenszel method. The Haploview 4.1 program wasused to compute pairwise LD statistics [28]. Conditional logistic
regression analysis was performed to assess the effect of each SNP
on the disease susceptibility using PLINK (http://pngu.mgh.
harvard.edu/purcell/plink/) [29].

region, we selected the Gly661Arg variant (56097365 G.A) foranalysis (Table 4). PCR primer pairs listed in Table 1 were used toamplify regions containing the SNPs mentioned above. The PCR
products were electrophoretically separated on a 1% agarose gel,and the PCR product bands were cut and frozen at 280uC in
Tris-EDTA buffer. The frozen samples were thawed, homogenized, and centrifuged at 15,000 g for 5 minutes. The supernatants
were subjected to cycle sequencing using the Big Dye terminator
sequencing kit (Life Technologies, Foster City, CA). The sequence
cycling reaction products were purified using the Qiagen Dye Ex
2.0 Spin Kit (QIAGEN, Hilden, Germany) according to the
manufacturer’s instructions. After drying, the DNA samples weremixed with high Dye Mix (Life Technologies) and were directly
sequenced using an ABI PRISM 310 genetic analyzer (LifeTechnologies). The sequence data were analyzed using Sequence
Scanner v. 1.0 (Life Technologies) and GENETYX-WIN v. 4.0(Genetyx, Tokyo, Japan). We did not find any novel DNA
sequences deposited in GenBank in this study.

Acknowledgments
We sincerely thank all the dog owners who have provided samples for thisstudy and all the veterinarians involved in sample collection and diagnosis.

Author Contributions
Conceived and designed the experiments: NK SK MS NI NM. Performed
the experiments: NK KTT MI. Analyzed the data: MI AM AY.
Contributed reagents/materials/analysis tools: NK KTT MI MS AM
AY NM. Wrote the paper: KTT MI AM.

Statistical Analysis
Hardy-Weinberg equilibrium was tested for each SNP amongglaucomatous and normal dogs. Differences in allele frequency

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Association between SRBD1 and ELOVL5 gene polymorphisms and primary
open-angle glaucoma. Invest Ophthalmol Vis Sci 52: 4626–4629.
11. Lovekin LG (1964) Primary glaucoma in dogs. J Am Vet Med Assoc 145: 1081–
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12. Kuchtey J, Olson LM, Rinkoski T, MacKay EO, Iverson TM, et al. (2011)
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࢖ࢾ࡟࠾ࡅࡿࣄࢺ࡬ࡢ㐺ྜᛶ࡟㛵ࡍࡿ㑇ఏᏊゎᯒ㸯≟ࡢࣄࢺ㐺ᛂᛶ࡟㛵ࡍࡿ≟✀ᕪࡢ᳨ฟ
㸰≟ࡢᐙ␆໬㐣⛬࡟࠾ࡅࡿ㑅ᢥᅽᅉᏊࡢ᥎ᐃ
㸱ࣄࢺ࡬ࡢ♫఍ㄆ▱⬟ຊ࡟࠾ࡅࡿ≟✀ᕪࡢ᳨ฟ
㸲≟ࡢ♫఍ㄆ▱⬟ຊ࡟㛵㐃ࡍࡿ㑇ఏᏊࡢྠᐃ
⳥Ỉ೺ྐ㸦㯞ᕸ኱Ꮫక౶ື≀Ꮫ◊✲ᐊ㸧ࠊⱱᮌ୍Ꮥ㸦㯞ᕸ኱Ꮫక౶ື≀Ꮫ◊✲ᐊ㸧ࠊỌ⃝⨾ಖ㸦⮬἞་⛉኱Ꮫ་Ꮫ㒊⏕⌮Ꮫㅮᗙ㸧㸯≟ࡢࣄࢺ㐺ᛂᛶ࡟㛵ࡍࡿ≟✀ᕪࡢ᳨ฟ
◊✲せ᪨᪥ᮏཬࡧ⡿ᅜ࡟࠾ࡅࡿ୍⯡ࡢ㣫࠸୺ཬࡧࣈ࣮ࣜࢲ࣮ࢆᑐ㇟࡜ࡋࡓ࢖ࢾࡢ⾜ື≉ᛶ࡟㛵ࡍࡿ࢔ࣥࢣ࣮ࢺㄪᰝࢆࠊ࢖ࣥࢱ࣮ࢿࢵࢺ፹యࢆ⏝࠸࡚ᐇ᪋ࡋࡓࠋ࢖ࢾࡢ⾜ືゎᯒࢩࢫ
ࢸ࣒ࡣࠊ&EDUT ࢆ⏝࠸ࡓࠋ㉁ၥࡣࠊ⡿ᅜ  ၥࠊ᪥ᮏ  ၥ࠿ࡽᡂࡾࠊᵝࠎ࡞ሙ㠃࡟࠾ࡅࡿ≟ࡢ⾜ືࢆ  ẁ㝵࡛ホ౯ࡍࡿෆᐜ࡛࠶ࡿࠋ≟✀ࢆ᪤࡟ሗ࿌ࡉࢀ࡚࠸ࡓ㑇ఏศᒱᅗࢆඖ࡟  ࡘࡢࢢ࣮ࣝࣉ࡟ศ㢮ࡋࠊ≟ࡢ⾜ື≉ᛶࡢ≟✀ࢢ࣮ࣝࣉẚ㍑࡟⏝࠸ࡓࠋ㉁ၥ㡯┠࡟ࡘ࠸࡚ᅉᏊศᯒཬࡧᖹ⾜ศᯒࢆ⾜࠸ࠊྛᅉᏊࡢᖹᆒ್ࢆᅉᏊᚓⅬ࡜ࡋ࡚ศᯒ࡟౑⏝ࡋࡓࠋ࢔ࣥࢣ࣮ࢺ⤖ᯝࢆᅉᏊศᯒࡋࡓ࡜ࡇࢁࠊ ࡢᅉᏊ࡟ศ㢮ࡉࢀࡓ㸦カ⦎ᛶࠊάⓎᗘࠊឡ╔ࠊศ㞳୙Ᏻࠊ౵ධ⪅࡟ᑐࡍࡿᨷᧁᛶࠊ㣫࠸୺࡟ᑐࡍࡿᨷᧁᛶࠊぢ▱ࡽࡠே࡟ᑐࡍࡿᨷᧁᛶࠊぢ▱ࡽࡠ≟࡟ᑐࡍࡿᨷᧁᛶࠊ㠀♫఍ⓗ่⃭࡟ᑐࡍࡿᜍᛧ཯ᛂࠊぢ▱ࡽࡠே࡟ᑐ
ࡍࡿᜍᛧ཯ᛂࠊぢ▱ࡽࡠ≟࡟ᑐࡍࡿᜍᛧ཯ᛂ㸧ࠋࡑࡢ࠺ࡕࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡢ࢖ࢾ
ࡣ࡝ࡢ≟✀ࢢ࣮ࣝࣉࡼࡾࡶࣄࢺ࡬ࡢឡ╔ࡀప࠸ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ㸦S㸧ࠋཎጞⓗ࡞≟ࡢឡ╔ࡣࡑࡢ௚ࡢ࡝ࡢ≟✀ࢢ࣮ࣝࣉࡼࡾࡶపࡃࠊඛ⾜◊✲࡛▱ࡽࢀ࡚࠸ࡿ㑇ఏศᒱᅗ࡜୍⮴ࡋࡓ⤖ᯝ࡛࠶ࡿࠋࡑࡢ௚ࡢ≟✀ࢢ࣮ࣝࣉ࡛ࡣࠊ࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࡣࠊぢ▱ࡽࡠே࡟ᑐࡍࡿᜍᛧ཯ᛂࠊぢ▱ࡽࡠ≟࡟ᑐࡍࡿᜍᛧ཯ᛂࠊ㠀♫఍ⓗ่⃭࡟ᑐࡍࡿᜍᛧ཯
ᛂࠊ㣫࠸୺࡟ᑐࡍࡿᨷᧁᛶࠊάⓎᗘࡀప࠸ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡲࡓࠊࣁ࣮ࢹ࢕ࣥࢢ
ࢢ࣮ࣝࣉࡣカ⦎ᛶࡀ㧗ࡃࠊࢺ࢖ࢢ࣮ࣝࣉࡣ㣫࠸୺࡟ᑐࡍࡿᨷᧁᛶࠊぢ▱ࡽࡠே࡟ᑐࡍࡿ
ᨷᧁᛶࠊ౵ධ⪅࡟ᑐࡍࡿᨷᧁᛶࡀ㧗࠿ࡗࡓࠋ◊✲┠ⓗ᥈⣴⾜ືࡸ㏨㑊⾜ືࠊ㐟ࡧࠊカ⦎ᛶ⬟ࠊ୙Ᏻഴྥ➼ࡢᗄࡘ࠿ࡢ࢖ࢾࡢ⾜ື≉ᛶ࡟ࡣࠊ≟✀㛫࡛ᕪࡀぢࡽࢀࠊ㑇ఏࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋ࢖ࢾࡢẼ㉁࣭⾜ືࡸ⬟ຊ࡟ࡘ࠸࡚≟✀㛫࡛ẚ㍑ࡍࡿࡇ࡜࡟ࡼࡾࠊࡑࢀࡽࡢ≟✀ࡀ࡝ࡢࡼ࠺࡟సᡂࡉࢀ࡚࠸ࡗࡓࡢ࠿ࡑࡢ㐣⛬ࢆ▱ࡿࡇ࡜ࡀ࡛ࡁࡿࠋ≉࡟ࠊࢤࣀ࣒࣡࢖ࢻ◊✲࡟ࡼࡗ࡚㑇ఏⓗ࡟᭱ࡶ࣑࢜࢜࢝࡟㏆࠸ࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡢ࢖ࢾࡢ⾜ື≉ᛶࡣࠊ♽ඛ✀࡟᭱ࡶ㏆࠸࡜⪃࠼ࡽࢀࠊ࢖ࢾࡢ

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ᐙ␆໬㸦㐍໬㸧ࢆ⪃࠼ࡿୖ࡛㔜せ࡜࡞ࡿࠋࡑࡇ࡛ࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡢ࢖ࢾࡢ୍⯡ⓗ࡞㣫⫱ୗ࡟࠾ࡅࡿ⾜ື≉ᛶ࡟ࡘ࠸࡚ㄪᰝࡋࡓࠋ
ᮦᩱ࡜᪉ἲ≟ࡢ⾜ື࡟㛵ࡍࡿㄪᰝࡣࠊ≟ࡢ⾜ືゎᯒࢩࢫࢸ࣒ࡢࡦ࡜ࡘ࡛࠶ࡿ Canine Behavioral
Assessment and Research Questionare (C-barq)ࢆ⏝࠸࡚⾜ࡗࡓࠋC-barq ࡣ࢙࢘ࣈࢧ࢖ࢺ
ୖ㸦⡿ᅜ http://www.cbarq.orgࠊ᪥ᮏ http://cbarq.inutokurasu.jp/㸧୍࡛⯡බ㛤ࡉࢀ࡚࠾ࡾࠊ
ࢹ࣮ࢱࡣ୍⯡ࡢ㣫࠸୺ཬࡧࣈ࣮ࣜࢲ࣮ࢆᑐ㇟࡟ࡋ࡚㞟ࡵࡽࢀࡓࠋ
ᮇ㛫ࡣࠊ⡿ᅜ 2006 ᖺ 4 ᭶ࠊ᪥ᮏ 2010 ᖺ 9 ᭶ࡼࡾ㛤ጞࡉࢀࠊ᪥ᮏࡢ࢙࢘ࣈࢧ࢖ࢺࡣ⡿ᅜࡢࡶࡢࢆ᪥ᮏ⏝࡟࢔ࣞࣥࢪࡋࡓ
ࡶࡢ࡜࡞ࡗ࡚࠸ࡿࠋ㉁ၥࡣࠊ⡿ᅜ 100 ၥࠊ᪥ᮏ 78 ၥ࠿ࡽᡂࡾࠊᵝࠎ࡞ሙ㠃࡟࠾ࡅࡿ≟ࡢ⾜ືࢆ 5 ẁ㝵࡛ホ౯ࡍࡿෆᐜ࡛࠶ࡿࠋࡲࡓࠊ≟ࡢᇶᮏ᝟ሗ࡜ࡋ࡚ࠊྡ๓ࠊ⏕ᖺ᭶᪥ࠊ≟✀ࠊᛶูࠊయ㔜ࠊ㑊ዷཤໃࡢᐇ᪋≧ἣࡸ᫬ᮇࠊ⑌ᝈࡢ᭷↓ࠊධᡭ᫬ᮇࠊධᡭඖࠊ㣫࠸୺ࡢ௒ࡲ
࡛ࡢ㣫⫱⤒㦂➼ࢆⓏ㘓ࡋ࡚ࡶࡽࡗࡓࠋ㉁ၥ㡯┠࡟ࡘ࠸࡚ᅉᏊศᯒࢆ⾜࠸ࠊྛᅉᏊࡢᖹᆒ್ࢆᅉᏊᚓⅬ࡜ࡋ࡚ศᯒ࡟౑⏝ࡋࡓࠋ≟✀ࡣ᪤࡟ሗ࿌ࡉࢀ࡚࠸ࡓ vonHoldt (2010)11 ࡟ࡼࡿ㑇ఏศᒱᅗࢆඖ࡟ 8 ࡘࡢࢢ࣮ࣝࣉ࡟
ศ㢮ࡋ࡚≟ࡢ⾜ື≉ᛶࡢ≟✀ࢢ࣮ࣝࣉẚ㍑࡟⏝࠸ࡓࠋゎᯒ࡟ࡣ୍⯡໬⥺ᆺࣔࢹࣝࢆ⏝࠸ࠊゎᯒࡣ SPSS ࢆ⏝࠸࡚⾜ࡗࡓ㸦SPSS v.19.0, SPSS Japan Inc., IBM company㸧ࠋࡲࡓࠊ≟✀ࢢ࣮ࣝࣉࡢ௚࡟ࡶࠊ≟ࡢ⾜ື≉ᛶ࡟ᙳ㡪ࢆཬࡰࡍ࡜ᛮࢃࢀࡿኚᩘ࡜ࡋ࡚ࠊᅜࠊᛶูࠊᖺ
㱋ࠊ㑊ዷཤໃࡢᐇ᪋≧ἣࠊయ㔜ࠊධᡭሙᡤࠊධᡭ᫬ᮇࠊ㣫࠸୺ࡢ௒ࡲ࡛ࡢ㣫⫱⤒㦂ࢆゎᯒ࡟ྵࡵࡓࠋࢡࣛࢫࢱ࣮ゎᯒࡣࠊSPSS ࢆ⏝࠸࡚ࠊ࢛࣮࢘ࢻἲ࡛⾜ࡗࡓࠋ⤖ᯝ࠾ࡼࡧ⪃ᐹ㸯㸬ࢹ࣮ࢱᵓ㐀᪥ᮏཬࡧ⡿ᅜ࡟ඹ㏻ࡋ࡚ぢࡽࢀࡓ 59 ≟✀ࢆ㑅ᢤࡋࠊᛶูཬࡧᅜࡢᙳ㡪ࢆ㝖ࡃࡓࡵ࡟ࡑࢀࡒ
ࢀࡢᅜ࡟࠾ࡅࡿ≟✀㢌ᩘ࡜ᛶู࡜ࢆྜࢃࡏࡓࠋࡑࡢ⤖ᯝࠊྛᅜ 1252 㢌࡜࡞ࡗࡓࠋ᪥ᮏཬࡧ
⡿ᅜ࡟ඹ㏻ࡋ࡚࠸ࡓ 63 ၥࡢ㉁ၥ࡟ࡘ࠸࡚ᅉᏊศᯒࢆ⾜ࡗࡓ⤖ᯝࠊ12 ࡢᅉᏊ࡟ศ㢮ࡉࢀࡓࠋ
ᅉᏊ㈇Ⲵ㔞ࡀ 0.4 ࡼࡾᑠࡉ࠸㡯┠ࢆ㝖ࡁࠊ෌ࡧᅉᏊศᯒࢆ⾜ࡗࡓ⤖ᯝࠊ෌ࡧ 12 ࡢᅉᏊ࡟ศ
㢮ࡉࢀࡓࠋᅉᏊ㈇Ⲵ㔞ࡀ 0.4 ࡼࡾᑠࡉ࠸㡯┠ࢆ෌ࡧ㝖ࡁࠊ෌ࡧᅉᏊศᯒࢆ⾜ࡗࡓ⤖ᯝࠊྠࡌ
ࡃᅉᏊᚓⅬ࡟࠾ࡅࡿඹ㏻ศᩓࡢ 55.14%ࢆ༨ࡵࡿ 12 ࡢᅉᏊ࡟ศ㢮ࡉࢀࡓࠋࡑࡢ࠺ࡕ 11 ࡢᅉᏊࡀࢡࣟࣥࣂࢵࢡࡢ࢔ࣝࣇ࢓್ 0.7 ௨ୖࢆ♧ࡋࡓࠋ11 ࡢᅉᏊࡣࠊぢ▱ࡽࡠே࡟ᑐࡍࡿᨷᧁᛶࠊぢ▱ࡽࡠே࡟ᑐࡍࡿᜍࢀࠊカ⦎ᛶࠊศ㞳୙Ᏻࠊ⯆ዧᛶࠊ㠀♫఍ⓗ࡞่⃭࡟ᑐࡍࡿᜍࢀࠊ
㣫࠸୺࡟ᑐࡍࡿᨷᧁᛶࠊぢ▱ࡽࡠ≟࡟ᑐࡍࡿᜍࢀࠊぢ▱ࡽࡠ≟࡟ᑐࡍࡿᨷᧁᛶࠊឡ╔ࠊ౵ධ⪅࡟ᑐࡍࡿᨷᧁᛶ࡛࠶ࡾࠊ11 ࡢᅉᏊ࡟ࡣ⥲ィ 50 ಶࡢ㉁ၥ㡯┠ࡀྵࡲࢀࡓࠋ
㸰㸬C-barq ᅉᏊᚓⅬ࡟ᑐࡍࡿ≟✀ࡢᙳ㡪≟✀ࢆࠊ᪤࡟ሗ࿌ࡉࢀ࡚࠸ࡓ vonHoldt (2010)11 ࡟ࡼࡿ㑇ఏศᒱᅗ㸦ᅗ 1-1㸧ࢆᇶ࡟ࠊձཎ
ጞⓗ࡞≟㸦࢔ࣥࢩ࢚ࣥࢺ㸧ࢢ࣮ࣝࣉࠊղࢺ࢖ࢢ࣮ࣝࣉࠊճࢫࣃࢽ࢚ࣝࠊࢭࣥࢺࣁ࢘ࣥࢻࠊ

171

ࣉ࣮ࢻࣝ࠿ࡽᡂࡿࢫࣃࢽ࢚ࣝࢢ࣮ࣝࣉࠊմ࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࠊյࢸࣜ࢔ࢢ࣮ࣝࣉࠊնࣁ
࣮ࢹ࢕ࣥࢢ࡜ࢧ࢖ࢺࣁ࢘ࣥࢻ࠿ࡽᡂࡿࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࠊշࣞࢺࣜࣂ࣮ࢢ࣮ࣝࣉࠊո
࣐ࢫࢳࣇࢢ࣮ࣝࣉࡢ 8 ࡘࡢࢢ࣮ࣝࣉ࡟ศ㢮ࡋ࡚ゎᯒࢆ⾜ࡗࡓࠋᰘ≟ࡣࠊvonHoldt (2010)࡟
ࡼࡿ㑇ఏศᒱᅗ࡟ྵࡲࢀ࡚࠸࡞࠸ࡓࡵࠊParker (2004)࡟ࡼࡿ㑇ఏศᒱᅗࢆᇶ࡟ཎጞⓗ࡞≟ࡢࢢ࣮ࣝࣉ࡟ศ㢮ࡋࡓࠋࡑࡢ௚ࡢ㑇ఏศᒱᅗ࡟ྵࡲࢀ࡞࠸≟✀࡟ࡘ࠸࡚ࡣࠊゎᯒ࠿ࡽ㝖እࡋࡓࠋ୍⯡໬⥺ᆺࣔࢹࣝࢆ⏝࠸࡚ゎᯒࢆ⾜ࡗࡓ⤖ᯝࠊ11 ࡢᅉᏊ඲࡚ࡀ≟✀ࢢ࣮ࣝࣉཬࡧࡑ
ࡢ௚ 8 ࡘࡢኚᩘ࡟ࡼࡗ࡚᭷ព࡟ㄝ᫂ࡉࢀࡓ㸦p<0.05, ⾲ 1-1, ᅗ 1-2 ཧ↷㸧ࠋぢ▱ࡽࡠே࡟ᑐࡍࡿᨷᧁᛶ㸸ࢺ࢖ࢢ࣮ࣝࣉࡀ᭱ࡶ㧗࠿ࡗࡓࠋࢺ࢖ࢢ࣮ࣝࣉࡣࠊࣁ࣮ࢹ࢕ࣥ
ࢢࢢ࣮ࣝࣉࠊࣞࢺࣜࣂ࣮ࢢ࣮ࣝࣉࠊ࣐ࢫࢳࣇࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟ᨷᧁⓗ࡛࠶ࡗࡓ㸦p<0.05㸧ࠋࡲࡓࠊࢫࣃࢽ࢚ࣝࢢ࣮ࣝࣉࡣࠊࣞࢺࣜࣂ࣮ࢢ࣮ࣝࣉཬࡧ࣐ࢫࢳࣇࢢ࣮ࣝࣉࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡣࣞࢺࣜࣂ࣮ࢢ࣮ࣝࣉࠊࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࡣࣞࢺࣜࣂ࣮ࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟
ᨷᧁⓗ࡛࠶ࡗࡓ㸦p<0.05㸧ࠋぢ▱ࡽࡠே࡟ᑐࡍࡿᜍࢀ㸸࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࡀ᭱ࡶపࡃࠊࡑࡢ௚ࡢ඲࡚ࡢ≟✀ࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟ప࠿ࡗࡓ㸦p<0.05㸧ࠋカ⦎ᛶ㸸ࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࡀ᭱ࡶ㧗࠿ࡗࡓࠋࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࡢカ⦎ᛶࡣࠊ࣮࣡
࢟ࣥࢢࢢ࣮ࣝࣉ௨እࡢ඲࡚ࡢ≟✀ࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟㧗࠿ࡗࡓ㸦p<0.05㸧ࠋศ㞳୙Ᏻ㸸≟✀ࢢ࣮ࣝࣉ࡟ࡼࡿ㐪࠸ࡣぢࡽࢀ࡞࠿ࡗࡓࠋ
⯆ዧᛶ㸸࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࡀ᭱ࡶప࠿ࡗࡓࠋ࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࡢ⯆ዧᛶࡣࠊ࣐ࢫࢳࣇ
ࢢ࣮ࣝࣉ௨እࡢ඲࡚ࡢ≟✀ࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟ప࠿ࡗࡓ㸦p<0.05㸧
ࠋࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝ
ࣉࡀ᭱ࡶ㧗ࡃࠊࢫࣃࢽ࢚ࣝࢢ࣮ࣝࣉࠊ࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࠊ࣐ࢫࢳࣇࢢ࣮ࣝࣉࡼࡾࡶ᭷ព
࡟㧗࠿ࡗࡓ㸦p<0.05㸧ࠋ㠀♫఍ⓗ࡞่⃭࡟ᑐࡍࡿᜍࢀ㸸࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࡀ᭱ࡶపࡃࠊࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࡀ
᭱ࡶ㧗࠿ࡗࡓࠋ࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࡢ㠀♫఍ⓗ࡞่⃭࡟ᑐࡍࡿᜍࢀࡣࠊ࣐ࢫࢳࣇࢢ࣮ࣝࣉ
௨እࡢ඲࡚ࡢ≟✀ࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟పࡃࠊࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࡢ㠀♫఍ⓗ࡞่⃭࡟ᑐࡍࡿᜍࢀࡣࠊࢫࣃࢽ࢚ࣝࢢ࣮ࣝࣉࠊ࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࠊ࣐ࢫࢳࣇࢢ࣮ࣝࣉࡼࡾࡶ᭷ព
࡟㧗࠿ࡗࡓ㸦p<0.05㸧ࠋ㣫࠸୺࡟ᑐࡍࡿᨷᧁᛶ㸸࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࡀ᭱ࡶపࡃࠊࢺ࢖ࢢ࣮ࣝࣉࡀ᭱ࡶ㧗࠿ࡗࡓࠋ࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࡢ㣫࠸୺࡟ᑐࡍࡿᨷᧁᛶࡣࠊࡑࡢ௚ࡢ඲࡚ࡢ≟✀ࢢ࣮ࣝࣉࡼࡾࡶప࠿
ࡗࡓ㸦p<0.05㸧
ࠋࢺ࢖ࢢ࣮ࣝࣉࡢ㣫࠸୺࡟ᑐࡍࡿᨷᧁᛶࡣࠊ࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࠊࣁ࣮ࢹ࢕
ࣥࢢࢢ࣮ࣝࣉࠊࣞࢺࣜࣂ࣮ࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟㧗࠿ࡗࡓ㸦p<0.05㸧ࠋࡲࡓࠊࢫࣃࢽ࢚ࣝࢢ
࣮ࣝࣉࡢ㣫࠸୺࡟ᑐࡍࡿᨷᧁᛶࡣࠊ࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉ࡜ࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࡼࡾࡶ᭷
ព࡟㧗࠿ࡗࡓ㸦p<0.05㸧ࠋぢ▱ࡽࡠ≟࡟ᑐࡍࡿᜍࢀ㸸࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࡀ᭱ࡶప࠿ࡗࡓࠋ࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉࡢぢ▱ࡽࡠ≟࡟ᑐࡍࡿᜍࢀࡣࠊࡑࡢ௚ࡢ඲࡚ࡢ≟✀ࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟ప࠿ࡗࡓ㸦p<0.05㸧ࠋぢ▱ࡽࡠ≟࡟ᑐࡍࡿᨷᧁᛶ㸸ࣞࢺࣜࣂ࣮ࢢ࣮ࣝࣉࡀ᭱ࡶపࡃࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࠊࢺ࢖
ࢢ࣮ࣝࣉࠊࢸࣜ࢔ࢢ࣮ࣝࣉཬࡧࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟ప࠿ࡗࡓ㸦p<0.05㸧ࠋ172ឡ╔㸸ཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡀ᭱ࡶప࠿ࡗࡓࠋཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡢឡ╔ࢫࢥ࢔ࡣࠊࡑࡢ௚
ࡢ඲࡚ࡢ≟✀ࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟ప࠿ࡗࡓ㸦p<0.05㸧ࠋ౵ධ⪅࡟ᑐࡍࡿᨷᧁᛶ㸸ࢺ࢖ࢢ࣮ࣝࣉࡀ᭱ࡶ㧗ࡃࠊࢺ࢖ࢢ࣮ࣝࣉࡢ౵ධ⪅࡟ᑐࡍࡿᨷᧁᛶ
ࡣࠊ࣐ࢫࢳࣇࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟㧗࠿ࡗࡓ㸦p<0.05㸧ࠋ㸱㸬C-barq ᅉᏊࠕឡ╔ࠖࡢ≟✀ࢡࣛࢫࢱ࣮ゎᯒཎጞⓗ࡞≟ࢢ࣮ࣝࣉ࡜ࡑࡢ௚ࡢ≟✀ࢢ࣮ࣝࣉ࡜࡛㐪࠸ࡀぢࡽࢀࡓឡ╔࡟ࡘ࠸࡚ࠊ≟✀ࢡࣛࢫࢱ࣮ゎᯒࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊ≟✀ࡣ኱ࡁࡃ 2 ࡘࡢࢡࣛࢫࢱ࣮࡟ศ㢮ࡉࢀࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡢ 5 ≟✀඲࡚ࡀࢡࣛࢫࢱ࣮1 ࡟ศ㢮ࡉࢀࡓ㸦ᅗ 1-3 ཧ↷㸧ࠋཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡢ
≟✀௨እ࡟ࡣࠊ࣍࣡࢖ࢺࢸࣜ࢔ࠊ࢘࢕࣌ࢵࢺࠊࢣ࢔ࣥࢸࣜ࢔ࠊ࣎ࣝࢰ࢖ࠊࢢ࣮ࣞࢺࢹ࣮ࣥ
ࡢ 5 ≟✀ࡀࢡࣛࢫࢱ࣮1 ࡟ศ㢮ࡉࢀࠊࡑࡢ௚ࡢ 36 ≟✀ࡣࢡࣛࢫࢱ࣮2 ࡟ศ㢮ࡉࢀࡓࠋ173

⾲ 1-1 ୍⯡໬⥺ᆺࣔࢹࣝࢆ⏝࠸ࡓᅉᏊᚓⅬࡢゎᯒ⤖ᯝ

F2: Fear of unfamiliar persons

F1:䚷Aggression to unfamiliar persons
䃦2df
p Pairwise comparison B5)
50.570 7 0.000 1>7, 2>6, 2>7, 2>8,
3>7, 3>8, 5>7, 6>7
1.015 1 0.314
1.683 1 0.195

Breed groups 1)
Country
Sex
Neutered status 2)
Source where aquired

3)

0.658

1 0.41724.1006 0.0012>3, 5>3

Dog-ownership experience4)
Body weight
Dog's age at evaluation
Dog's age when acquired
Breed groups*Country
Breed groups*Sex
Country*Sex
Breed groups*Country*Sex

1.361 1 0.243
0.571 1 0.450
0.000 1 0.996
14.696 1 0.000
61.631 15 0.000
63.198 15 0.000
12.030 3 0.007
79.827 31 0.000

Omnibus189.444 42 0.000

Breed groups 1)

df
p Pairwise comparison
䃦2
51.531 7 0.000
6>1, 6>2, 6>3,
6>5, 6>7, 6>8CountrySex

14.4880.749

1 0.000
1 0.387

Neutered status 2)

3.771

1 0.052

Source where aquired 3)

31.588

6 0.000

Dog-ownership experience4)
Body weight
Dog's age at evaluation
Dog's age when acquired
Breed groups*Country
Breed groups*Sex
Country*Sex
Breed groups*Country*Sex

15.057 1 0.000
3.725 1 0.054
22.534 1 0.000
5.055 1 0.025
89.920 15 0.000
59.386 15 0.000
15.663 3 0.001
104.545 31 0.000

Omnibus248.373 42 0.000

䃦2126.652
70.01069.123df
p Pairwise comparison
7 0.000
1>4, 2>4, 3>4,
5>4, 6>4, 7>4, 8>4
1 0.000
US>JPN
1 0.000
F>M

0.468

1 0.4941.940-0.003

6 0.9250.006
14.5180.627
3.223
139.576127.81485.804141.411

1
1
1
1
15
15
3
31

145.060

42 0.000

F3: Trainability

B

0.939
0.000
0.429
0.073
0.000
0.000
0.000
0.000

-0.029F4: Separation-related anxiety
䃦2
13.498df
p Pairwise comparison
7 0.061

US>JPN

31.0780.000

1 0.000
1 0.996

US>JPN4.368

1 0.037

I>N

3>4, 7>4

12.795

6 0.0462>1

10.161
4.925
5.444
1.083
96.17620.71731.078106.105

1
1
1
1
15
15
3
31

237.075

42 0.000

B

0.024
0.000

0.001
0.026
0.020
0.298
0.000
0.146
0.000
0.000

B

1>2
-0.012-0.0391, Ancient and spitz breeds: 1, Toy dogs: 2, Spaniels, scent hounds, and poodles: 3, Working dogs: 4, Small
terriers: 5, Sight hounds and herding dogs: 6, Retrievers: 7, Mastiff-like dogs: 82, neutered: N, intact: I
3, bred by owner: 1, friend of relative: 2, breeder: 3, pet store: 4, shelter: 5, stray: 6, other: 74, first ownership: 1, second and more ownership: 25, partial regression coefficient174F5: Energy and restless
Breed groups 1)

䃦2
df
p Pairwise comparison
44.844 7 0.000 1>4, 2>4, 3>4, 5>4,
6>4, 7>4, 6>3, 6>8Country
Sex

5.077
0.161

1 0.024
1 0.688

JPN>US

Neutered status 2)

5.631

1 0.018

N>I

Source where aquired 3)

10.706

6 0.098

Dog-ownership experience4)
Body weight
Dog's age at evaluation
Dog's age when acquired
Breed groups*Country
Breed groups*Sex
Country*Sex
Breed groups*Country*Sex

2.130 1 0.144
7.906 1 0.005
2.154 1 0.142
4.155 1 0.042
52.434 15 0.000
47.984 15 0.000
5.752 3 0.124
65.671 31 0.000

Omnibus148.076 42 0.000

Breed groups 1)

Ȥdf
p Pairwise comparison
84.719 7 0.000 1>4, 2>4, 2>6, 2>7,
3>4, 3>6, 5>4, 6>4,
7>4, 8>4Country
Sex

0.000
0.000

1 1.000
1 1.000

Neutered status 2)

0.000

Source where aquired 3)

0.000

F6: Fear of non-social stimuliB䃦2
44.844df
p Pairwise comparison
7 0.000 1>4, 2>4, 3>4, 6>3,
5>4, 6>4, 7>4, 6>8

5.077
0.161

1 0.024
1 0.688

JPN>US
N>I

5.631

1 0.018

10.706

6 0.098

2.130
7.906
2.154
4.155
52.43447.9845.752
65.671

1
1
1
1
15
15
3
31

148.076

42 0.000䃦2141.880df
p Pairwise comparison
7 0.000
1>4, 2>4, 3>4,
5>4, 6>4, 7>4, 8>4

80.13978.662

1 0.000
1 0.000

1 1.000

2.798

1 0.094

6 1.000

11.911

6 0.064

Dog-ownership experience4)
Body weight
Dog's age at evaluation
Dog's age when acquired
Breed groups*Country
Breed groups*Sex
Country*Sex
Breed groups*Country*Sex

0.000 1 1.000
0.023 1 0.881
0.000 1 0.986
2.897 1 0.089
89.609 14 0.000
85.619 14 0.000
20.113 3 0.000
91.284 29 0.000

1.017
5.361
1.257
0.008
153.605
142.858
98.852
155.422

1
1
1
1
15
15
3
31

Omnibus266.838 42 0.000

111.849

42 0.000-0.0120.001

F7: Aggression to household members

175

0.144
0.005
0.142
0.042
0.000
0.000
0.124
0.000

B

-0.0120.001

F8: Fear of unfamiliar dogs
B

0.313
0.021
0.262
0.929
0.000
0.000
0.000
0.000

B

US>JPN
F>M-0.017

F11: Attachment and attention-seeking

F9: Aggression to unfamiliar dogs
Breed groups 1)

䃦2
df
p Pairwise comparison
26.211 7 0.000 1>7, 2>7, 5>7, 6>7

Country
Sex

2.747
5.105

1 0.097
1 0.024B

䃦2
82.116136.912
3.030

M>F

df
p Pairwise comparison
7 0.000 2>1, 3>1, 4>1, 5>1,
6>1, 7>1, 8>1
1 0.000
US>JPN
1 0.082

Neutered status 2)

0.509

1 0.476

1.019

1 0.313

Source where aquired 3)

11.427

6 0.076

11.958

6 0.063

Dog-ownership experience4)
Body weight
Dog's age at evaluation
Dog's age when acquired
Breed groups*Country
Breed groups*Sex
Country*Sex
Breed groups*Country*Sex

0.780 1 0.377
1.718 1 0.190
16.415 1 0.000
2.756 1 0.097
36.747 15 0.001
38.165 15 0.001
9.449 3 0.024
52.726 31 0.009

0.042
7.023
11.1451.318
328.227
86.349145.751344.926

1
1
1
1
15
15
3
31

Omnibus104.112 42 0.000

385.296

42 0.000

0.069

F12: Aggression to persons passing near the
Breed groups1)

df
p Pairwise comparison
䃦2
19.286 7 0.007
2>8Country
Sex

3.724
0.984

1 0.054
1 0.321

Neutered status 2)

0.543

1 0.461

Source where aquired 3)

8.539

6 0.201

Dog-ownership experience4)
Body weight

0.335
0.007

1 0.563
1 0.934

Dog's age at evaluation

0.000

1 0.999

Dog's age when acquired

8.065

1 0.005

Breed groups*Country

29.034 15 0.016

Breed groups*Sex

26.774 15 0.031

Country*Sex

7.556

Breed groups*Country*Sex

47.057 31 0.032

Omnibus72.384 42 0.002

B-0.002

3 0.056

176

0.837
0.008
0.001
0.251
0.000
0.000
0.000
0.000

B

-0.005-0.018ᅗ 1-1 ≟✀ࡢศ㢮࡟⏝࠸ࡓ㑇ఏศᒱᅗ㸦vonHoldt 2010㸧

㯮࠸Ⅼࡣࠊࣈ࣮ࢺࢫࢺࣛࢵࣉ್ 95%௨ୖࢆ♧ࡍࠋ177

ᅗ 1-2 ≟✀ࢢ࣮ࣝࣉ࡟࠾ࡅࡿᅉᏊᚓⅬࡢᖹᆒࢫࢥ࢔

a vs b, p<0.05;

c vs d, p<0.05;

e vs f, p<0.05;

g vs h, p<0.05

Ancient and spitz breeds: Anci., Toy dogs: Toy, Spaniels, scent hounds, and poodles: Spa., Working
dogs: Work., Small terriers: Terr., Sight hounds and herding dogs: Herd., Retrievers: Retr.,
Mastiff-like dogs: Mast.

178

ᅗ 1-3 ឡ╔ࡢ≟✀ࢡࣛࢫࢱ࣮ゎᯒᶞᙧᅗ









179



㸰㸬≟ࡢᐙ␆໬㐣⛬࡟࠾ࡅࡿ㑅ᢥᅽᅉᏊࡢ᥎ᐃ◊✲せ᪨࣑࢜࢜࢝ࠊ⛅⏣≟ࠊᰘ≟ࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉ௨እࡢ࢖ࢾ㸦ࣛࣈࣛࢻ࣮ࣝࠊࢫࢱࣥࢲ࣮ࢻ
ࣉ࣮ࢻࣝ➼ࡢᵝࠎ࡞≟✀㸧ࡢ࢔࣑࣮ࣛࢮࢥࣆ࣮ᩘࢆㄪᰝࡋࡓࠋᰘ≟ࡣࠊ୍⯡ⓗ࡟࣌ࢵࢺ࡜
ࡋ࡚㣫ࢃࢀ࡚࠸ࡿᰘ≟࡜ኳ↛グᛕ≀ᰘ≟ಖᏑ఍ࡢཝࡋ࠸⟶⌮ࡢඖ࡛஺㓄ࡀ⾜ࢃࢀ࡚࠸ࡿ⦖
ᩥᰘࢆ⏝࠸ࡓࠋ
࣑࢜࢜࢝ࡢ࢔࣑࣮ࣛࢮࢥࣆ࣮ᩘࡣඛ⾜◊✲࡛ሗ࿌ࡉࢀ࡚࠸ࡿ࡜࠾ࡾࠊ2 ࢥࣆ࣮⛬ᗘ࡛࠶ࡗࡓࠋ
ࡲࡓࠊ࣑࢜࢜࢝࡬ࡢ㑇ఏⓗ㏆ࡉ࠿ࡽண᝿ࡋࡓ࡜࠾ࡾࠊ⛅⏣≟ࡢࢥࣆ࣮ᩘࡣ࣑࢜࢜࢝ࡼࡾࡶ
ከ࠸ࡀ୍⯡ⓗ࡞≟✀ࡼࡾᑡ࡞࠿ࡗࡓ㸦p<0.01㸧
ࠋ୍᪉ࠊ⛅⏣≟࡜ྠࡌࡃཎጞⓗ࡞≟ࢢ࣮ࣝࣉ࡟ྵࡲࢀࡿᰘ≟ࡢࢥࣆ࣮ᩘࡣࠊ୍⯡ⓗ࡞≟✀࡜ྠ⛬ᗘ࡛࠶ࡾࠊᰘ≟ࡢ࣑࢜࢜࢝࡬ࡢ㑇ఏⓗ㏆ࡉ࡜཯ࡋࡓ⤖ᯝ࡜࡞ࡗࡓࠋ⦖ᩥᰘࡢࢥࣆ࣮ᩘࡣࠊᰘ≟ࡼࡾᑡ࡞ࡃ㸦p<0.01㸧ࠊ⛅⏣≟ࡼࡾከ࠿ࡗࡓ㸦p<0.05㸧
ࠋ◊✲┠ⓗ2005 ᖺࡢ࢖ࢾࡢ඲ࢤࣀ࣒㓄ิࡢゎㄞ௨㝆ࠊ࢖ࢾࡢᐙ␆໬࡟㛵ࡍࡿࢤࣀ࣒࣡࢖ࢻ࡞◊✲ࡀάⓎ࡟⾜ࢃࢀࡓࠋࡑࢀࡽࡢ◊✲ᡂᯝ࡟ࡼࡾ࠶ࡆࡽࢀࡓ࢖ࢾࡢᐙ␆໬ࡢ㐣⛬࡛㑅ᢥᅽࡀ࠿࠿ࡗ
ࡓ࡜ᛮࢃࢀࡿ㑇ఏᏊࡢೃ⿵࡟ࡣࠊᾘ໬࣭௦ㅰ㛵㐃࡟㛵ࢃࡿࡶࡢࡀከࡃぢࡽࢀࡓ 12-14ࠋ≉࡟ࠊ࡛ࢇ࡫ࢇᾘ໬㓝⣲࡛࠶ࡿș-࢔࣑࣮ࣛࢮ㑇ఏᏊࡢࢥࣆ࣮ᩘࡀࠊ࣑࢜࢜࢝࡟ẚ࡭࡚࢖ࢾ࡛ࡣከ
ࡃ࡞ࡗ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡾࠊ࢖ࢾࡢᐙ␆໬࡟ࡣ࡛ࢇ࡫ࢇᾘ໬⬟ຊࡢኚ໬ࡀ኱ࡁࡃᙳ
㡪ࡋ࡚࠸ࡿࡢ࡛ࡣ࡞࠸࠿࡜᥎ ࡉࢀࡓࠋ୍᪉࡛ࠊ࣑࢜࢜࢝࠿ࡽ࢖ࢾ࡬࡜ᐙ␆໬ࡉࢀࡓࠕ㐍໬ࠖ㸦➨ 1 ࡢẁ㝵㸧ࡢ㑅ᢥᅽ࡜ࡋ࡚࡛ࢇ࡫
ࢇᾘ໬⬟ຊࡢኚ໬ࡀാ࠸ࡓࡢ࠿ࠊཪࡣࠊࣄࢺࡀ㎰⪔ࢆጞࡵࡿ࡜࡜ࡶ࡟࢖ࢾࡢ㣗⣊※ࡶኚ໬ࡋࡇࢀࡽࡢ㑇ఏᏊ࡟㑅ᢥᅽࡀ࠿࠿ࡗࡓࡢ࠿ࡣ᫂ࡽ࠿࡟࡞ࡗ࡚࠸࡞࠸ࠋᮏ◊✲࡛ࡣ➨ 1 ❶࠿ࡽ➨ 3 ❶ࡲ࡛ࡢ⤖ᯝ࡟ࡼࡾࠊ࢖ࢾࡢᐙ␆໬㸦㐍໬㸧࡟ࡣẼ㉁ࡀ㛵㐃ࡋ࡚࠸ࡓ࡜᥎ ࡋ࡚࠾ࡾࠊᮏ❶࡛ࡣࠊ࡛ࢇ࡫ࢇᾘ໬⬟ຊࡀ࢖ࢾࡢᐙ␆໬㸦㐍໬㸧㸦➨ 1 ࡢẁ㝵㸧ࡢ㑅ᢥᅽ࡜ࡋ࡚ാ
࠸ࡓࡢ࠿࡝࠺࠿ࢆㄪᰝࡍࡿࡇ࡜࡜ࡋࡓࠋ
ᮦᩱ࡜᪉ἲ࣑࢜࢜࢝ 12 㢌ࠊ
⛅⏣≟ 14 㢌ࠊᰘ≟ 34 㢌ࡢ⾑ᾮཪࡣ⫢⮚ࡢ⤌⧊∦ࡼࡾ DNA ࢆᢳฟࡋࡓࠋ
⾑ᾮࡢᢠจᅛ๣ࡣࠊ࢚ࢳࣞࣥࢪ࢔࣑ࣥࢸࢺࣛ㓑㓟㸦EDTA㸧ࢆ⏝࠸ࡓࠋ⫢⮚ࡢ⤌⧊∦ࡣࠊື≀⑓㝔࡟᳨࡚యࡉࢀࡓࡶࡢࢆ㒑㏦࠸ࡓࡔ࠸ࡓࠋࢧࣥࣉࣝࡣ඲࡚-20Υ࡟࡚෭෾ಖᏑࡋࡓࠋᰘ
≟ࡣࠊ୍⯡ⓗ࡟࣌ࢵࢺ࡜ࡋ࡚㣫ࢃࢀ࡚࠸ࡿᰘ≟ 17 㢌࡜ኳ↛グᛕ≀ᰘ≟ಖᏑ఍ࡢཝࡋ࠸⟶⌮ࡢඖ࡛஺㓄ࡀ⾜ࢃࢀ࡚࠸ࡿ⦖ᩥᰘ 17 㢌ࢆ⏝࠸ࡓࠋኳ↛グᛕ≀ᰘ≟ಖᏑ఍ࡣྂ௦ࡢ࢖ࢾࡢᙧ
ែࢆࡵࡊࡋ࡚ᰘ≟ࡢ஺㓄ࢆ⾜ࡗ࡚࠾ࡾࠊࡑࡢ⤖ᯝࠊ⦖ᩥᰘࡣ୍⯡ⓗ࡞ᰘ≟࡜ẚ࡭࡚ࠊὸ࠸

180㢠ẁࠊ୕ゅᙧࡢ㗦࠸┠ࠊ኱ࡁࡃ㗦࠸ṑ➼ࡢ⦖ᩥ᫬௦ࡢ㑇㊧࠿ࡽⓎ᥀ࡉࢀࡓ࢖ࢾ࡜㏆࠸ᙧែࢆᣢࡘࠋࡲࡓཎጞⓗ࡞≟ࢢ࣮ࣝࣉ௨እࡢ࢖ࢾࡢ௦⾲࡜ࡋ࡚ࠊࣛࣈࣛࢻ࣮ࣝࠊࢫࢱࣥࢲ࣮ࢻ
ࣉ࣮ࢻࣝ➼ࡢᵝࠎ࡞≟✀ࡢ࢖ࢾ 25 㢌ࢆ⏝࠸ࡓࠋ⾑ᾮཪࡣ⫢⮚ࡢ⤌⧊∦࠿ࡽࡢ DNA ᢳฟࡣ➨ 3 ❶࡜ྠᵝࡢᡭ㡰࡛⾜ࡗࡓࠋࢥࣆ࣮ᩘࡢ ᐃ࡟ࡣࠊTaqMan Genotyping Master Mix (Applied Biosystems)ࢆ⏝࠸ࡓ
TaqMan Copy Number Assay ཪࡣ SYBR® Premix Ex Taqτ II (TaKaRa) ࢆ⏝࠸ࡓ qPCR
ࢆ⾜ࡗࡓࠋࣉࣛ࢖࣐࣮ࡸࣉ࣮ࣟࣈࡢタィࡣ Axelsson ࡽࡢㄽᩥ 10 ࡟ᚑ࠸ࠊ᪉ἲࡣ࣓࣮࣮࢝
ࡢࣉࣟࢺࢥࣝ࡟ᚑࡗࡓࠋෆ㒊ᶆ‽࡟ࡣࠊ࢖ࢾࢤࣀ࣒ୖ࡛ 2 ࢥࣆ࣮࡛Ꮡᅾࡍࡿࡇ࡜ࡀ▱ࡽࢀ
࡚࠸ࡿ C7orf28B ࢆ⏝࠸ࠊࢧࣥࣉࣝࡣ 1 ಶయ 3 ᅇࡎࡘ ᐃࡋࡓࠋTaqMan ἲ࡜ qPCR ἲ࡜ࡢ஫᥮ᛶࢆࠊྠࡌࢧࣥࣉࣝࢆ ࡿࡇ࡜࡟ࡼࡾ☜ㄆࡋࡓࠋ⤫ィฎ⌮ࡣ SPSS statistic 17.0㸦SPSS Japan Inc.㸧ࢆ⏝࠸ࠊ᭷ពỈ‽ࡣ 5㸣࡜ࡋࡓࠋ
Kruskal-Wallis test ཬࡧ Mann Whitney U test㸦Bonferroni ⿵ṇ㸧ࢆ⾜ࡗࡓࠋ࣑࢜࢜࢝ࡢ
࠺ࡕ 1 ࡘࡢࢧࣥࣉࣝࡣࠊእࢀ್࡜ࡋ࡚㝖እࡋࡓࠋ⤖ᯝ࠾ࡼࡧ⪃ᐹ࣑࢜࢜࢝ࡢ࢔࣑࣮ࣛࢮࢥࣆ࣮ᩘࡣඛ⾜◊✲࡛ሗ࿌ࡉࢀ࡚࠸ࡿ࡜࠾ࡾࠊ2 ࢥࣆ࣮⛬ᗘ࡛࠶ࡗࡓࠋ
ࡲࡓࠊ࣑࢜࢜࢝࡬ࡢ㑇ఏⓗ㏆ࡉ࠿ࡽண᝿ࡋࡓ࡜࠾ࡾࠊ⛅⏣≟ࡢࢥࣆ࣮ᩘࡣ࣑࢜࢜࢝ࡼࡾࡶ
ከ࠸ࡀ୍⯡ⓗ࡞≟✀ࡼࡾᑡ࡞࠿ࡗࡓࠋ୍᪉ࠊ⛅⏣≟࡜ྠࡌࡃཎጞⓗ࡞≟ࢢ࣮ࣝࣉ࡟ྵࡲࢀ
ࡿᰘ≟ࡢࢥࣆ࣮ᩘࡣࠊ୍⯡ⓗ࡞≟✀࡜ྠ⛬ᗘ࡛࠶ࡾࠊᰘ≟ࡢ࣑࢜࢜࢝࡬ࡢ㑇ఏⓗ㏆ࡉ࡜཯ࡋࡓ⤖ᯝ࡜࡞ࡗࡓࠋ⦖ᩥᰘࡢࢥࣆ࣮ᩘࡣࠊᰘ≟ࡸ୍⯡ⓗ࡞≟✀ࡼࡾᑡ࡞ࡃࠊ⛅⏣≟ࡼࡾከ࠿ࡗࡓ㸦ᅗ 2-1 ཧ↷㸧ࠋ

181

Diploid Copy Number

ᅗ 2-1 ࢔࣑࣮ࣛࢮࢥࣆ࣮ᩘࡢ≟✀㛫࡞ࡽࡧ࡟࣑࢜࢜࢝࡜ࡢẚ㍑

40
30
20
10
0
Wolf

Akita

Jomon-Shiba

Shiba

Other
Breeds࣑࢜࢜࢝ Q  ⛅⏣≟ Q  ⦖ᩥᰘ Q  ᰘ≟ Q  ୍⯡ⓗ࡞≟✀ Q  
Mann Whitney U-tests wih Bonferroni correction **, p<0.01 *, p<0.05
ǘ2䠙48.3, df=4, p<0.01






182



㸱㸬ࣄࢺ࡬ࡢ♫఍ㄆ▱⬟ຊ࡟࠾ࡅࡿ≟✀ᕪࡢ᳨ฟ◊✲せ᪨ᵝࠎ࡞≟✀ࡢ࢖ࢾࢆ⏝࠸ࠊ࢖ࢾࡢㄆ▱⬟ຊࡢ≟✀ᕪࢆゎỴ୙ྍ⬟ㄢ㢟ཬࡧᣦᕪࡋ஧⪅㑅ᢥㄢ㢟࡟ࡼࡾホ౯ࡋࡓࠋゎỴ୙ྍ⬟ㄢ㢟࡛ࡣࠊ㣵ࢆᐜჾ࡛そ࠸ᐜჾࢆᅛᐃࡋࡓ≧ែ࡛ࠊ࢖ࢾࡀࣄࢺࢆぢࡿࡲ࡛ࡢ᫬㛫ࠊࣄࢺࢆぢ࡚࠸ࡿ᫬㛫ࠊࣄࢺࢆぢࡿᅇᩘࠊ஺஫จどࡢᅇᩘࢆᐃࡋࡓࠋᣦᕪࡋ஧⪅㑅ᢥㄢ㢟࡛ࡣࠊ㸰ࡘࡢ࢝ࢵࣉࡢ࡝ࡕࡽ࠿࡟㣵ࢆ㞃ࡋࡓୖ࡛ࠊࣄࣥࢺࢆฟ
ࡍࡇ࡜࡟ࡼࡾ࢖ࢾࡀ㣵ࡢධࡗ࡚࠸ࡿ᪉ࡢ࢝ࢵࣉࢆ㑅ࡪᅇᩘࢆᐃࡋࡓࠋࣄࣥࢺ࡜ࡋ࡚ࡣᐜ

ჾࢆ࡜ࢇ࡜ࢇ࡜ࡓࡓࡃ㸦ࢱࢵࣆࣥࢢ㸧
ࠊᐜჾ࡬ど⥺ࢆྥࡅࡿ㸦ど⥺㸧ࠊᐜჾࢆᣦᕪࡍ㸦ᣦᕪࡋ㸧ࡢ㸱✀㢮ࢆ⤌ࡳྜࢃࡏ࡚⏝࠸ࡓࠋゎỴ୙ྍ⬟ㄢ㢟࡛ࡣࠊཎጞⓗ࡞≟ࡣࠊ᭱ึ࡟ࣄࢺࢆぢࡿࡲ࡛ࡢ᫬㛫ࡀࢺ࢖ࢢ࣮ࣝࣉࠊࢫࣃ
ࢽ࢚ࣝࢢ࣮ࣝࣉࠊࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࡼࡾࡶ㛗ࡃࠊࣄࢺࢆぢ࡚࠸ࡿ᫬㛫ࡀࢫࣃࢽ࢚ࣝࢢ
࣮ࣝࣉࠊࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࠊࣞࢺࣜࣂ࣮ࢢ࣮ࣝࣉࡼࡾࡶ▷ࡃࠊࣄࢺࢆぢࡿᅇᩘࡀࢫࣃ
ࢽ࢚ࣝࢢ࣮ࣝࣉࠊࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࡼࡾࡶᑡ࡞ࡃࠊ஺஫จどࡢᅇᩘࡀࢺ࢖ࢢ࣮ࣝࣉࠊ
ࢫࣃࢽ࢚ࣝࢢ࣮ࣝࣉࠊࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࠊࣞࢺࣜࣂ࣮ࢢ࣮ࣝࣉࡼࡾࡶᑡ࡞࠿ࡗࡓࠋࡲ
ࡓࠊཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛ 2 ⩌ẚ㍑ࢆ⾜ࡗࡓ࡜ࡇࢁࠊ᭱ึ࡟ࣄࢺࢆぢࡿࡲ࡛ࡢ᫬
㛫ࠊࣄࢺࢆぢ࡚࠸ࡿ᫬㛫ࠊࣄࢺࢆぢࡿᅇᩘࠊ஺஫จどࡢᅇᩘࡢ඲࡚࡟࠾࠸࡚ࠊཎጞⓗ࡞≟ࡣ୍⯡ⓗ࡞≟✀ࡼࡾࡶ᭷ព࡟ࣄࢺࢆぢ࡞࠸⤖ᯝࡀᚓࡽࢀࡓ㸦p<0.01㸧ࠋᣦᕪࡋ஧⪅㑅ᢥㄢ㢟࡛ࡣࠊཎጞⓗ࡞≟ࡢᡂ⦼ࡣࠊど⥺㸩ᣦᕪࡋ㸩ࢱࢵࣆࣥࢢㄢ㢟ࠊど⥺㸩ᣦᕪࡋㄢ㢟ࠊᣦᕪࡋㄢ㢟ࡢ඲࡚࡟࠾࠸࡚ࡑࡢ௚ࡢ≟✀ࢢ࣮ࣝࣉ࡜ྠ➼࡛࠶ࡾࠊ≟✀ࢢ࣮ࣝࣉ࡟ࡼࡿ᭷ព࡞ᕪࡣぢࡽࢀ࡞࠿ࡗࡓࠋཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛㸰⩌ẚ㍑ࢆ⾜ࡗࡓ࡜ࡇࢁࠊど⥺㸩ᣦᕪࡋ㸩ࢱࢵࣆࣥࢢㄢ㢟࡟࠾࠸࡚ࠊཎጞⓗ࡞≟ࡣ୍⯡ⓗ࡞≟✀ࡼࡾࡶṇ⟅ᩘࡀ᭷ព࡟㧗࠿ࡗࡓ㸦p<0.05㸧ࠋど⥺㸩ᣦᕪࡋㄢ㢟ࠊᣦᕪࡋㄢ㢟࡛ࡣࠊ᭷ព࡞ᕪࡣぢࡽࢀ࡞࠿ࡗࡓࠋ◊✲┠ⓗ࢖ࢾࡢᣦᕪࡋ⌮ゎຊࡸゎỴ୙ྍ⬟࡞ሙ㠃࡛ࣄࢺࢆཧ↷ࡍࡿ⬟ຊࡣ㑇ఏⓗᇶ┙࡟ࡼࡿ࡜⪃࠼ࡽࢀࠊࡇࢀࡽࡢㄆ▱⬟ຊ࡟ࡘ࠸࡚≟✀㛫࡛ẚ㍑ࡍࡿࡇ࡜࡟ࡼࡾࠊࡑࢀࡽࡢ≟✀ࡀ࡝ࡢࡼ࠺
࡟సᡂࡉࢀ࡚࠸ࡗࡓࡢ࠿ࡑࡢ㐣⛬ࢆ▱ࡿࡇ࡜ࡀ࡛ࡁࡿࠋ≉࡟ࠊࢤࣀ࣒࣡࢖ࢻ◊✲࡟ࡼࡗ࡚㑇ఏⓗ࡟᭱ࡶ࣑࢜࢜࢝࡟㏆࠸ࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡢ࢖ࢾࡢ⾜ື≉ᛶࡣࠊ♽ඛ✀࡟᭱ࡶ㏆࠸࡜⪃࠼ࡽࢀࠊ࢖ࢾࡢᐙ␆໬㸦㐍໬㸧ࢆ⪃࠼ࡿୖ࡛㔜せ࡜࡞ࡿࡀࠊ≟✀ࡢ㑇ఏศᒱᅗ࡟࠾ࡅࡿ࣑࢜࢜࢝࠿ࡽࡢ㏆⦕ᗘ࡜ᣦᕪࡋ⌮ゎຊࡸゎỴ୙ྍ⬟ㄢ㢟࡟࠾ࡅࡿ♫఍ⓗㄆ▱⬟ຊ࡜ࡢ㛵㐃ᛶࡢ◊✲ࡣࡇࢀࡲ࡛࡟⾜ࢃࢀ࡚ࡁ࡚࠸࡞࠸ࠋ➨ 2 ❶࡛ࡣࠊ≟✀ࢆ
vonHoldt(2010)ࡢ㑇ఏศᒱᅗࢆᇶ࡟ 8 ࡘࡢ≟✀ࢢ࣮ࣝࣉ㸦ձཎጞⓗ࡞≟ࢢ࣮ࣝࣉࠊղࢺ࢖
ࢢ࣮ࣝࣉࠊճࢫࣃࢽ࢚ࣝࠊࢭࣥࢺࣁ࢘ࣥࢻࠊࣉ࣮ࢻࣝ࠿ࡽᡂࡿࢫࣃࢽ࢚ࣝࢢ࣮ࣝࣉࠊմ࣡

183

࣮࢟ࣥࢢࢢ࣮ࣝࣉࠊյࢸࣜ࢔ࢢ࣮ࣝࣉࠊնࣁ࣮ࢹ࢕ࣥࢢ࡜ࢧ࢖ࢺࣁ࢘ࣥࢻ࠿ࡽᡂࡿࣁ࣮ࢹ
࢕ࣥࢢࢢ࣮ࣝࣉࠊշࣞࢺࣜࣂ࣮ࢢ࣮ࣝࣉࠊո࣐ࢫࢳࣇࢢ࣮ࣝࣉ㸧࡟ศ㢮ࡋࠊ♫఍ⓗㄆ▱⬟ຊ࡟ࡘ࠸࡚ࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡢ≉ᚩࢆㄪᰝࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋᮦᩱ࡜᪉ἲ୍⯡ᐙᗞ࡛㣫⫱ࡉࢀ࡚࠸ࡿ࢖ࢾ 587 㢌ࢆᑐ㇟࡜ࡋ࡚⾜ືࢸࢫࢺࢆ⾜ࡗࡓࠋ࢖ࢾࡢເ㞟ࡣࠊ
බᅬࠊື≀⑓㝔ࠊࡋࡘࡅᩍᐊࠊࢻࢵࢢ࢝ࣇ࢙࡞࡝࡛ࢳࣛࢩࢆ㓄ᕸཪࡣᥖ♧ࡋ࡚⾜࠸ࠊ㣫࠸୺ᵝྠពࡢୖ࡛ࢸࢫࢺ࡟ཧຍࡋ࡚࠸ࡓࡔ࠸ࡓࠋ
୍⯡ᐙᗞ࡛㣫⫱ࡉࢀ࡚࠸ࡿ࢖ࢾ࡜≟⯋࡛㣫⫱ࡉࢀ࡚࠸ࡿ࢖ࢾ࡜ࡢẚ㍑࡛ࡣࠊື≀ᑓ㛛Ꮫᰯ࡛㣫⫱ࡉࢀ࡚࠸ࡿ࢖ࢾ 75 㢌ࢆᑐ㇟࡜ࡋ࡚⾜ືࢸࢫࢺࢆ⾜ࡗࡓࠋࢺ࣮ࣞࢽࣥࢢ⛉ࡢ࢖ࢾࡣ㐌࡟ 2-3 ᅇࢺ࣮ࣞࢽࣥࢢࢆཷࡅ࡚࠾ࡾࠊࢺ࣑ࣜࣥࢢ⛉ࡢ࢖ࢾࡣ㐌࡟ 1-2 ᅇࢺ࣑ࣜࣥࢢࢆཷࡅ
࡚࠸ࡿࠋ≟⯋࡛㣫ࢃࢀ࡚࠾ࡾࠊᩘ㢌ࡎࡘᗈ࠸㐠ືሙ࡟ᨺࡉࢀ࡚㐺ษ࡞㐠ືࡢᶵ఍ࢆ୚࠼ࡽ
ࢀ࡚࠸ࡿࠋධᡭඛࡣ࡯ࡰࣈ࣮ࣜࢲ࣮࡛࠶ࡾࠊ⮬ᐙ⦾Ṫࡢ࢖ࢾ࡜࣌ࢵࢺࢩࣙࢵࣉ࠿ࡽࡢ࢖ࢾ
ࡀᩘ㢌ྵࡲࢀ࡚࠸ࡿࠋ
ձゎỴ୙ྍ⬟ㄢ㢟࢛࣮࣑࢘ࣥࢢ࢔ࢵࣉヨ⾜㸸㣵ࢆᯈࡢୖ࡟⨨ࡁࠊ㣵ࢆᐜჾ࡛そࡗࡓ≧ែ࡛ࠊᯈࢆ࢖ࢾࡢ๓
࡟⨨࠸ࡓࠋ࢖ࢾࡀ⮬ศ࡛ᐜჾࢆ࡝ࡅ࡚୰ࡢ㣵ࢆ㣗࡭ࡿࡲ࡛ᚅࡗࡓࠋࡇࢀࢆ 6 ᅇ⧞ࡾ㏉ࡋࠊᐜჾࢆ࡝ࡅ࡚୰ࡢ㣵ࢆ㣗࡭ࡿࡇ࡜ࢆ࢖ࢾ࡟ぬ࠼ࡉࡏࡓࠋ
ࢸࢫࢺヨ⾜㸸㣵ࢆᯈࡢୖ࡟⨨ࡁࠊ㣵ࢆᐜჾ࡛そ࠸ࠊ௒ᗘࡣ࢖ࢾࡀᐜჾࢆ⮬ศ࡛࡝ࡅࡽࢀ
࡞࠸ࡼ࠺࡟ࠊᐜჾࢆᯈ࡟ᅛᐃࡋࡓࠋᯈࢆ࢖ࢾࡢ๓࡟⨨ࡁࠊ࢖ࢾࡢ⾜ືࢆ 2 ศ㛫ほᐹࡋࡓࠋ࢖ࢾࡢ⾜ືࢆࣅࢹ࣓࡛࢜࢝ࣛ᧜ᙳࡋࠊᚋ࡛ࣅࢹ࢜ゎᯒࢆ⾜࠸ࠊ࢖ࢾࡀ᭱ึ࡟ࣄࢺࢆぢࡿࡲ࡛ࡢ᫬㛫ࠊࣄࢺࢆぢ࡚࠸ࡿ᫬㛫ࠊࣄࢺࢆぢࡓᅇᩘࠊᐜჾ࡜ࣄࢺ࡜ࡢ஺஫จどࡢᅇᩘࢆィࡋࡓࠋղᣦᕪࡋ஧⪅㑅ᢥㄢ㢟

Hare ࡽ(2002) 21 ࡢᡭ㡰ࢆཧ⪃࡟⾜ࡗࡓࠋ࢛࣮࣑࢘ࣥࢢ࢔ࢵࣉヨ⾜㸸࢖ࢾࢆᯈࡢ┿ࢇ୰࡛ࠊᯈ࠿ࡽ 1m ࡢ㊥㞳࡟ᐇ㦂⪅࡜ᑐ㠃ࡍࡿࡼ࠺࡟ᗙࡽࡏࡓࠋ࢖ࢾࡀぢ࡚࠸ࡿ๓࡛㣵ࢆ 2 ࡘࡢᐜჾࡢ࡝ࡕࡽ࠿୍᪉࡟ධࢀࡓࠋ࢖ࢾ࡟㛤ᨺࡢྜᅗࢆฟࡋࠊᐜჾࢆ㑅ࡤࡏࡓࠋ࢖ࢾࡀ㣵ࡢධࡗ࡚࠸ࡿ᪉ࡢᐜჾ࡟᭱ึ࡟ゐࢀࡓሙྜ࡟ࡣࠊ
ᐜჾࢆᣢࡕୖࡆ࡚࢖ࢾ࡟㣵ࢆ㣗࡭ࡉࡏࡓࠋ࢖ࢾࡀ㣵ࡢධࡗ࡚࠸࡞࠸᪉ࡢᐜჾ࡟᭱ึ࡟ゐࢀࡓሙྜ࡟ࡣࠊᐜჾࢆᣢࡕୖࡆ࡚ࠊᐜჾࡢ୰࡟ఱࡶධࡗ࡚࠸࡞࠸ࡇ࡜☜ㄆࡉࡏࡓࠋࡉࡽ࡟ࠊ
㣵ࡢධࡗ࡚࠸ࡿ᪉ࡢᐜჾࢆᣢࡕୖࡆ࡚ࠊ㣵ࡀධࡗ࡚࠸ࡿࡇ࡜ࢆ࢖ࢾ࡟☜ㄆࡉࡏࡓࠋ࢛࣮࢘
࣑ࣥࢢ࢔ࢵࣉヨ⾜ࡣࠊ࢖ࢾࡀ㐃⥆ࡋ࡚ 3 ᅇࠊ㣵ࡀධࡗ࡚࠸ࡿ᪉ࡢ࢝ࢵࣉࢆ㑅ࡪࡲ࡛⥆ࡅࡽ
ࢀࡓࠋ10 ศ㛫⤒ࡗ࡚ࡶ࢛࣮࣑࢘ࣥࢢ࢔ࢵࣉヨ⾜ࢆ㏻㐣࡛ࡁ࡞࠿ࡗࡓሙྜ࡟ࡣࠊࢸࢫࢺࡣ⤊஢࡜ࡋࠊࡑࡢ࢖ࢾࡣ㝖እࡉࢀࡓࠋ
ࢸࢫࢺヨ⾜㸸࢖ࢾࢆᯈࡢ┿ࢇ୰࡛ࠊᯈ࠿ࡽ 1m ࡢ㊥㞳࡟ᐇ㦂⪅࡜ᑐ㠃ࡍࡿࡼ࠺࡟ᗙࡽࡏࡓࠋ
࢖ࢾࡢྡ๓ࢆ࿧ࡧࠊ㣵ࢆぢࡏࡓᚋ࡟ࠊ⾪❧ࡢ⿬࡛࢖ࢾ࠿ࡽぢ࠼࡞࠸ࡼ࠺࡟㣵ࢆ 2 ࡘࡢᐜჾ184

ࡢ࡝ࡕࡽ࠿୍᪉࡟ධࢀࡓࠋ㣵ࢆ㞃ࡍᐜჾࡣࠊ࠶ࡽ࠿ࡌࡵ↓సⅭ࡟Ỵࡵࡽࢀࡓࠋᐜჾࢆᯈࡢ
୧➃࡟ࢫࣛ࢖ࢻࡉࡏࡓᚋ࡟ࠊ⾪❧ࢆ࡝ࡅࡓࠋ࢖ࢾࡢྡ๓ࢆ࿧ࡧὀ┠ࡉࡏ࡚࠿ࡽࠊᣦᕪࡋ➼ࡢࣄࣥࢺࢆฟࡋࡓࠋࣄࣥࢺࡣࠊᐜჾࢆ࡜ࢇ࡜ࢇ࡜ࡓࡓࡃ㸦ࢱࢵࣆࣥࢢ㸧ࠊᐜჾࢆᣦᕪࡍ㸦ᣦᕪࡋ㸧ࠊᐜჾ࡬ど⥺ࢆྥࡅࡿ㸦ど⥺㸧ࠊࡢ୕✀㢮ࢆ⤌ࡳྜࢃࡏ࡚⏝࠸ࡓࠋࣄࣥࢺࡢ⤌ࡳྜࢃ
ࡏࡣ 4 ࣃࢱ࣮ࣥ⾜࠸ࠊど⥺㸩ᣦᕪࡋ㸩ࢱࢵࣆࣥࢢ(GPT)ㄢ㢟ࠊど⥺㸩ᣦᕪࡋ(GP)ㄢ㢟ࠊᣦᕪ
ࡋ(P)ㄢ㢟ࠊࢥࣥࢺ࣮ࣟࣝ(Cࠊࣄࣥࢺ࡞ࡋ)ࡢ㡰␒࡛⾜ࡗࡓࠋྛࣃࢱ࣮ࣥࢆ 18 ᅇࡎࡘ⾜࠸ࠊィ 72 ᅇࡢヨ⾜ࢆ⾜ࡗࡓ㸦ᅗ 2-1 ཧ↷㸧ࠋ⤫ィゎᯒࡣࠊ
ࡲࡎ≟✀ࢆ᪤࡟ሗ࿌ࡉࢀ࡚࠸ࡓ㑇ఏศᒱᅗࢆඖ࡟ 8 ࡘࡢࢢ࣮ࣝࣉ࡟ศ㢮ࡋࠊ࢖ࢾࡢ♫఍ⓗㄆ▱⬟ຊࡢ≟✀ࢢ࣮ࣝࣉ㛫ẚ㍑࡟⏝࠸ࡓࠋ࡞࠾ࠊ࣮࣡࢟ࣥࢢࠊࢸࣜ࢔ࠊ࣐ࢫ
ࢳࣇࡢࢢ࣮ࣝࣉ࡟ࡘ࠸࡚ࡣࠊ౛ᩘࡀᑡ࡞࠿ࡗࡓࡓࡵࠊศᯒ࠿ࡽࡣ㝖እࡋࠊ5 ࡘࡢࢢ࣮ࣝࣉ࡛
ẚ㍑ࡋࡓࠋḟ࡟ࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉ௨እࡢ 7 ࡘࡢࢢ࣮ࣝࣉࡢ࢖ࢾࢆ୍⯡ⓗ࡞≟✀ࢢ࣮ࣝࣉ࡜ࡋ࡚ࡲ࡜ࡵ࡚ࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉ࡜୍⯡ⓗ࡞≟✀ࢢ࣮ࣝࣉ࡜ࡢ 2 ⩌ẚ㍑ࢆ⾜ࡗࡓࠋ⤫ィฎ⌮ࡣ SPSS statistic 17.0㸦SPSS Japan Inc.㸧ࢆ⏝࠸ࠊ᭷ពỈ‽ࡣ 5㸣࡜ࡋࡓࠋ
Kruskal-Wallis test ཬࡧ Mann Whitney U test㸦Bonferroni ⿵ṇ㸧ࢆ⾜ࡗࡓࠋᣦᕪࡋ஧⪅㑅ᢥㄢ㢟࡜ゎỴ୙ྍ⬟ㄢ㢟ࡢ㛵㐃ᛶࡢ᳨ウࡢ⤫ィฎ⌮ࡣ SPSS statistic 17.0㸦SPSS Japan
Inc.㸧ࢆ⏝࠸ࠊ᭷ពỈ‽ࡣ 5㸣࡜ࡋࠊ┦㛵ゎᯒࢆ⾜ࡗࡓࠋ⤖ᯝ࠾ࡼࡧ⪃ᐹձゎỴ୙ྍ⬟ㄢ㢟᭱ึ࡟ࣄࢺࢆぢࡿࡲ࡛ࡢ᫬㛫ࡣࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡀࠊࢺ࢖ࢢ࣮ࣝࣉࠊࢫࣃࢽ࢚ࣝࢢࣝ
࣮ࣉࠊࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟㐜࠿ࡗࡓ㸦p<0.05㸧ࠋࣄࢺࢆぢ࡚࠸ࡿ᫬㛫ࡣࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡣࠊࢫࣃࢽ࢚ࣝࢢ࣮ࣝࣉࠊࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࠊࣞࢺࣜࣂ࣮ࢢ࣮ࣝࣉ
ࡼࡾࡶ᭷ព࡟ᑡ࡞࠿ࡗࡓ㸦p<0.05㸧ࠋࣄࢺࢆぢࡿᅇᩘࡣࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡣࢫࣃࢽ࢚ࣝ
ࢢ࣮ࣝࣉࠊࣁ࣮ࢹ࢕ࣥࢢࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟ᑡ࡞ࡃ㸦p<0.05㸧ࠊࢺ࢖ࢢ࣮ࣝࣉࡣࢫࣃࢽ࢚
ࣝࢢ࣮ࣝࣉࡼࡾࡶ᭷ព࡟ᑡ࡞࠿ࡗࡓ㸦p<0.05㸧
ࠋ஺஫จどࡢᅇᩘࡣࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡣࠊ
௚ࡢ 4 ࡘࡢࢢ࣮ࣝࣉ඲࡚࡜ẚ㍑ࡋ࡚᭷ព࡟ᑡ࡞ࡃ㸦p<0.05㸧
ࠊࢺ࢖ࢢ࣮ࣝࣉࡣࣁ࣮ࢹ࢕ࣥࢢ
ࢢ࣮ࣝࣉࡢ࢖ࢾࡼࡾࡶ᭷ព࡟ᑡ࡞࠿ࡗࡓ㸦p<0.05㸧ࠋ㸦ᅗ 3-1 ཧ↷㸧 ཎጞⓗ࡞≟✀ࢢ࣮ࣝࣉ࡜ࡢẚ㍑࡛ࡣࠊ᭱ึ࡟ࣄࢺࢆぢࡿࡲ࡛ࡢ᫬㛫ࠊࣄࢺࢆぢ࡚࠸ࡿ᫬㛫ࠊࣄࢺࢆぢࡿᅇᩘࠊ஺஫จどࡢᅇᩘࠊ඲࡚࡟࠾࠸࡚ࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡢ࢖ࢾࡣ୍⯡ⓗ࡞≟✀ࡼࡾࡶ᭷ព࡟ࣄࢺࢆぢ࡞࠸⤖ᯝࡀᚓࡽࢀࡓࠋ
㸦p<0.01, ᅗ 3-2 ཧ↷㸧ղᣦᕪࡋ஧⪅㑅ᢥㄢ㢟≟✀ࢢ࣮ࣝࣉ࡟ࡼࡿ㐪࠸ࡣぢࡽࢀ࡞࠿ࡗࡓࠋ
㸦p>0.05, ᅗ 3-3 ཧ↷㸧 ཎጞⓗ࡞≟✀ࢢ࣮ࣝࣉ࡜ࡢẚ㍑࡛ࡣࠊど⥺㸩ᣦᕪࡋ㸩ࢱࢵࣆࣥࢢㄢ㢟࡟࠾࠸࡚ࠊཎጞⓗ࡞≟ࢢ࣮ࣝࣉࡢ࢖ࢾࡣ୍⯡ⓗ࡞≟✀ࡼࡾࡶṇ⟅ᩘࡀ᭷ព࡟㧗࠿ࡗࡓ㸦p<0.05㸧ࠋど⥺㸩ᣦᕪ
ࡋㄢ㢟ࠊᣦᕪࡋㄢ㢟࡛ࡣࠊ᭷ព࡞ᕪࡣぢࡽࢀ࡞࠿ࡗࡓࠋ
㸦p>0.05, ᅗ 3-4 ཧ↷㸧185

ᅗ 3-1 ゎỴ୙ྍ⬟ㄢ㢟࡛ࣄࢺࢆཧ↷ࡍࡿ⬟ຊ࡟ࡘ࠸࡚≟✀ࢢ࣮ࣝࣉ㛫ẚ㍑**, p<0.01*, p<0.05

Mann-Whitney U-test with Bonferroni correction᭱ึ䛻䝠䝖䜢ぢ䜛䜎䛷䛾᫬㛫㻌 㻌 ǘ2䠙19.732, df=4, p<0.01䝠䝖䜢ぢ䛶䛔䜛᫬㛫㻌 ǘ2䠙19.659, df=4, p<0.01䝠䝖䜢ぢ䜛ᅇᩘ㻌 ǘ2䠙28.023, df=4, p<0.01஺஫จど䛾ᅇᩘ㻌 ǘ2䠙36.956, df=4, p<0.01186ᅗ 3-2 ゎỴ୙ྍ⬟ㄢ㢟࡛ࣄࢺࢆཧ↷ࡍࡿ⬟ຊࡢཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀ࡢẚ㍑**, p<0.01
Mann-Whitney U-test᭱ึ䛻䝠䝖䜢ぢ䜛䜎䛷䛾᫬㛫㻌 㻌 ǘ2䠙17.691, df=1, p<0.01䝠䝖䜢ぢ䛶䛔䜛᫬㛫㻌 ǘ2䠙16.122, df=1, p<0.01䝠䝖䜢ぢ䜛ᅇᩘ㻌 ǘ2䠙21.007, df=1, p<0.01஺஫จど䛾ᅇᩘ㻌 ǘ2䠙26.659, df=1, p<0.01187ᅗ 3-3 ᣦᕪࡋ஧⪅㑅ᢥㄢ㢟ࡢṇ⟅ᩘ࡟ࡘ࠸࡚≟✀ࢢ࣮ࣝࣉ㛫ẚ㍑

p>0.05Mann-Whitney U-test with Bonferroni correctionど⥺䠇ᣦᕪ䛧䠇䝍䝑䝢䞁䜾(GPT)ㄢ㢟㻌 㻌 ǘ2䠙8.624, df=4, p>0.05
ど⥺䠇ᣦᕪ䛧(GP)ㄢ㢟㻌 ǘ2䠙2.918, df=4, p>0.05
ᣦᕪ䛧(P)ㄢ㢟㻌 ǘ2䠙4.771, df=4, p>0.05
䝁䞁䝖䝻䞊䝹(Control)ㄢ㢟㻌 ǘ2䠙1.055, df=4, p>0.05

188ᅗ 3-4 ᣦᕪࡋ஧⪅㑅ᢥㄢ㢟ࡢṇ⟅ᩘࡢཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜ࡢẚ㍑*, p<0.05
Mann-Whitney U-testど⥺䠇ᣦᕪ䛧䠇䝍䝑䝢䞁䜾(GPT)ㄢ㢟㻌 㻌 ǘ2䠙6.662, df=1, p<0.05
ど⥺䠇ᣦᕪ䛧(GP)ㄢ㢟㻌 ǘ2䠙1.461, df=1, p>0.05
ᣦᕪ䛧(P)ㄢ㢟㻌 ǘ2䠙3.299, df=1, p>0.05
䝁䞁䝖䝻䞊䝹(Control)ㄢ㢟㻌 ǘ2䠙1.111, df=1, p>0.05189㸲㸬≟ࡢ♫఍ㄆ▱⬟ຊ࡟㛵㐃ࡍࡿ㑇ఏᏊࡢྠᐃ
◊✲せ᪨ࢥ࣑ࣗࢽࢣ࣮ࢩࣙࣥ⬟ຊ࡟㛵ࢃࡿ࣍ࣝࣔࣥ࡜ࡋ࡚ࠊ࢜࢟ࢩࢺࢩࣥ࡜ࢥࣝࢳࢰ࣮ࣝ࡟╔┠ࡋࠊ࣓ࣛࣀࢥࣝࢳࣥ㸰ཷᐜయ㸦0&5㸧࡜࢜࢟ࢩࢺࢩࣥࠊ࢜࢟ࢩࢺࢩࣥཷᐜయࡢ㑇ఏᏊࢆ㑅ᢥࡋࡓࠋࡉࡽ࡟ࢤࣀ࣒࣡࢖ࢻゎᯒ࡟ࡼࡗ࡚࢖ࢾࡢ㐍໬࡟㛵ࢃࡿ࡜ሗ࿌ࡉࢀ࡚࠸ࡿೃ
⿵㑇ఏᏊ࠿ࡽ :%6&5 ࢆ㑅ᢤࡋࠊࡇࢀࡽ࡟㛵㐃ࡍࡿ㑇ఏᏊࡢከᆺࢆㄪ࡭ࠊ≟✀ᕪࡸ⾜ືᐇ㦂ࡢ⤖ᯝ࡜ࡢ㛵㐃ᛶࢆㄪ࡭ࡓࠋ
࢖ࢾࡢ㐍໬ೃ⿵㑇ఏᏊ࡛࠶ࡿ :%6&5㸦࢘࢕ࣜ࢔࣒ࢬ⑕ೃ⩌㛵㐃㑇ఏᏊ㸧࡟࠾࠸࡚ࠊ
ཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛ฟ⌧㢖ᗘࡢ␗࡞ࡿ୍ሷᇶከᆺ㸦F&!7㸧ࡀ᳨ฟࡉࢀࠊ
ཎጞⓗ࡞≟࡛ࡣ୍⯡ⓗ࡞≟✀࡟ẚ࡭ 7 ࢆᣢࡘ㢖ᗘࡀ㧗࠿ࡗࡓࠋࡲࡓࡇࡢ 7 ᆺࡢ㑇ఏᏊᆺࢆᣢࡘಶయ࡛ࡣࠊᣦᕪࡋ஧⪅㑅ᢥㄢ㢟ࡢど⥺㸩ᣦᕪࡋ㸩ࢱࢵࣆࣥࢢࡢㄢ㢟ࡢṇ⟅ᩘࡀ㧗࠸⤖ᯝ࡜࡞ࡗࡓ㸦S㸧ࠋ0&5㸦࣓ࣛࣀࢥࣝࢳࣥ  ཷᐜయ㑇ఏᏊ㸧࡟࠾࠸࡚ࠊཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛ฟ⌧㢖ᗘࡢ␗࡞ࡿ୍ሷᇶከᆺ㸦F*!$9DO,OH㸧ࡀ᳨ฟࡉࢀࠊཎጞⓗ࡞≟࡛ࡣ୍⯡ⓗ࡞≟✀࡟ẚ࡭ $ ࢆᣢࡘ㢖ᗘࡀ㧗࠿ࡗࡓࠋࡲࡓࡇࡢ $ ᆺࡢ㑇ఏᏊᆺࢆᣢࡘಶయࢆ୍⯡ⓗ࡞≟✀ෆ࡛ẚ㍑ࡋࡓ⤖ᯝࠊᣦᕪࡋ஧⪅㑅ᢥㄢ㢟ࡢど⥺㸩ᣦᕪࡋ㸩ࢱࢵࣆࣥࢢㄢ㢟ࠊど⥺㸩ᣦᕪࡋㄢ㢟ࠊᣦᕪࡋㄢ㢟ࡢṇ⟅ᩘࡀప࠸⤖ᯝ࡜࡞ࡗࡓ
㸦S㸧ࠋ27㸦࢜࢟ࢩࢺࢩࣥ㑇ఏᏊ㸧࡟࠾࠸࡚ࠊཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛ฟ⌧㢖ᗘࡢ␗࡞ࡿ୍ሷᇶከᆺ㸦F&!$㸧࡜཯᚟ᩘከᆺ㸦FBUHS****&&㸧ࡀ᳨ฟࡉࢀࠊཎጞⓗ࡞≟࡛ࡣ୍⯡ⓗ࡞≟✀࡟ẚ࡭୍ሷᇶከᆺ࡛ࡣ $ ࢆᣢࡘ㢖ᗘࡀ㧗ࡃࠊ཯᚟ᩘ
ከᆺ࡛ࡣ  ሷᇶཬࡧ  ሷᇶࡢ㛗ࡉࡢ㓄ิࢆᣢࡘ㢖ᗘࡀప࠿ࡗࡓࠋཎጞⓗ࡞≟࡟ከ࠸$$ ᆺࡢ୍ሷᇶከᆺࢆᣢࡘಶయ࡛ࡣࠊ$& ᆺࡢ㑇ఏᏊᆺࢆᣢࡘಶయࡼࡾࡶࠊゎỴ୙ྍ⬟ㄢ㢟ࡢࣄࢺࢆぢ࡚࠸ࡿ᫬㛫ࠊࣄࢺࢆぢࡿᅇᩘࠊ஺஫จどࡢᅇᩘ࡟࠾࠸࡚ࣄࢺࢆぢ࡞࠸⤖ᯝ࡜࡞ࡗࡓ㸦S㸧ࠋ཯᚟ᩘከᆺ࡛ࡣ  ሷᇶཪࡣ  ሷᇶࡢ㛗ࡉࡢ㓄ิࢆ࣊ࢸ࡛ࣟᣢ
ࡘಶయ࡟࠾࠸࡚ࠊᤄධࢆᣢࡓ࡞࠸ಶయࡼࡾࡶゎỴ୙ྍ⬟ㄢ㢟࡛ࣄࢺࢆぢࡿᅇᩘࡀከ࠸ഴྥࡀぢࡽࢀࡓ㸦S 㸧
ࠋ275㸦࢜࢟ࢩࢺࢩࣥཷᐜయ㑇ఏᏊ㸧࡟࠾࠸࡚ࡣࠊཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛ฟ⌧㢖ᗘࡢ␗࡞ࡿ୍ሷᇶከᆺࡀ᳨ฟࡉࢀࡓࡶࡢࡢࠊ⾜ືᐇ㦂ࡢ⤖ᯝ࡜ࡣ㛵㐃ᛶࡀぢࡽࢀ࡞࠿ࡗࡓࠋ
◊✲┠ⓗ ᖺ࡟࢖ࢾࡢ඲ࢤࣀ࣒㓄ิࡀබ⾲ࡉࢀࡓ ࠋゎㄞࡉࢀࡓࡢࡣ࣓ࢫࡢ࣎ࢡࢧ࣮
ࠕࢱ࣮ࢩࣕࠖ
ࡢࢤࣀ࣒࡛࠶ࡿࠋࡑࡢᚋࠊࡇࡢࠕࢱ࣮ࢩࣕࠖࡢࢤࣀ࣒ゎㄞ⤖ᯝ㸦⿕そᗘ  ಸ㸧ࠊ ᖺ࡟ሗ࿌ࡉࢀ࡚࠸ࡓࢫࢱࣥࢲ࣮ࢻࣉ࣮ࢻࣝࡢࢤࣀ࣒ゎㄞ⤖ᯝ㸦⿕そᗘ  ಸ㸧 ཬࡧࠊ ≟✀㸦⿕そᗘ  ಸ㸧ࠊ࣑࢜࢜࢝ࡸࢥ࣮ࣚࢸ࠿ࡽᡂࡿ  ✀㢮ࡢ࢖ࢾ⛉ື≀㸦⿕そᗘ  ಸ㸧ࡢሷᇶ㓄ิࢆỴᐃࡋࡓ⤖ᯝࢆẚ㍑ࡍࡿࡇ࡜࡟ࡼࡾࠊ ୓ಶ௨ୖࡢ  ሷᇶከᆺ㸦613V㸧ࡀྠᐃ

190ࡉࢀࡓ ࠋ࢖ࢾࡢᐙ␆໬ࡢࣉࣟࢭࢫ࡟ࡣఱࡽ࠿ࡢ⾲⌧ᆺ࡟ࡼࡿᙉ࠸㑅ᢥᅽࡀ࠿࠿ࡗ࡚࠾ࡾࠊ
㑅ᢥᅽࡢࢩࢢࢼࣝࡀࢤࣀ࣒ୖ࡟ṧࡉࢀ࡚࠸ࡿ࡜ᛮࢃࢀࡿࠋࡑࡢࢩࢢࢼࣝࢆ᥈ࡿࡓࡵ࡟ࠊ
ᖺ࡟ࢤࣀ࣒࣡࢖ࢻ࡞ 613 ࢪ࢙ࣀࢱ࢖ࣆࣥࢢ࢔ࣞ࢖ࢆ⏝࠸ࡓゎᯒࡀሗ࿌ࡉࢀࡓ ࠋࡑࡢ⤖ᯝࠊࣄࢺ࡟࠾࠸࡚⫗యⓗ࣭⢭⚄ⓗ࡟Ⓨ㐩ࡢ㐜ࢀࢆ♧ࡍⓎ㐩㞀ᐖ࡛࠶ࡿ࢘࢕ࣜ࢔࣒ࢬ⑕ೃ⩌࡟㛵㐃ࡋ࡚࠸ࡿ࡜ࡉࢀ࡚࠸ࡿ㑇ఏᏊ࡛࠶ࡿ :%6&5 ࡢ㏆ࡃ࡟㑅ᢥᅽࡢࢩࢢࢼࣝࡀぢࡽࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ࢘࢕ࣜ࢔࣒ࢬ⑕ೃ⩌ࡢேࠎࡣࠊ཯ࡾ㏉ࡗࡓ㰯࡜ᑠࡉ࡞㢡ࢆᣢࡘዿ⢭ࡢࡼ࠺࡞㢦ࡘࡁࢆࡶࡕࠊ⫗యⓗ࡟ࡶ⢭⚄ⓗ࡟ࡶⓎ㐩ࡢ㐜ࢀࢆ♧ࡍࠋ⬻ᐜ✚ࡀᑠࡉࡃࠊ▱⬟పୗࡀぢࡽࢀࡿࠋどぬ✵㛫ㄆ▱ࡢാࡁ࡟ⴭࡋ࠸㞀ᐖࢆᣢࡘ୍᪉࡛ࠊゝㄒ⬟ຊ࡟ࡣၥ㢟ࡀ࡞ࡃࠊከᘚ࡛ࠊேࢆᜍࢀࡎ♫஺ⓗ࡞ᛶ᱁ࢆᣢࡘ ࠋ⬻ᐜ✚ࡀᑠࡉ࠸ࡇ࡜ࡸࠊከᘚ࡛࠶ࡿࡇ࡜ࠊ
ேࢆᜍࢀࡎ♫஺ⓗ࡞ᛶ᱁➼ࡣࠊ࢖ࢾ࡟ࡶඹ㏻ࡋ࡚ぢࡽࢀࡿ≉ᚩ࡛࠶ࡾࠊ:%6&5 㑇ఏᏊࡣ࢖ࢾࡢᐙ␆໬㸦㐍໬㸧࡜㛵㐃ࡋ࡚࠸ࡿ࠿ࡶࡋࢀ࡞࠸࡜⪃࠼ࠊᮏ㑇ఏᏊࢆ♫఍ⓗㄆ▱⬟ຊ࡟㛵㐃ࡍࡿ㑇ఏᏊࡢ➨  ೃ⿵࡜ࡋࡓࠋ
ࡉࡽ࡟ࠊྠࡌࡃࢤࣀ࣒࣡࢖ࢻ࡞ 613 ࢪ࢙ࣀࢱ࢖ࣆࣥࢢ࢔ࣞ࢖ࢆ⏝࠸ࡓ &KDVH ࡽࡢ◊✲ᡂᯝ࡛ࡣࠊᐙ␆໬㛵㐃㑇ఏᏊ࡜ࡋ࡚ 0&5㸦࣓ࣛࣀࢥࣝࢳࣥ  ཷᐜయ㸧ࡀᣲࡆࡽࢀ࡚࠸ࡿ ࠋ&KDVHࡽࡢ◊✲࡟࠾࠸࡚ࠊ
ࣁ࣮ࢹ࢕ࣥࢢ㛵㐃㑇ఏᏊ࡜ࡋ࡚ᣲࡆࡽࢀ࡚࠸ࡿ 0&5 ࡣࠊ๪⭈࡟࠶ࡿ $&7+ཷᐜయ࡛ࢥࣝࢳࢰ࣮ࣝࡢ⏘⏕࡟㛵୚ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋ࢟ࢶࢿࡢᐙ␆໬ᐇ㦂࡟࠾࠸࡚ே࡬ࡢᨷᧁᛶ࡟ࡼࡗ࡚㑅ᢥⓗ࡟⦾Ṫࡋ࡚࠸ࡃࡇ࡜࡟ࡼࡾࠊ+3$ ㍈ࡢάᛶࡀపୗࡍࡿࡇ࡜ࡸ
⾑₢୰ࢥࣝࢳࢰ࣮ࣝ㔞ࡀపୗࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ ࠋࡲࡓࠊLQYLWUR ࡢᐇ㦂࡟࠾࠸࡚ࠊࢢࣝࢥࢥࣝࢳࢥ࢖ࢻࡣ⣽⬊ࡢศ⿣ቑṪࡢ᫬㛫ࢆㄪ⠇ࡋࠊ⣽⬊ࡢศ໬ࢆㄏᑟࡍࡿࡇ࡜ࡀ
ሗ࿌ࡉࢀ࡚࠾ࡾ ࠊࢥࣝࢳࢰ࣮ࣝࡣⓎ㐩㏿ᗘ࣭๭ྜࡢㄪᩚ࡟㛵୚ࡍࡿྍ⬟ᛶࡀ࠶ࡿࠋ࢖ࢾ
ࡢᐙ␆໬㸦㐍໬㸧࡟࠾࠸࡚ࠊே࡬ࡢᨷᧁᛶࡸᜍᛧᚰ࡟ࡼࡿ㑅ᢥᅽࡀാࡁࠊࡑࡢ⤖ᯝࠊⓎ㐩
㏿ᗘࡸ๭ྜ࡟ኚ໬ࡀ⌧ࢀ⌧ᅾࡢᵝࠎ࡞≟✀ࡀసᡂࡉࢀࡓ࡜ࡶ⪃࠼ࡽࢀࡿࠋ0&5 㑇ఏᏊࡣ࢖ࢾࡢᐙ␆໬㸦㐍໬㸧࡜㛵㐃ࡋ࡚࠸ࡿ࠿ࡶࡋࢀ࡞࠸࡜⪃࠼ࠊᮏ㑇ఏᏊࢆㄆ▱⬟ຊ㛵㐃㑇ఏᏊࡢ➨  ࡢೃ⿵࡜ࡋࡓࠋ
ᮦᩱ࡜᪉ἲ
࢖ࢾ 443 㢌ཬࡧ࣑࢜࢜࢝ 12 㢌ࡢ⾑ᾮࠊཱྀෆ⢓⭷ཪࡣ⫢⮚ࡢ⤌⧊∦࠿ࡽ DNA ࢆᢳฟࡋ࡚
㑇ఏᏊゎᯒࢆ⾜ࡗࡓࠋ⾑ᾮࡢᢠจᅛ๣ࡣࠊ࢚ࢳࣞࣥࢪ࢔࣑ࣥࢸࢺࣛ㓑㓟㸦EDTA㸧ࢆ⏝࠸ࡓࠋཱྀෆ⢓⭷ࡢ᥇ྲྀ࡟ࡣࠊEndo Cervex ࣈࣛࢩ㸦࢔ࢬ࣡ࣥ㸧ࢆ⏝࠸ࡓࠋ⫢⮚ࡢ⤌⧊∦ࡣࠊື≀⑓㝔࡟᳨࡚యࡉࢀࡓࡶࡢࢆ㒑㏦࠸ࡓࡔ࠸ࡓࠋࢧࣥࣉࣝࡣ඲࡚-20Υ࡟࡚෭෾ಖᏑࡋࡓࠋ
⾑ᾮཬࡧཱྀෆ⢓⭷࠿ࡽࡢ DNA ࡢᢳฟ࡟ࡣ QIAamp® DNA Mini Kit㸦QIAGEN㸧ࢆ⏝࠸ࡓࠋᢳฟ᪉ἲࡣ࣓࣮࣮࢝ࡢࣉࣟࢺࢥࣝ࡟ᚑࡗࡓࠋ⫢⮚ࡢ⤌⧊∦࠿ࡽࡢ DNA ࡢᢳฟࡣࠊ
DNAzol(Invitrogen)ࢆ⏝࠸࡚⾜ࡗࡓࠋ⣽⬊ࡢ◚○࡟ࡣࠊࣅ࣮ࢬ᪉ᘧࡢ㧗㏿⣽⬊◚○ࢩࢫࢸ
ࠋDNA ࡢᢳฟ
࣒࡛࠶ࡿ Precellys24 (Bertin Technologies) ࢆ⏝࠸ࡓ㸦5000rpm, 20sec2㸧᪉ἲࡣࠊ࣓࣮࣮࢝ࡢࣉࣟࢺࢥࣝ࡟ᚑࡗࡓࠋ
 Ensembl ཬࡧ National Center for Biotechnology Information (NCBI)ࡢ࢖ࣥࢱ࣮ࢿࢵ

191

ࢺࢹ࣮ࢱ࣮࣋ࢫࢆ⏝࠸࡚ 1 ሷᇶከᆺ㸦SNPs㸧ࢆ᳨⣴ࡋࡓࠋ࢚ࢡࢯࣥ㡿ᇦ࡟࠶ࡿ SNPs ࢆᑐ㇟࡜ࡋࠊWBSCR17 ࡛ࡣࠊ࢖ࢾࢤࣀ࣒➨ 6 ᰁⰍయࡢ 2168245 ␒┠ሷᇶᑐ࡟࠶ࡿྠ⩏✺↛ኚ␗(rs24319159)ࢆࢱ࣮ࢤࢵࢺ࡜ࡋࡓࠋMC2R ࡛ࡣࠊ➨ 1 ␒ᰁⰍయࡢ 24388879 ␒┠ሷᇶᑐ࡟࠶ࡿྠ⩏✺↛ኚ␗(rs21898857, SNP1)࡜➨ 1 ␒ᰁⰍయࡢ 24389137 ␒┠ሷᇶᑐ࡟࠶ࡿྠ
⩏✺↛ኚ␗(rs21898855, SNP2)ࢆࢱ࣮ࢤࢵࢺ࡜ࡋࡓࠋOTR ࡛ࡣࠊ➨ 20 ␒ᰁⰍయࡢ 9359598
␒┠ሷᇶᑐ࡟࠶ࡿ࣑ࢫࢭࣥࢫኚ␗㸦rs22927823, SNP1㸧
ࠊ9359610 ␒┠ሷᇶᑐ࡟࠶ࡿ࣑ࢫ
ࢭࣥࢫኚ␗㸦rs22927826, SNP2㸧
ࠊ9360368 ␒┠ሷᇶᑐ࡟࠶ࡿྠ⩏✺↛ኚ␗㸦rs22927829,
SNP3㸧ࠊ9378164 ␒┠ሷᇶᑐ࡟࠶ࡿྠ⩏✺↛ኚ␗㸦rs8679682, SNP4㸧ཬࡧ 9378224 ␒┠ሷᇶᑐ࡟࠶ࡿྠ⩏✺↛ኚ␗㸦rs22896457, SNP5㸧ࢆࢱ࣮ࢤࢵࢺ࡜ࡋࡓࠋOT ࡛ࡣࠊ➨ 24 ␒ᰁⰍయࡢ 18193483 ␒┠ሷᇶᑐ࡟࠶ࡿྠ⩏✺↛ኚ␗/ࢫࣉࣛ࢖ࢩࣥࢢ㡿ᇦኚ␗㸦rs23181992,
SNP1 㸧 ཬ ࡧ 18193484 ␒ ┠ ሷ ᇶ ᑐ ࡟ ࠶ ࡿ ྠ ⩏ ✺ ↛ ኚ ␗ / ࢫ ࣉ ࣛ ࢖ ࢩ ࣥ ࢢ 㡿 ᇦ ኚ ␗
㸦rs23181995, SNP2㸧ࢆࢱ࣮ࢤࢵࢺ࡜ࡋࡓ㸦ᅗ 4-1 ཧ↷㸧ࠋࡇࢀࡽࡢࢱ࣮ࢤࢵࢺࢆቑᖜࡍ
ࡿࡼ࠺࡟ࣉࣛ࢖࣐࣮ࢆタィࡋࡓࠋ ୖグࡢ SNPs ࡟ࡘ࠸࡚ࢩ࣮ࢡ࢚ࣥࢫࡋࡓ㝿࡟ MC2R ࡜ OT ࡟࡚᪂ࡓ࡟ぢࡘ࠿ࡗࡓࢹ࣮
ࢱ࣮࣋ࢫ࡛ࡣබ㛤ࡉࢀ࡚࠸࡞࠸ከᆺࡶࢱ࣮ࢤࢵࢺ࡜ࡋ࡚㏣ຍࡋࡓࠋMC2R ࡛ࡣࠊࢥ࣮ࢹ࢕
ࣥࢢ㡿ᇦࡢ 871 ␒┠ሷᇶᑐࡢ㠀ྠ⩏✺↛ኚ␗㸦c.871G>A, Val291Ile, SNP3)ࠊཬࡧ OT ࡛ࡣ࢖ࣥࢺࣟࣥ㡿ᇦࡢ 1 ሷᇶከᆺ㸦c.322+50C>A, intron-SNP)࡜ྠࡌࡃ࢖ࣥࢺࣟࣥ㡿ᇦࡢ཯᚟ᩘከᆺ㸦c.322+51_52ins, intron-repeat)࡛࠶ࡿ㸦ᅗ 4-1 ཧ↷㸧ࠋ࡞࠾ࠊOT ࡣ 3 ࡘࡢ࢚ࢡࢯࣥ㡿ᇦ࡜ 2 ࡘࡢ࢖ࣥࢺࣟࣥ㡿ᇦ࠿ࡽᡂࡾࠊ3 ࡘࡢ࢚ࢡࢯࣥ㡿ᇦࡣࠊ࢜࢟ࢩࢺࢩࣥࡢ࣏ࣜ࣌
ࣉࢳࢻཬࡧ࢜࢟ࢩࢺࢩࣥࡢ㍺㏦ࢱࣥࣃࢡ㉁࡛࠶ࡿࢽ࣮ࣗࣟࣇ࢕ࢪࣥࢆࢥ࣮ࢻࡋ࡚࠸ࡿࠋ
PCR ࡣ My Cycler τ thermal Cycler㸦BIO RAD㸧ཪࡣ PCR ࢧ࣮࣐ࣝࢧ࢖ࢡࣛ̿Dice
Touch(Takara)ࢆ⏝࠸ࡓࠋ㓝⣲ࡣࠊKOD FX-Neo㸦TOYOBO㸧ࢆ⏝࠸࡚ࠊ཯ᛂᾮࡢ⤌ᡂࡣ࣓࣮࣮࢝ࡢࣉࣟࢺࢥࣝ࡟ᚑࡗࡓࠋPCR ᮲௳ࡣࠊ94Υ2 ศࢆ 1 ࢧ࢖ࢡࣝࠊ98Υ10 ⛊ࠊ65Υ30⛊ࢆ 40 ࢧ࢖ࢡࣝࠊ72Υ7 ศࢆ 1 ࢧ࢖ࢡࣝࠊࡑࡢᚋࡣ 4Υ࡛⥔ᣢࡋࡓࠋࡲࡓࠊOT ࡢ PCR ࡛ࡣ୍㒊ࡢࢧࣥࣉࣝ࡟࠾࠸࡚ࠊLA-Taq㸦takara㸧ࢆ⏝࠸ࡓࠋ཯ᛂᾮࡢ⤌ᡂࡣ࣓࣮࣮࢝ࡢࣉࣟ
ࢺࢥࣝ࡟ᚑ࠸㸦GCI buffer ౑⏝㸧ࠊPCR ᮲௳ࡣࠊ95Υ2 ศࢆ 1 ࢧ࢖ࢡࣝࠊ95Υ30 ⛊ࠊ65Υ
30 ⛊ࠊ74Υ1min ࢆ 40 ࢧ࢖ࢡࣝࠊ74Υ10 ศࢆ 1 ࢧ࢖ࢡࣝࠊࡑࡢᚋࡣ 4Υ࡛⥔ᣢࡋࡓࠋPCR⏘≀ࡢ⢭〇࡟ࡣ Nucleo Spin® Gel and PCR Clean-up㸦Takara㸧ࢆ⏝࠸࡚ࠊ᪉ἲࡣ࣓࣮࢝
࣮ࡢࣉࣟࢺࢥࣝ࡟ᚑࡗࡓࠋ
ࣉࣛ࢖࣐࣮ΰྜ῭ࡳ㗪ᆺ࠿ࡽࡢ㎿㏿ࢩ࣮ࢣࣥࢫཷクࢧ࣮ࣅࢫ࡛࠶ࡿࣉ࣑ࣞࢵࢡࢫࢩ࣮ࢣ
ࣥࢫゎᯒ㸦takara㸧ࢆ฼⏝ࡋࡓࠋPCR ⏘≀ཬࡧࣉࣛ࢖࣐࣮ࡢ㔞ࡣࠊ࣓࣮࣮࢝ࡢࣉࣟࢺࢥࣝ
࡟ᚑࡗࡓࠋPCR ⏘≀ࡢ⃰ᗘࡣࠊ㟁ẼὋື࡟ࡼࡾ☜ㄆࡋࡓࣂࣥࢻࡢ᫂ࡿࡉࢆᇶ࡟⟬ฟࡋࡓࠋࢧࣥࣉࣝࡣ-20Υ࡟࡚࠶ࡽ࠿ࡌࡵ෭෾ࡋࠊࢻࣛ࢖࢔࢖ࢫࢆධࢀ࡚ࠊࢡ࣮ࣝᏯᛴ౽㸦෭෾ࢱ࢖ࣉࠊ࣐ࣖࢺ㐠㍺㸧࡟࡚㒑㏦ࡋࡓࠋ
OT-repeat ከᆺࡀ࣊ࢸࣟࡢಶయ࡛ࡣࠊࢩ࣮ࢡ࢚ࣥࢫࢆࡍࡿ࡜ 2 ࡘࡢἼᙧࡀ㔜࡞ࡗ࡚ࡋࡲ࠸
࠺ࡲࡃุᐃࡍࡿࡇ࡜ࡀ࡛ࡁ࡞࠿ࡗࡓࠋࡑࡇ࡛ࠊGenetic Analyzer㸦AB Applied Biosystems㸧

192࡟ࡼࡿ ᐃࢆ⾜ࡗࡓࠋPCR ࢆ⾜ࡗࡓᚋ࡟ࠊࢧࣥࣉࣝࢆࢡ࣮ࣝᏯᛴ౽㸦෭ⶶࢱ࢖ࣉࠊ࣐ࣖࢺ
㐠㍺㸧࡟࡚㒑㏦ࡋࠊᐃࡣி㒔኱Ꮫࡢ㔝⏕ື≀◊✲ࢭࣥࢱ࣮࡟౫㢗ࡋࡓࠋPCR 㓝⣲ࡣࠊ

LA-Taq㸦takara㸧ࢆ⏝࠸࡚ࠊ཯ᛂᾮࡢ⤌ᡂࡣ࣓࣮࣮࢝ࡢࣉࣟࢺࢥࣝ࡟ᚑࡗࡓ㸦GCI buffer౑⏝㸧ࠋPCR ᮲௳ࡣࠊ95Υ2 ศࢆ 1 ࢧ࢖ࢡࣝࠊ95Υ30 ⛊ࠊ65Υ30 ⛊ࠊ74Υ1min ࢆ 35 ࢧ
࢖ࢡࣝࠊ74Υ10 ศࢆ 1 ࢧ࢖ࢡࣝࠊࡑࡢᚋࡣ 4Υ࡛⥔ᣢࡋࡓࠋཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛ከᆺ㢖ᗘࢆẚ㍑ࡋࡓࠋከᆺ㢖ᗘࡢẚ㍑ࡣࠊ࢚ࢡࢭࣝ⤫ィ
㸦Square Test㸧ࢆ⏝࠸࡚⾜ࡗࡓࠋࡲࡓࠊ㑇ఏᏊᆺ࡜➨ 2 ❶ࡢ♫఍ㄆ▱⬟ຊ࡜ࡢ㛵㐃ゎᯒࢆ⾜ࡗࡓࠋ㛵㐃ゎᯒ࡟ࡣࠊSPSS statistic 17.0㸦SPSS Japan Inc.㸧ࢆ⏝࠸࡚ࠊMann Whitney
U test ࢆ⾜ࡗࡓࠋ᭷ពỈ‽ࡣ 5㸣࡜ࡋࡓࠋ⤖ᯝ࠾ࡼࡧ⪃ᐹձ  ከᆺ㢖ᗘࡢẚ㍑
 WBSCR17 ࡢከᆺ㢖ᗘࡣཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛᭷ព࡞ᕪࡀぢࡽࢀࠊཎጞⓗ࡞≟࡟࠾࠸࡚ T/T ᆺࡢ࢖ࢾࡀከ࠸⤖ᯝ࡛࠶ࡗࡓ㸦p<0.01, ᅗ 4-2 ཧ↷㸧ࠋࡲࡓ࣑࢜࢜࢝ 6 㢌࡛ࢪ࢙ࣀࢱ࢖ࣆࣥࢢࢆ⾜ࡗࡓ࡜ࡇࢁࠊC/C ᆺࡀ 4 㢌ࠊT/C ᆺࡀ 2 㢌࡛࠶ࡗࡓࠋWBSCR17 ࡟ࡘ࠸࡚ࡣࠊ࢚ࢡࢯࣥ㡿ᇦ඲㛗࡟ࡘ࠸࡚ 11 㢌ࡢ࢖ࢾ࡛ࢩ࣮ࢡ࢚ࣥࢫࢆ⾜࠸ከᆺࡢ᳨⣴ࢆ⾜ࡗࡓࡀࠊ࢖ࣥࢺࣟࣥ㡿ᇦ࡟ࡣ௚࡟ࡶ࠸ࡃࡘ࠿ከᆺࡀぢࡽࢀࡓࡶࡢࡢ㸦ᅗ 4-1 ཧ↷㸧ࠊ࢚ࢡࢯࣥ㡿
ᇦ࡟ࡣ௚࡟ከᆺࡣぢࡽࢀ࡞࠿ࡗࡓࠋ
 MC2R ࡢከᆺ㢖ᗘࡣ SNP1 ཬࡧ SNP3 ࡟࠾࠸࡚ཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛᭷ព࡞ᕪࡀぢࡽࢀࠊ୍⯡ⓗ࡞≟✀࡟࠾࠸࡚ G/G ᆺࡢ࢖ࢾࡀከ࠸⤖ᯝ࡛࠶ࡗࡓ
㸦p<0.01, ᅗ 4-3 ཧ↷㸧ࠋ࣑࢜࢜࢝ 12 㢌࡛ࡣࠊSNP1 ࡛ࡣ G/G ᆺࡀ 1 㢌ࠊG/C ᆺࡀ 4 㢌ࠊC/C ᆺࡀ 7 㢌࡛ࠊSNP3
࡛ࡣ G/G ᆺࡀ 2 㢌ࠊA/G ᆺࡀ 9 㢌ࠊA/A ᆺࡀ 1 㢌࡛࠶ࡗࡓࠋSNP2 ࡛ࡣࠊከᆺࡣぢࡽࢀࡿࡶࡢࡢࠊཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛᭷ព࡞ᕪࡣぢࡽࢀ࡞࠿ࡗࡓ㸦p>0.05㸧
ࠋࡲࡓࠊ࢖ࢾ
࡛ࡣ඲࡚ࡢಶయࡀ C/C ᆺ࡛࠶ࡿࡢ࡟ᑐࡋ࡚ࠊ࣑࡛࢜࢜࢝ࡣ 12 㢌୰ࠊC/C ᆺ 9 㢌ࠊC/T ᆺ 2
㢌࡜ T/T ᆺ 1 㢌࡛࠶ࡿྠ⩏✺↛ኚ␗ࡀ SNP1 ࡜ SNP2 ࡢ㛫࡟㸯ࡘぢࡽࢀࡓ㸦➨ 1 ␒ᰁⰍయࡢ 24389032 ␒┠ሷᇶᑐ, SNP1.5㸧
ࠋ OTR ࡢከᆺ㢖ᗘࡣ SNP4 ࡟࠾࠸࡚ཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛᭷ព࡞ᕪࡀぢࡽࢀࠊཎጞⓗ࡞≟࡟࠾࠸࡚ T/T ᆺࡢ࢖ࢾࡀከ࠸⤖ᯝ࡛࠶ࡗࡓ㸦p<0.01, ᅗ 4-4 ཧ↷㸧ࠋ࣑࢜࢜࢝ 5 㢌࡛ࡣࠊC/C ᆺࡀ 4 㢌ࠊT/T ᆺࡀ 1 㢌࡛࠶ࡗࡓࠋSNP1 ࡜ 2 ࡛ࡣࠊ඲࡚ࡢ࢖ࢾࡀ G/G ᆺ࡛࠶ࡾࠊከᆺࡣぢࡽࢀ࡞࠿ࡗࡓࠋSNP3 ࡜ SNP5 ࡛ࡣࠊࢃࡎ࠿࡟ከᆺࡀぢࡽࢀࡿࡶࡢࡢࠊ95%௨ୖࡢ࢖ࢾࡀ T/T ᆺ࡛࠶ࡗࡓࠋ
OT ࡢከᆺ㢖ᗘࡣ intron-SNP ࡟࠾࠸࡚ཎጞⓗ࡞≟࡜୍⯡ⓗ࡞≟✀࡜࡛᭷ព࡞ᕪࡀぢࡽࢀࠊ୍⯡ⓗ࡞≟✀࡟࠾࠸࡚ C/C ᆺࡢ࢖ࢾࡀከ࠸⤖ᯝ࡛࠶ࡗࡓ㸦p<0.01, ᅗ 4-5 ཧ↷㸧ࠋ࣑࢜࢜࢝
7 㢌࡛ࡣࠊC/C ᆺࡀ 2 㢌ࠊC/A ᆺࡀ 2 㢌ࠊA/A ᆺࡀ 3 㢌࡛࠶ࡗࡓࠋࡲࡓࠊOT ࡢ intron-repeatከᆺ࡛ࡣࠊ6 ሷᇶḞኻࠊᤄධ࡞ࡋࠊ6 ሷᇶᤄධࠊ25 ሷᇶᤄධࠊ37 ሷᇶᤄධᆺࡀぢࡽࢀࡓࠋ6ሷᇶḞኻᆺ࡟ࡘ࠸࡚ࡣࠊࢫࢱࣥࢲ࣮ࢻࣉ࣮ࢻࣝࡢ୍㒊ࡢಶయ࡛ࡢࡳぢࡽࢀࡓࠋཎጞⓗ࡞≟193࡜୍⯡ⓗ࡞≟✀࡜࡛ẚ㍑ࡋࡓ࡜ࡇࢁࠊཎጞⓗ࡞≟࡟࠾࠸࡚ 25 ሷᇶᤄධ࡜ 37 ሷᇶᤄධᆺࡀ
ᑡ࡞࠸ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ㸦ᅗ 4-5 ཧ↷㸧ࠋࡲࡓࠊ࣑࡛࢜࢜࢝ࡣ࢖ࢾ࡜ࡣ␗࡞ࡿ㛗ࡉࡢᤄධ㸦12 ሷᇶᤄධ࡜ 18 ሷᇶᤄධ㸧ࡀぢࡽࢀࡓࠋࡉࡽ࡟ࠊ࣑࡛࢜࢜࢝ࡣ 2 ␒┠ࡢ࢚ࢡࢯࣥ㡿ᇦࡢᮎᑿ 6 ሷᇶ࠿ࡽ࢖ࣥࢺࣟࣥ㡿ᇦࡢ᭱ึ 11 ሷᇶࡲ࡛ࡢ㡿ᇦ࡛ࠊ࢖ࢾ࡛ࡣぢࡽࢀ࡞࠸ከᆺࡀぢࡽࢀࡓࠋ࢖ࢾ࡛ࡣࢭࣜࣥࠊࣉࣟࣜࣥࡢ࡜ࡇࢁࡀࠊ࢔ࢫࣃࣛࢠࣥࠊ࢔ࢫࣃࣛࢠࣥ㓟࡟࡞ࡿ㠀ྠ⩏ኚ␗࡛࠶ࡿࠋᮏኚ␗ࡣ࣑࢜࢜࢝ 11 㢌୰ 5 㢌࡟ぢࡽࢀࡓࠋSNP1 ࡜ 2 ࡛ࡣࠊ඲࡚ࡢ
࢖ࢾࡀ A/A ᆺ㸦SNP1㸧
ࠊC/C ᆺ㸦SNP2㸧࡛࠶ࡾࠊከᆺࡣぢࡽࢀ࡞࠿ࡗࡓ㸦ᅗ 4-5 ཧ↷㸧ࠋղ  ㄆ▱⬟ຊ࡜㑇ఏᏊᆺ࡜ࡢ㛵㐃ゎᯒ WBSCR17 ࡟࠾࠸࡚ࠊ㑇ఏᏊᆺ࡜ᣦᕪࡋ஧⪅㑅ᢥㄢ㢟ࡢど⥺㸩ᣦᕪࡋ㸩ࢱࢵࣆࣥࢢࡢㄢ
㢟࡛㛵㐃ᛶࡀ᭷ព࡟ぢࡽࢀࡓ㸦p<0.05, ᅗ 4-6 ཧ↷㸧ࠋཎጞⓗ࡞≟࡟ከ࠸ T/T ᆺࡢ࢖ࢾࡢṇ⟅ᩘࡀ㧗࠸⤖ᯝ࡜࡞ࡗࡓࠋど⥺㸩ᣦᕪࡋࡢㄢ㢟࡜ᣦᕪࡋࡢㄢ㢟࡛ࡣࠊ᭷ព࡞ᕪࡣぢࡽࢀ࡞࠿ࡗࡓ㸦p>0.05㸧
ࠋࡲࡓࠊゎỴ୙ྍ⬟ㄢ㢟ࡢ⤖ᯝ࡜ࡢ㛵㐃ᛶࡶぢࡽࢀࠊࣄࢺࢆぢ࡚࠸ࡿ᫬㛫ࠊࣄࢺࢆぢࡿᅇᩘ࡟࠾࠸࡚ࠊ୍⯡ⓗ࡞≟✀࡟ከ࠸ C/C ᆺࡢ࢖ࢾ࡛ࣄࢺࢆࡼࡃぢࡿ⤖ᯝࡀᚓࡽࢀࡓ㸦p>0.05, ᅗ 4-7 ཧ↷㸧ࠋ
 MC2R ࡛ࡣࠊ㑇ఏᏊᆺ࡜ㄆ▱⬟ຊࢸࢫࢺ࡜ࡢ㛫࡟᭷ព࡞㛵㐃ᛶࡣぢࡽࢀ࡞࠿ࡗࡓ
㸦p>0.05,
ᅗ 4-8 ཧ↷㸧
ࠋSNP3 ࡟࠾࠸࡚ࠊᣦᕪࡋ஧⪅㑅ᢥㄢ㢟ࡢᣦᕪࡋㄢ㢟࡛㛵㐃ᛶࡢഴྥ(p=0.064)
ࡀぢࡽࢀࡓࠋ୍⯡ⓗ࡞≟✀࡟ከ࠸ G/G ᆺ࡛ṇ⟅ᩘࡢ㧗࠸ഴྥ࡛࠶ࡗࡓࠋ
 OTR ࡛ࡣࠊ㑇ఏᏊᆺ࡜ㄆ▱⬟ຊࢸࢫࢺ࡜ࡢ㛫࡟᭷ព࡞㛵㐃ᛶࡣぢࡽࢀ࡞࠿ࡗࡓ
㸦p>0.05㸧
ࠋ OT ࡛ࡣࠊintron-SNP ࡢ㑇ఏᏊᆺ࡜ゎỴ୙ྍ⬟ㄢ㢟࡛㛵㐃ᛶࡀ᭷ព࡟ぢࡽࢀࡓ㸦p<0.05,
ᅗ 4-9 ཧ↷㸧ࠋཎጞⓗ࡞≟࡟ከ࠸ intron-SNP A/A ᆺࡢ࢖ࢾࡣࠊࣄࢺࢆぢ࡚࠸ࡿ᫬㛫ࡀ▷ࡃࠊࣄࢺࢆぢࡿᅇᩘ࡜஺஫จどࡢᅇᩘࡀᑡ࡞࠸⤖ᯝ࡜࡞ࡗࡓ㸦p<0.05㸧
ࠋᣦᕪࡋ஧⪅㑅ᢥㄢ㢟࡛ࡣ㛵㐃ᛶࡀぢࡽࢀ࡞࠿ࡗࡓ㸦p>0.05㸧
ࠋintron-repeat ከᆺ࡛ࡣࠊ࠸ࡎࢀࡢ⾜ືᐇ㦂ࡢ⤖ᯝ࡜ࡶ᭷ព࡞㛵㐃ᛶࡀぢࡽࢀ࡞࠿ࡗࡓ㸦p>0.05㸧ࡶࡢࡢࠊᤄධ࡞ࡋ࡜ 25 ሷᇶᤄධࠊᤄධ࡞ࡋ
࡜ 37 ሷᇶᤄධࡢ࣊ࢸࣟࡢಶయ࡟࠾࠸࡚ࠊゎỴ୙ྍ⬟ㄢ㢟࡛ࣄࢺࢆぢࡿᅇᩘࡀከ࠸ࡼ࠺࡟ᛮ
࠼ࡓ㸦p>0.05, ᅗ 4-10 ཧ↷㸧ࠋࡑࡇ࡛ࠊᤄධ࡞ࡋࡢ࣍ࣔࠊᤄධ࡞ࡋ࡜ 25 ሷᇶᤄධཪࡣᤄධ
࡞ࡋ࡜ 37 ሷᇶᤄධࡢ࣊ࢸࣟࠊ
25 ሷᇶᤄධཪࡣ 37 ሷᇶᤄධࡢ࣍ࣔࡢ 3 ⩌࡛ẚ㍑ࡋࡓ࡜ࡇࢁࠊ
ᤄධ࡞ࡋࡢ࣍ࣔࡢಶయࡣ࣊ࢸࣟࡢಶయࡼࡾࡶࣄࢺࢆぢࡿᅇᩘࡀᑡ࡞࠸ഴྥࡀᚓࡽࢀࡓ
(p=0.078)ࠋ

194

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195

ᅗ 4-2 WBSCR17 ከᆺ㢖ᗘ

6TXDUH7HVWǘ2䠙38.5, df=2, p<0.01

196ᅗ 4-3 MC2R ከᆺ㢖ᗘ

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6TXDUH7HVWǘ2䠙47.2, df=2, p<0.01

197ᅗ 4-4 OTR ከᆺ㢖ᗘ

6TXDUH7HVWǘ2䠙1.94, df=2, p>0.05

6TXDUH7HVWǘ2䠙43.8, df=2, p<0.01

6TXDUH7HVW
6TXD
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198ᅗ 4-5 OT ከᆺ㢖ᗘ199

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ᣦᕪ䛧(P)ㄢ㢟㻌 ǘ2䠙0.109, df=1, p>0.05
䝁䞁䝖䝻䞊䝹(Control)ㄢ㢟㻌 ǘ2䠙0.138, df=1, p>0.05

201

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᭱ึ䛻䝠䝖䜢ぢ䜛䜎䛷䛾᫬㛫㻌 㻌 ǘ2䠙1.083, df=1, p>0.05
䝠䝖䜢ぢ䛶䛔䜛᫬㛫㻌 ǘ2䠙4.647, df=1, p<0.05䝠䝖䜢ぢ䜛ᅇᩘ㻌 ǘ2䠙5.297, df=1, p<0.05஺஫จど䛾ᅇᩘ㻌 ǘ2䠙1.047, df=1, p>0.05

202ᅗ 4-8 MC2R SNP3 ࡜ᣦᕪࡋ஧⪅㑅ᢥㄢ㢟

p=0.064㻌

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ど⥺䠇ᣦᕪ䛧(GP)ㄢ㢟㻌 ǘ2䠙1.469, df=1, p>0.05
ᣦᕪ䛧(P)ㄢ㢟㻌 ǘ2䠙3.421, df=1, p=0.064
䝁䞁䝖䝻䞊䝹(Control)ㄢ㢟㻌 ǘ2䠙0.020, df=1, p>0.05

203ᅗ 4-9 OT intron-SNP ࡜ゎỴ୙ྍ⬟ㄢ㢟

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Mann-Whitney U-test with Bonferroni correction᭱ึ䛻䝠䝖䜢ぢ䜛䜎䛷䛾᫬㛫㻌 㻌 ǘ2䠙5.013, df=2, p>0.05
䝠䝖䜢ぢ䛶䛔䜛᫬㛫㻌 ǘ2䠙9.345, df=2, p<0.05䝠䝖䜢ぢ䜛ᅇᩘ㻌 ǘ2䠙9.508, df=2, p<0.05஺஫จど䛾ᅇᩘ㻌 ǘ2䠙6.371, df=2, p<0.05

204ᅗ 4-10 OT intron-repeat ࡜ゎỴ୙ྍ⬟ㄢ㢟

p>0.05Mann-Whitney U-test with Bonferroni correction᭱ึ䛻䝠䝖䜢ぢ䜛䜎䛷䛾᫬㛫㻌 㻌 ǘ2䠙4.215, df=6, p>0.05
䝠䝖䜢ぢ䛶䛔䜛᫬㛫㻌 ǘ2䠙8.368, df=6, p>0.05
䝠䝖䜢ぢ䜛ᅇᩘ㻌 ǘ2䠙11.039, df=6, p=0.087
஺஫จど䛾ᅇᩘ㻌 ǘ2䠙7.258, df=6, p>0.05

205

◊✲Ⓨ⾲
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2. Ꮫ఍Ⓨ⾲1)እụள⣖Ꮚࠊᑎෆ㇦ࠊỌ⃝⨾ಖࠊⱱᮌ୍Ꮥࠊ⳥Ỉ೺ྐ㸸࢖ࢾࡢࣄࢺ࡜ࡢࢥ࣑ࣗࢽࢣ࣮
ࢩࣙࣥ⬟ຊࡢရ✀ᕪ㸪㛵㐃㑇ఏᏊࡢ᥈⣴. ᪥ᮏື≀ᚰ⌮Ꮫ఍➨ 73 ᅇ኱఍ࠊ⟃Ἴ኱Ꮫࠊ9
᭶ 2013 ᖺ

2)∦ᒣ┿ᕼࠊỌ⃝⨾ಖࠊⱱᮌ୍Ꮥࠊ⳥Ỉ೺ྐ㸸య఩ࡸ᝟ືࡢኚ໬ࡀ࢖ࢾࡢᚰᢿኚື࡟ཬ
ࡰࡍᙳ㡪. ᪥ᮏື≀ᚰ⌮Ꮫ఍➨ 73 ᅇ኱఍ࠊ⟃Ἴ኱Ꮫࠊ9 ᭶ 2013 ᖺ

3)ᑎෆ㇦ࠊỌ⃝⨾ಖࠊእụள⣖Ꮚࠊᆏ⏣᪥㤶⌮ࠊⱱᮌ୍Ꮥࠊ⳥Ỉ೺ྐ㸸࢖ࢾࡢࣄࢺ࡜ࡢど⥺ࢥ࣑ࣗࢽࢣ࣮ࢩࣙࣥࡢ≟✀ᕪ. ᪥ᮏື≀ᚰ⌮Ꮫ఍➨ 73 ᅇ኱఍ࠊ⟃Ἴ኱Ꮫࠊ9 ᭶ 2013ᖺ4)ᑠᕝ⨾㔛ࠊᅭྐ⥴⌮ࠊỌ⃝⨾ಖࠊⱱᮌ୍Ꮥࠊ⳥Ỉ೺ྐࠊ: ࢜࢟ࢩࢺࢩࣥᢞ୚࡟ࡼࡿࣄࢺ࡬ࡢど⥺ቑᙉࡢ᪥ᮏ≟✀࡜ὒ≟✀ࡢẚ㍑. ᪥ᮏື≀ᚰ⌮Ꮫ఍➨ 73 ᅇ኱఍ࠊ⟃Ἴ኱Ꮫࠊ9
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206

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Behavioural Processes 125 (2016) 96–100

Contents lists available at ScienceDirect

Behavioural Processes
journal homepage: www.elsevier.com/locate/behavproc

Owners’ direct gazes increase dogs’ attention-getting behaviors
Midori Ohkita a,c,∗ , Miho Nagasawa b,c , Mogi Kazutaka c , Takefumi Kikusui caSenshu University, Japan
Jichi Medical University, Japanc
Azabu University, Japanba r t i c l e

i n f oArticle history:
Received 3 August 2015
Received in revised form 24 January 2016
Accepted 19 February 2016
Available online 23 February 2016
Keywords:
Gaze
AttentionDogs

a b s t r a c t
This study examined whether dogs gain information about human’s attention via their gazes and whether
they change their attention-getting behaviors (i.e., whining and whimpering, looking at their owners’
faces, pawing, and approaching their owners) in response to their owners’ direct gazes. The results showedthat when the owners gazed at their dogs, the durations of whining and whimpering and looking at theowners’ faces were longer than when the owners averted their gazes. In contrast, there were no differences in duration of pawing and likelihood of approaching the owners between the direct and averted
gaze conditions. Therefore, owners’ direct gazes increased the behaviors that acted as distant signals anddid not necessarily involve touching the owners. We suggest that dogs are sensitive to human gazes,
and this sensitivity may act as attachment signals to humans, and may contribute to close relationships
between humans and dogs.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Dogs, Canis familiaris, were domesticated at least 14,000 years
ago (Druzhkova et al., 2013; Nobis, 1979; Vilá et al., 1997). Since
then, humans and dogs have established close relationships. Communication by means of visual information is a crucial feature inthese close relationships between humans and dogs. It is especially
important for working dogs, such as hunting dogs, to understandhuman visual communicative information to aid them in making
cooperative movements. Previous studies have revealed that dogs
understand visual information given by humans very well (Hareet al., 2002; Miklósi et al., 1998).
In addition, dogs are able to understand the visual attentionof humans as indicated by body orientation, the turning of thehead, and gaze (Hare and Tomasello, 1999). In the current theoryof human cognitive development, understanding the visual attention of others links to more complex social-cognitive skills, suchas understanding intentions, or “theory of mind” (Baron-Cohen,
1995). Similarly, dogs’ understanding of humans’ visual attention
may contribute to the communication between the two. This ability∗ Corresponding author at: School of Human Science, Senshu University, 2-1-1
Higashimita, Tama-ku, Kawasaki-shi, Kanagawa 214-8580, Japan.
E-mail address: m.ohkita.animalcognition@gmail.com (M. Ohkita).

of dogs might be supported by the co-habitation between humanand dogs.
Studies have examined whether dogs understand humans’attention based on their gazes (Call et al., 2003; Schwab and Huber,
2006). For example, Call et al. conducted a series of trials in whichdogs were forbidden to take a piece of visible food (i.e., the experimenters spoke a “Don’t take it” command). In some trials, thehumans continued to look at the dogs throughout the trial (direct
gaze condition), whereas in other trials, the humans closed theireyes (no-gaze condition). Call et al. reported that the dogs retrieved
less food in the direct gaze condition than in the no-gaze condition,suggesting dogs respond to commands in different ways dependingon whether humans gaze directly the dogs or not. Furthermore, thedogs understood the humans’ attention as it was communicated bytheir gazes.
In Call et al.’s study, however, there are differences between the
direct gaze and closed eyes condition in not only the directions of
the gaze but also in the opening and closing of the eyes. Therefore, the true value of humans’ gaze directions for the dogs was notclear. In humans, infants are sensitive to gaze direction, and they
understand from the gaze direction whether others have directedattention to them (Farroni et al., 2002, 2000; Samuels, 1985).
Therefore, in the current study, in order to examine whether dogs
understand their owners’ attention according to gaze direction, we

http://dx.doi.org/10.1016/j.beproc.2016.02.013
0376-6357/© 2016 Elsevier B.V. All rights reserved.

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investigated whether they change their behaviors in different ways
depending on owners’ gazes.
In particular, we assessed dogs’ attention-getting behaviors:
whining and whimpering, looking at the owners’ faces, pawing, andapproaching. For the reason mentioned bellow, we focused on theattention-getting behaviors.
Studies of children with autism have revealed that their frequencies of affective expression and spontaneous social behaviors
for others, such as looking at others or making physical contact withthem, were lower than those observed among typically developingchildren in communicative situations (e.g., Baranek, 1999; Adrien
et al., 1993). Studies have suggested that the lower frequencies
found in communicative situations made it difﬁcult for children
with autism to establish closer relationships with others. Accordingly, spontaneous social behaviors, including the attention-getting
behaviors, were important in subsequent reciprocal communication (Leavens et al., 2005).
In the present study, we examine whether dogs change
their attention-getting behaviors according to humans’ gazes incommunicative situations. We compared the durations of theattention-getting behaviors in the Direct Gaze condition with thosein the Avert Gaze condition. We predicted that if dogs are sensitive
to humans’ gazes and they understand humans’ attention according to gaze, then the durations of the attention-getting behaviors
would be longer in the Avert Gaze condition compared to the Direct
Gaze condition.
2. Materials and methods
2.1. Subjects
The subjects were 20 pairs of owners and healthy household
samples. The samples consisted of three Yorkshire Terriers, two
Bolognese, two Chihuahuas, and two Pekingeses, as well as one ofeach of these breeds: Beagle, Bernese Mountain Dog, Italian Greyhound, Boston Terrier, Labrador Retriever, Shin-tzu, Papillon, andWelsh Corgi. In addition, three mixed breed dogs (two Miniature
Dachshund and Toy Poodle, two Chihuahua and Toy Poodle). Tendogs were male, and ten were female, with a mean age of 4.2 [SD:3.1] years. One owner was male, and nineteen were female, with amean age of 35.5 [SD: 12.8] years.
This study was approved by the Ethics Committee of Azabu University, Japan. Informed written consent was obtained from eachparticipant.
2.2. Experimental procedures
The experiment was conducted in a room at Azabu University(Japan). The room was divided into partitions, yielding a space forthe dog of 125 × 250 cm (Fig. 1a). The front of this space was a blankwall with an acrylic window. The owner sat outside the space infront of the window, and the dog was able to view the owner’s facethrough the window (Fig. 1b).
The experiment consisted of two conditions. In the Direct Gazecondition, the owner faced the window and gazed directly at thedog on the other side. In the Avert Gaze condition, the owner
faced the window but did not gaze at the dog (i.e., they gazed toeither the left or the right). We asked the owners to have a neutral
facial expression in both conditions. Each condition was conducted
two times (i.e., 2 trials × 2 conditions), resulting in a total of 4 trials per dog. The order of the trials was an ABBA design and wascounterbalanced within subjects. Each trial was lasted for 1 min.
The interval between trials was 3 min. In the inter-trial interval,
owners were able to interact freely with their dogs. The total experiment duration was 16 min. All trials were video-recorded (Sony

209

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video camera HDR-CX180 seated on a tripod) to analyze the dogs’
behaviors.
2.3. Data analysis
Attention-getting behaviors, including standing in close proximity to the owner from the acrylic window (i.e., within an area of
70 × 70 cm from the windows; Fig. 1a), looking at the owner’s face,whining and whimpering, and pawing, were recorded using an alloccurrence sampling. We analyzed the durations of these behaviors
in the Direct Gaze and Avert Gaze conditions, using an Excel VBAbased event recorder. One of the authors (Midori Ohkita) analyzed
all the videotaped data. In addition, a blind observer who did notknow the purpose of the study analyzed randomly selected subjects (20%). Agreement reliability between the author and the naïve
observer was excellent (rs = .98). We calculated sum durations foreach behavior in each condition for each subject. Statistical analyses were performed using Wilcoxon signed-ranks test with an alphalevel of .05.
3. Results
Fig. 2 shows the means of the sum durations (in seconds) for
each attention-getting behaviors in each condition for each subject. The durations of whining and whimpering (Z = 2.62, p = .01,
r = .59) and looking at the owners’ faces (Z = 4.43, p < .001, r = .99)
were longer in the Direct Gaze condition than in the Avert Gazecondition. There were no differences between the two conditions
in durations of pawing (Z = 1.47, p = .14, r = .33) or standing close to
the acrylic window (Z = 0.71, p = .48, r = .16).
4. Discussion
We investigated whether dogs change some of their attentiongetting behaviors in different ways depending on owners’ direct
gazes by comparing their behavior under the avert gaze. Dogs
usually perform attention-getting behaviors in order to get attention from humans. If dogs are sensitive to humans’ gazes andthey understand humans’ attention according to gaze, then the
durations of the attention-getting behaviors would be longer inthe Avert Gaze condition compared to the Direct Gaze condition.
An interesting ﬁnding, contrary to our expectations, was that the
durations of whining and whimpering and looking at their owners’
face were longer in the Direct Gaze condition than in the Avert Gazecondition. These results suggest that the dogs were sensitive to thedirection of their owners’ gazes. Subsequently, the dogs showed
an increase of their own attention-getting behaviors. The question,
then, arose; Why did the owners’ attention behaviors—that
is, the direct gaze—increase their dogs’ attention-getting
behaviors.
One possible explanation for this question is that attentiongetting behaviors act as appetitive behavior whereas being touchedby the owners or given a reward like food acts as a consummatory behavior. In general, owners’ tend to touch their dogs aftergazing at them directly (attending to them) in response to thedogs’ attention-getting behaviors, dogs are more likely to act out
attention-getting behaviors in response to their owners’ gazes until
these behaviors are reinforced by their owners’ touches (Bentosela
et al., 2008). In the present study, in the Direct Gaze condition, thedogs did perform attention-getting behaviors in response to theirowners’ gazes. In contrast, in the Avert Gaze condition, dogs did act
out these behaviors a little, because their owners gazes were notpresent. In order to ensure these behaviors mainly occurred in the
Direct Gaze condition, the durations of these behaviors were longer
in that condition than in the Avert Gaze condition.

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M. Ohkita et al. / Behavioural Processes 125 (2016) 96–100

Fig. 1. Experimental setup.
(a) Schematic of the enclosed space for the dog and the position of the owner outside the enclosure. (b) Actual image of an owner behind the acrylic window with the dog.

Another possibility for the reasons why the owners’ attention
behaviors increased their dogs’ attention-getting behaviors is thatowners’ gazes provide social rewards for dogs and increase dogs’
behaviors toward the owners. Nagasawa et al. (2009, 2015) examined whether direct gaze contributes to the establishment of therelationships between humans and dogs. Dogs’ behaviors of gazing at and touching their owners was longer when the owners
interacted with their dogs (interaction experiment) than when
the owners did not look at the dogs (control experiment). Thissuggested that the owners’ gazes increased the dogs’ attachment
behaviors (dogs’ gazes at their owners, dogs’ approaching theirowners, and dogs’ touching their owners); therefore, there werepositive loops of direct gaze and attachment behaviors between
owners and their dogs. In this regards, it is feasible in the present
study that owners’ eye gaze enhance their dogs’ whining and whimpering and looking at their owners’ face as attachment behavior, as
well as Nagasawa et al.’s studies.
Positive loops have been studied in mother-infant communication (Kendrick, 2004; see Nagasawa et al., 2013 for a review). Thesestudies showed that mothers’ parental behaviors (e.g., stroking)facilitated infants’ attachment behaviors, which in turn increasingly facilitated the mothers’ parental behaviors. Previous studies
have also suggested that this positive loop stimulates the release ofcentrally acting oxytocin, and this oxytocin acts as a social reward
within the nucleus accumbens. As a result, this positive loop might
contribute to mother-infant bonding. In Nagasawa et al. (2009,2015) studies, the positive loop of direct gaze between owners
and dogs stimulated the release of oxytocin. That is, the positive loop of direct gaze functioned as a social reward for bothowners and dogs (i.e., a primary reinforcer). This positive loopof direct gaze may establish a closer relationship between dogs
and humans. Therefore, the ﬁndings of this present study arein line with those of Nagasawa et al., suggesting that owners’gazes provide social rewards for their dogs and increase the dogs’
attachment behaviors, gazing at their owners. Further research isneeded to examine whether dogs’ increased behaviors according
to humans’ gazes are attention-getting behaviors or attachment
behaviors.
In Nagasawa et al.’s studies, the durations of not only the dogs’gazes but also the dogs’ touches, including pawing and approaching the owners, were longer when the owners interacted with thedogs in their usual way (eye gaze condition) than when the owners did not look at their dogs (non-eye gaze condition). The resultsof the present study are not consistent with these ﬁndings. In ourstudy, the owner’s direct gaze did not facilitate the dog’s pawing or
approaching the owner. Our experimental conditions were different from those used in Nagasawa et al. study (2009, 2015). In thatstudy, the owners could touch their dogs. In the present study, apartition wall kept the owners from being able to touch their dogs.
This difference in experimental conditions might lead to differencesin the dogs’ behaviors in response to their humans’ gazes. This may
be because ﬂoor and ceiling effects prevented differences between
the conditions in terms of pawing and approaching their owners,
respectively. It might be difﬁcult for the dogs to paw the smoothacrylic window, so the dogs might not have wanted to paw in either
condition. In addition, because each condition was shorter in thepresent study than in Nagasawa et al.’s studies, the duration might
not have been long enough for the between-condition approachingdifferences to occur.
Even if a human’s direct gaze signals rewards (i.e., being touchedby humans) for dogs, they are nevertheless sensitive to humangazes. For many species, it is known that perceiving a direct gazeby a member of another species arouses an aversive response(Emery, 2000), probably because it is a salient signal of potential210

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99

Fig. 2. Means of sum durations for attention-getting behaviors.
Box plots show means of sum durations (in seconds) for attention-getting behaviors in each condition for each subject. *p < .05, ** p < .01, *** p < .001 (Wilcoxon signed-rankstest).

threat. In contrast, humans’ direct gazes at dogs may contribute toclose relationships between the two, although humans and dogs
are phylogenetically very distant species. Therefore, these behavioral changes might be involved in convergent evolution of humans
and dogs. By using adult dogs and their owners as subjects inthe present study, however, we could not eliminate the possibility of prior learning. We intend to address these issues in future
research.
Acknowledgments
This experiment was approved by the Animal Care and Use committee of Azabu University and was carried out according to Azabu
University’s Guidelines for Animal Research.
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212ǤǤ Ȁ  

OPENR ǣͶͼ͸ͶͷͻA ǣͶͺ͸ͶͷͻPǣͷ; ͸ͶͷͻComparison of owner-reported
behavioral characteristics among
genetically clustered breeds of dog(Canis familiaris).
Akiko Tonoikeͷ, Miho Nagasawaͷ,͸, Kazutaka Mogiͷ, James A. Serpell͹, Hisashi Ohtsukiͺ& Takefumi KikusuiͷDuring the domestication process, dogs were selected for their suitability for multiple purposes,
resulting in a variety of behavioral characteristics. In particular, the ancient group of breeds that  ơ    
to other breed groups. Here, we used questionnaire evaluations of dog behavior to investigate   ơ  Ǥ
A standardized questionnaire, the Canine Behavioral Assessment and Research QuestionnaireȋǦȌǡǡơ Ǧ ȋ=͸ǡͿͻͷȌand the United States (n =ͷͶǡ͹;ͿȌǤ  
spitz breed group showed low attachment and attention-seeking behavior. This characteristic
distinguished the ancient group from any other breed groups with presumed modern European
origins, and may therefore, be an ancestral trait.

The dog (Canis familiaris) was the first animal to be domesticated1 and today hundreds of different
breeds are recognized. Breeds seem to be different in several aspects of their behavior due to the effects
of artificial selection2–5. Although breeds are traditionally classified by the jobs they were originallyselected to perform, parallel selection for other traits, such as suitability as pets, has also affected modern
breed-typical behavior6. With the remarkable improvement of technologies available for genetic analysis,
genetic relationships in dog breeds have recently been studied and genetic classifications of dog breeds
have been constructed7,8. As a result, although dog breeds have traditionally been classified by their roles
in human activities, historical records, and physical phenotypes, it is now possible to classify them basedon patterns of genetic variation9–11.
Cladogram analysis of dog genes showed the separation of several breeds with supposedly ancientorigins from a large group of breeds with presumed modern European origins7,8. Modern European
breeds are the products of controlled breeding practices since the Victorian era, and because they haveoriginated recently and lack deep histories, the genetic groups have short internodes and low bootstrap
support. On the other hand, ancient breeds are highly divergent and are distinct from modern European
breeds. Since the dogs from these ancient breeds are genetically related most closely to wolves, they may
exhibit remnants of wolves’ behavioral, morphological and physiological characteristics.
The Canine Behavioral Assessment and Research Questionnaire (C-BARQ) is designed to provide
dog owners and professionals with standardized evaluations of canine temperament and behavior12. Theͷ

Department of Animal Science and Biotechnology, Azabu University, Sagamihara, Kanagawa, Japan. ͸Departmentof Physiology, Jichi Medical University, Shimotsuke, Tochigi, Japan. ͹School of Veterinary Medicine, University ofPennsylvania, Philadelphia, Pennsylvania, USA. ͺDepartment of Evolutionary Studies of Biosystems, School of
Advanced Sciences, The Graduate University for Advanced Studies (SOKENDAI). Correspondence and requests
for materials should be addressed to M.N. (email: nagasawa@carazabu.com)

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C-BARQ has also been translated for use in Japan13,14 after examination of the validity of questionnaire items15. In this study, we used C-BARQ evaluations of dogs to investigate whether the behavioral characteristics of dogs are different among genetically clustered breed groups. Although C-BARQ
scores are obtained from dog owners and may therefore be influenced by subjective biases, the use ofthis instrument allows the standardized assessment of behavior in very large numbers of dogs, and has
proven useful for studying breed differences in behavior16–18. Several studies comparing wolves, dogs, andother canids, suggest that behavioral changes were critical during the early stages of the domestication
process19–21. We investigated the behavioral characteristics of breeds, especially those belonging to theancient group, to understand the characteristics of this highly divergent group of ancient breeds.

Materials and Methods
Questionnaire. Behavioral data in the present study were collected from dog owners using the
C-BARQ, which included 100 questions that asked owners to indicate how their dogs have responded in
the recent past to a variety of common events and stimuli using a series of 0–4 rating scales. The C-BARQ
is a standardized questionnaire that is widely used to assess the prevalence and severity of behavioral
problems in dogs. The various C-BARQ item and subscale scores have also been shown to provide an
accurate measure of canine behavioral phenotypes. Seven of the original 11 subscales were validated
using a panel of 200 dogs previously diagnosed with specific behavior problems12. More recently, otherstudies have provided criterion validation of the C-BARQ by demonstrating associations between factor
and item scores and training outcomes in working dogs22, the performance of dogs in various standardized behavioral tests23–26, and neurophysiological markers of canine anxiety and compulsive disorders27,28.
The original C-BARQ was translated into Japanese by two behavioral professionals and reviewed by two
professors15. Twenty-two out of 100 questions were eliminated due to the cultural and environmental
differences between Japan and the USA, resulting in 78 questions for the Japanese version.
C-BARQ data were collected via the freely accessible websites http://www.cbarq.org (US, from April2006) and http://cbarq.inutokurasu.jp/(JPN, from September 2010). Before answering the questionnaire,dog owners were asked to provide information about their dogs, such as its breed, age, sex, neuter
status, body weight, age when acquired, where acquired, and the presence of any health problems. The
online survey was advertised via articles in newspapers, magazines, online news, etc., in each country.
The C-BARQ database was used for different purpose 29. The Ethical committee of Azabu University
approved this study. We obtained the informed consent from all respondents and our methods werecarried out in accordance with the approved guidelines.
Statistical analyses. Data from the completed questionnaires were subjected to factor analysis.
Parallel analysis was used to determine the number of interpretable factors that could be extracted, andvarimax rotation was used to identify empirical groupings of items that measured different behavioral
traits. The Cronbach’s α coefficient was calculated to assess internal consistency (reliability) of extracted
factors; this coefficient describes how well a group of questionnaire items focuses on a single idea orconstruct. For comparison of the factors, we calculated the average of item scores composing each factor,
which was analyzed as a factor score. The factor scores were then analyzed using generalized linear models These were analyzed by SPSS v.19.0 (SPSS Japan Inc., IBM company), except for the parallel analysis(R v. 3.0.0, 2013-04-03, The R Foundation for Statistical Computing).

Results

Subjects. A total of 5,377 C-BARQ questionnaires were completed in Japan. Dogs that were < 1 or
> 7 years of age or had severe or chronic health problems were excluded, leaving a total of 3,098 completed questionnaires (57.76%) that were considered valid. The age cut-off was chosen to eliminate dogs
whose behavior might have been affected by immaturity or senescence (in the case of some large or giant
breeds). The response rates for each of the 78 questions in the questionnaire ranged from 39.22% to99.86% (median, 98.39%, mode, 99.15%). The low response rates obtained for some questionnaire items
were primarily due to the fact that the questionnaire’s focus on events and stimuli occurring in recent
past tended to exclude uncommon events and situations. Among the 14,481 questionnaires completed
in the United States, 10,500 satisfied the requirements above (72.51%). The response rates for each of
the 100 questions in the questionnaire ranged from 81.57% to 99.72% (median, 97.85%, mode, 98.04%).
Fifteen questions with response rates < 85.0% either in Japanese or US data were excluded for further
analyses. Any questionnaires that had < 75.0% response rates were also excluded, leaving 2,951 (54.88%)
and 10,389 (71.74%) completed questionnaires that could be used in analyses in Japan and the UnitedStates, respectively.
Factor analysis. For the factor analysis we selected 59 breeds that were common to both countries
and then matched the samples for sex and number of dog for each country in order to eliminate any sex
or country biases (n = 1,252 each, Supplementary Table 1). Sixty-three of the questionnaire items common to both countries were analyzed by factor analysis and parallel analysis, and these items were sorted
into 12 factors. After removing the items with factor loadings of < 0.4, the remaining items were analyzed
by factor analysis again, and yielded 12 factors. After removing the items with factor loadings of < 0.4
again, the remaining items were analyzed by factor analysis, and again yielded 12 factors that accounted
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Factorloadings

Factors & questionnaire items
Aggression to unfamiliar persons
When approached directly by an unfamiliar adultwhile being walked or exercised on a leash

0.836

When approached directly by an unfamiliar childwhile being walked or exercised on a leash

0.744

When an unfamiliar person approaches theowner or a member of the owner’s family at
home

0.670

When an unfamiliar person approaches theowner or a member of the owner’s family awayfrom home

0.792

When mailmen or other delivery workers
approach the home

0.604

When an unfamiliar person tries to touch or pet
the dog

0.806

Toward unfamiliar persons visiting the home

0.717

Fear of unfamiliar persons
When approached directly by an unfamiliar adultwhile away from the home

0.823

When approached directly by an unfamiliar childwhile away from the home

0.747

When unfamiliar persons visit the home

0.689

When an unfamiliar person tries to touch or pet
the dog

0.761

Trainability
Returns immediately when called while off leash

0.667

Obeys a sit command immediately

0.676

Obeys a stay command immediately

0.717

Seems to attend to or listen closely to everything
the owner says or does

0.693

Not slow to respond to correction or punishment

0.619

Not easitly distracted by interesting sights,
sounds, or smell

0.447

Separation-related anxiety
Excessive salivation when left or about to be left
on its owner

0.497

Whining when left or about to be left on its
owner

0.714

Barking when left or about to be left on its owner

0.735

Howling when left or about to be left on its
owner

0.608

Chewing/scratching at doors, floor, windows,
curtains, etc.

0.462

Loss of appetite when left or about to be left on
its owner

0.403

Energy and restless
Not shaking, shivering, or trembling when left orabout to be left on its owner

0.441

Restlessness, agitation, or pacing when left orabout to be left on its owner

0.477

When a member of the household returns home
after a brief absence

0.690

When visitors arrive at its home

0.480

Playful, puppyish, boisterous

0.694

Active, energetic, always on the go

0.493

SS loadings

Proportion
Var

Cumulative
Var

Cronbach’s α

5.13

0.10

0.10

0.92

2.79

0.05

0.15

0.90

2.77

0.05

0.20

0.81

2.73

0.05

0.25

0.76

2.65

0.05

0.30

0.72

Continued

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Factorloadings

Factors & questionnaire items
Fear of non-social stimuli
In response to sudden or loud noises

0.665

In response to strange or unfamiliar objects on ornear the sidewalk

0.671

During thunderstorms

0.422

When first exposed to unfamiliar situations

0.468

In response to wind or wind-blown objects

0.699

Aggression to household members
When toys, bones, or other objects are taken
away by a member of the household

0.577

When approached directly by a member of thehousehold while it is eating

0.773

When food is taken away by a member of thehousehold

0.849

When a member of the household retrieves foodor objects stolen by the dog

0.626

Fear of unfamiliar dogs
When approached directly by an unfamiliar dogof the same or larger size

0.769

When approached directly by an unfamiliar dog
of a smaller size

0.792

When barked, growled, or lunged at by
unfamiliar dog

0.756

Aggression to unfamiliar dogs
When approached directly by an unfamiliar maledog while being walked or exercised on a leash

0.849

When approached directly by an unfamiliar
female dog while being walked or exercised ona leash0.840

When barked, growled, or lunged at by
unfamiliar dog

0.642

Attachment and attention-seeking
Tends to follow a member of household fromroom to room about the house

0.541

Tends to sit close to or in contact with a memberof the household when that individual is sitting
down

0.672

Tends to nudge, nuzzle, or paw a member of thehousehold for attention when that individual issitting down

0.701

Becomes agitated when a member of thehousehold shows affection for another person

0.466

Aggression to persons passing near the house
When strangers walk past the home while thedog is in the yard

0.652

When joggers, cyclists, roller skaters, orskateboarders pass the home while the dog is in
the yard

0.607

SS loadings

Proportion
Var

Cumulative
Var

Cronbach’s α

2.27

0.04

0.34

0.75

2.25

0.04

0.38

0.84

2.21

0.04

0.42

0.88

2.10

0.04

0.46

0.89

1.78

0.03

0.53

0.70

1.15

0.02

0.55

0.89

Table 1. Factor loading of questionnaire items constituting each factor.

for 54.96% of the common variance in item scores. Out of these twelve factors, eleven were found to haveadequate Cronbach’s α values (≥ 0.7) (Table 1). The following eleven factors were extracted: aggression
to unfamiliar persons (F1), fear of unfamiliar persons (F2), trainability (F3), separation-related behavior(F4), energy and restlessness(F5), fear of non-social stimuli (F6), aggression to household members (F7),fear of unfamiliar dogs (F8), aggression to unfamiliar dogs (F9), attachment and attention-seeking (F11),
and aggression to persons passing near the house (F12). These results are shown in Table 1.

ƪ Ǧ  Ǥ Using the generalized linear model, the influence of breeds and various demographic variables on C-BARQ factor scores were examined. The dog
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Breed groups

BreedsAncient and spitz breeds(152)

Basenji(JPN4, US4), Shiba Inu(64, 64), Akita(4, 4), Siberian Husky(2, 2), Samoyed(2, 2)

Toy dogs (612)

Shih Tzu(40, 40), Chihuahua(110, 110), Pug(46, 46), Papillon(24, 24), Pomeranian(50, 50), Miniature
Pinscher(26, 26), Brussels Griffon(2, 2), Pekingese(8, 8)

Spaniels, scent hounds,
and poodles (388)American Cocker Spaniel(14, 14), English Cocker Spaniel(10, 10), English Springer Spaniel(4, 4), CavalierKing Charles Spaniel(36, 36), Brittany(2, 2), Beagle(42, 42), Bichon Frise(10, 10), Maltese(32, 32) , Toy
Poodle(22, 22), Miniature Poodle(12, 12), Standard Poodle (10, 10)

Working dogs (32)

Doberman Pinscher(6, 6), German Shepherd(10, 10)

Small Terriers (236)

Cairn Terrier(4, 4), Jack Russell Terrier(58, 58), West Highland White Terrier(10, 10), Yorkshire Terrier(46,46)

Sight hounds andherding dogs (408)

Italian Greyhound(18, 18), Whippet(6, 6), Borzoi(2, 2), Pembroke Welsh Corgi(48, 48), AustralianShepherd(6, 6), Border Collie(74, 74), Shetland Sheepdog(50, 50)

Retrievers (304)

Labrador Retriever(82, 82), Flat-Coated Retriever(8, 8), Golden Retriever(44, 44), Great Dane(2, 2), Bernese
Mountain Dog(16, 16)

Mastiff-like dogs (112)

Boston Terrier(32, 32), Boxer(4, 4), Bulldog(2, 2), French Bulldog(18, 18)

Table 2. Genetically clustered breed groups used for statistical analysis. The numbers of selected dogs
are shown in parentheses.

breeds were separated into eight breed groups according to the cladogram suggested by vonHoldt (2010).
The eight groups consist of 1) ancient and spitz breeds, 2) toy dogs, 3) spaniels, scent hounds, andpoodles, 4) working dogs, 5) small terriers, 6) sight hounds and herding dogs, 7) retrievers, and 8)
mastiff-like dogs. As the Shiba Inu breed was not included in vonHoldt’s cladogram, we classified theminto the ancient and spitz breed group according to the cladogram suggested by Parker (2004). Other
dog breeds not shown in vonHoldt’s cladogram were eliminated from further analyses. The dog breeds
in each breed group are shown in Table 2.
We analyzed the relationships between breed groups and C-BARQ factor scores while taking into
account the possible intervening effects of the following 8 variables: country, sex, spay/neuter status,
source from which dogs were acquired, owner’s experience of dog-ownership, dog’s age when acquired,dog’s body weight, dog’s age at the time of evaluation. These variables have previously been shown to
influence the expression of behavior in dogs16,30–32. All of the factor scores were explained significantly
by the variables, although most of the variance was explained by breed groups, country, sex, spay/neuter
status, and source from which dogs were acquired. Significant interactions between breed group andother variables were also found. Results are shown in Table 3 and Fig. 1.
Toy dogs obtained the highest scores for Factor 1 (aggression to unfamiliar persons), and were significantly more aggressive in this context than sight hounds and herding dogs, retrievers, and mastiff-likedogs. Spaniels, scent hounds, and poodles were significantly more aggressive to unfamiliar persons thanretrievers and mastiff-like dogs. The ancient and spitz breed group and sight hounds and herding dogs
were significantly more aggressive to unfamiliar persons than retrievers. For F2 (fear of unfamiliar persons), working dogs obtained the lowest scores, and were significantly lower than all other breed groups.
For F3 (trainability), sight hounds and herding dogs obtained the highest scores, and were significantly
more trainable than all other breed groups except working dogs. For F4 (separation-related anxiety),
there were no breed group differences. For F5 (energy and restlessness), working dogs obtained thelowest scores, and were significantly lower than all other breed groups except mastiff-like dogs. Sight
hounds and herding dogs obtained the highest scores, and were significantly higher than the spaniels,
scent hounds, and poodles breed group and mastiff-like dogs. For F6 (fear of non-social stimuli), sighthounds and herding dogs obtained the highest scores, and were significantly higher than the spaniels,
scent hounds, and poodles breed group and mastiff-like dogs. Working dogs obtained the lowest scores,
and were significantly lower than all other breed groups except mastiff-like dogs. For F7 (aggression tohousehold members), working dogs obtained the lowest scores, and were significantly less aggressive inthis context than all other breed groups. Toy dogs obtained the highest scores, and were significantly
more aggressive to household members than the sight hounds and herding dogs breed group and retrievers. The spaniels, scent hounds, and poodles breed group was significantly more aggressive to household
members than the sight hounds and herding dogs breed group. For F8 (fear of unfamiliar dogs), working
dogs obtained the lowest score, and were significantly lower than all other breed groups. For F9 (aggression to unfamiliar dogs), retrievers obtained the lowest scores, and were significantly less aggressive inthis context than ancient and spitz breeds, toy dogs, the small terriers, and the sight hounds and herding dogs breed group. For F11 (attachment and attention-seeking), the ancient and spitz breed groupobtained the lowest scores, and were significantly lower than all other breed groups. For F12 (aggression
to persons passing near the house), toy dogs obtained the highest scores, and were significantly moreaggressive in this context than mastiff-like dogs.

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F1: Aggression to unfamiliar persons

χ2

df

Pairwisecomparisonp

F2: Fear of unfamiliar persons

B5)

df

p

Pairwisecomparison

126.6527

0.000

1 > 4, 2 > 4,
3 > 4, 5 > 4,
6 > 4, 7 > 4,
8> 4

0.314

70.0101

0.000

US > JPN
F> M

Breed groups1)

50.5707

0.000Country

1.015

1

1 > 7, 2 > 6,
2 > 7, 2 > 8,
3 > 7, 3 > 8,
5 > 7, 6 > 7

χ2

F3: Trainability

B

Pairwisecomparison

χ2

df

p

51.531

7

0.000

6 > 1, 6 > 2,
6 > 3, 6 > 5,
6 > 7, 6 > 8

14.4881

0.000

US > JPN

B

Sex

1.683

1

0.195

69.123

1

0.000

0.749

1

0.387

Neutered status2)

0.658

1

0.417

0.468

1

0.494

3.771

1

0.052

24.100

6

0.001

1.940

6

0.925

31.5886

0.000

3 > 4, 7 > 4

15.057

1

0.000

2> 1

3.725

1

0.054

22.534

1

0.000

0.024
0.000

Source where aquired3)
Dog-ownership experience4)

2 > 3, 5 > 3

1.361

1

0.243

0.006

1

0.939

Body weight

0.571

1

0.450

14.5181

0.000

Dog’s age at evaluation

0.000

1

0.996

0.627

1

0.429

− 0.003− 0.029

Dog’s age when acquired

14.696

1

0.000

3.223

1

0.073

5.055

1

0.025

Breed groups*Country

61.63115

0.000

139.57615

0.000

89.92015

0.000

Breed groups*Sex

63.19815

0.000

127.81415

0.000

59.38615

0.000Country*Sex

12.0303

0.007

85.8043

0.000

15.663

3

0.001

31

0.000

141.41131

0.000

104.54531

0.000

189.44442145.060420.000

248.37342

0.000

Breed groups*Country*Sex
Omnibus

0.000

F4: Separation-related anxiety
χ2

df

p

Breed groups1)

13.4987

0.061Country

31.078

1

0.000

Sex

0.000

1

0.996

4.368

1

0.037

12.7956

0.046

10.1611

0.001

Neutered status2)
Source where aquired3)

Dog-ownership experience4)

F5: Energy and restless

Pairwisecomparison

B

US > JPN

I> N

1> 2

F6: Fear of non-social stimuli

χ2

df

p

Pairwisecomparison

χ2

df

p

Pairwisecomparison

44.8447

0.000

1 > 4, 2 > 4,
3 > 4, 5 > 4,
6 > 4, 7 > 4,
6 > 3, 6 > 8

44.8447

0.000

1 > 4, 2 > 4,
3 > 4, 6 > 3,
5 > 4, 6 > 4,
7 > 4, 6 > 8

JPN > US

5.077

1

0.024

JPN > US

0.161

1

0.688

5.631

1

0.018

B

5.077

1

0.024

0.161

1

0.688

5.631

1

0.018

10.7066

0.098

10.706

6

0.098

2.130

1

0.144

2.130

1

0.144

7.906

1

0.005

2.154

1

0.142

4.155

1

0.042

Body weight

4.925

1

0.026

− 0.012

7.906

1

0.005

Dog’s age at evaluation

5.444

1

0.020

− 0.039

2.154

1

0.142

Dog’s age when acquired

1.083

1

0.298

4.155

1

0.042

N> I

− 0.0120.001

Breed groups*Country

96.17615

0.000

52.43415

0.000

52.43415

0.000

Breed groups*Sex

20.71715

0.146

47.98415

0.000

47.98415

0.000Country*Sex

31.078

3

0.000

5.752

3

0.124

5.752

3

0.124

Breed groups*Country*Sex

106.10531

0.000

65.67131

0.000

65.67131

0.000

Omnibus237.075420.000

148.076420.000

148.076

42

0.000

F7: Aggression to household members
χ2

df

p

Pairwisecomparison

Breed groups1)

84.7197

0.000

1 > 4, 2 > 4,
2 > 6, 2 > 7,
3 > 4, 3 > 6,
5 > 4, 6 > 4,
7 > 4, 8 > 4Country

0.000

1F8: Fear of unfamiliar dogs

N> I

− 0.0120.001

F9: Aggression to unfamiliar dogs

χ2

df

p

Pairwisecomparison

141.880

7

0.000

1.000

80.139

1

B

B

χ2

df

p

Pairwisecomparison

1 > 4, 2 > 4,
3 > 4, 5 > 4,
6 > 4, 7 > 4,8> 426.2117

0.000

1 > 7, 2 > 7,
5 > 7, 6 > 7

0.000

US > JPN

2.747

1

0.097

F> M

B

Sex

0.000

1

1.000

78.662

1

0.000

5.105

1

0.024

Neutered status2)

0.000

1

1.000

2.798

1

0.094

0.509

1

0.476

Source where aquired3)

0.000

6

1.000

11.9116

0.064

11.427

6

0.076

Dog-ownership experience4)

0.000

1

1.000

1.017

1

0.313

0.780

1

0.377

Body weight

0.023

1

0.881

5.361

1

0.021

1.718

1

0.190

− 0.017

B

M> F

Continued

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Dog’s age at evaluation

0.000

1

0.986

1.257

1

0.262

16.415

1

0.000

Dog’s age when acquired

2.897

1

0.089

0.008

1

0.929

2.756

1

0.097

Breed groups*Country

89.609140.000

153.60515

0.000

36.74715

0.001

Breed groups*Sex

85.619140.000

142.85815

0.000

38.16515

0.001Country*Sex

20.11330.000

98.852

3

0.000

9.449

3

0.024

Breed groups*Country*Sex

91.28429

0.000

155.42231

0.000

52.72631

0.009

Omnibus266.83842

0.000

111.849420.000

104.112420.000

F11: Attachment and attention-seeking

0.069

F12: Aggression to persons passing near the house

χ2

df

p

Pairwisecomparison

Breed groups1)

82.1167

0.000

2 > 1, 3 > 1,
4 > 1, 5 > 1,
6 > 1, 7 > 1,
8> 1Country136.9121

0.000

US > JPN

3.724

1

0.054

Sex

3.030

1

0.082

0.984

1

0.321

Neutered status2)

1.019

1

0.313

0.543

1

0.461

Source where aquired3)

11.958

6

0.063

8.539

6

0.201

Dog-ownership experience4)

0.042

1

0.837

0.335

1

0.563

B

χ2

df

p

Pairwisecomparison

19.286

7

0.007

2> 8

Body weight

7.023

1

0.008

− 0.005

0.007

1

0.934

Dog’s age at evaluation

11.14510.001− 0.018

0.000

1

0.999

Dog’s age when acquired

1.318

1

0.251

8.065

1

0.005

Breed groups*Country328.22715

0.000

29.03415

0.016

Breed groups*Sex

86.349150.000

26.77415

0.031Country*Sex

145.751

3

0.000

7.556

3

0.056

Breed groups*Country*Sex

344.92631

0.000

47.05731

0.032

Omnibus385.29642

0.000

72.384

42

0.002

B

− 0.002

Table 3. Results for the analysis of factor scores using generalized linear models. 1) Ancient and spitz
breeds: 1, Toy dogs: 2, Spaniels, scent hounds, and poodles: 3, Working dogs: 4, Small terriers: 5, Sight
hounds and herding dogs: 6, Retrievers: 7, Mastiff-like dogs: 8. 2) neutered: N, intact: I. 3) bred by owner:
1, friend of relative: 2, breeder: 3, pet store: 4, shelter: 5, stray: 6, other: 7. 4) first ownership: 1, second andmore ownership: 2. 5) partial regression coefficient”.

Some factors were different between US and Japan (US > JPN; F2, F3, F4, F8, F11 JPN > US; F5, F6),and between male and female (male > female; F9 female > male; F2, F8). Some factors were affectedby spay/neuter status (intact > neutered; F4 neutered > intact; F5, F6), source from which dogs wereacquired (F1; friend or relative > breeder, shelter > breeder F3; breeder > pet store, other > pet store)
and owner’s experience of dog-ownership (first ownership > second and more ownership; F4 second andmore ownership > first ownership; F3). Body weight, dog’s age at the time of evaluation and dog’s age
when acquired also influenced some factors (body weight; F2, F4, F5, F6, F8, F11 dog’s age at evaluation; F3, F4, F9, F11 dog’s age when acquired; F1, F3, F5, F6, F12). The environment for dogs and theirowners are different in Japan and US. For example, pet stores are a popular source of dog acquisition in
Japan compared with the US where most purebred dogs are acquired directly from breeders. In order
to investigate the effect of country on breed group differences, we separated the questionnaire data into
two groups -dogs living in Japan and the USA- and analyzed for the breed group differences separately
in each group. There were some differences between countries, the primary breed group differences
remained the same in both countries, especially with respect to F11 (attachment and attention-seeking),
even though there were large differences in the environment surrounding the dogs in two countries. The
results are shown in Supplemental Table 2.
Additionally, we conducted cluster analyses of the factors using breed medians and Ward’s method.
All of the breeds of the ancient and spitz breed group are clustered in one group in the dendrogram
branches associated with F2 (fear of unfamiliar persons), F4 (separation-related anxiety) and F11 (attachment and attention-seeking). Four out of five breeds of the ancient and spitz breed group are also clustered in one group in the branch associated with F8 (fear of unfamiliar dogs). In F11 (attachment andattention-seeking), two clusters were identified and all of five breeds of ancient and spitz breed groupwere classified into the same cluster. Five other breeds also clustered as showing low levels of attachment and attention-seeking, including two terriers (West Highland White Terrier, Cairn Terrier), twosight hounds (Whippet, Borzoi), and the Great Dane. In F4 (separation-related anxiety), two clusters
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Figure 1. Average factor scores for breed groups. The dog breeds were separated into eight breed groups
according to the cladogram, Ancient and spitz breeds: 1, Toy dogs: 2, Spaniels, scent hounds, and poodles: 3,
Working dogs: 4, Small terriers: 5, Sight hounds and herding dogs: 6, Retrievers: 7, Mastiff-like dogs: 8. a vs b,
p < 0.05; c vs d, p < 0.05; e vs f, p < 0.05; g vs h, p < 0.05.

were identified and all of five breeds of ancient and spitz breed group were classified into the samecluster. Fifteen other breeds are also clustered as showing low levels of separation anxiety. In F2 (fearof unfamiliar persons), two clusters were identified and eleven breeds (Chihuahua, Poodle (Toy), Boxer,
Yorkshire Terrier, Shetland Sheepdog, Whippet, Maltese, Miniature Pinscher, Great Dane, Cocker Spaniel(American), and Italian Greyhound) were in one cluster and all other breeds were in the other. The trees
for F2, F4 and F11 are shown in Supplementary Figure 1, 2 and 3.

Discussion
Using a validated online behavioral evaluation system (C-BARQ), we collected data on the behavioral
characteristics of dogs in Japan and the United States, and investigated differences among geneticallyclassified breed groups. Overall, most of the variance in C-BARQ factor scores was explained by thevariables; breed group, country, sex, spay/neuter status, and source from which dogs were acquired.
Significant interactions between breed group and other variables were also found, indicating that the
behaviors evaluated by C-BARQ were influenced by genetic origins, hormonal status and environmental
factors, such as country. Some factors were clearly explained by breed group differences. These differences may be related to the effects of direct selection for behavioral characteristics or due to differences in
the conditions of life that different breeds experience during development. Since it is hard to believe that
all ancient breeds grew up in similar environments that were distinct from those of all modern breeds,
it appears unlikely that the observed breed group differences are due solely to environmental factors.
Furthermore, when we separated the questionnaire data into two groups—dogs living in Japan and theUnited States—and analyzed for breed group differences separately in each group (Supplementary Table 2),
we identified similar breed group differences in behavior in both countries. This finding supports theview that these differences are primarily due to genetic factors. The most unique among the eight breed
groups is the working dog group, which shows the lowest levels of fear of unfamiliar persons, non-social
stimuli, and unfamiliar dogs, and the lowest scores for energy, hyperactivity, and aggression to household
members. Working dogs are used as police dogs, military dogs, watch dogs, and may be under strong
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selection for these characteristics. Even those that live as family pets, may still retain the the effects ofpast selection for working roles. Since the data for working dogs are from only two breeds, Doberman
Pinscher and German Shepherd, there is a possibility that the uniqueness of the working dog group isrelated to the small size of this group. Similarly, the trainability of sight hounds and herding dogs may
be high because of direct selection for this characteristic. Most interestingly, the scores for attachment
and attention-seeking of the ancient and spitz breeds group is different from the scores of any otherbreed group with presumed modern European origins. Some studies suggested that even hand-reared
wolves did not show attachment-like behavior like dogs21,33; therefore, the unique characteristic of thisbreed group may be one of the remnants of wolves’ behavioral characteristics and may be very informative of understanding the dog domestication processes. “Attachment” in C-BARQ is defined by owners’
responses to questions concerning the dog’s tendency “to follow members of the household from room
to room about the house,” “to sit close to or in contact with a member of the household when that individual is sitting down,” “to nudge, nuzzle, or paw a member of the household for attention when that
individual is sitting down,” and “to become agitated when a member of the household shows affection
for another person or animal.” Considering the history of these breeds, it seems unlikely that dogs in
the ancient and spitz breed group were selected for low degrees of attachment and attention seeking.
Rather, it is more likely that the capacity to form attachments for humans was an important componentof the evolution of modern dogs. Furthermore, we believe that the cluster analysis of the attachment
and attention-seeking traits, in which all five breeds in the ancient and spitz breeds groups clustered ina small tightly clustered group of 10 breeds, clearly separated from the 36 other breeds, supports ourinterpretation that low levels of attachment and attention-seeking are a distinctive behavioral characteristic of the ancient and spitz breed groups. The five other breeds that clustered with the ancient and spitz
breeds for attachment and attention-seeking may have developed lower levels of attachment secondarily
as adaptations for hunting independently of human guidance. We also investigated the influence of thetwo different grouping methods, of vonHoldt et al.8 and Parker et al.11 on the low attachment tendency
in the ancient and spitz breed group. The low attachment tendency in the ancient and spitz breed groupwas stable for both grouping methods.
Although some of the breeds in the ancient and spitz group have practical functions such as pulling
sleds (e.g. Siberian husky), their C-BARQ scores for attachment and attention-seeking are not different
from the other breeds in the ancient and spitz group. This may be because they are primarily motivatedto run in groups without formal training or the need to attend to or follow instructions from a humanhandler1. We also need to be careful about interpreting the close relationship of these breeds to wolves
in the cladogram because there may be an influence of recent crossing with wolves.
Previous discussions of the behavioral changes associated with the domestication of the dog havetended to emphasize the role of selection for the trait of “tameness” (i.e. loss of fearful or aggressive
responses toward humans)34,35. However, a previous study of species differences in behavior towards
humans between hand-reared dogs and wolf pups also revealed that even wolves that have been intensively socialized do not show the same levels of attachment behavior towards humans that dogs do 33. Thissuggests that in addition to tameness, dogs may acquired high levels of attachment and attention-seeking
behavior toward humans during the domestication process. In a famous series of experiments involving
farmed foxes (Vulpes vulpes), individuals with low aggressive-fearful reactions to humans were selectively
bred for over forty generations. This led to a unique population of foxes that also gradually showed highattachment behaviors, such as actively seeking contact with humans, tail-wagging in anticipation of social
contact, licking experimenters’ faces and hands, and following them like dogs36. Considering the resultsof our study, together with the results of such experiments, we believe that one of the earliest stages ofdog domestication may have involved selection for not only low aggressive-fearful tendencies in ancestral
wolves toward humans, but also the early development of human-directed attachment behavior.
Despite their low attachment and attention-seeking tendencies, the aggressive and fearful reactions
towards humans were relatively low in the ancient and spitz breed group. It is possible that domestication
may have involved a two-stage process, with selection for low aggressive and fearful tendencies occurring in the first stage, and selection for prosociality (attachment and attention-seeking) occurring later,
perhaps in association with the development of more specialized working roles. As a result, the ancientand spitz breeds may retain the low aggressive and fearful tendencies associated with stage 1, but lack thestrong prosocial traits associated with stage 2 and more modern breeds of dog. Viewed in this light, theaggressiveness toward humans characteristic of toy dogs may be a secondary development concomitantwith their small body size which renders them less of a threat to humans. Or it may be that toy breeds
are less adequately socialized by their owners. This association between small body size and aggression
in dogs confirms the findings of previous studies17.
The findings of the present study, namely that the ancient and spitz breed group shows the lowest
attachment levels and is significantly different from other breed groups, confirms the idea that selective
processes may have taken place during domestication on genetic changes affecting the attachment system, and that the consistently low attachment levels found in this group of breeds may be a remnant of an
earlier stage of dog evolution. Since we could not fully describe the contribution of environmental factors
to these observed breed differences in behavior, future investigations will need to take into account thepossible effects of breed-specific environmental influences.

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Acknowledgements
This work was financially supported by MEXT*-Supported Program for the Strategic Research Foundation
at Private universities, 2011–2015, and Grant-in-Aid for Scientific Research on Innovative Areas No.
25118007 “The Evolutionary Origin and Neural Basis of the Empathetic Systems”.

Author Contributions
A.T., M.N. and T.K. wrote the main manuscript text and A.T. prepared Fig. 1 and supplementary Figures
1–3. T.K., M.N. and H.O. conducted statistical analysis and prepared all tables. T.K., K.M. and J.A.S.
collected data and prepared experimental design. All authors reviewed the manuscript.

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Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Tonoike, A. et al. Comparison of owner-reported behavioral characteristics
among genetically clustered breeds of dog (Canis familiaris). Sci. Rep. 5, 17710; doi: 10.1038/srep17710(2015).
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FULL PAPER

Ethology

Comparison of behavioral characteristics of dogs in the United States and Japan
Miho NAGASAWA1,2), Shunichi KANBAYASHI2, Kazutaka MOGI2), James A. SERPELL3) and Takefumi KIKUSUI2)*
1)The Department of Physiology, Jichi Medical University, 3311–1 Yakushiji, Shimotsuke, Tochigi 329–0498, Japan
2)The Department of Animal Science and Biotechnology, Azabu University, 1–17–71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa

252–5201, Japan
3)School of Veterinary Medicine, University of Pennsylvania, 3900 Delancey Street, Philadelphia, PA 19104–6010, U.S.A.

(Received 28 April 2015/Accepted 14 September 2015/Published online in J-STAGE 27 September 2015)
This study examined the difference in dog owning between Japan and the United States, and the effect of these differenceson dogs’ behavioral characteristics. Behavioral evaluations of privately-owned dogs were obtained by using online questionnaire. We
compared background and demographic information from the two countries and analyzed the effects of these differences on behavioral
characteristics in dogs. The results indicated that there was a bias in the dog breeds kept in Japan compared to the United States and thatJapanese dogs’ body weight was lower than the US dogs. The main source of dog acquisition was pet stores in Japan and breeders and/or
shelters in the United States. Multiple linear regression analysis found that Japanese dogs showed more aggression to household members
and higher energy, restlessness and fear of non-social stimuli than US dogs, while US dogs showed more fear of unfamiliar persons,separation-related behavior and excitability. US dogs also showed higher levels of trainability and attachment to owners. The lower dog’s
body weight was, the higher the behavioral scores except for trainability were. When dogs that were obtained under 3 months of age wereanalyzed, the younger the dogs were when their owners obtained them, the higher the scores on some behavioral problem factors w ere. The
higher rates of problem behaviors among Japanese dogs compared with US dogs suggest that the preference for small breed dogs an d poor

ABSTRACT.

KEY WORDS:

behavioral characteristics, canine, C-BARQ, questionnaire
doi: 10.1292/jvms.15-0253; J. Vet. Med. Sci. 78(2): 231–238, 2016

It is estimated that there are 10,346,000 pet dogs in Japan[7]. Japanese dogs are treated as “family members” andprovided with the kind of socialization and training more
typical of western countries, such as the United States. On
the other hand, as dogs have become increasingly integrated
into human habitats, behavioral problems have also become
more apparent, and these behaviors can sometimes lower thedog owners’ quality of life.
In order to solve and prevent these behavior problems,
evaluation methods have been developed to help understanddogs’ behavioral characteristics [4, 8, 18, 21, 23]. The Canine Behavioral Assessment and Research Questionnaire(C-BARQ) is designed to provide dog owners and professionals with standardized evaluations of canine temperamentand behavior [6]. The C-BARQ is also used in Japan [11, 17]
after examination of the validity of questionnaire items [16].
The goal of the present study was to use C-BARQ evaluations of dogs from Japan and the United States to investigate
whether cultural differences in dog ownership between

the two countries may be important for understanding
variation in dog behavior. First, there are differences in the
typical household living environment. Japanese homes are
generally smaller than in the United States, making someparticular small breed dogs more desirable in Japan. Second, the practice of dog acquisition from breeders is not yet
typical among Japanese dog owners, and it is still common
to acquire dogs from pet stores. A recent study in the United
tend to display higher rates of behavioral problems as adults
than those acquired from non-commercial breeders [14]. The
Japanese tendency to acquire puppies from pet stores may
therefore result in higher rates of undesirable behavior thanin the US. In light of these differences, we hypothesized thatJapanese and American dogs would display consistent differences in the prevalence of particular behavior problems.
The second goal of this study was to examine factors thatthe behavioral characteristics of adults in human and nonhuman animals, including dogs [1, 2, 5, 12, 19, 25]. Therefore,
we focused primarily on those factors related to experiences
in youth.

of their dogs. A number of general differences between*CORRESPONDENCE TO: K IKUSUI, T., Department of Animal Scienceand Biotechnology, Azabu University, 1–17–71 Fuchinobe, Chuoku, Sagamihara, Kanagawa 252–5201, Japan.
e-mail: kikusui@azabu-u.ac.jp©2016 The Japanese Society of Veterinary Science

MATERIALS AND METHODS
Questionnaire: Behavioral data in the present study werecollected from dog owners using the C-BARQ, which includes 100 items that ask owners to indicate how their dogs
have responded “in the recent past” to a variety of common

This is an open-access article distributed under the terms of the Creative
Commons Attribution Non-Commercial No Derivatives (by-nc-nd)
License <http://creativecommons.org/licenses/by-nc-nd/3.0/>.

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M. NAGASAWA ET AL.

events and stimuli using a series of 0–4 rating scales (seesupplemental data). The C-BARQ is a standardized questionnaire designed to assess the prevalence and severity of
behavioral problems in dogs. The validation and reliabilityof C-BARQ have been described elsewhere [3, 6, 20]. The
original C-BARQ was translated into Japanese by 2 behavioral professionals and corrected by 2 professors.
Subjects: C-BARQ data were collected via the freely
accessible websites http://www.cbarq.org (US, from April
2006 to November 2012) and http://cbarq.inutokurasu.jp/(JPN, from September 2010 to November 2012). Before
answering the questionnaire, dog owners were asked to provide information about their dogs, such as its breed, age, sex,
neuter status, body weight, age when acquired, where acquired and the presence of any health problems. The online
survey was advertised via articles in newspapers, magazines,
online news and so on, in each country. The C-barq database
was used for different purpose [24].
Statistical analyses: Demographic data on the dogs were
2 tests and Kruskalanalyzed by Mann-Whitney U
ference was found in a Kruskal-Wallis test, we conducted
Mann-Whitney U-tests for multiple comparisons, applying
the Bonferroni correction.
Data from the completed questionnaires were subjected
to factor analysis. Kaiser’s eigenvalue rule was used todetermine the number of interpretable factors that could be
extracted, and varimax rotation was used to identify empirical groupings of items that measured different behavioral

data of response rates <75.0% were also excluded, leaving
2,789 (54.61%) and 10,389 (71.74%) completed questionnaires that could be used in analyses in Japan and the UnitedStates, respectively.
Demographic data: There were 113 and 194 pure breeds
represented, respectively, in the Japanese and US data, andthere were mixed or unknown breeds in the both countries.
In Japan, approximately half of all dogs belonged to themost popular 9 breeds, of which the only large breed was theLabrador Retriever. In the United States, the most popular 27
breeds accounted for 50% of all dogs (Supplementary Table1). The median age of the dogs was 3.23 ± 1.46 years inJapan and 3.00 ± 1.50 years in the United States. There was
the 2 countries. However, the proportions of dogs that were2 (1)=226.85,
P<0.001) with a greater proportion of dogs in the UnitedStates being neutered/spayed (JPN: 63.8%, US: 77.8%;
U=12467505, P<0.001). The body weights of dogs in Japan± 3.75 kg, US: 24.00 ± 9.50 kg; U=4911742, P<0.001). The
lower in Japan than in the United States (JPN: 8.00 ± 8.00
weeks; US: 12.00 ± 22.00 weeks; U=13310688, P<0.001).
The proportion of sources from which dogs were acquired
most common answer was “pet stores” (40.7%), and thesecond was “breeders” (25.3%), while “breeders” (42.4%)
States. This information is shown in Table 1a.
For the Factor Analysis, we selected 59 breeds that werecommon to both countries and then matched the samples forsex and number of dog of each breed in order to eliminate
any breed or sex biases (n=1,234 each, Supplementary Table
ferent in the 2 countries, but the dogs’ body weights in Japan
were lower than in the United States even after standardization of the numbers and sex ratios of each breed (JPN: 8.0 ±
4.5, US: 9.0 ± 5.5; U=691119.5, P<0.001). Dog’s age when
acquired in Japan was younger than in the United States(JPN: 10 ± 3.5 weeks, US: 12 ± 21.5 weeks; U=591855.5,
P<0.001). The proportion of sources from which dogs wereacquired showed the same tendencies as the analysis abovein both countries (Table 1b). The median body weights oftion in both countries (except for “Bred by owners”, “other”

internal consistency (reliability) of extracted factors; this cofocuses on a single idea or construct. For comparison of thefactors, we calculated the average of item scores composing
each factor, which was analyzed as a factor score. The factor
scores were analyzed by multiple linear regression analysis
and Mann-Whitney U-tests to investigate the association
with the demographic characteristics of the dogs. Results are
expressed as medians ± interquartile ranges (SPSS v.19.0,
SPSS Japan Inc., IBM company, Tokyo, Japan), and theresults of post-hoc tests were described by the corrected Pvalues.
RESULTS
There were 5,107 C-BARQ questionnaires completed inJapan. Dogs that were <1 or >7 years of age or had severeor chronic health problems were excluded, leaving a totalof 2,933 completed questionnaires (57.43%) that were considered valid. The response rates for each of the 78 itemsin the questionnaire ranged from 39.22 to 99.86% (median,
97.48%, mode, 98.84%). Among the 14,481 questionnaires
ments above (72.51%). The response rates for each of the
100 items in the questionnaire ranged from 81.57% to99.72% (median, 97.85%, mode, 98.04%). Fifteen itemswith response rates <85.0% either in Japanese or US data
were excluded for further analyses. In each dog case, thelower body weight than dogs from friends or relatives, breeders and shelters. In the United States, dogs acquired fromand shelters. The median ages of dogs when acquired were
In both countries, dogs from shelters and adopted as strays
breeders and pet stores. However, in the United States, dogs
from breeders. This information is summarized in Table 2.
Factor analysis: Sixty-three of the questionnaire items

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COMPARISON OF CHARACTERISTICS OF DOGS
Table 1.

The source from which dogs were acquireda. All dogs
Bred byowner

Friend or
relative

Breeder

Pet store

Shelter

Stray

Other

N/A

Total

(n)
(%)

22
0.8

237
8.5

707
25.3

1,136
40.7

194
7.0

32
1.1

70
2.5

391
14.0

2,789
100.0

The United States (n)
(%)

519
5.0

716
6.9

4,407
42.4

308
3.0

3,488
33.6

463
4.5

488
4.7

0
0.0

10,389
100.0

Total

541
4.1

953
7.2

5,114
38.8

1,444
11.0

3,682
27.9

495
3.8

558
4.2

391
3.0

13,178100.0

Breeder

Pet store

Shelter

Stray

Other

N/A

Total

389
31.5

575
46.6

32
2.6

10
0.8

30
2.4

94
7.6

1,234
100.0

Japan

(n)
(%)

2 (7)=4,086.466, P<0.001.

b. Dogs after breed- and sex-matched selection for factor analysis
Bred by
owner
6
0.5

Friend or
relative
98
7.9

The United States (n)
(%)

52
4.2

110
8.9

596
48.3

73
5.9

304
24.6

34
2.8

65
5.3

0
0.0

1,234
100.0

Total

58
2.4

208
8.4

985
39.9

648
26.3

336
13.6

44
1.8

95
3.8

94
3.8

2,468
100.0

Japan

(n)
(%)

(n)
(%)

2 (7)=713.144, P<0.001.

Table 2.

The comparison of dog’s current body weight and age when acquired among the source in acquisition
Friends or relatives

Breeders

Pet stores

Shelters

Japan
Body weight
Dog’s age when acquired

8.00 ± 8.25 a)
10.00 ± 7.50 a)

9.00 ± 8.88 a)
9.00 ± 5.88 a)

6.00 ± 2.50 b)
11.00 ± 3.50 a)

The United States
Body weight
Dog’s age when acquired

8.00 ± 7.00
12.00 ± 25.50 a)

10.00 ± 9.38 a)
9.00 ± 5.88 b)

7.00 ± 2.50 b)
8.00 ± 4.50 b)
10.00 ± 4.00 a,b) 78.00 ± 67.50 c)

Stray

9.00 ± 7.25 a)
10.50 ± 6.50
89.00 ± 68.75 b) 116.00 ± 82.38 b)
9.00 ± 5.88
77.00 ± 48.00 c)

2 (4)P-value

60.71291.008

<0.01
<0.01

20.161392.48

<0.01
<0.01

the United States, Age when acquired: Friends or relatives vs. Stray in Japan, P<0.05.

common to the 2 countries were analyzed by factor analysis
in each country to compare the behavioral characteristics ofthe 2 countries, and these items were sorted into 16 factors.
After removing the items with factor loadings of <0.4, theremaining items were analyzed by factor analysis again andyielded 12 factors that accounted for 55.47% of the common
variance in item scores. Eleven factors were found to haveing factors were extracted: Aggression to unfamiliar persons(F1), Fear of unfamiliar persons (F2), Separation-related behavior (F3), Aggression to household members (especiallyresource guarding) (F4), Energy and restlessness (F5), Fearof non-social stimuli (F6), Fear of unfamiliar dogs (F7),
Trainability (F8), Aggression to unfamiliar dogs (F9), Excitability (F10), Attachment and attention-seeking (F11), andAggression to persons passing near the house (F12). These
results are shown in Table 3. We compared these results withthe previous study (11 factors) [16] and found that 8 factors (F2, F3, F4, F6, F7, F8, F9 and F11) were almost thesame as the previous analysis. Other 2 factors in the previous

study split respectively into 2 factors (F1 and F12, and F5and F10). The other factor in the previous study was chasing, however, the questionnaire items about chasing did notmeet the criterion of response rate and were excluded in thisanalysis.
Multiple linear analyses of factor scores: We examinedvariables on C-BARQ factor scores using multiple linear
regression analysis. We analyzed each factor score using thefollowing 7 explanatory variables: country, sex, spay/neuter
status, owner’s experience of dog-ownership, the source inacquisition of dogs, dog’s age and body weight at the timeof evaluation, and dog’s age when acquired. Regarding thesource in acquisition of dogs, the cases acquired from breeders were set as the baseline, and the standardized partial
baseline. In addition, we limited dog’s age when acquired
under 3 months of age and reanalyzed the same explanatory
-

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Table 3.

M. NAGASAWA ET AL.
Factor loading of questionnaire items constituting each factor
Factorloading

Factors & questionnaire items
Aggression to unfamiliar persons
When approached directly by an unfamiliar adult while being walked or exercised on a leash
When an unfamiliar person tries to touch or pet the dog
When an unfamiliar person approaches the owner or a member of the owner’s family away from home
When approached directly by an unfamiliar child while being walked or exercised on a leash
Toward unfamiliar persons visiting the home
When an unfamiliar person approaches the owner or a member of the owner’s family at home
When mailmen or other delivery workers approach the home
Fear of unfamiliar persons
When approached directly by an unfamiliar male adult while away from the home
When an unfamiliar person tries to touch or pet the dog
When approached directly by an unfamiliar child while away from the home
When unfamiliar persons visit the home
Separation-related behavior
Barking when left or about to be left on its owner
Whining when left or about to be left on its owner
Howling when left or about to be left on its owner
Excessive salivation when left or about to be left on its owner
Loss of appetite when left or about to be left on its ownerAggression to members of the household (especially related to possession)
When food is taken away by a member of the household
When approached directly by a member of the household while it is eating
When a member of the household retrieves food or objects stolen by the dog
When toys, bones or other objects are taken away by a member of the household
When verbally corrected or punished by a member of the household
Energy and restless
Playful, puppyish, boisterous
When a member of the household returns home after a brief absence
Active, energetic, always on the go
When visitors arrive at its home
Restlessness, agitation or pacing when left or about to be left on its owner
Fear of non-social stimuli
In response to wind or wind-blown objects
In response to strange or unfamiliar objects on or near the sidewalk
In response to sudden or loud noises
During thunderstorms
Fear of unfamiliar dogs
When approached directly by an unfamiliar dog of the same or larger size
When barked, growled or lunged at by unfamiliar dog
When approached directly by an unfamiliar dog of a smaller size
Trainability
Obeys a stay command immediately
Obeys a sit command immediately
Seems to attend to or listen closely to everything the owner says or does
Returns immediately when called while off leash
Aggression to unfamiliar dogs
When approached directly by an unfamiliar male dog while being walked or exercised on a leash
When approached directly by an unfamiliar female dog while being walked or exercised on a leash
When barked, growled or lunged at by unfamiliar dog
Excitability
Just before being taken on a car trip
Just before being taken on a walk
When playing with you or other familiy member of your house
Displays a strong attachment for one particular member of the household
Attachment and attention-seeking
Tends to nudge, nuzzle or paw a member of the household for attention when that individual is sitting down
Tends to sit close to or in contact with a member of the household when that individual is sitting down
Tends to follow a member of household from room to room about the house
Becomes agitated when a member of the household shows affection for another person
Aggression to persons passing near the house
When strangers walk past the home while the dog is in the yard
When joggers, cyclists, roller skaters or skateboarders pass the home while the dog is in the yard

227

Eigenvalue

Contribution
ratio

9.367

9.798

0.918

4.218

5.375

0.900

3.838

5.055

0.804

3.314

5.009

0.723

2.275

4.861

0.841

2.142

4.432

0.758

1.887

4.114

0.883

1.734

4.028

0.800

1.537

4.026

0.897

1.370

3.443

0.678

1.255

3.368

0.705

1.016

2.239

0.877

0.837
0.803
0.788
0.752
0.713
0.664
0.599
0.823
0.770
0.752
0.693
0.736
0.711
0.616
0.490
0.476
0.405
0.831
0.772
0.689
0.642
0.401
0.709
0.685
0.526
0.489
0.462
0.699
0.677
0.664
0.478
0.427
0.810
0.759
0.755
0.769
0.731
0.622
0.609
0.853
0.841
0.641
0.680
0.640
0.433
0.422
0.710
0.656
0.532
0.473
0.662
0.599

235

COMPARISON OF CHARACTERISTICS OF DOGS

F1 Aggression to unfamiliar
persons

Country a)
Sex b)
Neutered status c)
Dog–ownership experience d)
The source in acquisition e)
Bred by owner
Friend or relative
Pet store
Shelter
Stray
Body weight
Dog’s age at evaluation
Dog’s age when acquired
Adj R2F4 Aggression to house member

Acquired under
3 months of age

All

Acquired under
3 months of age

All

Acquired under
3 months of age

All

Acquired under
3 months of age–0.031–0.058 **0.031–0.012–0.043–0.072 **0.014–0.0200.127 **
0.065 **
–0.016–0.0020.089 **
0.039
–0.022–0.0240.249 **
–0.0300.033–0.093 **0.267 **
–0.0270.009–0.105 **–0.633 **–0.0190.015–0.025–0.655 **–0.0200.009–0.020–0.0130.108 **
0.058 *
0.094 **
0.053 *
–0.160 **0.014–0.079 **0.046 **

–0.060 *0.135 **
0.086 **
0.053 *
0.020
–0.181 **0.035–0.104 **0.071 **

0.003
–0.001
0.023
0.051 *
0.003
–0.178 **–0.0380.054 *
0.063 **

–0.010
0.006
0.052
0.037
–0.012–0.182 **–0.028–0.067 *0.043 **

–0.0110.039
0.040
0.096 **
0.078 **
–0.148 **–0.057 **–0.0320.112 **

–0.060 *
0.043
0.053
0.091 **
0.047
–0.179 **–0.040–0.092 **0.130 **

–0.0040.048 **
0.057 **
0.043 *
0.038 *
–0.023–0.145 **–0.056 **0.461 **

–0.0390.057 **
0.040
0.023
0.007
–0.033–0.137 **–0.061 **0.465 **

All

Acquired under
3 months of age–0.165 **–0.082 **0.041 *
–0.039–0.0270.047 *
0.158 **
0.059 *
0.034
–0.106 **–0.022–0.0320.099 **

F6 Fear for non-social objects
All

Acquired under
3 months of age–0.187 **–0.078 **0.025–0.046–0.103 **0.027–0.048 *–0.045 *–0.111 **0.021
0.149 **
0.024
–0.004–0.144 **–0.034–0.147 **0.127 **

–0.028–0.0270.048–0.0160.017–0.139 **0.048 *
0.044
0.048 **

F9 Aggression to
unfamiliar dogs

Country a)
Sex b)
Neutered status c)
Dog–ownership experience d)
The source in acquisition e)
Bred by owner
Friend or relative
Pet store
Shelter
Stray
Body weight
Dog’s age at evaluation
Dog’s age when acquired
Adj R2F3 Separation- related anxiety

All

F5 Energy & restless

Country a)
Sex b)
Neutered status c)
Dog–ownership experience d)
The source in acquisition e)
Bred by owner
Friend or relative
Pet store
Shelter
Stray
Body weight
Dog’s age at evaluation
Dog’s age when acquired
Adj R2F2 Fear for unfamiliar persons

F7 Fear for unfamiliar dogs

F8 Trainability

All

Acquired under
3 months of age

All

Acquired under
3 months of age–0.132 **0.038–0.033–0.057 *0.007
0.089 **
–0.044 *–0.019–0.007
0.096 **
–0.015–0.0190.153 **
0.036
–0.063 **0.083 **

0.148 **
0.057 *
–0.0390.091 **

–0.050–0.0350.060 *
0.024
0.002
–0.149 **0.064 *
–0.0270.063 **

–0.014–0.0110.086 **
0.020
–0.015–0.136 **0.005–0.0050.038 **

–0.051–0.0110.110 **
0.011
–0.030–0.154 **0.007–0.0570.052 **

–0.023–0.037–0.153 **–0.063 *–0.0290.156 **
0.105 **
–0.091 **0.113 **

–0.005–0.041–0.145 **–0.0130.010
0.164 **
0.095 **
0.045
0.107 **F10 Excitability

F11 Attachment andattention-seeking

F12 Aggression to passers
around house

All

Acquired under
3 months of age

All

Acquired under
3 months of age

All

Acquired under
3 months of age

All

Acquired under
3 months of age

0.035–0.078 **–0.014–0.0240.039–0.077 **–0.029–0.0200.255 **
–0.049 *0.077 **
–0.044 *0.270 **
–0.0370.080 **
–0.0290.395 **
–0.0160.021
0.017

0.395 **
–0.0240.017
0.021

0.077 **
–0.0220.029–0.0020.070 *
–0.0220.019–0.0010.007
0.076 **
0.029
0.105 **
0.053 *
–0.078 **0.115 **
–0.055 *0.034 **

–0.0180.113 **
0.072 *
0.036
0.016
–0.079 **0.135 **
–0.072 *0.043 **

–0.0090.047 *
0.048 *
0.058 *
0.037
–0.053 **0.012–0.093 **0.066 **

–0.046
0.041
0.046
0.045
0.001
–0.063 *0.027–0.070 *0.075 **

–0.041 *
0.023
0.036
0.058 *
0.036
–0.068 **–0.079 **–0.0180.156 **

–0.094 **
0.018
0.016
0.016
0.023
–0.094 **–0.061 *–0.075 **0.149 **

0.005
0.083 **
0.045
0.066 *
0.029
–0.133 **0.014–0.096 **0.027 **

–0.016
0.109 **
0.053
0.026
–0.010–0.161 **0.035–0.0480.035 **

1/ Not acquired from there: 0.
factor score compared to this baseline. *P<0.05, **P<0.01.

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236

M. NAGASAWA ET AL.

sources from which dogs were acquired in the United States
were breeders and shelters. This might be due to the easy
access to animal shelters as a source for acquiring dogs in
the United States. The ratio of neutered dogs was higher in
the United States than in Japan, suggesting a cultural difference in attitudes to surgical sterilization between the twoence more properly. The results are shown in Table 4. In thecase that all data were included, all of the factor scores except
in F4, F5 and F6 indicated that dogs in Japan tended todisplay higher factor scores than dogs in the US. Sex differences explained F1, F2, F5, F7, F9 and F10, and male dogs
tended to obtain higher factor scores than female dogs on F1,
F5, F9 and F10. Neuter/Spay status explained F5, F6, F7, F8
and F10, and intact dogs showed higher scores on F6, F7 andF8. Previous experience of dog-owning explained F3, F6, F8
and F10, and the score of F8 was higher when owners had
previous dog-owning experience. In the source in acquisition of dogs, acquisition from a shelter explained 10 factors(except for F6 and F7). The case of friend or relative andpet store explained 6 factors respectively (Friend or relative:
F1, F4, F5, F9, F10 and F12, pet store: F1, F4, F5, F7, F8
and F10). These factor scores except for F8 were higher incomparison to the acquisition from breeders. Body weight
explained all factors except F4, and the lower dog’s bodyweights were, the higher their factor scores were except for
F8. Age at evaluation explained F3, F4, F6, F8, F9 and F11,and older dogs obtained higher factor scores for F6, F8 andF9. Age when dogs were acquired explained F1, F2, F4, F8,
F9, F10 and F12, and the younger the dogs were when theirowners obtained them, the higher their scores on these factors except for F2 were. In the case of only dogs that wereobtained under 3 months of age (n=1,516), the source in acquisition of dogs which explained the factors mostly is frompet stores (F1, F5, F6, F7, F8 and F9), of which factor scores
except for F8 were higher in comparison to the acquisition
from breeders. Dogs from friend or relative showed highest
scores in the factors related with aggression (F1, F4, F9 andF12). Age when dogs were acquired explained the factor
scores except for F6, F7, F8 and F12, and the younger thedogs were when their owners obtained them, the higher theirscores on these factors were.

in the United States than in Japan. This result may be due tothe larger proportion of dogs acquired from shelters in the
US sample.
After breed- and sex-matched selection for Factor Analysis, we compared the demographic information again. We
body weight differences remained despite breed-matched selection between two countries. Second, with respect to dog’s
tween dogs from breeders and those from pet stores in eachcountry. Considering the time-lag from weaning to acquisition from pet stores [15], dogs from pet stores may be takenfrom their litters earlier than dogs from breeders. Third, thethan that from breeders in each country, suggesting that
small breeds were acquired mainly from pet stores.
In order to investigate which demographic variables affected the dog’s behavioral characteristics, we analyzed
C-BARQ factor scores using multiple linear regression
analysis. We found that the variable ‘country’ affected 9 outof 12 factors. Dogs in Japan showed the more aggression
toward household members, more energy and restlessness,and more fear of non-social stimuli than dogs in the UnitedStates. On the other hand, dogs in the United States showed
more fear of unfamiliar persons, more separation-related
behavior and greater excitability, while showing also higherscores for trainability and attachment and attention-seeking.
Further research will be needed to identify the reasons forthese differences in behavior between the two countries.
In all sources in acquisition of dogs except for “bred byowner”, most factor scores were higher than those of breeder(except for trainability). The source in acquisition of dogs
that explained the factors mostly was from pet stores when
dog’s age when acquired was limited under 3 months of age,although that was from shelter in the case of all data. The
factor scores except for trainability of dogs from pet stores
were higher than from breeders, suggesting the possibility
that the early developmental experience was not appropriate in pet stores. In addition, it is notable that acquisition
from friend or relative was strongly related to aggression.
We found also that dog’s body weight correlated with mostbehavioral characteristics, indicating that small breed dogs
tended to display a greater potential for problem behaviors

DISCUSSION
Using an online behavioral evaluation system, we collected data on the behavioral characteristics of dogs in Japanand the United States, and investigated differences between
the 2 countries. First, we compared background and demographic information for the dogs using all valid data. The
results showed that there was a bias in the dog breeds thatare chosen by the owners in Japan compared to the UnitedStates, which has a wide variety in the breeds that are comlower than those of US dogs even after standardization
for the number and sexes of each breed, indicating both aJapanese preference for small-sized dogs and a tendency for
US dogs to be heavier irrespective of breed and sex. From ahistorical perspective, most breeds were produced in Europe
and the United States, while there were few breeds native
to Japan; lack of diversity of breeds in Japan might bringa bias towards owning particular breeds. Japanese owners
commonly acquire their dogs from pet stores, while the mainfrom a previous study that found that dogs’ skull shape,
height and bodyweight co-vary with their behavior [13]. The
We hypothesized that dog’s age at time of acquisition was
related to their behavioral characteristics. Multiple linear
regression analysis in this study did not show a large effect229

COMPARISON OF CHARACTERISTICS OF DOGSof age of acquisition on factor scores, because these data
included dogs that were acquired in adulthood. Analysis
which limited dogs that were acquired under 3 months ofage revealed that the effect of age of acquisition on factor
scores became higher than the analysis which included all
dogs in most factors. This suggests that the earlier the dogs
were acquired in their developmental period (that is, dogs
were separated from their mother too early), the worse the

3.

4.

5.

models, it has been demonstrated that the maltreatment ofpups in early developmental periods, such as early weaning,

6.

that early weaning caused exposure to high levels of glucocorticoids in the central nervous system of mouse pups [9].
These can increase the stress response of pups and decreasenutritional factors in the central nervous system, leading toexcessive anxiety and aggression and decreased learning
capacity, among other effects [10]. From these previous
studies, the current data suggested that the high fear, highaggression, high excitability and low trainability found indogs from pet stores were related to early weaning. In addition, Slabbert and Rasa [22] found that dogs that had been
separated from their mothers and nest sites (but not theirlittermates) at 6 weeks of age were prone to exhibit loss ofappetite and weight and increased distress, mortality andsusceptibility to disease compared to dogs that had remainedat the nest sites with their mothers until 12 weeks of age.
It can be inferred that while it is important for dogs to be
exposed to human society during the early developmental
period for socialization, very early mother-infant separation
could cause adverse effects in dogs during adulthood.
In conclusion, the comparison of demographic data andbehavioral characteristics of dogs between Japan and theUnited States, using an online questionnaire, showed thatJapanese owners were more likely to have acquired theirdogs from pet stores and had preferences for a few particular small breeds. We also found a greater potential for thedevelopment of problem behaviors in dogs in Japan compared with the United States. The Japanese preference foracquiring small breed dogs from pet stores may have been
responsible for these negative behavioral effects, but further
research is needed to clarify the precise causal mechanism(s) involved.

7.

8.

9.

10.

11.

12.

13.

14.

15.

ACKNOWLEDGMENT
ported by MEXT*-Supported Program for the Strategic
Research Foundation at Private universities.
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麻布大学雑誌

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Akiko TONOIKE1, Go TERAUCHI1, Miho INOUE-MURAYAMA2,
Miho NAGASAWA1, Kazutaka MOGI1, Takefumi KIKUSUI1
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Graduate School of Veterinary Sciences, Azabu University,
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Wildlife Research Center, Kyoto University, 2-24 Tanaka-Sekiden-cho, Sakyo, Kyoto 606-8203, Japan1
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236

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 年Fig. 1 Genotype frequency and Allele frequency for the SNPs, rs8679682 and rs22896457, in wolves and dogs.
Genotype frequency and allele frequency for the SNPs rs8679682 and rs22896457, in wolves and dogs and in dog
breed groups (Ancient and spitz, Toy, Spaniels, Scent hounds, and Poodles, Sight hounds and herding, Retrievers,
Small terriers and Mastiﬀ-like) were compared. The frequencies were signiﬁcantly diﬀerent for both SNPs between
wolves and dogs and also among dog breed groups (p<0.05, chi-square test). (a) Genotype frequency of rs8679682(b) Genotype frequency of rs22896457 (c) Allele frequency of rs8679682 (d) Allele frequency of rs22896457

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237

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Acknowledgements   'SFFENBO ")  (SPOBV  *  4DIXFJ[FS  3. 7FDDIZP %0%  )BO  &  4JMWB  1.  (BMBWFSOJ  .  'BO  ; 

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SHORT COMMUNICATION

doi: 10.1111/age.12344

Copy number variations in the amylase gene (AMY2B) in Japanesenative dog breeds
A. Tonoike*, Y. Hori†, M. Inoue-Murayama†, A. Konno†, K. Fujita†, M. Miyado‡, M. Fukami‡,
M. Nagasawa*§, K. Mogi* and T. Kikusui**Companion Animal Research, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252-5201, Japan. †Department of
Psychology, Kyoto University, Sakyo, Kyoto 606-8501, Japan. ‡Department of Molecular Endocrinology, National Research Institute forChild Health and Development, Tokyo 157-8535, Japan. §Department of Physiology, Jichi Medical University, Shimotsuke, Tochigi 3290498, Japan.

Summary

A recent study suggested that increased copy numbers of the AMY2B gene might be a
crucial genetic change that occurred during the domestication of dogs. To investigate
AMY2B expansion in ancient breeds, which are highly divergent from modern breeds ofpresumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy
numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those inwolves. However, compared to a group of various modern breeds, Akitas had fewer copy
numbers, whereas Shibas exhibited the same level of expansion as modern breeds.
Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shibathat has been bred to maintain their appearance resembling ancestors of native Japanese
dogs and that originated in the same region as the Akita, were lower than those in the
Shiba. These differences may have arisen from the earlier introduction of rice farming to theregion in which the Shiba originated compared to the region in which the Akita andthe Jomon-Shiba originated. Thus, our data provide insights into the relationship between
the introduction of agriculture and AMY2B expansion in dogs.
Keywords agriculture, AMY2B expansion, ancient breeds, domestication, Shiba, wolf

Recently, whole-genome resequencing in dogs and wolves
has identiﬁed genomic regions that were under selection
during dog domestication. From these analyses, researchers
found that copy numbers of the AMY2B locus increased byseveral fold in dogs compared to wolves (Axelsson et al.
2013). The AMY2B gene encodes a protein with amylase
activity that is crucial to the digestion of starch-rich diets,
and studies have suggested that increased AMY2B copy
numbers allowed ancestors of dogs to thrive on starch-richdiets. This ﬁnding also suggested that the development ofagriculture might have catalysed dog domestication. Previous phylogenetic studies in dogs and wolves revealed thatbreeds of dogs are genetically distinct and that some breeds
with Asian and African origins are highly divergent frommodern breeds of presumed European origins (Parker et al.
2004; vonHoldt et al. 2010). Despite these previous studies,

Address for correspondence
K. Mogi, Companion Animal Research, School of Veterinary Medicine,
Azabu University, Sagamihara, Kanagawa 252-5201, Japan.
E-mail: mogik@azabu-u.ac.jp
Accepted for publication 02 July 2015580

it is still not known whether ancient breeds have only a few
AMY2B copy numbers, as is the case in wolves. Moreover,the inﬂuence of regional agricultural histories on AMY2B
expansion is unclear.
Japan is an island country located in the Paciﬁc Ocean;
the western end of Japan is close to the Korean peninsula. It
is believed that rice farming was brought to Japan from the
Korean peninsula along with human migration in the
fourth to third century BC, with expansion then occurringto the north-east regions (Hudson 1999). Japanese native
dog breeds, the Shiba and the Akita, have been classiﬁed as
ancient breeds (Parker et al. 2004; vonHoldt et al. 2010).
The Shiba is believed to have originated in the western partof Japan (from Shimane to Nagano prefectures), whereas
the Akita is believed to have originated in the north-east
region (Akita prefecture) (Tanabe 1991). Here, we compared variations in AMY2B copies among Shibas, Akitas, agroup of various modern breeds and wolves. We also
analysed a unique line of Shibas, known as the JomonShiba, which originated in the eastern part of Japan.
Compared to the Shiba, Jomon-Shibas have skulls withmore acute orbital angles and larger teeth, resembling dog
skulls excavated from archaeological sites belonging to the© 2015 Stichting International Foundation for Animal Genetics, 46, 580–583

240

AMY2B copy numbers in Japanese dog breeds
Jomon period (14 500–300 BC) in Japan (Tanabe 1991).
The breeding of Jomon-Shibas has been strictly controlled
by the Japan Society for Preservation of the NaturalMonument Shiba-Dog, founded in 1959, through the use ofmorphological characteristics, such as a shallower stop andlarger teeth when compared to the Shiba, as indexes(Fig. 1a,b).
Blood samples were collected, and DNA was extracted
from wolves (n = 12; Table 1), Akitas (n = 14), JomonShibas (n = 17), Shibas (n = 17) and dogs of various modern
breeds (n = 25). Variations in AMY2B copy numbers wereanalysed by Multiplex TaqMan assays, as described by
Axelsson et al. (2013), or standard qPCR assays using SYBR
Premix Ex Taq II (TaKaRa). For the Multiplex TaqMan
assays, reference assays were designed to amplify C7orf28B,
which is present in two copies in the canine genome. The
standard qPCR assays were performed on a Thermal Cycler
Dice Real Time System II (TaKaRa), according to the
manufacturer’s protocol. Relative copy numbers were
calculated according to the DDCt method using C7orf28B
as a reference gene. The results for several samples analysedusing both the methods were comparable. For each sample,
three replications were performed. Statistical analysis was
carried out using Kruskal–Wallis tests with post hoc Mann–
Whitney U-tests with Bonferroni correction (SPSS v. 19.0;
SPSS Japan Inc.). One of the wolf samples was excluded as
result of outlier statistics.
The average AMY2B copy number in wolves was low(mean  standard deviation: 2.7  0.9; Fig. 1c), consistent
with previous studies (Axelsson et al. 2013; Freedman et al.

2014). However, the copy number in Akitas was signiﬁcantly higher (8.1  3.2) than that in wolves but lower
than those of modern breeds (15.0  6.7). When considered together with a recent report showing that the average
AMY2B copy number in the Samoyed, another ancient
breed, was similar to that of the Akita (Arendt et al. 2014),
these data suggest that expansion of AMY2B copy numbers
occurring in ancient breeds was less than that in modern
breeds. We also found that the average copy number in the
Shiba was similar (17.1  4.2) to that of modern breeds(Fig. 1c), despite the observation that Shibas are highly
divergent from modern breeds of presumed European
origins (Parker et al. 2004). This observation may berelated to the agricultural history of Japan, in which rice
farming was introduced earlier to the region where the
Shiba originated than to the region where the Akita
originated. This is supported by another analysis focusing
on other breeds (Freedman et al. 2014), which suggested
that breeds historically bred in areas of intensive agriculture
seem to have more AMY2B copy numbers than do breeds
historically associated with nomadic hunter gatherers. This
also raises the possibility that agriculture accelerated
AMY2B expansion even after the domestication of dogs aswell as during the process of domestication. Interestingly,
the AMY2B copy number in the Jomon-Shiba was significantly lower (11.9  3.6) than that in the Shiba (Fig. 1c).
Because the Jomon-Shiba originated in the eastern part ofJapan, these dogs may have been inﬂuenced less byagriculture than the Shiba was, as was observed with the
Akita. Alternatively, selective breeding to maintain the

(a)

(b)

(c)

Figure 1 Facial views of Jomon-Shiba (a) andShiba (b) from the left angle. AMY2B diploid
copy number analysis (c). Box plot showing
the diploid amylase copy number in wolves(n = 11), Akitas (n = 14), Jomon-Shibas(n = 17), Shibas (n = 17) and other breeds(n = 25). Signiﬁcance was determined using
Mann–Whitney U-tests with Bonferroni correction: **P < 0.01; *P < 0.05.
© 2015 Stichting International Foundation for Animal Genetics, 46, 580–583

241

581

582

Tonoike et al.
Table 1 Diploid copy numbers in individual dogs and wolves.

Table 1 (continued)

Wolf/dog

Diploid copy number

Wolf/dog

Diploid copy number

Wolf 1
Wolf 2
Wolf 3
Wolf 4
Wolf 5
Wolf 6
Wolf 7
Wolf 8Wolf 9Wolf 10
Wolf 11
Wolf 12
Akita 1
Akita 2
Akita 3
Akita 4
Akita 5
Akita 6
Akita 7
Akita 8
Akita 9Akita 10
Akita 11
Akita 12
Akita 13
Akita 14
Jomon-Shiba 1
Jomon-Shiba 2
Jomon-Shiba 3
Jomon-Shiba 4
Jomon-Shiba 5
Jomon-Shiba 6
Jomon-Shiba 7
Jomon-Shiba 8
Jomon-Shiba 9
Jomon-Shiba 10
Jomon-Shiba 11
Jomon-Shiba 12
Jomon-Shiba 13
Jomon-Shiba 14
Jomon-Shiba 15
Jomon-Shiba 16
Jomon-Shiba 17
Shiba 1
Shiba 2
Shiba 3
Shiba 4
Shiba 5
Shiba 6
Shiba 7
Shiba 8
Shiba 9Shiba 10
Shiba 11
Shiba 12
Shiba 13
Shiba 14
Shiba 15
Shiba 16
Shiba 17
Labrador 1
Labrador 2

3
3
5
3
3
2
2
3
2
2
2
10
13
8
10
9
9
6
4
10
12
11
9
6
5
2
18
15
11
11
9
14
12
20
11
10
15
9
6
13
8
10
11
21
21
15
22
12
13
15
19
17
25
19
15
23
11
13
14
16
16
34

Labrador 3
Labrador 4
Labrador 5
Labrador 6
Labrador 7
Labrador 8
Labrador 9
Labrador 10
Labrador 11
Labrador 12
Labrador 13
Labrador 14
Labrador 15
Standard Poodle 1
Standard Poodle 2
Miniature Dachshund 1
Shih Tzu 1
Bolognese 1
Beagle 1
Miniature Dachshund 9 Toy Poodle 1
Miniature Dachshund 9 Toy Poodle 2
Mongrel 1
Mongrel 2

29
10
12
7
12
15
15
10
17
8
15
15
9
14
12
20
15
24
3
14
20
14
16

appearance of the Jomon-Shiba may have prevented the
expansion of AMY2B copy numbers. Further studies are
required to examine these possibilities.
In conclusion, this analysis revealed that AMY2B
expansion occurred in ancient dogs in Japan. The level of
AMY2B expansion in Japanese native breeds was equal toor lower than that of modern breeds. Such differences may
be attributed to the timing of the introduction of agriculturein the regions of origin.

Acknowledgements
This work was supported by JSPS KAKENHI (grant numbers
25118007, 25290082, and 25118005), a research project
grant MEXT*-Supported Program for the Strategic Research
Foundation at Private Universities, a research project grantawarded by Azabu University and the Cooperation Research
Program of Wildlife Research Center, Kyoto University. We
thank Mrs. Maki Kuroi and Dr. Eri Iwata for kindly
providing us dog photos.

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The dilution hypothesis provides an alternative
framework with which to explain observations of
the apparent recalcitrance of DOC and lends aphysiological meaning to the operationally defined “semi-labile” and “semi-refractory” fractions(16, 17). We hypothesize that under the dilution
hypothesis, very heterogeneous mixtures of labile
compounds appear semirefractory, whereas increasingly less diverse DOM assemblages containing larger concentrations of some substrates
will present higher microbial growth and DOC
turnover rates, resulting in increasing degrees ofapparent lability. The microbial generation of apparently recalcitrant material (18) from labile
substrates in a process recently dubbed the “microbial carbon pump” (19) can also be explainedwith the dilution hypothesis. Microbial utilizationof abundant, labile compounds results in hundredsof different metabolites (20), which are subsequently consumed down to the lowest utilizable
concentration. This mechanism explains observations of relatively concentrated, labile materials
being transformed into apparently recalcitrant
matter through microbial consumption (18) but
does not necessarily imply the formation of structurally recalcitrant molecules. Indeed, “recalcitrant”
DOC is not defined structurally, but operationally,as the DOC pool remaining after long experimental incubations or as the fraction transported in
an apparently conservative manner with theocean circulation (1). Thus, the dilution hypothesis
severely limits the feasibility of geoengineering
efforts to enhance carbon storage in the deep
ocean (21) by using the microbial carbon pump.
FT-ICR-MS characterization of DOC from different oceans (13, 14, 22, 23) and also from thisstudy (fig. S5) shows no indication of prevalent,
intrinsically recalcitrant compounds accumulating in substantial amounts. Conversely, FT-ICRMS data show that oceanic DOC is a complex
mixture of minute quantities of thousands of organic molecules, which is in good agreement withthe dilution hypothesis. Mean radiocarbon agesof deep oceanic DOC in the range of 4000 to 6000
years have been considered as evidence for its recalcitrant nature (24, 25). However, these are average ages of a pool containing a mixture of very
old molecules >12,000 years old but also featuring
a large proportion of contemporary materials (26).
Moreover, elevated radiocarbon ages only demonstrate that these old molecules are not being
newly produced at any appreciable rate—becausethat would lower their isotopic age—but does notnecessarily imply that they are structurally recalcitrant. Furthermore, it is unlikely that naturalorganic molecules can accumulate in the ocean in
substantial concentrations and remain recalcitrant
or be preserved for millennia when degradation
pathways for novel synthetic pollutants evolve soonafter these compounds are released in nature (27).
Although there might be a truly recalcitrant component in deep oceanic DOC, our results clearlyshow that the concentration of individual labile
molecules is a major factor limiting the utilization of a substantial fraction of deep oceanic DOC.
These results provide, therefore, a robust andparsimonious explanation for the long-term preSCIENCE sciencemag.org

servation of labile DOC into one of the largest
reservoirs of organic carbon on Earth, opening a
new avenue in our understanding of the global
carbon cycle.
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Matter, D. A. Hansell, C. A. Carlson, Eds. (Academic Press, San
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26. C. L. Follett, D. J. Repeta, D. H. Rothman, L. Xu, C. Santinelli,Proc. Natl. Acad. Sci. U.S.A. 111, 16706–16711 (2014).
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AC KNOWL ED GME NTSThis is a contribution to the Malaspina 2010 Expedition project,funded by the CONSOLIDER-Ingenio 2010 program of the fromthe Spanish Ministry of Economy and Competitiveness (Ref.
CSD2008-00077). J.M.A. was supported by a “Ramón y Cajal”
research fellowship from the Spanish Ministry of Economy andCompetitiveness. E.M. was supported by a fellowship from the Junta
para la Ampliación de Estudios program of CSIC. G.J.H. and R.L.H.
were supported by the Austrian Science Fund (FWF) projects I486B09 and P23234-B11 and by the European Research Council (ERC)
under the European Community’s Seventh Framework Programme(FP7/2007-2013)/ERC grant agreement 268595 (MEDEA project).
We thank A. Dorsett for assistance with DOC analyses, participants in
the Malaspina Expedition and the crews of the BIO Hespérides, and
RV Pelagia and the personnel of the Marine Technology Unit of CSIC
for their invaluable support. Original data sets are available online at
http://digital.csic.es/handle/10261/111563. J.M.A. designed theexperimental setup, carried out part of the experiments, measured
prokaryotic abundance, analyzed the data, and wrote the manuscript.
E.M. carried out part of the experiments and data analysis. C.M.D.
designed the Malaspina 2010 Expedition, was responsible for DOC
analyses, and together with G.J.H. contributed to the design of theexperiments and discussion of results. R.L.H. and T.D. analyzed the
FT-ICR-MS samples. All authors discussed the results and contributed
to the manuscript.
SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/348/6232/331/suppl/DC1
Materials and Methods
Figs. S1 to S9Tables S1 and S2References (28–35)
18 July 2014; accepted 4 March 2015
Published online 19 March 2015;10.1126/science.1258955

SOCIAL EVOLUTION

Oxytocin-gaze positive loop and thecoevolution of human-dog bonds
Miho Nagasawa,1,2 Shouhei Mitsui,1 Shiori En,1 Nobuyo Ohtani,1 Mitsuaki Ohta,1Yasuo Sakuma,3 Tatsushi Onaka,2 Kazutaka Mogi,1 Takefumi Kikusui1*
Human-like modes of communication, including mutual gaze, in dogs may have beenacquired during domestication with humans. We show that gazing behavior from dogs,
but not wolves, increased urinary oxytocin concentrations in owners, which consequently
facilitated owners’ affiliation and increased oxytocin concentration in dogs. Further, nasallyadministered oxytocin increased gazing behavior in dogs, which in turn increased urinaryoxytocin concentrations in owners. These findings support the existence of an interspecies
oxytocin-mediated positive loop facilitated and modulated by gazing, which may havesupported the coevolution of human-dog bonding by engaging common modes ofcommunicating social attachment.

D

ogs are more skillful than wolves andchimpanzees, the closest respective relatives of dogs and humans, at using humansocial communicative behaviors (1). Morespecifically, dogs are able to use mutual
gaze as a communication tool in the context ofneeds of affiliative help from others (2). Conver-gent evolution between humans and dogs may
have led to the acquisition of human-like communication modes in dogs, possibly as a byproduct of temperament changes, such as reduced
fear and aggression (1). This idea yields interesting
implications that dogs were domesticated bycoopting social cognitive systems in humans that244

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Corrected 12 June 2015; see full text.

Corrected 12 June 2015; see full text.

1
Department of Animal Science and Biotechnology, Azabu
University, Sagamihara, Kanagawa, Japan. 2Department of
Physiology, Jichi Medical University, Shimotsuke, Tochigi, Japan.
3
University of Tokyo Health Sciences, Tama, Tokyo, Japan.

*Corresponding author. E-mail: kikusui@azabu-u.ac.jp334

17 APRIL 2015 • VOL 348 ISSUE 6232

100

**

80
60

*

***

60

**

40

20

0

Pre

Post

500

200

*

400

*

300
200
100
0

LG

SG Wolf40
200Dog or wolf’s oxytocin (pg/mg)

Duration (s)

120

***
***

ing behaviors was measured during the interaction: “dog’s gaze at owner (dog-to-owner gaze),”“owner’s talking to dog (dog-talking),” and “owner’s touching of dog (dog-touching).” Dog owners
were assigned to one of two groups: long gazeor short gaze (fig. S1). Wolves were tested withthe same procedure and were compared withthe two dog groups. Dogs in the long-gaze groupgazed most at their owners among the threegroups. In contrast, wolves rarely showed mutual
gazing to their owners (Fig. 1A and fig. S2). Aftera 30-min interaction, only owners in the longgaze group showed a significant increase inurinary oxytocin concentrations and the highest
change ratio of oxytocin (Fig. 1, B and C). The
oxytocin change ratio in owners correlated significantly with that of dogs, the duration of dogto-owner gaze, and dog-touching. Moreover, theduration of the dog-to-owner gaze correlatedwith dog-talking and dog-touching (table S2A);
however, through multiple linear regression analysis, we found that only the duration of dog-toowner gaze significantly explained the oxytocinchange ratio in owners. The duration of dogtouching showed a trend toward explaining
oxytocin concentrations in owners (Table 1A).
Similarly, a significantly higher oxytocin change
ratio was observed in the dogs of the long-gazegroup than in those of the short-gaze group(Fig. 1, D and E). The duration of dog-to-owner
gaze also significantly explained the oxytocinchange ratio in dogs, and the duration of dogtouching showed a trend toward explaining
oxytocin concentrations in dogs by multiple linear regression analysis (Table 1A). In wolves, incontrast, the duration of wolf-to-owner gaze did

The change ratio of owner (%)

140

Owner’s oxytocin (pg/mg)

Further, social interaction initiated by a dog’s gazeincreases urinary oxytocin in the owner, whereas
obstruction of the dog’s gaze inhibits this increase(19). These results demonstrate that the acquisition of human-like social communication improves
the quality of human-dog affiliative interactions,leading to the establishment of a human-dog
bond that is similar to a mother-infant relationship. We hypothesized that an oxytocin-mediatedpositive loop, which originated in the intraspecies exchange of social affiliation cues, acts on
both humans and dogs, is coevolved in humans
and dogs, and facilitates human-dog bonding.
However, it is not known whether an oxytocinmediated positive loop exists between humans
and dogs as has been postulated between motherand infants, and whether this positive loop emerged
during domestication.
We tested the hypothesis that an oxytocinmediated positive loop exists between humans
and dogs that is mediated by gaze. First, we
examined whether a dog’s gazing behavior affected urinary oxytocin concentrations in dogs
and owners during a 30-min interaction. We
also conducted the same experiment using handraised wolves, in order to determine whether thispositive loop has been acquired by coevolution
with humans. Second, we determined whether
manipulating oxytocin in dogs through intranasal administration would enhance their gazing
behavior toward their owners and whether thisgazing behavior affected oxytocin concentrationsin owners.
In experiment 1, urine was collected from thedogs and owners right before and 30 min after
the interaction, and the duration of the follow-

to Do
-o g
wn o
er r w
ga olfze Do
gto or wuc ohi lfng
Do
g
or
ta wo
lki lfng

are involved in social attachment. The development of human-unique social cognitive modes
may depend on specific temperament and social
affiliation changes and may have consequently
evolved differently from those of chimpanzees
and bonobos (3). Thus, although humans anddogs exist on different branches of the evolutionary tree, both may have independently acquired tolerance of one another because ofalterations in neural systems that mediate affiliation (1). These alterations may be related
to paedomorphic characteristics in dogs, whichenabled them to retain a degree of social flexibility and tolerance similar to that of humans(4, 5); therefore, it is plausible that a specific affiliative relationship developed between humans
and dogs despite interspecies differences. This
common social relationship change may haveenabled cohabitation between humans and dogs
and the eventual development of human-likemodes of social communication in dogs.
Gaze plays an important role in human communication. Gaze not only facilitates the understanding of another’s intention but also theestablishment of affiliative relationships withothers. In humans, “mutual gaze” is the mostfundamental manifestation of social attachment
between a mother and infant (6), and maternal
oxytocin is positively associated with the duration of mother-to-infant gaze (7). Oxytocin plays
a primary role in regulating social bonding between mother and infants and between sexual
partners in monogamous species (8, 9). Moreover, activation of the oxytocin system enhances
social reward (10) and inhibits stress-induced
activity of the hypothalamic-pituitary-adrenal
axis (11). It has therefore been suggested thatthese functions may facilitate dyadic interaction,such as an oxytocin-mediated positive loop of
attachment and maternal behaviors between
mother and infant (12, 13): Maternal nurturing
activates the oxytocinergic system in the infant,thus enhancing attachment; this attachment thenstimulates oxytocinergic activity in the mother,which facilitates further maternal behavior (9).
Because the establishment of such an oxytocinmediated positive loop requires the sharing ofsocial cues and recognition of a particular partner, the study of oxytocin-mediated bonding has
been restricted to intraspecies relationships.
The human-dog relationship is exceptional
because it is an interspecies form of attachment.
Dogs can discriminate individual humans (14, 15).
Furthermore, dogs show distinctly different behavior toward caregivers as compared with handraised wolves (14), and interaction with dogs
confers a social buffering effect to humans. Likewise, dogs also receive more social buffering
effects from interacting with humans than fromconspecifics (16). Tactile interaction between
humans and dogs increases peripheral oxytocin
concentrations in both humans and dogs (17, 18).

The change ratio of dog or wolf (%)

R ES E A RC H | R E PO R TS

300

200

100

*** ***

150

**

100
50

Fig. 1. Comparisons of behavior and uri00nary oxytocin change among long gaze
Pre
Post
LG SG Wolf
dogs (LG, n = 8, black bars and circles),short gaze dogs (SG, n = 22, white bars
and circles), and wolves (wolf, n = 11, gray bars and square). (A) Behavior during the first 5-mininteraction. (B) and (D) Changes of urinary oxytocin concentrations after a 30-min interaction.
Urinary oxytocin concentrations in owners (B) and dogs or wolves (D) collected before and after a
30-min interaction are shown. (C) and (E) Comparisons of the change ratio of urinary oxytocin
among LG, SG, and wolf for owners (C) and dogs or wolves (E). The results of (A), (B), and (D) are
expressed as mean T SE. (C) and (E) reflect median T quartile. ***P < 0.001, **P < 0.01, *P < 0.05.

245

sciencemag.org SCIENCECorrected 12 June 2015; see full text.

(A) Experiment 1
Oxytocin change ratio
Owners–0.107–0.264

†

†

0.321

**
200

100

Duration of Proximity (sec)

300

1500

100

50

0

OW
UP
Male dogs

OW
UP
Male dogs

OW
UP
Female dogs

OW
UP
Male dogs

OW
UP
Female dogs

400

80

***
60

40

*** ***

20*
300

Owners of male dogs / oxytocin
Owners of male dogs / saline
Owners of female dogs / oxytocin
Owners of female dogs / saline

200

100

0

0

Pre

Owners of Owners ofmale dogs female dogs

Post
200

Dogs

Dog’s sex
0.090
0.138
Oxytocin
0.202
0.234
administration
Dog-to-owner gaze
0.458** 0.030
Dog touching owner –0.040 –0.054
Proximity to owner
0.048 –0.023
R
0.574
0.275
Adjusted R2
0.248 –0.046
P
0.005
0.686
Sex: Female = 1, male = 0; oxytocin administration:oxytocin = 1, saline = 0.

The change ratio of dog (%)

0.388*
0.575
0.247
0.020

Oxytocin change ratio

SCIENCE sciencemag.org

500

0.335

(B) Experiment 2

Owners

1000

0

OW
UP
Female dogs

150

0.458*
0.619
0.306
0.008

duration of gazing behavior significantly explained the oxytocin change ratio in owners(Table 1B). Thus, oxytocin administration enhances the gazing behavior of female dogs, which
stimulates oxytocin secretion in their owners.
Conversely, when interaction from humans waslimited, no significant difference in urinary oxytocin concentrations in dogs was observed after
the interaction in either the oxytocin or thesaline conditions, and no significant oxytocinchange ratio was found in dogs (Fig. 2, F and
G). These results thus suggest that, althoughoxytocin administration may enhance dog gazing
behavior and lead to an oxytocin increase inowners, limited owner-to-dog interaction may
prevent the increased oxytocin secretion in dogs
by breaking the oxytocin-mediated positive loop.

150

Duration of Touch (sec)

Duration of Gaze (sec)

***
**

**

0

Dog’s oxytocin (pg/mg)

Owner talking
to dogOwnertouching dog
Dog-to-owner gazeR
Adjusted R2
P

Dogs

400

The change ratio of owner (%)

Table 1. Results of multiple linear regression
analysis of oxytocin change ratio and behavioral variables in owners and dogs. *P < 0.05,†
P < 0.1; R, multiple correlation coefficient;**, P < 0.01 .

touch the dog voluntarily. Urine samples fromthe owner and the dog were collected before andafter the interaction and were later compared.
The total amount of time that the dog gazed at,
touched, and was close to the owner and theunfamiliar participants was also measured.
Oxytocin administration to dogs significantly
increased the duration that the dog gazed at theowner in female dogs but not male dogs (Fig.
2A). Further, urinary oxytocin concentration significantly increased in the owners of female dogs
that received oxytocin versus saline, even thoughoxytocin was not administered to the owners (Fig.
2D). No significant effect of oxytocin administration was observed in the other measured dog
behaviors (Fig. 2, B and C). Furthermore, multiple linear regression analysis revealed that the

Owner’s oxytocin (pg/mg)

not correlate with the oxytocin change ratio ineither owners or wolves, and wolf-to-owner gaze
did not explain the oxytocin change ratio inowners and wolves (tables S2B and S3). These
results suggest that wolves do not use mutual
gaze as a form of social communication withhumans, which might be expected because wolves
tend to use eye contact as a threat among conspecifics (20) and avoid human eye contact (21).
Thus, dog-to-owner gaze as a form of social communications probably evolved during domestication and triggers oxytocin release in the owner,
facilitating mutual interaction and affiliative
communication and consequently activation ofoxytocin systems in both humans and dogs in apositive loop.
In experiment 2, we evaluated the direct evidence of whether oxytocin administration enhanced dog gazing behavior and the subsequent
increase in urinary oxytocin concentration inowners. This experiment involved 27 volunteers
and their dogs, and participants unfamiliar tothe dogs. A solution containing oxytocin or saline
was administered to the dog and the dog thenentered the experimental room, where the ownerand two unfamiliar people were seated (fig. S4).
Human behavior toward dogs was restricted toprevent the influence of extraneous stimuli on dog
behavior and/or urinary oxytocin concentration.
They were forbidden to talk to each other or to

RESEAR CH | R E P O R T S

100

50

150

Male dog / oxytocin
Male dog / saline
Female dog / oxytocin
Female dog / saline

100

50

0

0

Pre

Post

Male dogs Female dogs

Fig. 2. Comparisons of behavior and urinary oxytocin between oxytocin and saline treatment
conditions. (A) to (C) The effects of oxytocin administration on dog behaviors. Panels show the mean
duration of dogs’ gaze at participants (A), touching participants (B), and time spent in the proximity ofless than 1 m from each participant (C). Black and white bars indicate, respectively, oxytocin- and saline
treatment conditions. OW, owner; UP, unfamiliar person. (D) to (G) Change in urinary oxytocin concentrations after a 30-min interaction after oxytocin or saline administration. Urinary oxytocin concentrations of owners (D) and dogs (F) before and after a 30-min interaction are shown for oxytocin andsaline groups. The change ratio of urinary oxytocin in owners (E) and dogs (G) is compared between
male and female dogs. ***P < 0.001, **P < 0.01, *P < 0.05. The results of (A) to (D) and (F) are
expressed as mean T SE. (E) and (G) reflect median T quartile.

246

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R ES E A RC H | R E PO R TS

Interestingly, oxytocin administration only
increased mutual gaze duration in female dogs,
whereas sex differences were not observed inexperiment 1, which did not include unfamiliar
individuals. Sex differences in the effects of intranasal oxytocin have been reported in humans
as well (22), and it is possible that females are
more sensitive to the affiliative effects of oxytocinor that exogenous oxytocin may also be activating the vasopressin receptor system preferentiallyin males. Oxytocin and the structurally related
vasopressin affect social bonding and aggression
in sexually dimorphic manners in monogamous
voles (8, 9), and oxytocin possibly increases aggression (23, 24). Therefore, the results of experiment 2 may indicate that male dogs were attending
to both their owners and to unfamiliar people
as a form of vigilance. The current study, despite
its small sample size, implies a complicated rolefor oxytocin in social roles and contexts in dogs.
In human infants, mutual gaze represents
healthy attachment behavior (25). Human functional magnetic resonance imaging studies showthat the presentation of human and canine family members’ faces activated the anterior cingulate cortex, a region strongly acted upon byoxytocin systems (26). Urinary oxytocin variation in dog owners is highly correlated with thefrequency of behavioral exchanges initiated by
the dogs’ gaze (19). These results suggest thathumans may feel affection for their companion
dogs similar to that felt toward human family
members and that dog-associated visual stimuli,
such as eye-gaze contact, from their dogs activate
oxytocin systems. Thus, during dog domestication, neural systems implementing gaze communications evolved that activate the humans’ oxytocin
attachment system, as did gaze-mediated oxytocin release, resulting in an interspecies oxytocinmediated positive loop to facilitate human-dog
bonding. This system is not present in the closest
living relative of the domesticated dog.
In the present study, urinary oxytocin concentrations in owners and dogs were affected by the
dog’s gaze and the duration of dog-touching. In
contrast, mutual gaze between hand-raised wolves
and their owners was not detected, nor was there
an increase of urinary oxytocin in either wolves ortheir owners after a 30-min experimental interaction (experiment 1). Moreover, the nasal administration of oxytocin increased the total amount oftime that female dogs gazed at their owners and,in turn, urinary oxytocin concentrations in owners(experiment 2). We examined the association between our results and early-life experience withhumans in dogs and wolves in order to test thepossibility that our results were due to differencesin early-life experience with humans. The results
did not indicate a significant association between
the animals’ early-life experiences with humans
and the findings of the current study (see thesupplementary methods). Moreover, there wereno significant differences between dogs in thelong-gaze group and wolves in either the durationof dog/wolf-touching and dog/wolf-talking, suggesting that the shorter gaze of the wolves was
not due to an unstable relationship. These re336

17 APRIL 2015 • VOL 348 ISSUE 6232

Corrected 12 June 2015; see full text.

sults support the existence of a self-perpetuatingoxytocin-mediated positive loop in human-dog
relationships that is similar to that of humanmother-infant relations. Human-dog interaction
by dogs’ human-like gazing behavior brought onsocial rewarding effects due to oxytocin releasein both humans and dogs and followed the
deepening of mutual relationships, which led tointerspecies bonding.
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AC KNOWL ED GME NTSThis study was supported in part by the Grant-in-Aid for Scientific
Research on Innovative Areas (No. 4501) from the Japan Society for
the Promotion of Science, in Japan. We thank all human and canine
participants, Howlin' Ks Nature School, U.S. Kennel, R. Ooyama andN. Yoshida-Tsuchihashi from Azabu University, and Drs. Kato and
Takeda from University of Tokyo Health Sciences. We are also gratefulto Cody and Charley for their significant contributions. The analyzed
data are included in the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/348/6232/333/suppl/DC1
Materials and Methods
Figs. S1 to S5
Tables S1 to S4
References (27 –30)
Movies S1 to S3
Data Tables 1 and 2
9 September 2014; accepted 3 March 201510.1126/science.1261022

PLANT ECOLOGY

Anthropogenic environmental
changes affect ecosystemstability via biodiversity
Yann Hautier,1,2,3* David Tilman,2,4 Forest Isbell,2 Eric W. Seabloom,2Elizabeth T. Borer,2 Peter B. Reich5,6
Human-driven environmental changes may simultaneously affect the biodiversity, productivity,and stability of Earth’s ecosystems, but there is no consensus on the causal relationships
linking these variables. Data from 12 multiyear experiments that manipulate important
anthropogenic drivers, including plant diversity, nitrogen, carbon dioxide, fire, herbivory, andwater, show that each driver influences ecosystem productivity. However, the stability of
ecosystem productivity is only changed by those drivers that alter biodiversity, with a given
decrease in plant species numbers leading to a quantitatively similar decrease in ecosystemstability regardless of which driver caused the biodiversity loss. These results suggestthat changes in biodiversity caused by drivers of environmental change may be a major factor
determining how global environmental changes affect ecosystem stability.

Human domination of Earth’s ecosystems,
especially conversion of about half of the
Earth’s ice-free terrestrial ecosystems into
cropland and pasture, is simplifying ecosystems via the local loss of biodiversity(1, 2). Other major global anthropogenic changes
include nutrient eutrophication, fire suppression

247

and elevated fire frequencies, predator decimation, climate warming, and drought, which likely
affect many aspects of ecosystem functioning,
especially ecosystem productivity, stability, andbiodiversity (1, 3–7). However, to date there has
been little evidence showing whether or how thesethree ecosystem responses may be mechanisticallysciencemag.org SCIENCESHORT COMMUNICATION
Communicative & Integrative Biology 8:3, e1017157; May/June 2015; Published with license by Taylor & Francis Group, LLC

Intranasal administration of oxytocin promotessocial play in domestic dogs
Teresa Romero1,2,*, Miho Nagasawa3, Kazutaka Mogi3, Toshikazu Hasegawa1, and Takefumi Kikusui3
1

Department of Cognitive and Behavioral Sciences; Graduate School of Arts and Science; The University of Tokyo; Tokyo, Japan; 2Japanese Society for the Promotion of Sciences;
Tokyo, Japan; 3Department of Animal Science and Biotechnology; Azabu University; Kanagawa, Japan

Downloaded by [Azabu University] at 22:17 18 February 2016

Keywords: companion animals, domestic dogs, oxytocin, social play, social bonds

In a recent paper,1 we examined whether oxytocin in the domestic dog modulates the maintenance of close social
bonds in non-reproductive contexts. We found that exogenous oxytocin promotes positive social behaviors not only
toward conspeciﬁcs, but also toward human partners. Here we examined in further detail the effect that oxytocin
manipulation has on social play. When sprayed with oxytocin, subjects initiated play sessions more often and played for
longer periods of time than when sprayed with saline. Furthermore, after oxytocin nasal intake dogs displayed playsignals more often than after saline administration, suggesting that oxytocin enhances dogs’ play motivation. To ourknowledge, this study provides the ﬁrst evidence that oxytocin promotes social play in the domestic dog. We use theseresults to hypothesize on the potential therapeutic use of oxytocin for promoting social behaviors and treating social
deﬁcits in the domestic dog.

Over the past several decades, extensive animal research has
shown that the hypothalamic neuropeptide oxytocin plays animportant role in the regulation of several behaviors associatedwith sociality, such as sexual behavior, pair-bonding, parental
care, peer recognition, and social memory (for reviews see
refs.2,3). More recent studies in humans have revealed that oxytocin is also involved in aspects of social cognition including social
perception, emotion recognition, sensibility to the experiences ofothers, and pro-social behaviors (for reviews see refs.4,5).
Building on these ﬁndings, researchers have suggested that the
manipulation of the oxytocin system may be used as a tool for
improving socio-cognitive abilities in individuals with social deﬁcit disorders (e.g. autism spectrum disorder, social anxiety disorder, and borderline personality disorder; for reviews see refs.6,7).
In fact, in the last decade the effect of administered oxytocin has
been tested in clinical populations, with several of these studies
reporting beneﬁcial effects of oxytocin on social attention andemotion recognition in autistic individuals, and a reduction ofsocial anxiety in patients with social phobia and borderline personality disorder (e.g., refs.8-11). While at this point muchremains unknown about the lasting effects of these beneﬁts andthe extent to which personality and personal history mitigate
these beneﬁcial effects, such ﬁndings are clearly grounds for optimism about the therapeutic potential of oxytocin.
A similar therapeutic use of oxytocin could be relevant to animal
health. This clinical approach could be particularly interesting forcompanion animals showing behavioral problems related to social
deﬁcits. Each year, millions of dogs and cats arrive in animal shelters
after abandonment, abuse, or relinquishment by their previous
caregivers. The most widely reported reasons for relinquishment ofdogs in USA are behavioral problems due to lack of proper socialization and habituation (i.e. fear and aggression directed to strangers
or other dogs).12,13 Although in many countries there are approved
drugs for the treatment of anxiety related disorders in domestic animals,14 to date there is no drug for the treatment of social deﬁcits.
Thus, the development of pharmacotherapies that promote social
integration in companion animals may be helpful in the treatment
of selected behavior problems.
In a recent paper,1 we showed that nasal intake of oxytocin
promotes social bonding in domestic dogs. A total of 16 adultdogs from different breeds participated as subjects in a randomized placebo-controlled experiment (females D 8; male D 8;
mean age 6.1 y (SEM D 0.7)). Each dog received a nasal spray of
100ml of oxytocin (40 IU, Peptide institute, Japan) or 100ml ofsaline solution, depending on the testing condition. All subjects
received both conditions and each condition was carried out on
different days. After spray intake, dogs stay in the experimentalroom (11.5 £ 6.5m) with their owners and a familiar dog partner, and their behaviors were video recorded for 60 minutes.
Owners were instructed to sit quietly in the experimental roomand not to actively interact with their dogs; while dogs could
move freely in the room (for further details, see1). We found that

© Teresa Romero, Miho Nagasawa, Kazutaka Mogi, Toshikazu Hasegawa, and Takefumi Kikusui*Correspondence to: Teresa Romero; Email: tromero@darwin.c.u-tokyo.ac.jp
Submitted: 11/13/2014; Revised: 01/06/2015; Accepted: 01/09/2015
http://dx.doi.org/10.1080/19420889.2015.1017157
This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The
moral rights of the named author(s) have been asserted.

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the animals. Recent evidence shows that through play animals
reduce tension around stressful situations,27 or turn a stranger
into a familiar individual.28 Furthermore, play is frequently usedas part of therapies to correct behavioral problems in dogs.29
Thus, the combination of behavioral interventions with pharmacotherapies that promote afﬁliation in general and play in particular might be a potential fruitful strategy for the treatment of
behavior problems in companion animals.
In the present study, all dogs that participated as partners had afriendly relationship with the subjects. Thus, we did not observe
any agonistic behavior during the experimental sessions and noneof the play sessions resembled or escalated into aggression. A playsession started when an individual directed a play signal (including
play bow, face paw, and exaggerated approaches and retreats18,21,30)
or any playful behavior (including nipping, inhibited bite, playchasing, mounting, play-ﬁghting, and play-tackling18,21,30) toward
the partner, and ended when their activity ceased or one of the subjects moved away (>1 m). A new play session was recorded if at
Figure 1. Duration of play bouts during the oxytocin and the saline conditions. The box plots represent the median and upper and lower quarleast 5 seconds elapsed between the end of the ﬁrst bout and the starttiles; and the whiskers indicate the values within 1.5 times theof the new bout. Only play sessions that lasted more than 5 seconds
interquartile range. *P < 0.05
were included in the analysis (N D 74). All subjects but one wereinvolved in at least one social play session (mean duration in secoxytocin caused dogs to engage in higher levels of afﬁliation and onds D 161.3 § 52.6 SE), and only one subject engaged in solitary
approach with their dog partners, suggesting that the experimen- play. Thus, solitary play sessions were excluded from the analyses.
tal intranasal administration of oxytocin in dogs promotes afﬁlia- The type of treatment dogs were administered with did not affecttive motivation and facilitates bond formation and maintenance.
how often subjects were involved in social play (Wilcoxon signed
Here, we analyze in more detail the episodes of social play that rank test: z D ¡1.749, N D 15, p D 0.08), nor how often they
took play during the experimental sessions in order to address the engaged in play after receiving a play invitation (z D 0.479, N D 15,
question of whether intranasal administration of oxytocin enhan- p D 0.6384). However, after oxytocin nasal spray intake, dogs initices social play in dogs. Play is a highly plastic and versatile ated play sessions signiﬁcantly more often than after saline treatbehavior that generally occurs when animals are free from envi- ment (z D ¡1.997, N D 15, p D 0.046). We then examined theronmental and social stressors.15 Although play is characteristic total amount of time dogs spent playing with their partners. Weof early life stages, in many species social play continues into found that oxytocin treatment was associated with longer play sesadulthood (e.g. rodents,16 canids,17,18 primates19,20). Social play sions (z D ¡2.040, N D 15, p D 0.041, Fig. 1).
is thought to help animals to develop social and emotional ﬂexiGiven that social play is an interaction that requires the active
bility, conﬂict resolution skills and appropriate reactions to unex- involvement of both partners, the total time that 2 individuals playpected situations.15,21-24 It could also serve to assess social together depends on both individuals’ motivation. Thus, we evalurelationships and/or increase social afﬁnity between individuals. ated the subject’s relative role in maintaining the play session by
19,25,26
Social play is also associated with immediate beneﬁts for examining the production of play signals that are known to promote and/or facilitate play interactions.18,31 We did not ﬁnd any effect oftreatment type on the frequency of playsignals received (z D ¡0.255, N D 15, p
D 0.799, Fig. 2A). However, after oxytocin nasal intake dogs directed play signals
to their play-mates signiﬁcantly more
often than after saline treatment (z D
¡2.090, N D 15, p D 0.037, Fig. 2B).
These ﬁndings suggest that the experimental administration of oxytocin had
positive effects on dogs’ motivation tointeract in a playful way with conspeciﬁcs.
Play has long been identiﬁed as a potential
welfare indicator,15 a facilitator of socialFigure 2. Frequency of play signals received (A) and given (B) during the oxytocin and the salineemotional learning,22 and an implicitly
experimental conditions. The box plots represent the median and the upper and lower quartiles; andthe whiskers indicate the values within 1.5 times the interquartile range. *P < 0.05.
rewarding behavior.32 Thus, increasing

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play interactions could be beneﬁcial for dogs. Previous results1 haveprovided behavioral evidence that exogenous oxytocin promotes
positive social behaviors in the domestic dog. The present study
extends this data showing that oxytocin manipulation also enhances
play motivation, suggesting that this pharmacological intervention
may be used to help manage a range of animal behavioral problems.
The reader should note, however, that the positive effects of oxytocin on social approach, general afﬁliation1 and play (this study)
were observed in healthy dog participants. Hence, a key question iswhether administration of oxytocin can inﬂuence social behaviors
in animals with behavioral disorders. Caution is therefore warrantedin the interpretation of our results, and further studies on whether
and how oxytocin inﬂuences social interactions in clinic populations are clearly necessary.
In summary, this study provides the ﬁrst evidence that administration of oxytocin increases dogs’ motivation to play with conspeciﬁcs and reﬁnes our knowledge about the behavioral effects ofexogenous oxytocin in dogs. A more thorough understanding of
this peptide and its behavioral consequences in healthy individuals
may help to design pharmacological interventions aimed at promoting social behavior in companion animals whose social skills
have been compromised, such as dogs with poor socialization at
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Disclosure of Potential Conﬂicts of Interest

No potential conﬂicts of interest were disclosed.
Acknowledgments

We thank the dogs and their owners for their participation in
the study. We are also grateful to Darby Proctor for useful comments on a previous version of the manuscript.
Funding

The present study was approved by the Ethics Committee of
Azabu University (Japan) (No. 130304–2). This research was
supported by the JSPS Research Fellowships for Foreign
Researchers (No. P10311) and the MEXT Grant-in-aid for Scientiﬁc Research (No. 26380981) to T. R., the MEXT Grant-inaid for Challenging Exploratory Research (No. 23650132) to T.
H., and the Grant-in-Aid for Scientiﬁc Research on Innovative
Areas (No. 4501) to T.K. and T. H.

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ᙜึࡣࠊࣅ࣮ࢢࣝ≟ࡢ➨୍࢚ࢡࢯࣥ࠾ࡼࡧ࢖ࣥࢺࣟࣥࢆྵࡴᗈ࠸㡿ᇦࢆࢥ࣮ࢻࡍࡿ 71)$㑇ఏᏊゎᯒࢆ 3&5 ἲ࡟࡚ᐇ᪋ࡋࡓࠋࡑࡢ⤖ᯝࠊྠ୍≟✀࡟࡟ࡶ࠿࠿ࢃࡽࡎሷᇶ㛗ࡢ␗࡞ࡿࣂ
ࣥࢻࡀ᳨ฟࡉࢀࣁࣉࣟࢱ࢖ࣉࡀᏑᅾࡍࡿࡶࡢ࡜ண᝿ࡉࢀࡓ ᅗ  ࠋࡇࡢணഛ◊✲ࡢゎᯒ⤖251ᯝࢆ௨࡚ࠊ௚≟✀࡟࠾࠸࡚ࡶྠᵝࡢሷᇶ㛗ࡢኚ໬ࡀ࠶ࡿࡇ࡜ࢆண᝿ࡋ 71)$ 㑇ఏᏊゎᯒࢆᐇ᪋ࡋࡓࠋ◊✲ᑐ㇟࡜ࡋ࡚ࠊࣅ࣮ࢢࣝࠊࢳ࣡࣡ࠊ࣑ࢽࢳࣗ࢔ࢲࢵࢡࢫࠊࢺ࢖ࣉ࣮ࢻࣝࠊࢥ࣮ࢠ࣮࠾ࡼ
ࡧᰘ≟ࠊྛ  㢌௨ୖ࠿ࡽ࡞ࡿྜィ  㢌ࡢ '1$ ヨᩱࢆ⏝࠸ࡓࠋ➨㸯࢖ࣥࢺࣟࣥෆࡢ 715 ከᆺ ࢆ ุ ᐃ ࡍ ࡿ ࡓ ࡵ ࡟ ࠊ ⣙  ሷ ᇶ ᑐ ࡢ ⏘ ≀ ࡀ ᚓ ࡽ ࢀ ࡿ 71)$675)  ̓
**$$*$7*&7&$7**$77*&7̓ ࡜ 71)$6755 ̓7$&&&$&$&&&&$&$7&7&7̓ ࣉࣛ࢖࣐
࣮ࢆタィࡋࡓ ᅗ  ࠋ3&5 ⏘≀ࡣ 㸣࢔࣮࢞ࣟࢫࢤ࡛ࣝ㟁ẼὋື࠾ࡼࡧ⺯ග≀㉁ )$0 ࡛ᶆ㆑ࡋ*HQHWLFDQDO\]HUࢆ⏝࠸ࡓ㑇ఏᏊᆺุᐃ࡟౪ࡋࡓࠋ
⤖ᯝ࠾ࡼࡧ⪃ᐹㄪᰝࡋࡓ  ಶయ࠿ࡽࡍ࡭࡚ࡢ≟✀࡟࠾࠸࡚ ᅇ㸪 ᅇ㸪 ᅇ㸪 ᅇᆺࡢ  ᑐ❧㑇ఏᏊࡀ᳨ฟࡉࢀࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊ࢖ࢾ 71)$ 㑇ఏᏊࡢ➨㸯࢖ࣥࢺࣟࣥ࡟ *$$7 ࢆ཯᚟༢఩࡜ࡍࡿ
WHWUDQXFOHRWLGHUHSHDWV௨ᚋ 715 ࡀᏑᅾࡋࠊ⧞ࡾ㏉ࡋᩘከᆺࡀᏑᅾࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓ ᅗ  ࠋࡇࡢ≉ᚩⓗ࡞㓄ิࡣࠊ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺ࡜࿧ࡤࢀ┿᰾⏕≀ࡢࢤࣀ࣒ୖ࡟ࡣࠊ
࠸ࡓࡿᡤ࡟ᵝࠎ࡞ᩘࡢ཯᚟ '1$ 㓄ิࡀᏑᅾࡍࡿࠋྠ୍ಶయࡢࢤࣀ࣒ෆ㒊࡛࠶ࡗ࡚ࡶ཯᚟ᅇᩘ
ࡣሙᡤࡈ࡜࡟␗࡞ࡾࠊಶయ㛫࡛ࡶ␗࡞ࡿࠋࡇࡢ✀ࡢ཯᚟㓄ิࡢ࠺ࡕ a ሷᇶᑐࢆ཯᚟༢఩࡜
ࡍࡿࡶࡢࢆ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺ཯᚟㓄ิ࡜࠸࠸㸦▷ሷᇶ⦪ิ㓄ิ675 ࡜ࡶ࠸࠺㸧ࠊ࢖ࣥࢺࣟࣥ࡟Ꮡᅾࡋ࡚࠸ࡿࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊࣅ࣮ࢢࣝ✀࠿ࡽࡣ  ᅇᆺ࠿ࡽ  ᅇᆺࡲ࡛඲࡚ࡢᑐ❧㑇ఏᏊࡀ᳨ฟࡉࢀࡓ
ࡀࠊ௚ࡢ  ရ✀࠿ࡽࡣ  ᅇᆺࡣ᳨ฟࡉࢀ࡞࠿ࡓࠋ࣊ࢸࣟ᥋ྜᗘࡢᮇᚅ್㸦+H㸧࠾ࡼࡧከᆺ᝟
ሗ㔞 3,& ࡣࠊရ✀࡟ࡼࡗ࡚ࠊࡑࢀࡒࢀ ࡜ ࡢ㛫࡛ᕪ␗ࡀㄆࡵ
ࡽࢀࡓࠋࡇࡢ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺ࣐࣮࣮࢝ࡣࠊከᆺᛶ࡟ᐩࡴࡤ࠿ࡾ࡛࡞ࡃྠ᫬࡟ᆺุᐃࡀᐜ
࡛᫆࠶ࡿࡓࡵࠊ࢖ࢾ 71)$ 㑇ఏᏊࡢࣛࣥࢻ࣐࣮ࢡ࡜ࡋ࡚ࠊఏ㐩୙ᖹ⾮ࢸࢫࢺ࡞࡝ࡢ⑌ᝈ┦㛵ゎᯒ࡟฼⏝ྍ⬟࡛࠶ࡿࠋ
ᮏ◊✲ࡢ⤖ᯝ࠿ࡽ≟ࡢ 71)$ 㑇ఏᏊ࡟࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺࡀぢฟࡉࢀࠊࡇࢀࡽࡀච␿௓ᅾᛶ⑌ᝈࡸ⭘⒆⑌ᝈࡢ⑓ែⓎ⏕ࡸデ᩿ἲࡢ☜❧࡟ᐤ୚ࡍࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋ
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ච␿௓ᅾᛶ⑌ᝈ࠾ࡼࡧ⭘⒆⑌ᝈ࡟࠾ࡅࡿ 71)1 ࡢ㑇ఏᏊࡢゎᯒஂᮎṇᬕ㸦㯞ᕸ኱Ꮫෆ⛉Ꮫ➨஧㸧◊✲せ᪨࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺࡣࠊ≟ࡢච␿௓ᅾᛶ⾑ᾮ⑌ᝈ࠾ࡼࡧ⭘⒆ᛶ⑌ᝈࡢデ᩿ᛂ⏝࡟᭷ຊ࡜⪃࠼᥈⣴ࢆ⾜ࡗࡓࠋච␿௓ᅾᛶ⑌ᝈ࡛ࡣࠊ᭱⤊ⓗ࡟  ౛ࡢ≟ࡢච␿௓ᅾᛶ⾑ᑠᯈῶᑡ࡛ゎᯒࢆᐇ᪋ࡋࡓࡀࠊ≉␗ⓗ࡞ 613 ࡢゎᯒ࡟ࡣ⮳ࡽ࡞࠿ࡗࡓࠋࡉࡽ࡟ࠊࣜࣥࣃ⭘࠾ࡼࡧᝏᛶ㯮Ⰽ⭘ࡢィ  ౛࡟࠾࠸࡚ 71)$ 㑇ఏᏊࡢ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺゎᯒࢆᐇ᪋ࡋ࡚࠸ࡓࡀࠊኚ␗ࡣぢฟࡔࡍࡇ࡜ࡣᅔ㞴࡛≉␗ⓗ࡞ 613 ࡢゎᯒ࡟ࡣ⮳ࡽ࡞࠿ࡗࡓࠋᮏ◊✲࡛ࡣࠊ≟ࡢ௚ࡢච␿௓ᅾᛶ⾑ᾮ⑌ᝈ࠾ࡼࡧ⭘⒆ᛶ⑌ᝈ࡟ࡘ࠸࡚ゎᯒ୰࡛㐩ᡂᗘࡣ 㸣࡛࠶ࡗࡓࠋࡇࡢཎᅉ࡜ࡋ࡚ࠊච␿௓ᅾ
ᛶ⾑ᾮ⑌ᝈࡣࡑࡶࡑࡶẕ㞟ᅋࡀᑡ࡞ࡃࠊ࠿ࡘ☜ᐃデ᩿せ௳ࢆ‶ࡓࡍࡶࡢࡀᑡ࡞ࡗࡓࡇ࡜ࡀᣲ
ࡆࡽࢀࡿࠋࡲࡓ⭘⒆ᛶ⑌ᝈ࡟࠾࠸࡚ࡣࠊ஧ḟデ⒪᪋タ࡟᮶㝔ࡍࡿࣜࣥࣃ⭘࠾ࡼࡧᝏᛶ㯮Ⰽ⭘ࡢ᳨యᩘࡀῶᑡഴྥ࡛࠶ࡿࡇ࡜ࡶᣲࡆࡽࢀࡿࠋࡇࡢࡼ࠺࡟ゎᯒᩘࡀࡈࡃᑡᩘ࡛࠶ࡿࡓࡵࠊኚ
␗ࡢ᭷↓ࢆ☜ㄆ࡛ࡁ࡞࠸ࡇ࡜࡜ࣉࣛ࢖࣐࣮ࡢタィ⮬య࡟ၥ㢟ࡀ࠶ࡗࡓྍ⬟ᛶࡀ࠶ࡾࠊ௒ᚋ᳨
ウࡍࡿᚲせࡀ࠶ࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋ
◊✲┠ⓗ๓㏙ࡋࡓࡼ࠺࡟≟࡛ࡣච␿௓ᅾᛶ⑌ᝈ࠾ࡼࡧ⭘⒆⑌ᝈࡢ⮫ᗋ౛ࡀከࡃᏑᅾࡍࡿࠋච␿௓ᅾ
ᛶ⾑ᑠᯈῶᑡ⑕ ,07 ࡣࠊ≟࡛ẚ㍑ⓗከⓎࡍࡿ⾑ᾮ⑌ᝈ࡛࠶ࡾ୰࡟ࡣ㞴἞ᛶࡢ⑕౛ࡶᏑᅾࡍࡿࠋ
἞⒪ࡣࣉࣞࢻࢽࢰࣟࣥࡢᢞ୚ࡀ୍⯡ⓗ࡛ࡣ࠶ࡿࡀࠊ᭱㏆἞⒪᢬ᢠᛶࡢ ,07 ࡢⓎ⏕౛ࡶከࡃぢཷࡅࡽࢀࠊච␿ᢚไ๣ࡸ⭁⮚᦬ฟ࡞࡝ࡢ἞⒪ࡶ⾜ࢃࢀ࡚࠸ࡿࠋ౛࠼ࡤࠊ⅖⑕ᛶ⭠⑌ᝈ ,%' ࠊ≉࡟ࣜࣥࣃ⌫ᛶᙧ㉁⣽⬊ᛶ⭠⅖ /3( ࡛ࡣ,/,/,/,/,/ ࡢ௚࡟ 71)Șࡢ⾑୰ࣞ࣋ࣝࡢୖ᪼ࡀሗ࿌ࡉࢀ࡚࠸ࡿ

ࡉࡽ࡟ࠊࣜࣥࣃ⭘࡞࡝ࡢ⾑ᾮ⭘⒆࡟ࡼࡿ⑌ᝈࡣ≟⊧࡟࠾࠸࡚Ⓨ⏕⋡ࡀ㧗ࡃࠊ⮫ᗋୖ㔜せ࡞⑌ᝈ࡛࠶ࡿࠋ⌧ᅾࡢ⾑ᾮ⭘⒆ࡢ᳨ᰝࡣ⾑ᾮ᳨ᰝࡸ⣽⬊デࡀ୺࡛࠶ࡿࠋࡋ࠿ࡋࠊࡇࢀࡽࡢ᳨ᰝ
᪉ἲ࡟ࡣࡶࡕࢁࢇ᳨ฟ㝈⏺ࡀ࠶ࡾࠊ⑓⌮᳨ᰝࡣ౵くᛶࡀ㧗࠸ࡇ࡜ࡶ࠶ࡾ⤒㐣ほᐹ࡟㢗ࡽ࡞ࡃ࡚ࡣ࡞ࡽ࡞࠸ࠋࡑࡇ࡛ὀ┠ࡉࢀ࡚࠸ࡿࡢࡀ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺࢆ⏝࠸ࡓ㑇ఏᏊデ᩿ἲ࡛࠶ࡿࠋ
࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺゎᯒ࡟ࡼࡿ⭘⒆デ᩿ࡣࠊ㔪⏕᳨ᮦᩱ࡞࡝ࡢࡈࡃᑡ㔞ࡢ᳨య࡛ࡢ᳨ᰝࡀྍ
⬟࡛࠶ࡾࠊࡲࡓᐈほⓗ࡟⾑ᾮ⭘⒆ࡢᝏᛶࢆホ౯࡛ࡁࡿ࡞࡝ࡢ฼Ⅼࡀᣲࡆࡽࢀࡿࠋࡇࡢデ᩿᪉ἲࢆ☜❧ࡍࡿ࡟ࡣ୙Ᏻᐃᛶࢆ♧ࡍ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺ࣐࣮࣮࢝ࡢ⤠ࡾ㎸ࡳࡀㄢ㢟࡜࡞ࡗ࡚࠸
ࡿࠋ
㏻ᖖࠊ⣽⬊ࡣศ໬࣭௦ㅰࡢ㐣⛬࡛␗ᖖࡀ⏕ࡌࡿ࡜࢔࣏ࢺ࣮ࢩࢫ࡟ࡼࡗ࡚␗ᖖ࡞⣽⬊ࡣᾘ⁛ࡍࡿࠋࡋ࠿ࡋࠊᝏᛶᗘࡢ㧗࠸⒴⣽⬊࡛ࡣ࢔࣏ࢺ࣮ࢩࢫࡀ⏕ࡌࡎ࡟⒴⣽⬊ࡣቑṪࡋ࡚࠸ࡃࠋ୍
᪉ࠊⓑ⾑⑓࡟࠾࠸࡚ࡣࡇࡢ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺ㡿ᇦ࡟࠾࠸࡚㑇ఏᏊḞᦆ㸦/2+㸧ࡸ㛗ࡉࡢኚ໬ࡀ⏕ࡌ࡚ࡔࢇࡔࢇ⵳✚ࡉࢀࠊ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺࡢ୙Ᏻᐃᛶࡀぢཷࡅࡽࢀࡿࡼ࠺࡟࡞ࡿࠋࡍ
࡞ࢃࡕࠊ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺ࣐࣮࣮࢝ࢆㄪ࡭࡚ࢤࣀ࣒ࡢ୙Ᏻᐃᛶࡀぢࡽࢀࢀࡤࠊࡑࡢ⤌⧊ࡀ⭘⒆ᛶ࡛࠶ࡿࡇ࡜ࡢドᣐ࡟࡞ࡿࠋ

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ࡇࢀࡽࡢ࣐࢖ࢡࣟࢧࢸࣛ࢖ࢺࡣࠊ≟ࡢච␿௓ᅾᛶ⾑ᾮ⑌ᝈ࠾ࡼࡧ⭘⒆ᛶ⑌ᝈࡢデ᩿ᛂ⏝࡟᭷ຊ࡜⪃࠼᥈⣴ࢆ⾜ࡗࡓࠋᮦᩱ࡜᪉ἲ
ච␿௓ᅾᛶ⑌ᝈ࡛ࡣࠊ᭱⤊ⓗ࡟  ౛ࡢ≟ࡢච␿௓ᅾᛶ⾑ᑠᯈῶᑡ࡛ゎᯒࢆᐇ᪋ࡋࡓࠋ,07 ࡢ⑕౛ࡣࠊ㯞ᕸ኱Ꮫ௜ᒓື≀⑓㝔ࡶࡋࡃࡣᮾி኱Ꮫື≀་⒪ࢭࣥࢱ࣮࡟᮶㝔ࡋࡓ≟  㢌࡛࠶ࡗࡓࠋ
඲⑕౛࡛ࠊ⾑ᑠᯈᩘῶᑡࡢཎᅉ࡜࡞ࡿᇶ♏⑌ᝈࡣㄆࡵࡽࢀࡎࠊࡉࡽ࡟จᅛ⣔᳨ᰝࢆᐇ᪋ࡋࡓࡀࠊ
37ࠊ$377ࠊࣇ࢕ࣈࣜࣀ࣮ࢤࣥཬࡧ )'3 ࡢ࠸ࡎࢀ࡟࠾࠸࡚ࡶ᫂ࡽ࠿࡞␗ᖖ್ࢆ♧ࡋࡓಶయࡣ࡞ࡃ ',&ࡢⓎ⏕ࡣ␲ࢃࢀ࡞࠿ࡗࡓࠋ
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1)

Horimoto T, Gen F, Murakami S, Iwatsuki-Horimoto K, Kato K, Hisasue M, Sakaguchi M,
Nidom CA, Kawaoka Y. : Cats as a potential source of emerging influenza virus infections.
Virol Sin. 30, 221-3, 2015.

2)

Kawarai S, Hisasue M, Matsuura S, Ito T, Inoue Y, Neo S, Fujii Y, Madarame H, Shirota K,
Tsuchiya R. : Canine pemphigus foliaceus with concurrent immune-mediated thrombocytopenia.
Am Anim Hosp Assoc. 51, 56-63, 2015.

3)

Watanabe M, Tanaka K, Takizawa T, Segawa K, Neo S, Tsuchiya R, Murata M, Murakami M,
Hisasue M.: Characterization of a canine tetranucleotide microsatellite marker located in the first
intron of the tumor necrosis factor alpha gene. J Vet Med Sci. 76, 119-22, 2014.

4)

Kamiie J, Shimoyama N, Aihara N, Hisasue M, Naya Y, Ogihara K, Shirota K. : Quantitative
DQDO\VLVRI&'İLQDFORQHGFDQLQHO\PSKRPDFHOOOLQHE\VHOHFWHGUHDFWLRQPonitoring assay.
Biosci Biotechnol Biochem. 78, 271-5, 2014.

5)

Horimoto T, Gen F, Murakami S, Iwatsuki-Horimoto K, Kato K, Akashi H, Hisasue M,
Sakaguchi M, Kawaoka Y, Maeda K. : Serological evidence of infection of dogs with human
influenza viruses in Japan. Vet Rec. 174:96, (Epub 2013 Dec 11) 2014.

6)

Watanabe S, Ito J, Baba T, Hiratsuka T, Kuse K, Ochi H, Anai Y, Hisasue M, Tsujimoto H,
Nishigaki K.J : Notch2 transduction by feline leukemia virus in a naturally infected cat. Vet
Med Sci. 76, 553-7. 2014.

7)

Kurihara Y, Suzuki T, Sakaue M, Murayama O, Miyazaki Y, Onuki A, Aoki T, Saito M, Fujii Y,
Hisasue M, Tanaka K, Takizawa T. : Valproic acid, a histone deacetylase inhibitor, decreases
proliferation of and induces specific neurogenic differentiation of canine adipose tissue-derived
stem cells. J Vet Med Sci. 76:15-23. 2014.

8)

Fujimoto A, Neo S, Ishizuka C, Kato T, Segawa K, Kawarai S, Ogihara K, Hisasue M, Tsuchiya
R. : Identification of cell surface antigen expression in canine hepatocellular carcinoma cell lines.
Vet Med Sci. 75:831-5. 2013.

9)

Kato T, Hisasue M, Segawa K, Fujimoto A, Makiishi E, Neo S, Yasuno K, Kobayashi R,
Tsuchiya

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in12'6&,'ȖF QXOO PLFHZLWKDFXWHKHSDWLWLVLQGXFHGE\&&O Vet Med Sci. 75:847-55. 2013.
10) Segawa K, Kondo T, Kimura S, Fujimoto A, Kato T, Ishikawa T, Neo S, Hisasue M, Yamada T,
Tsuchiya R. : Effects of prostaglandin E1 on the preparation of platelet concentrates in dogs. J
Vet Intern Med. 26:370-6. 2012.
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258

NOTE

Biochemistry

Characterization of a Canine Tetranucleotide Microsatellite Marker Locatedin the First Intron of the Tumor Necrosis Factor Alpha Gene
Masashi WATANABE1)**, Kazuaki TANAKA2)**, Tatsuya TAKIZAWA2), Kazuhito SEGAWA1), Sakurako NEO1),
Ryo TSUCHIYA 1), Michiko MURATA3), Masaru MURAKAMI3) and Masaharu HISASUE1)*
1)Laboratory of Veterinary Internal Medicine II, Faculty of Veterinary Medicine, Azabu University, Chuo-ku, Sagamihara, Kanagawa

252–5201, Japan
2)Laboratory of Animal Biotechnology, Faculty of Veterinary Medicine, Azabu University, Chuo-ku, Sagamihara, Kanagawa 252–5201,

Japan3)Laboratory of Molecular Biology, Faculty of Veterinary Medicine, Azabu University, Chuo-ku, Sagamihara, Kanagawa 252–5201,

Japan(Received 18 June 2013/Accepted 21 August 2013/Published online in J-STAGE 13 September 2013)
ABSTRACT. A polymorphic tetranucleotide (GAAT) n
TNFA) gene wascharacterized in this study; 139 dogs were analyzed: 22 Beagles, 26 Chihuahuas, 20 Miniature Dachshunds, 24 Miniature Poodles, 22 Pem5, (GAAT) 6, (GAAT) 7 and (GAAT) 8, including 9He
PIC

TNFA gene.
KEY WORDS:

canine TNFA gene, tetranucleotide microsatellite.
doi: 10.1292/jvms.13-0316; J. Vet. Med. Sci. 76(1): 119–122, 201418]. Microsatellites were thought to be evolutionarily neutral
monocytes and macrophages (Online Mendelian Inheritancein Man (OMIM, 191160),. In humans, genetic variations in
the TNFA gene are associated with various disorders, e.g.,

however, many reports have indicated that microsatellites in-

syndrome [8].

analyzing genetic polymorphisms.
n
TNFA gene and demonstrated that this microsatellite locus was highly polymorphic,

TNFA mRNA in colonic mucosa has been reported on dogs
with idiopathic lymphocytic-plasmacytic colitis [16].
The canine TNFA gene is located on dog chromosome 12
NW_003726081), and it
morphisms in the canine TNFA*C ORRESPONDENCE TO: H ISASUE

grooming shops in Japan. The 139 samples obtained were
24 Miniature Poodles, 22 Pembroke Welsh Corgis and 25
Shiba Inus. This study was approved by the Institutional
Animal Care and Use Committee (Permission number:
1306094) and carried out according to the Azabu University-(Macherey-Nagel, Duren, Germany), according to the manu-

252–5201, Japan.
e-mail: hisasue@azabu-u.ac.jp

ative Commons Attribution Non-Commercial No Derivatives (by-ncnd) License <http://creativecommons.org/licenses/by-nc-nd/3.0/>.

TNFA
TNFA

259

-

120

M. WATANABE ET AL.

TNFA
TNFA

TNFA gene, respectively. (B) The variations detected in the partial TNFA
gene in 7 Beagles.

TNFA). PCR was
μl reaction volumes, according to the

He
5–8

TNFA
TNFA

(GAT) (GAAT) 2]
-

25 μl
TNFA
dog-TNFA

were electrophoresed on a 3.0% agarose gel and visualized

the dog_TNFA_R1 primer side. As a result, we determined(GAAT)5, (GAAT)6, (GAAT)7 and (GAAT)8, were detected
TNFA-STR1 tetranucleotide microsatellite

TNFA-

TNFA-STR1were determined automatically using an ABI 3130 genetic
TNFA
Table 1 shows the genotypic and allele distribution in 6 dog
nucleotide databases under accession numbers AB819627–
AB819629.

breeds. This is because the (GAAT)8
Chihuahuas, Miniature Dachshunds, Miniature Poodles,

260

121

TETRANUCLEOTIDE MICROSATELLITE IN DOG TNFATNFA gene on a 3.0% agarose gel. The genotypes relate to
Osaka, Japan).
n tetranucleotide microsatellite in canine TNFA gene among dog breeds

Genotype
Beagle (22)
Miniature Dachshund d) (20)
Chihuahua d) (26)
Miniature Poodle (24)Pembroke Welsh Corgi (22)
Shiba Inu (25)

5

6

7

8

5/6

5/7

5/8

6/7

6/8

7/8

5

6

7

8

1
8
12
6
8
13

0
0
2
1
1
0

2
2
0
0
0
0

4
0
0
0
0
0

5
2
9
6
5
2

5
7
3
11
4
10

1
0
0
0
0
0

1
1
0
0
4
0

0
0
0
0
0
0

3
0
0
0
0
0

0.295
0.625
0.692
0.604
0.568
0.760

0.136
0.075
0.250
0.167
0.250
0.040

0.295
0.300
0.058
0.229
0.182
0.200

0.273
0.000
0.000
0.000
0.000
0.000

He a)

PIC b)

Deviations

0.749
0.527
0.464
0.566
0.595
0.389

0.682
0.438
0.391
0.493
0.516
0.333

P<0.05NS e)
NS
P<0.05NSNSc)Pembroke Welsh Corgis and Shiba Inus. (GAAT)5 was the
H
(PIC
(P

receptor gene. J. Mol. Biol. 267: 229–236. [Medline]
2. Botstein, D., White, R. L., Skolnick, M. and Davis, R. W. 1980.
Am. J. Hum. Genet. 32:
314–331. [Medline]
3. Buranawuti, K., Boyle, M. P., Cheng, S., Steiner, L. L., McDou-He
He was observed in
He and PIC

J. Med. Genet. 44: 209–214.
[Medline]

the canine TNFA
rosatellite alleles could be associated with susceptibility toanalysis. Evol. Bioinform. Online 1: 47–50.
5. Gardezi, A. Z., Ziaei, Y. Z. and Marashi, S. M. 2008. MicrosatelJ. Crit. Care 23: 440–444. [Medline]

variation and canine diseases.

Matta, M. A., Souza, S. M., Lana, M., Chiari, E., Galvão, L. M.

ACKNOWLEDGMENTS. We thank Dr. Hidekatsu ShiVet. Immunol. Immunopathol. 130: 43–52. [Medline]
7. Hearne, C. M., Ghosh, S. and Todd, J. A. 1992. Microsatellites
Trends Genet. 8: 288–294.
[Medline]nine DNA samples. This work was supported by the MEXT
Universities, 2011–2015. The authors would like to thank

261

122

M. WATANABE ET AL.

8. Ishii, T., Hirose, H., Saito, I., Nishikai, K., Maruyama, H. and-in young, older, and diabetic Japanese men. Metabolism 49:
1616–1618. [Medline]
Trends Genet.
13: 74–78. [Medline]
10. Klesert, T. R., Otten, A. D., Bird, T. D. and Tapscott, S. J. 1997.

TNF2
with septic shock susceptibility and mortality: a multicenter
study. JAMA 282: 561–568. [Medline]
Amos, C. I., Zhu, D. K., Ward, R. H., Clegg, D. O., Shanahan,tibility to rheumatoid arthritis. Am. J. Hum. Genet. 59: 676–683.
[Medline]
16. Ridyard, A. E., Nuttall, T. J., Else, R. W., Simpson, J. W. and-

DMAHP. Nature Genet. 16: 402–406.
[Medline]
Genes

Vet.

Immun. 1: 185–190. [Medline]

Immunol. Immunopathol. 86: 205–214. [Medline]

J. 1998. A tetranucleotide polymorphic microsatellite, locatedVet. Immunol. Immunopathol. 146: 143–149. [Medtranscription regulatory element in vitro. Hum. Mol. Genet. 7:
423–428. [Medline]
13. Menezes-Souza, D., Corrêa-Oliveira, R., Guerra-Sá, R., Giunchet-line]
polymorphisms which can be typed using the polymerase chain
reaction. Am. J. Hum. Genet. 44: 388–396. [Medline]
19. Wei, J., Ramchand, C. N. and Hemmings, G. P. 1995. AssociationTHphrenia. Psychiatr. Genet. 5: 83–88. [Medline]

Leishmania (Leishmania)
chagasi presenting distinct cutaneous parasite density and clinicalstatus. Vet. Parasitol. 177: 39–49. [Medline]

262

Bioscience, Biotechnology, and Biochemistry, 2014
Vol. 78, No. 2, 271–275

Note

Quantitative analysis of CD3ε in a cloned canine lymphoma cell line byselected reaction monitoring assay
Junichi Kamiie1, Naoto Shimoyama1, Naoyuki Aihara1, Masaharu Hisasue2, Yuko Naya3, Kikumi
Ogihara3,* and Kinji Shirota1,41

Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, Sagamihara, Japan;
Laboratory of Veterinary Internal Medicine II, School of Veterinary Medicine, Azabu University, Sagamihara,
Japan; 3Laboratory of Pathology, School of Life and Environmental Science, Azabu University, Sagamihara, Japan;
4
Research Institute of Biosciences, Azabu University, Sagamihara, Japan
2

Received August 7, 2013; accepted October 28, 2013

Downloaded by [Azabu University] at 20:54 19 April 2016

http://dx.doi.org/10.1080/09168451.2014.878216

Several quantitative proteomics techniques that
employ mass spectrometry have been reported
recently.7–9) In particular, absolute quantitative proteomics in the selected reaction monitoring (SRM) mass
spectrometry mode is suitable for the highly sensitive
and accurate quantiﬁcation of such proteins.9) A quantitative approach by SRM has been applied to the studyof integral membrane proteins, including drug transporters10) and G protein-coupled receptors.11)
In this study, we aimed to develop a quantitative
method of determining the levels of canine CD3ε incells. To verify the usefulness of the method with biological samples, we applied it to establish a novel
canine T-cell lymphoma cell line, and we determinedthe expression level of CD3ε in these cells.
To establish a novel CD3ε-expressing canine T-cell
lymphoma cell line, we analyzed cells from the intestinal
mass of a 5-year-old male beagle. Cytological examination of the aspirates revealed that the mass wascomposed of large heteromorphic lymphoid cells withnumerous mitoses. Based on these ﬁndings, we diagnosed the intestinal mass as a lymphoma. One monthafter cytological examination, the beagle died at theowner’s home and a necropsy was performed with theowner’s approval. The intestinal mass was ﬁxed in 10%
phosphate-buffered formalin pH 7.4, processed routinely,
embedded in parafﬁn, and cut into sections by 3 μm
thick. The parafﬁn-embedded sections were stained withhematoxylin and eosin or subjected to immunohistochemistry. The de-waxed sections were pretreated by
microwaving them in citrate buffer and then incubatingthem with mouse anti-human-CD3ε antibody (clone
F7.2.38, DAKO Japan, Tokyo) for 1 h at room temperature. The signals were visualized with horseradish peroxidase-conjugated rabbit anti-mouse IgG (Nichirei
Biosciences, Tokyo) and 3,3′-diaminobenzidine.
The surface of the intestinal tumor mass was sterilizedwith 70% ethyl alcohol and pipetted into a single-cell

We established a mass spectrometry-based quantitative method of assaying CD3ε, a component of the
T-cell receptor complex. It revealed a CD3ε level of
1 mol per cell in a newly derived canine T-cell lymphoma cell line. Our results suggest that thismethod has sufﬁcient sensitivity to quantify CD3εlevels in canine lymphoma cells reliably.
Key words:

CD3; lymphoma; quantitative proteomics;
selected reaction monitoring; mass
spectrometry

CD3ε, a component of the CD3 complex, is one of
the most abundantly expressed cell-surface proteins of
the T-cell lineage. The CD3 complex is composed offour polypeptide chains (δ, ε, γ, and ζ) and is associated with the T-cell receptor (TCR). These chains form
δε and γε heterodimers and ζζ homodimers. The δε andγε heterodimers are associated with TCR-α and –β,
respectively, and generate intermediate complexes.1,2)
The ζζ homodimers bind to TCR complexes and aretransported to the cell surface.3,4)
In T-cell lineage differentiation, TCR complexes are
expressed on thymocytes beginning at the early stages ofmaturation, and expression of them increases during thematuration process. In addition, each component of
the TCR complex changes its expression level during thedevelopment of the T-cell lineage.5,6) Stoichiometricanalyses based on absolute quantiﬁcation of the levels of
TCR components are important in understanding theirroles in development, but no such quantitative measurements of TCR components yet been reported. Although
TCR components are integral membrane proteins, it is difﬁcult to develop quantitative immunological assays, suchas the enzyme-linked immunosorbent assay (ELISA),
due to the insolubility of these membrane proteins.

*Corresponding author. Email: ogiwarak@azabu-u.ac.jp
Abbreviations: TCR, T-cell receptor; ELISA, enzyme-linked immunosorbent assay; SRM, selected reaction monitoring; PBS, phosphate-bufferedsaline; CV, coefﬁcient of variation.
© 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry263

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272

J. Kamiie et al.

the cells expressed CD3ε (Fig. 1(C)), but not CD4,
CD8α, or CD79α (data not shown). By Western blotting,expression of CD3ε in the cells was also detected(Fig. 1(D)), whereas CD79α, a B-cell marker, was notdetectable in them (data not shown).
We established a quantitative method of detecting
CD3ε-derived peptide by mass spectrometry in SRM
mode. First, a target peptide sequence was selectedfrom among 27 tryptic peptides derived from thecanine CD3ε sequence (SWISS-PROT: P27597)
according to previously described peptide selection criteria.10) Brieﬂy, the target peptide was selected by thefollowing criteria: (i) the target peptide sequence isunique for canine CD3ε, (ii) it does not contain chemically reactive residues (cysteine or methionine), and(iii) it does not contain post-translational modiﬁcations
or continuous sequences of K or R. The sequence
NDNLVEGASNR, a canine-speciﬁc sequence, wasselected as quantitative target. An isotope-labeled peptide of the target sequence that contained a labeled alanine residue (13C3, 15N) was synthesized by
Thermoelectron Corporation (Sedanstrabe, Germany),
as reference for the quantitative assay.
SRM transitions were determined with high sensitivity. Seven transitions derived from the y3 to y9 productions were identiﬁed for the endogenous and isotopelabeled forms of NDNLVEGASNR. To verify that, wecommanded sufﬁcient sensitivity to detect each of thetransitions, SRM analysis of the cultured cell sample
was done for the 14 transitions. Tryptic digestion of
the cultured cells was done as described previously.12)
We solubilized the cultured cells in 7 M guanidine
HCl, 0.5 M Tris–HCl (pH 8.5), and 50 mM EDTA. The
protein concentration of the lysate was measured with aprotein assay kit (DC Protein Assay, Bio-Rad) withbovine serum albumin as standard. One μg of proteins
was reduced with equal amounts of dithiothreitol andS-carbamoylmethylated and with 2.5 times the amountof iodoacetamide. The proteins were precipitated withmethanol and chloroform (4:1/v:v). The precipitatedproteins were dissolved in 1.2 M urea and 500 mM
Tris–HCl (pH 8.0), and then digested with trypsin (Promega, Fichburg, WI) at an enzyme to substrate ratio of
1:100 at 37 °C over 16 h. The efﬁciency of tryptic
digestion was checked by SDS-PAGE followed byWestern blotting for CD3ε. The speciﬁc band of CD3εdisappeared in the digested sample (Fig. 1(D)). We
spiked the tryptic digest with 200 fmol of isotopelabeled peptide. The tryptic digests were acidiﬁed withformic acid to a pH of < 2.0. The acidiﬁed samples
were centrifuged at 21,500 × g for 15 min, and then the
supernatants were injected into a nano-LC system (Paradigm MS4, AMR, Tokyo) connected to an ESI-triplequadrupole mass spectrometer (TSQ vantage, Thermo
Fisher Scientiﬁc, Yokohama, Japan). The nano-LC system was run with a reversed phase column (L-column
ODS; 0.1 mm inner diameter × 50 mm, 3 μm particles)with a gradient starting at 5% B to 45% B in 45 min,at a ﬂow rate of 500 nL/min. The gradient was generated by mixing water containing 0.1% formic acid (A)and acetonitrile containing 0.1% formic acid (B). The
spectrometer was operated in SRM mode with a 1 s
cycle time. Collision energies for each transition weresuspension in culture medium. The cell suspension wascentrifuged and resuspended in fresh culture medium.
This procedure was repeated several times. Primaryculture of the cells obtained was carried out in an
RPMI-1640 growth medium containing 18% heat-inactivated fetal bovine serum (Gibco BRL, Cergy-Pontoise,France), 0.1% glutamine, 1 mM sodium pyruvate, 0.1%
spectinomycin, and 0.035% NaHCO3 in 60-mm Petridishes at 37 °C in a CO2 incubator. The medium was
replaced approximately twice per week, and conﬂuent
cultures were subcultured. The cell line was subjected tocloning by limiting dilution in 96-well plates in an
RPMI-1640 medium containing 10% fetal bovine serum.
At 4 to 7 days after cultivation, the clones that had
grown were trypsinized and subjected to a further limiting dilution step. The cloned cell line was eventually
established after three limiting dilution steps.
Flow cytometry analysis of the CD antigens
expressed by the cloned cell line was done with antibodies for mouse anti-canine CD3 (clone CA17.2A12,
Serotec, Oxford, UK), mouse anti-canine CD4 (clone
CA13.1E4, Serotec), or mouse anti-canine CD8α (clone
CA9.JD3, Serotec) diluted to 1:200, 1:20, and 1:20,
respectively, in Dulbecco’s phosphate-buffered saline(PBS) with 1% bovine albumin. We incubated 1 ×
106 cells/100 μL for 1 h at 4 °C with 10 μL of each primary antibody. After they were washed twice in PBS,
the cells were ﬁxed with 0.5% paraformaldehyde in
PBS for 1 h at 4 °C. After further washing in PBS,
FITC-conjugated goat anti-mouse IgG (Cappel, Durham, NC) was added, and the mixture was incubatedfor 1 h at 4 °C. The cells were then washed and ﬁxed
with 0.5% paraformaldehyde in PBS. After washing in
PBS, the cells were resuspended in PBS and analyzed
with a ﬂow cytometer (Epics XL/XL–MCL, Beckman
Coulter, San Jose, CA).
For western blotting, 2 × 108 cells were washed threetimes with PBS and then centrifuged at 250 × g for 10
min. The cell pellets were homogenized with 10 times
the initial volume of 0.1% SDS and 10 mM Tris–HCl(pH 8.0). Protein concentrations were measured with a
DC Protein Assay Kit (Bio-Rad Laboratories, Hercules,CA) with bovine serum albumin (Bio-Rad) as standard.
A total of 10 μg of protein from the cells was resolved
by 5–20% SDS-polyacrylamide gel electrophoresis andelectro-transferred to PVDF membranes (Bio-Rad). After
treatment with a blocking buffer (4% skim milk in 25
mM Tris–HCl pH 8.0, 125 mM NaCl, and 0.1% Tween20), the membranes were incubated with anti-humanCD3ε antibody at 4 °C for 16 h. They were then washedwith the blocking buffer three times and incubated withhorseradish peroxidase-conjugated polyclonal rabbit
anti-mouse immunoglobulin (Dako Japan, Tokyo).
Signals were visualized with a chemiluminescence kit(ECL-plus; GE Healthcare UK, Buckinghamshire).
According to a histopathological analysis, the intestinal mass of the beagle was comprised of a monoclonal
cluster of large round proliferating tumor cells. The
tumor cells were 5–20 μm in diameter with a sharp, irregularly indented nucli (Fig. 1(A)). Mitosis was observed
to have occurred frequently. Immunohistochemistry
revealed strong expression of CD3ε in the cytoplasm of
the tumor cells (Fig. 1(B)). Flow cytometry revealed that264

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Quantitative assay of CD3ε in canine lymphoma

273

Fig. 1. Morphological ﬁndings and CD3ε expression in intestinal tumor cells and cultured cells.
Note: (A) Hematoxylin and eosin staining of the intestinal tumor. Tumor cells were 5–20 μm in diameter with sharp, irregularly indented nuclei.
Mitosis was observed to have occurred frequently. Scale bar represents 50 μm. (B) Immunohistochemical detection of CD3ε in the intestinal tumor.
Tumor cells showed strong CD3ε expression. Scale bar represents 25 μm. (C) Detection of CD3ε in the cultured cells by ﬂow cytometry. (D)Detection of CD3ε in cultured canine T-cell lymphoma cells. A total of 10 μg of protein from cells (lane 1) and a tryptic digest of cells (lane 2)
were analyzed by Western blotting to detect CD3ε.

are suitable for sensitive and accurate quantiﬁcation ofCD3ε levels. We used a total of six transitions that contained three transitions derived from y5, y6, and y7 ofthe endogenous and isotope-labeled peptides toquantify the CD3ε levels.
To assess the linearity of this method, dilution seriesof the cells were analyzed. The cell suspension was
diluted with 1% BSA containing PBS to dilutions of
1:100, 1:1,000, and 1:10,000 in low-binding tubes. The
cell concentration in each tube was measured with a
Neubauer counting chamber under light microscopy. Aliquots of 1 × 103, 1 × 105, and 1 × 106 cells were transferred to new tubes from the dilutions of 1:100, 1:1000,
and 1:10,000, respectively. For each sample, the quantitative value of CD3ε per cell was calculated as the average of nine quantitative values for the y5, y6, and y7transitions from three different experiments. Quantitativevalues were correlated with cell numbers ranging from5000 to 50,000, and the regression line was represented
by the equation y = 0.0005 x 1.1 (R2 = 1.0; Fig. 2(C)). Our
results indicate that the established method has a
100-fold dynamic range with excellent linearity.
The established method measured 0.82 ± 0.06 fmol ofCD3ε in 5 × 103 cells in linearity (Fig. 2(C)). This result
suggest that the limit of quantiﬁcation of the established method is femto mol per assay, and that method
nearly equaled that of ELISA in sensitivity, thesensitivity of which is approximately 0.1 to 1 fmol.13)

determined by Pinpoint software, version 1.0 (Thermo
Fisher Scientiﬁc).
We generated SRM chromatograms of the endogenous and isotope-labeled peptides in 1 μg of proteinfrom the cultured cells spiked with 200 fmol of the isotope-labeled peptides. All transitions showed signal
peaks at the same retention time of 10.95 min(Fig. 2(A)). Identity conﬁrmation of the targeted peptide relied on the composite MS/MS spectra displayed.
The consistency of the SRM fragmentation patterns is
illustrated in Fig. 2(B). The transitions, 594.8[M+H]2+/
504.3[M+H]+, 594.8[M+H]2+/633.3[M+H]+, and 594.8[M+H]2+/732.4[M+H]+, derived from the y5, y6, andy7 endogenous peptides respectively, exhibited morethan 2 to 5-fold higher signal intensities than the otherfour transitions (Fig. 2(B)). The order of the peak areas
derived from the y-series ions in the SRM analysis was
reﬂected the MS/MS spectrum signal intensity results(y6 > y7 > y5 > y3 > y8 > y9 > y4) (Fig. 2(B)).
Quantitative values were calculated based on thepeak area ratio (endogenous/isotope-labeled peptide).
We calculated the quantitative values for each production in 0.1 μg of protein from cultured cells spiked with200 fmol of isotope-labeled peptide. The transitions fory5, y6, and y7 showed excellent accuracy, with a coefﬁcient of variation (CV) < 5%, although the other fourtransitions had CVs of 8.5–13.9% (Table 1). These
results suggest that the transitions for y5, y6, and y7

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274

J. Kamiie et al.

Fig. 2. Relative signal intensities of transitions representing endogenous and stable isotope-labeled CD3ε peptides in cultured cells and linearityof the quantitatively measured CD3ε levels.
Note: Stable isotope-labeled peptide (200 fmol) was spiked into 1 μg of protein from cultured canine cells (A and B) or, a dilution series of thecultured cells (C). LC/MS/MS with SRM transitions was done to quantify endogenous and stable isotope-labeled CD3ε peptides. (A) Chromatograms of transitions for y-series ions derived from endogenous and isotope-labeled peptides. Y-axes represent relative signal intensity, and the highest signal intensity in each chromatogram was deﬁned as 100%. (B) Signal intensities of SRM transitions for the NDNLVEGASNR peptide as aspectrum imitation (lower layer) as compared to the corresponding MS/MS spectrum of the doubly charged precursor mass of NDNLVEGASNR
peptide (upper layer). Y-axes represent relative signal intensity, and the highest signal intensity in each chromatogram was deﬁned as 100%. (C)The amount of CD3ε was determined by the ratio of the peak area of the unlabeled and stable isotope-labeled peptides (unlabeled/labeled) in adilution series of a cell line. Each point represents the mean value ± SD (n = 3).

We calculated the CD3ε levels per cell that we determined based on three transitions that we replicated inthree experiments with a digest of 5000 cells. There
was no signiﬁcant difference (ANOVA, p > 0.05) inmean values based on the Q1/Q3 transitions 594.8/
504.3, 594.8/633.3, and 594.8/732.4. The calculated
values were 1.08, 1.12, and 1.15 amol/cell, respectively.
Finally, the amount of protein in each sample wasdetermined to be 1.12 amol/cell with a CV of 1.35% asaverage of the calculated values for the three transitions. These quantitative results indicate the high sensitivity and reproducibility of the method.

Table 1. Quantitative values for transitions in 0.1 μg of Proteinfrom cultured canine T-Cell lymphoma.
Quantitative values (fmol/assay)
Productions

Assay 1

Assay 2

Assay 3

Mean ± SD

C.V. (%)

y3
y4
y5
y6
y7
y8
y9

11.1
7.25
8.62
8.19
8.18
7.42
7.54

12.0
9.49
9.30
8.62
8.63
8.58
9.75

10.1
9.10
9.25
8.86
8.87
9.15
8.09

11.1 ± 0.94
8.62 ± 1.20
9.06 ± 0.38
8.56 ± 0.34
8.56 ± 0.35
8.38 ± 0.88
8.46 ± 1.15

8.46
13.9
4.20
3.94
4.08
10.5
13.6

266

Quantitative assay of CD3ε in canine lymphoma

In conclusion, we established a quantitative assay formeasuring CD3ε levels by SRM-based mass spectrometry. This assay measured the absolute expression levelof CD3ε in a canine T-cell lymphoma cell line. It
should be possible to develop quantitative assays forother components of the TCR by the approachdescribed here. In addition, 300 SRM transitions wererun in a single analysis by means of one of several
modern mass spectrometers. Therefore, it should bepossible to develop simultaneous quantitative assays
for TCR components by mass spectrometry in the
SRM mode. Analysis of the quantitative proﬁles ofcomponents of the TCR should contribute to a better
understanding of lymphocyte biology.

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heterodimer formation is a prerequisite for association of CD3zeta 2 into functionally competent TCR.CD3 complexes. J. Biol.
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cytometric analysis: implications for differentiation. J. Immunol.
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pre-TCR and CD3 complex during thymus development. Int.
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Quantitative analysis of complex protein mixtures using isotopecoded afﬁnity tags. Nat. Biotechnol. 1999;17:994–999.
[8] Ong SE, Foster LJ, Mann M. Mass spectrometric-based approaches
in quantitative proteomics. Methods. 2003;29:124–130.
[9] Picotti P, Rinner O, Stallmach R, Dautel F, Farrah T, Domon B,
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K, Sekine Y, Uchida Y, Ito S, Terasaki T. Quantitative atlas ofmembrane transporter proteins: development and application of ahighly sensitive simultaneous LC/MS/MS method combined withnovel in-silico peptide selection criteria. Pharm. Res.
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[13] Harlow ED, Lane D. Antibodies a laboratory manual. New
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578–583.

Acknowledgments

Downloaded by [Azabu University] at 20:54 19 April 2016275

We would like to thank Ms Ai Uchida and Mr
Hirotaka Kawakami for technical assistance. This study
was partially supported by a Grant-in-Aid for thedevelopment of intellectual property by Azabu
University.

References[1] Alarcon B, Berkhout B, Breitmeyer J, Terhorst C. Assembly ofthe human T cell receptor-CD3 complex takes place in the endoplasmic reticulum and involves intermediary complexes between
the CD3-gamma.delta.epsilon core and single T cell receptoralpha or beta chains. J. Biol. Chem. 1988;263:2953–2961.
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the TCR/CD3 complex: CD3 epsilon/delta and CD3 epsilon/
gamma dimers associate indistinctly with both TCR alpha andTCR beta chains. Evidence for a double TCR heterodimer
model. Eur. J. Immunol. 1998;28:12–21.
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Virology

Notch2 Transduction by Feline Leukemia Virus in a Naturally Infected Cat
Shinya WATANABE1), Jumpei ITO2), Takuya BABA2), Takahiro HIRATSUKA2), Kyohei KUSE2), Haruyo OCHI 2),
Yukari ANAI2), Masaharu HISASUE3), Hajime TSUJIMOTO4) and Kazuo NISHIGAKI1,2)*
1)Laboratory of Molecular Immunology and Infectious Disease, United Graduate School of Veterinary Science, Yamaguchi University,

1677–1 Yoshida, Yamaguchi 753–8515, Japan
2)Laboratory of Molecular Immunology and Infectious Disease, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677–1

Yoshida, Yamaguchi 753–8515, Japan
3)Laboratory of Veterinary Internal Medicine II, School of Veterinary Medicine, Azabu University, 1–17–71 Fuchinobe, Chuo-ku,

Sagamihara-shi, Kanagawa 252–5201, Japan4)Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1–1–1 Yayoi,

Bunkyo-ku, Tokyo 113–8657, Japan(Received 3 July 2013/Accepted 25 November 2013/Published online in J-STAGE 9 December 2013)
ABSTRACT.

Feline leukemia virus (FeLV) induces neoplastic and nonneoplastic diseases in cats. The transduction of cellular genes by FeLVof feline Notch2 transduction by FeLV in an infected cat with multicentric lymphoma and hypercalcemia. We cloned recombinant FeLVs
harboring Notch2 in the env gene. Notch2 was able to activate expression of a reporter gene, similar to what was previously reported in cats
Notch2 strongly correlates with FeLVinduced lymphoma.
KEY WORDS: feline leukemia virus, hypercalcemia, lymphoma, Notch2, transduction.
doi: 10.1292/jvms.13-0344; J. Vet. Med. Sci. 76(4): 553–557, 2014

Feline leukemia virus (FeLV), a gammaretrovirus thatcan cause a variety of both proliferative and degenerative
diseases, is a major pathogen of feline lymphoma [4, 6].
The transduction and activation of cellular proto-oncogenes
by FeLV are mechanisms associated with the occurrenceof lymphomas and sarcomas. Some recombinant FeLVs
harboring cellular sequences, such as the transcription factor
myc
tcr [10], have been cloned from cats with naturally occurring
lymphoma. FeLV, which transduces the intracellular regionof Notch2, has been cloned from cats with experimental
FeLV-induced thymic lymphoma [24].
Notch2 is a single-spanning transmembrane receptor thatbelongs to the Notch family of proteins, which play a role incell differentiation and generation of tumors. The physicalcontact between cells expressing Notch ligands (e.g., deltalike ligands DLL1, 3 and 4 and Jagged1 and 2) and cells
expressing the Notch protein induces proteolytic cleavageof Notch. This leads to release of the intracellular region of
Notch into the nucleus, resulting in activation of responsive
gene expression [reviewed in 11]. The active forms of Notch
receptors have been reported in human patients with lymphoma and leukemia [8, 15, 27, 30]. Here, we report, for the

Notch2 sequence by FeLV
(Notch2-FeLV) in a naturally infected cat with multicentric
lymphoma and hypercalcemia.
A 2-year-old, 2.0-kg, spayed female Japanese domestic
cat was referred to the Veterinary Medical Center, The
University of Tokyo, in 1995 with consecutive debilitation,
dehydration and leanness. The cat was tested positive for
FeLV p27-Gag antigen and diagnosed with multicentric
lymphoma. Although the tumor had temporally gone into
remission after chemotherapy, relapse occurred, and severe
hypercalcemia was observed in its blood biochemistry proscapula and humerus. Despite treatment with furosemide,
infusion of sodium chloride saline, porcine calcitonin (4 IU/kg) and salmon calcitonin (4 IU/kg) for hypercalcemia, little
effective palliation was observed, and the cat died with neural manifestations on day 21. Marked invasion of the tumor
cells was seen in the multiple tissues at necropsy.
the entire env gene of the FeLV provirus using two PCRs

*C ORRESPONDENCE TO: N ISHIGAKI, K., Laboratory of Molecular Im munology and Infectious Disease, Joint Faculty of Veterinary
Medicine, Yamaguchi University, 1677–1 Yoshida, Yamaguchi
753–8515, Japan. e-mail: kaz@yamaguchi-u.ac.jp©2014 The Japanese Society of Veterinary Science[28]. Amplicons were cloned using Zero Blunt PCR Cloning
Kit (Invitrogen, Carlsbad, CA, U.S.A.). Two FeLV clones(KeyN2-1 and KeyN2-2) contained feline Notch2-like sequences; the nucleotide sequences of the clones were deposited in GenBank under accession numbers AB818695 andAB818696, respectively (Fig. 1). Both sequences containedThis is an open-access article distributed under the terms of the Creative
Commons Attribution Non-Commercial No Derivatives (by-nc-nd)
License <http://creativecommons.org/licenses/by-nc-nd/3.0/>.

268

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S. WATANABE ET AL.
Table 1.

Blood tests for the cat with lymphoma
Complete blood countRBC (×10 6/ )Ht (%)Hb (g/dl)
TP (g/dl)
PLT (×10 3/ )WBC (×10 3/ )Eos (%)
Band (%)
Seg (%)
Lym (%)
Mono (%)

Patient

Reference range

6.59
28
9.5
6.8
95
15.8
1
0
72
26
1

5.00–10.00
24–45
8.0–15.0
5.7–7.8
300–800
4.9–20.0
2–10
0–2
35–75
20–55
1–4

BUN (mg/dl)
Cre (mg/dl)
ALT (U/l)
ALP (U/l)
LDH (U/l)
Ca (mg/dl)
P (mmol/l)Na (mmol/l)
K (mmol/l)
Cl (mmol/l)Patient

Reference range

45.0
1.8
199
1
711
17.3
8.0
148
4.2
115

17.6–32.8
0.8–2.4
12–130
14–111
0–798
8.8–11.9
2.6–6.0
147–156
3.4–4.6
107–120

ALP, alkaline phosphatase; ALT, alanine aminotransferase; Band, banded neutrophil; BUN, blood urea
nitrogen; Ca, calcium concentration; Cl, chloride; Cre, creatinine; Eos, eosinophil; RBC, red blood cells;
Ht, hematocrit; Hb, hemoglobin; K, potassium; LDH, lactate dehydrogenase; Lym, lymphocyte; Mono,monocyte; Na, sodium; P, phosphate; PLT, platelet; Seg, segmented neutrophil; TP, total protein; WBC,
white blood cells.

Fig. 1. Genetic structures of Notch2-FeLV. Schematic structures of the two clones of Notch2-FeLV (KeyN2-1 and KeyN2-2), feline Notch2
and prototype FeLV provirus. Notch2 contains EGF (blue) and Lin-12-Notch repeats (LNR; pink) in its extracellular region and ANK repeats(orange), two NLSs (green) and proline/glutamic acid/serine/threonine-rich motifs (PEST; purple) in its intracellular region. TM; Notch2transmembrane. Triangle indicates the primers used for cloning the two Notch2-FeLVs. sp, signal peptide.

nal sequences derived from FeLV env gene, and an intracellular region harboring transmembrane (TM) and ankyrin(ANK)-repeats of the feline Notch2 gene. Both clones had
short 23-amino-acid open reading frame (ORF), possibly
derived from the FeLV env gene (Fig. 2A). A second ORF
contained a sequence with a frame-shifted env sequence at
its C-terminal, and this ORF possibly expresses viral Notch2(v-Notch2) fusion protein (Fig. 2B). Other researchers havereported similar Notch2 transduction during experimental
infection of cats with FeLV 61E, a cloned virus, and haveisolated four clones of Notch2-FeLV from two cats [24].

with functional ANK repeats and two nuclear location signals (NLSs). Such truncated expression of Notch receptors
can lead to the constitutive activation of Notch signaling[reviewed in 12]. Direct repeat sequences of the enhanceror the upstream of the enhancer (URE) [21] were not seen in
the LTR of KeyN2-2.
We further analyzed activation of Notch signaling pathway by the v-Notch2 protein using transient luciferase reporter assays. The predicted second ORF of clone KeyN2-2

223R), tagged with Myc at the C-terminus and then cloned
The second ORF of the recombinant v-Notch2 protein istranslated by using the internal ribosome entry site (IRES)
activity within the TM region of Notch2 [14]. All variants
isolated to date include the intracellular region of Notch2

was transiently expressed in HEK293T cells using ScreenFect A (Wako, Osaka, Japan) as manufacturer’s instructions(Fig. 3A). Co-transfection of our plasmid with pGa981-6

269

NOTCH2 TR ANSDUCTION IN FELV

555

Fig. 2. The sequence alignment of Notch2
env gene and the recombinant Notch2 are underlined and in bold. FeLV clone 33 (GenBank accession no. AB060732) [22] was used as aenv gene was frame-shifted (red). *Stop codon. TM; Notch2transmembrane.

Fig. 3.

Expression and activation of v-Notch2 protein. (A) Expression of Myc-tagged v-Notch2

and total cell lysates were subjected to Western blotting analysis using mouse anti-Myc (Wako, Osaka,
HEK293T cells in 24-well plate were co-transfected with pGa981-6 (50 ng), phRL-CMV (5 ng) andv-Notch2 expressing plasmids (v-N2). Luciferase assay was performed in triplicate, and the relative
luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega) at 48 hr
post transfection. The relative luciferase unit (RLU) is shown relative to the negative control (vector).
Error bars denote the standard deviation (SD). **P<0.01 using unpaired t-test.

270

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S. WATANABE ET AL.
[CrossRef]
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V. and Mullins, J. I. 1989. Structure, origin, and transforming
activity of feline leukemia virus-myc recombinant provirus FTT.
J. Virol. 63: 2108–2117. [Medline]
6. Dorn, C. R., Taylor, D. O. and Hibbard, H. H. 1967. Epizootiologic characteristics of canine and feline leukemia andlymphoma. Am. J. Vet. Res. 28: 993–1001. [Medline]
7. Elliott, J., Dobson, J. M., Dunn, J. K., Herrtage, M. E. and Jackson, K. F. 1991. Hypercalcaemia in the dog: A study of 40 cases.
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Sakano, S., Bigas, A., Jimi, E. and Okabe, K. 2008. The association of Notch2 and NF-kappaB accelerates RANKL-induced
osteoclastogenesis. Mol. Cell. Biol. 28: 6402–6412. [Medline][CrossRef]
10. Fulton, R., Forrest, D., McFarlane, R., Onions, D. and Neil, J.
C. 1987. Retroviral transduction of T-cell antigen receptor betachain and myc genes. Nature 326: 190–194. [Medline] [CrossRef]
11. Greenwald, I. 1994. Structure/function studies of lin-12/Notch
proteins. Curr. Opin. Genet. Dev. 4: 556–562. [Medline] [CrossRef]
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216–233. [Medline] [CrossRef]
13. Kurooka, H., Kuroda, K. and Honjo, T. 1998. Roles of theankyrin repeats and C-terminal region of the mouse notch1
intracellular region. Nucl. Acids Res. 26: 5448–5455. [Medline][CrossRef]
14. Lauring, A. S. and Overbaugh, J. 2000. Evidence that an IRES
within the Notch2 Coding Region Can Direct Expression of a
Nuclear Form of the Protein. Mol. Cell Biol. 6: 939–945. [Medline] [CrossRef]
15. Lee, S., Kumano, K., Nakazaki, K., Sanada, M., Matsumoto,A., Yamamoto, G., Nannya, Y., Suzuki, R., Ota, S., Ota, Y.,
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Fukayama, M., Seto, M., Kurokawa, M., Ogawa, S. and Chiba,
S. 2009. Gain-of-function mutations and copy number increasesof Notch2 in diffuse large B-cell lymphoma. Cancer Sci. 100:920–926. [Medline] [CrossRef]
16. Levy, L. S., Gardner, M. B. and Casey, J. W. 1984. Isolation ofa feline leukaemia provirus containing the oncogene myc from afeline lymphosarcoma. Nature 308: 853–856. [Medline] [CrossRef]
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binding promoter and phRL-CMV reference plasmid (Promega, Madison, WI, U.S.A.) that constitutively expresses
renilla luciferase showed dose-dependent activation of thev-Notch2 protein (Fig. 3B).
Lymphoma and refractory hypercalcemia was observedin our cat. Hypercalcemia is commonly linked to malignancy in dogs and humans, especially in lymphoma [7, 23].
In most cases of feline lymphoma, elevation of the serum
calcium concentration or parathyroid hormone-related peptide (PTHrP) is uncommon [2, 25]. Recently, an association
between bone metabolism and Notch signaling has been
revealed; RANKL-induced association of Notch2 and Jagged1 in pre-osteoclasts can lead to the differentiation into
osteoclasts and activates osteoclastgenesis in them throughtracellular region of Notch2. Therefore, v-Notch2 likely possesses the potential to activate osteoclastgenesis independentof RANKL stimulation when overexpressed in osteoclast/monocyte lineage cells.
Because of the recombination-prone property of gammaretroviruses, we observed numerous recombination eventsand various recombinant forms of FeLVs transducing cellular genes, including endogenous FeLV (enFeLV) [28] andERV-DC [1]. The emergence of such recombinant viruses
FeLV-infected cats. Additionally, various numbers of protooncogenes, which function as a key regulator of proliferation
recombinant gammaretroviruses. Although Notch2 has beenrecognized as tumor suppressor in some human tumors [17],transduction of Notch2 gene seems to be a feasible mechanisms for FeLV-induced lymphomagenesis. Finally, our report may provide a new insight into the relationship between
the Notch signaling pathway and humoral hypercalcemia.
ACKNOWLEDGMENTS. The pGa981-6 reporter construct
was provided by RIKEN BRC, which participates in the National Bio-Resources Project of the Ministry of Education,
Culture, Sports, Science and Technology (MEXT), Japan.
This study was supported by a grant from the MEXT, Japan(Grant# 22380168).
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sequence of a transduced myc gene from a defective feline leukemia provirus. J. Virol. 55: 177–183. [Medline]
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from feline leukemia virus. J. Virol. 76: 1527–1532. [Medline][CrossRef]
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1996. Transduction of Notch2 in feline leukemia virus-induced
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De Wolf-Peeters, C. and Delabie, J. 2008. NOTCH2 mutations
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NOTE

Clinical Pathology

Ayumi FUJIMOTO1), Sakurako NEO1), Chinatsu ISHIZUKA1), Takashi KATO1), Kazuhito SEGAWA1),
Shinpei KAWARAI2), Kikumi OGIHARA3), Masaharu HISASUE1)* and Ryo TSUCHIYA1)
1)Laboratory of Veterinary Internal Medicine II, School of Veterinary Medicine, Azabu University, 1–17–71 Fuchinobe, Chuoku,

Sagamihara, Kanagawa 252–5201, Japan2)Department of Veterinary Teaching Hospital, School of Life and Environmental Science, Azabu University, 1–17–71 Fuchinobe, Chuoku,

Sagamihara, Kanagawa 252–5201, Japan
3)Laboratory of Pathology, School of Life and Environmental Science, Azabu University, 1–17–71 Fuchinobe, Chuoku, Sagamihara,

Kanagawa 252–5201, Japan(Received 17 December 2012/Accepted 30 January 2013/Published online in J-STAGE 13 February 2013)
ABSTRACT.

study was to investigate surface antigens, which are considered as stem/progenitor or cancer cell markers, in cHCC cell lines. Expression of
CD133 and Dlk-1 expression was detectable in all cell lines, and three cell lines expressed CD29. These results indicate that CD29, CD44,
KEY WORDS:

canine, hepatocellular carcinoma, liver, stem cell, tumor marker.
J. Vet. Med. Sci.

Canine hepatocellular carcinoma (cHCC) is an uncommon condition, accounting for <1% of all canine tumors.
However, cHCC is the most common primary liver tumor,

hepatic stem/progenitor cells is required to perform analysisof hepatocyte differentiation and maturation. Hepatic stem
and/or progenitor cells not only play a pivotal role in liver
regeneration but are also able to accumulate mutations in theand dogs in general aged >10 years are over-represented inon the basis of their expression of immunohistochemicalmarkers including CD13, CD44, CD90, CD133, epithelial
diseases. Hepatic tumors are usually recognized only in theadvanced stages or when metastasis has already been established. Characterization of hepatic progenitor cells (HPCs)
is one of the key parameters in understanding the cellularmechanisms involved in tumorigenesis, along with the establishment of early diagnostic tools and the development of atherapeutic method. In human medicine, it is widely accepted that cancer cells arise from stem/progenitor cells, as these

et al. reported that CD44 and keratin
19 were expressed in most cHCC cases, while CD133 wasers has not been investigated, and analytical research into
hepatocellular stem/progenitor cell markers, has not been
conducted. Alison maintained that the rodent counterparts
to HPCs described in human models are often referred toas oval cells, which express certain antigens traditionallytime to acquire the requisite number of genetic changes forthat various antigens expressed in liver stem/progenitor cells
match with hepatocellular carcinoma (HCC) cell surface
markers, including cancer stem cells, controversy continues-

tissue, and it will contribute to the diagnosis of cHCC using immunohistochemistry of biopsy samples obtained
from clinical case. In the present study, we investigated theexpression level of some surface antigens recognized as*C ORRESPONDENCE TO: HISASUE
nal Medicine II, School of Veterinary Medicine, Azabu University,

All tumors were removed from dogs with cHCC andJapan.

transplantation to Rowett nude rats and cultured through-©2013 The Japanese Society of Veterinary Science273

A. FUJIMOTO ET AL.

-

those >70, 40 and 10%, respectively. All experiments wereconducted independently and in triplicate. Statistical analy-l
mg/l streptomycin (Gibco Brl) and 2 mmol/ltion). Data regarding expression of surface antigens on mature hepatocytes are expressed as mean ± standard deviation.
All four cHCC cell lines expressed CD44, CD133 andDlk-1 (Fig. 2 and Table 1). Marked CD29 expression was

2with the range of the expression level being between 0.13).
The medium was changed every 3 days, and cells were used-of culture.
mature hepatocyte population. The left lateral lobe (approximately 30 g) was surgically removed from clinically healthyments were conducted with humane care and in compliancewith the guidelines of Azabu University for the treatmentof experimental animals (100323-1). The portal branch wascannulated and perfused with 300 ml of Ca2+- and Mg2+-freerespectively). Expression of all surface antigens on mature
hepatocytes of liver tissue derived from normal healthy dogsl) and EGTA (0.19 g/l). Tissues were then incubatedat 37°C for 20 min with a perfusate of 300 ml collagenase

In this study, CD44, CD133 and Dlk-1 were expressedin all cHCC cell lines, but not in normal hepatocytes. All
cell lines showed high expression of CD44, a member ofl) and CaCl2l
was perfused again with the addition of 300 ml serum-free

relatively consistent with that of cHCC studies, indicatingthat CD44+ cell populations are involved as stem/progenitor

cell suspension was collected. The cells were centrifuged at
g for 3 min, and the separated hepatocytes were used for+ hepatocytes

7/ml andincubated at 4°C for 30 min with monoclonal antibodies,-

hematopoietic stem cells, was observed in cHCC. In addition, another recent study demonstrated that hepatocellularcancer stem cells expressed CD133, and the CD133+ population had both in vitro and in vivo tumourigenic potential
et al. investigated CD133

anti-human CD29, IgG
CD34, IgG
IgG
ed rat anti-canine CD90, IgG

pression level in the present study was caused by the methodof immunohistochemical analysis used, because antigen--

CD133, IgG
1goya, Japan). Isotype-matched immunoglobulins serving as
negative controls were as follows: PE-labeled mouse IgG

hepatic stem/progenitor cell marker, and in the present study,
it was expressed among all cell lines analyzed. This surface
antigen was found to be associated with early liver tissue development, because it was strongly expressed in mouse fetal

rat IgG
PE-labeled rat IgG
FITC-labeled rat IgG1

et al. reported that Dlk-1 was

274

μm.

In the present study, 3 of 4 cell lines showed high CD29
expression, suggesting that this might be a progenitor sur-investigation will be needed to measure CD90 expression in
hepatic cancer stem cells.
CD34, a hematopoietic stem cell antigen that is also
considered a stem cell marker, was not expressed among allcell lines studied, suggesting that it would not be a suitablebiochemical signals from the extracellular environment,
mainly with respect to growth, differentiation, invasivenessand metastatic aspects of malignant cells. Suzuki et al. demonstrated that fetal mouse liver cell populations expressing
CD29 and CD49f differentiated to form colonies containing mature hepatocytes and cholangiocytes, indicating thatthese markers have potential as hepatic stem cell markers

CD34 was found to be valuable in distinguishing HCC fromet al. reported
CD34+ sinusoidal endothelial cells in 27/30 (90%) cases ofhuman HCC, but this antigen was either not or poorly exet al.
demonstrated that murine liver stem cells are Sca+, CD34 ,+ and albumin , suggesting that hepatic

regenerative liver tissue after hepatectomy, and this marker
has shown potential as a stem/progenitor cell marker (datanot shown).
The presence of CD90+ cells was found to be associatedwith a high incidence of distant organ metastasis, suggest-

There is a possibility that CD29, CD44, CD133 and Dlk-1
are expressed only in situations where liver regeneration is
to distinguish between tumor and normal tissue using only
these antigens. In the present study, these surface antigens
were not assessed in liver tissue after hepatectomy or hepatitis, and further research is required to determine appropri-

CD90, while expression was absent in the other cell lines
investigated. However, because the proportion of cancer
stem cells involved in HCC is relatively low, further analysis
will be required to elucidate whether CD90 is suitable forthe purpose of determining cHCC stem cells. In addition,
Arends et al. reported that progenitor cells derived fromhealthy mature canine liver expressed CD90, suggesting its

pathogenic role of these antigens were not investigated inthis study. Furthermore, the expression of surface antigens
in cHCC cell lines may be transformed by abundant repeated275

A. FUJIMOTO ET AL.

Fig. 2.

Flow cytometry was performed to analyze the expression of CD29, CD34, CD44, CD90, CD133 and Dlk-1 in four cHCC cell

Table 1.

Expression status of surface antigens in cHCC cell lines and hepatocyte
Hepatocyte

CD44
CD133
Dlk-1
CD29
CD90
CD34

29.07%
20.97%
20.90%

19.70%
21.17%
94.30%
1.79%

99.30%

4.00 ± 0.04%
3.01 ± 0.02%

0.13%

passage in vitro. In particular, transplantation experiment andcharacterization of cell populations established using thesetion of expression of these markers in biopsy samples may
be helpful in establishing cHCC diagnosis. However, todetermine the usefulness of this diagnostic tool in veterinary

and investigation of gene m-RNA/protein expression ofother oncogenes, transcription factors and factors associatedwith hepatocyte differentiation/maturation will be required.
In conclusion, CD29, CD44, CD133 and Dlk-1 wereexpressed in cHCC cell lines, suggesting that these surface
markers have potential as diagnostic tools and will be helpful in cHCC characterization. In addition, they have thepotential to detect hepatocellular stem/progenitor cells incanine, similar to those in humans. We expect that evalua-antigens should be investigated among cHCC, hepatitis andhyperplastic tissues from clinical cases.
. This study was supported by the
Science Research Promotion Fund of the Promotion and
Mutual Aid Corporation for Private Schools of Japan and a
Grant-in-Aid for Matching Fund Subsidy for Private Universities.

276

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2. Arends, B., Spee, B., Schotanus, B. A., Roskams, T., Van den
In vitro differentiation of liver progenitor cells derived from healthy dog
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3. Chiba, T., Zheng, Y. W., Kita, K., Yokosuka, O., Saisho, H.,
Onodera, M., Miyoshi, H., Nakano, M., Zen, Y., Nakanuma,
Y., Nakauchi, H., Iwama, A. and Taniguchi, H. 2007. Enhanced
self-renewal capability in hepatic stem/progenitor cells drives
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pCEA, CD10, and CD34 expression in separating hepatocellularcytology. Diagn. Cytopathol. 30
Bouvet, M. and Hoffman, R. M. 2010. Simultaneous color-coded
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4. Cogliati, B., Aloia, T. P., Bosch, R. V., Alves, V. A., Hernandezkao, K., Nakauchi, H. and Taniguchi, H. 2000. Flow-cytometricseparation and enrichment of hepatic progenitor cells in thedeveloping mouse liver. Hepatology 32

stem/progenitor cells in canine hepatocellular and cholangiocellular carcinoma. Vet. Comp. Oncol. 8-

murine resident liver stem cell. Cell Death Differ. 15

of Dlk/Pref-1. J. Cell Sci. 116-

lecular markers in the diagnosis of hepatocellular carcinoma.
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neoplasia in dogs. Vet. Radiol. 28
7. Kawarai, S., Hashizaki, K., Kitao, S., Nagano, S., Madarame,
H., Neo, S., Ishikawa, T., Furuichi, M., Hisasue, M., Tsuchiya,

Tsuchiya, R., Shida, T. and Kobayashi, K. 1999. Serum alphafetoprotein values in dogs with various hepatic diseases. J. Vet.
Med. Sci. 61
20. Yamashita, T., Honda, M., Nakamoto, Y., Baba, M., Nio, K.,
Hara, Y., Zeng, S. S., Kondo, T. H., Takatori, H., Yamashita,T., Mizukoshi, E., Ikeda, H., Zen, Y., Takamura, H., Wang, X.
W. and Kaneko, S. 2013. Discrete nature of EpCAM(+) andCD90(+) cancer stem cells in human hepatocellular carcinoma.
Hepatology 57

characterization of primary canine hepatocellular carcinoma celllines producing alpha-fetoprotein. Vet. Immunol. Immunopathol.
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inhibition by imatinib mesylate attenuates progenitor cell expansion and inhibits liver tumor formation in mice. Gastroenterology 135
Bachand, A. M., Kenney, J. G. and Withrow, S. J. 2004. Massive
J. Am.
Vet. Med. Assoc. 225

Ishikawa, Y., Shinozaki, E., Mizunuma, N., Hatake, K. andpatic stem/progenitor cells, is expressed in hepatocellular, colon,
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the promise of liver cancer stem cell makers. Stem Cells Dev. 20:

terization of tumorigenic liver cancer stem/progenitor cells.
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2010. Cancer stem/progenitor cells are highly enriched in
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277

FULL PAPER

SurgerycMice with Acute Hepatitis Induced by CCl4

null

Takashi KATO1), Masaharu HISASUE1)*, Kazuhito SEGAWA1), Ayumi FUJIMOTO1), Eri MAKIISHI1), Sakurako NEO1),
Kyohei YASUNO2), Ryosuke KOBAYASHI2) and Ryo TSUCHIYA 1)
1)Laboratory of Internal Medicine II, School of Veterinary Medicine, Azabu University, 1–17–71 Fuchinobe, Chuoku, Sagamihara,

Kanagawa 252–5201, Japan
2)Research Institute of Biosciences, School of Veterinary Medicine, Azabu University, 1–17–71 Fuchinobe, Chuoku, Sagamihara,

Kanagawa 252–5201, Japan(Received 7 December 2012/Accepted 2 February 2013/Published online in J-STAGE 15 February 2013)
ABSTRACT. Bone marrow cell infusion (BMI) has recently been suggested as an effective therapy for refractory liver disease; however, thenull(NOG)
in a mouse model of acute liver failure. Acute hepatitis was induced by carbon tetrachloride (CCl 4cmice and wild-type (WT) C57BL mice, and the characteristics of liver dysfunction and the degree of hepatic injury and regeneration werecompared between the two mouse models. Next, female CCl 4-treated NOG mice were xenotransplanted with male PKH26-labeled cBMCs,
and the potential of cBMCs to accumulate in injured liver tissue compartments was examined. Fluorescence microscopy was perform ed tohistologically detect the infused cBMCs, and DNA polymerase chain reaction was performed for detection of the male Y chromosome (SRYgene) in the recipient female NOG mice. The number of PKH26-positive cBMCs transplanted in the liver tissue gradually increased in the
NOG mice. The infused cBMCs were located in the necrotic area of the liver at an early stage after transplantation, and most had accumu -differences were observed concerning the extent liver injury and regeneration between the cBMC-transplanted and saline control mice.

KEY WORDS:

null(NOG) mouse.
ccanine bone marrow cell, carbon tetrachloride (CCl 4

doi: 10.1292/jvms.12-0530; J. Vet. Med. Sci. 75(7): 847–855, 2013

wherein it was shown that BMCs administered through aperipheral vein improved liver function in patients with livercirrhosis [13, 22]. Therefore, BMC infusion may prove to bea curative therapy for liver cirrhosis in the future [27].
Recent studies have shown that the effects of BMC ther-

Chronic hepatitis (CH) and cirrhosis have long been recognized in many dog breeds by both primary and referral
veterinary practices [18, 21]. These disorders are often perTherefore, more effective therapies are needed. In human
medicine, liver transplantation is the only effective cure for
CH and cirrhosis, but the limitations of transplantation, suchas a lack of donors, surgical complications, rejection andhigh cost, have led to proposals for less invasive regenerative therapy procedures.
Recent reports demonstrated that murine bone marrowcells (BMCs) transplanted via peripheral vein populated anddifferentiated into albumin-producing hepatocytes via hepatoblast intermediates [1, 26, 29]. Interestingly, BMC infusion improves hepatic function, including elevation of serum

accumulation ability, immunomodulation, trophic support,
differentiation (plasticity), revascularization and tissue
ability is one of the most important factors in cell transplant
therapy [4, 28]. In murine models, accumulation ability has
creas [24]. In another study of liver cirrhosis patients withsplenomegaly, splenectomy enhanced the repopulation of
BMCs into the cirrhotic liver microenvironment and tended
to result in greater improvement of liver function [12]. These

corrects liver dysfunction and improves survival rate in human cirrhosis patient [10, 12]. On the basis of these results,
a clinical trial of autologous BMC infusion was conducted,

the disordered liver is important for effective results.
We previously demonstrated the capacity of canine
BMCs (cBMCs) for differentiation into albumin-producing
hepatocyte-like cells [15]. It has also been shown thatbone marrow stromal cells (BMSCs), which are one of thecomponents of BMCs, have various therapeutic potentials
such as matrix metalloproteinase activity [7]. Furthermore,
xenotransplantation using canine BMSCs may be effective
4-treated im-*C ORRESPONDENCE TO: H ISASUE , M., Laboratory of Veterinary Internal Medicine II, School of Veterinary Medicine, Azabu University,
1–17–71 Fuchinobe, Chuoku, Sagamihara, Kanagawa 252–5201,
Japan.
e-mail: hisasue@azabu-u.ac.jp©2013 The Japanese Society of Veterinary Science

278

848

T. KATO ET AL.

autologous transplantation of BMCs in liver injured models,studies demonstrating the ability of cBMCs to accumulate
in the disordered liver have been limited. In addition, thesystemic effects of infused BMC populations are not wellknown in both dogs and rodents. Haraguchi et al. reported
that the donor genomic DNAs of xenotransplanted canine
BMSCs were detectable in recipient nude mice administeredcyclosporin A (CSA) [8]. Cho et al. described that infused

extracted, and single-cell suspensions were prepared bypassing the cells through a 100-μinto new tubes. Mononuclear cells (MNCs) were isolatedwith Ficoll solution (Lymphoprep; Axis-Shield, Oslo, Norway) and washed and concentrated three times. The isolated
Red Fluorescent Cell Linker Kit (Sigma–Aldrich, St Lous,
MO, U.S.A.) according to the manufacturer’s protocol. Incubation was performed for 5 min at room temperature with 1
× 107 MNCs/ml, followed by washing in phosphate-bufferedsaline (PBS) to remove proteins from the culture medium
for optimal staining. The staining reaction was stopped with

CCl4-injected recipient mice [4]. However, these studies
have not elucidated the localization of transplanted cells ininjured liver tissue. To analyze the accumulation of BMCs innull(NOG) mice in this studycbecause this model accepts heterologous cells much morein hepatocyte growth medium as previously described [15].
Transplantation: Fifteen female NOG mice were grouped
as follows: CCl4 + cBMC (n=5), oil + cBMC (n=5) and CCl4
+ saline (n=5). At 1.5–2 hr after BMC harvesting, prepared
MNCs (1 × 106) or saline was administered to the tail veinsof CCl4
after 1 week, and the livers, lungs and spleens were removed
to evaluate the systemic distribution of the administered
cBMCs (Fig. 1A). To investigate the time course of BMC
accumulation in the injured liver, 32 female NOG mice weregrouped into CCl4 + cBMC (n=16) and CCl4 + saline (n=16)[11].
Therefore, the present study used an NOG mouse modelwith acute hepatitis induced by CCl4 treatment to investigate
the ability of xenotransplanted cBMCs to accumulate in inaccumulation in the liver.
MATERIALS AND METHODS
Animals: Six-week-old NOG mice were purchased fromthe Central Institute for Experimental Animals (Kawasaki,
Japan). Same age C57BL6J Jcl mice were purchased from
CLEA Japan, Inc. (Kawasaki, Japan) to serve as the wildtype (WT) murine control. All mice were shipped to the
Research Institute of Biosciences of Azabu University andhandled with humane care under pathogen-free conditions.
The mice were housed in a room under controlled temperature (25°C), humidity and lighting (12-hr light/dark cycle).
Access to food and tap water was ad libitum throughout the
study period. All of male NOG and C57BL mice were usedin research for an experimental model of liver injury, andfemale NOG mice were used for cBMC transplant experimentation. A 2-year-old male beagle dog that was clinically
healthy was used as a donor of cBMCs for transplantation
into NOG mice. All experiments in this study were performed in accordance with the Animal Protection Guidelinesof Azabu University (authorization no. 10–79).
Experimental model of liver injury: Forty male NOG
mice and 20 male WT mice were divided into three groups:
NOG + CCl4 (n=20), NOG + oil (n=20) and WT + CCl4
(n=20). CCl4 (Wako Pure Chemical Industries, Osaka, Japan) dissolved in olive oil (1 ml/kg) was administered via
intraperitoneal injection in the NOG + CCl4 and WT + CCl4groups, and the extent of liver injury and regeneration was

and the livers were removed at 0 hr, 24 hr, 48 hr and 1 week
after BMC transplantation (Fig. 1B).
Laboratory analysis: The mice were anesthetized withpentobarbital (Somnopentyl; Kyoritu Seiyaku Corporation,
Tokyo, Japan), and their blood was collected from the heart
and centrifuged at 15,00G for 15 min at 4°C. Albumin, alanine aminotransferase (ALT) and aspartate amino transferase (AST) levels were analyzed with a Hitachi 9,000 series
automatic analyzer.
Histological examination
μm-thick sections were stainedwith hematoxylin and eosin (HE). The relative necrotic
area, expressed as a percentage of the total liver area, was(http://rsb.info.nih.gov/ij).
Immunohistochemistry: Tissue sections were subjected
to immunohistochemistry using rat monoclonal antibody
to the Ki-67 antigen (1:200; Dako, Tokyo, Japan), which
detects proliferating cells. Microwave antigen retrieval was
performed in citrate buffer (pH 6.0) for 10–20 min. The sec2O2 in methanol for 20 min.
For the primary antibody reaction, slides were incubated in a
wet chamber overnight at 4°C. The immunoreactive materi-

24 hr, 48 hr, 72 hr and 1 week after CCl4 injection, and theformaldehyde for histological examination.
Preparation of donor BMCs: cBMCs were harvested fromthe humerus and femur of the dog under general anesthesia with intravenous injection of medetomidine (20 μg/kg)
and pentobarbital (10 mg/kg). The cells were collected in a
heparin-containing syringe. After fat removal, cBMCs were

Simple Stain MAX PO; Nichirei, Tokyo, Japan) and diamisections were counterstained with hematoxylin. The number

279

XENOTRANSPLANTATION OF CANINE BONE MARROW CELLS

Fig. 1.

849

Experimental design of cBMC transplantation into NOG mice. (A) The cBMCs were collectedliver injury induced by CCl4 treatment. The mice were grouped as CCl4 + cBMC, oil + cBMC andCCl4 + saline groups (n=5, respectively). The systemic distribution of the transplanted male cBMCs
was evaluated 1 week after transplantation. (B) Sampling times after cBMC transplantation. The CCl4
+ cBMC (n=12) and CCl4
hr, 24 hr, 48 hr and 1 week after BMC transplantation (n=4, respectively).

the ratio of Ki-67-positive cells was calculated.

-

freshly isolated lungs, spleens and livers were embedded in
optimal cutting temperature medium and frozen. Frozen sections (5 μm) were obtained using a cryostat (model HM505;
stained with phalloidin (Alexa Fluor 488 phalloidin, Invitrogen, Carlsbad, CA, U.S.A.). Vectashield mounting medium
with DAPI (Vector Laboratories Inc., Burlingame, CA,
U.S.A.) was used for nuclear staining. The lung and spleen
sections were nuclear stained with DAPI alone. Images(FSX100; Olympus, Tokyo, Japan). Computer-assisted image analysis was performed using ImageJ software version
1.41.
: Genomic DNA
was isolated from NOG mouse liver tissue homogenates using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).
The presence or absence of the sex-determination region of
the male Y chromosome (SRY gene) in the recipient female
NOG mice was assessed by PCR as previously described[25]. Primer sequences for the SRY gene were obtained from280

conditions were as follows: incubation at 94°C for 2 min; 40
cycles of incubation at 94°C for 30 sec, 57°C for 30 sec androse gel electrophoresis and stained with ethidium bromide.
Positive (male canine genomic DNA) and negative (female
canine genomic DNA) controls were included in each assay.
Statistical analysis: Data are expressed as means ± standard error of the mean (SEM). The Mann–Whitney U test
was used to compare the different groups as appropriate.
P
Analyses were performed using GraphPad Prism 5.
RESULTS
Characterization of CCl4-induced acute liver dysfunctionin NOG mice: The degree of liver injury was assessed bymeasuring serum liver enzymes (Fig. 2A). Serum albumin
4 injection, after which the levels improved to within the normal
range in both NOG and WT mice. Both serum ALT and AST

850

T. KATO ET AL.

Fig. 2. CCl4-induced acute liver injury and regeneration in NOG and WT mice. (A) Serum albumin, ALT and AST levels before and 7
days after CCl4
4 injection in both WT and NOG mice. Scale bar=200 μm. (C) Regeneration of livertissue was estimated by detection of Ki-67-positive cells. The ratios of Ki-67-positive cells were calculated in 2,000 hepatocytes. Scalebar=100 μgroup; P
4 treatment of NOG and WT mice (n=4 per group; P<0.05).

increased markedly 24 hr after CCl4 injection; however, they
decreased to within the normal range 48 hr after CCl4 injection, with no differences between the NOG and WT mice. To
evaluate liver damage and regeneration, the necrotic areasof the liver were measured in NOG and WT mice injectedwith CCl4
tent of liver necrosis between the NOG and WT mice (Fig
2B). Immunohistochemistry using Ki-67, a marker for cellproliferation, was performed to assess hepatocyte regeneration (Fig. 2C). Ki-67-positive hepatocytes were limited 24 hr
after CCl4 injection; however, there were many positive cells
48 hr after CCl4 injection, with no differences between the
NOG and WT mice.
Migration of PKH26-positive cBMCs into injured liver
lobules: PKH26-positive cBMCs (1 × 106) were injected

into liver-damaged and control (undamaged) female NOG
microscopy a week after transplantation (Fig. 3). A few
PKH26-positive cells were detected in sections of theliver from undamaged controls (Fig. 3A), and there wereno PKH26-positive cells in CCl4-damaged mice without
cBMC transplantation. In contrast, in the transplanted CCl4treated mice, the number of PKH26-positive cells gradually
increased, spreading into the liver lobules (Fig. 3A, 3B).
Some PKH26-positive cells were observed in the lung tissueof both liver-damaged and control mice (Fig. 3A, 3B), while
no PKH26-positive cells were detected in spleen tissue fromany of the groups (Fig. 3A).
Detection of the SRY gene in transplanted NOG mice:281

XENOTRANSPLANTATION OF CANINE BONE MARROW CELLS

Fig. 3.

851

Engraftment of donor cBMCs in liver, lung, and spleen tissues was examined 1 week after transplanta-of PKH26-positive cBMCs were detected in livers of the CCl4 + cBMC group, but only a few cBMCs weredetected in livers of the oil + cBMC group. In both the CCl4 + cBMC and oil + cBMC groups, some PKH26positive cells were detected in the lung tissue, but not in the spleen tissue. Scale bar=200 μm (B) Quantitative
increased in the CCl 4 + cBMC group compared with the oil + cBMC group, but the number of PKH26Oil+cBMCs group (n=4 per group; P
exclusively in the livers of the female xenotransplanted mice. Lane 1, molecular weight standard; lane 2,
cBMC-infused liver tissue; lane 3, saline-treated liver tissue; lane 4, male dog liver tissue (positive control);lane 5, female dog liver tissue (negative control).

chromosome, demonstrated that transplanted cBMCs were
present in the livers of the transplanted female mice, indicating that male cBMCs accumulated in the liver tissue of thefemale mice (Fig. 3C).
Localization of infused cBMCs in the injured Liver: Phalloidin staining of frozen liver sections was performed to further elucidate the localization of cBMCs in the mouse liver.

The actin cytoskeleton was disordered in necrotic tissue,and PKH26-positive cells were slightly accumulated in the
necrotic areas 24 and 48 hr after transplantation (Fig. 4A).
One week after transplantation, the number of accumulated
cBMCs (PKH26-positive cells) gradually increased andspread into the liver lobules (Fig. 4A, 4B).
Effects of cBMC transplantation on CCl4-induced acute

282

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T. KATO ET AL.

Fig. 4. Combined analysis of actin phalloidin (green), PKH26 (red) and DAPI (blue). (A) The left liver lobes of
NOG mice were analyzed 24 hr, 48 hr and 1 week after cBMC transplantation. The dotted lines indicated theand 48 hr after transplantation, cBMCs were sparsely distributed, corresponding to the necrotic areas. Thesecells had accumulated 1 week after transplantation. (B) Quantitative comparison of PKH26-positive cBMCs inwith that in the oil + cBMC group (n=4 per group; P<0.05). Scale bar=Small bar=200 m, Large Bar=50 m(Fig. A).

liver dysfunction in the NOG mice: To clarify the therapeutic

DISCUSSION

model, the degrees of hepatic injury and regeneration werecompared between the cBMC-transplantation and saline(control) groups (Fig. 5). As parameters of hepatic injury,
serum albumin and hepatic enzyme levels (Fig. 5A) andnecrotic areas (Fig. 5B) were measured. As a measure ofhepatocyte regeneration, the Ki-67-positive ratios were
measured in each group (Fig. 5C). However, none of theseIn the present study, the infused cBMCs not only accumulated in the injured mouse liver at an early stage after

and transplantation groups (P=0.872).

Transplantation of cBMCs into recipient mice was success-tissue. Although several investigators have reported the ability of BMCs to engraft in experimental models, including
study to investigate whether canine BMCs can accumulate in283

XENOTRANSPLANTATION OF CANINE BONE MARROW CELLS

853

between the saline and cBMC groups. Scale bar=200 μm. (C) Hepatocyte regeneration was measured by immunohistochemistry of Ki-67
48 hr after CCl4 injection. Regeneration was unchanged at each time point in both the saline and cBMC groups. Scale bar=100 μm. Bars

ful, as indicated by the detection of both PKH26-labeled
cBMCs and the canine SRY gene in the liver tissue of female
NOG mice. In particular, infused cBMCs were located in the
necrotic area of the liver an early stage after transplantation,with the largest accumulation at 1 week after transplantation.
CCl4-induced acute liver injury in NOG mice peaked at 24hr, and most liver regeneration occurred at 48 hr after CCl4
injection. However, the time course of cBMC migration to
the injured liver tissue did not correspond to the time courseof liver injury or regeneration, because most cBMC accumulation occurred more than 48 hr after transplantation. Previously, Thomas et al. demonstrated that macrophage therapy

for their study, which they were established by a long-term
protocol of CCl4 injection on two consecutive days per week
for at least 6 weeks. In the present study, NOG mice were injected with CCl4 a single time and used as an acute hepatitis
model, so its pathogenesis might be the cause of the different
time course of cell accumulation in chronic hepatic injury.
Therefore, we suggested that transplanted cell accumulationin the injured liver might be affected by the difference intion.
have been developed for heterograft experimentation [2].
NOG mice can accept heterologous cells much more readily
though the immune system plays an important role in liver

detected in liver tissue from 1 to 7 days after transplantation, but the number of cells was not increased [28]. Chronic

284

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T. KATO ET AL.

regeneration [5, 19], it is not known whether NOG mice
are an appropriate model for spontaneous acute hepatitis.

Science Research Promotion Fund of the Promotion and
Mutual Aid Corporation for Private Schools of Japan, by aMatching Fund Subsidy for Private Universities and by agrant from the Ministry of Education, Culture, Sports, Science and Technology (No 80333144).

comparing CCl4-induced hepatitis and liver regeneration between NOG and WT mouse models of acute hepatitis, whichsuggested that NOG mice can be used as an acute liver injury
model similar to WT mice. Both serum hepatic enzyme levels and necrotic areas showed maximum increases 24 hr after
CCl4 injection, and Ki-67-positive hepatocytes appeared 48
hr after CCl4
tent with those of a previous study [20]. It is well known thatthe hepatocyte growth factor (HGF) is the most importantfactor for hepatic regeneration [23]. We expect that liver

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J Vet Intern Med 2012;26:370–376

Eﬀects of Prostaglandin E 1 on the Preparation of Platelet
Concentrates in Dogs
K. Segawa, T. Kondo, S. Kimura, A. Fujimoto, T. Kato, T. Ishikawa, S. Neo, M. Hisasue,
T. Yamada, and R. Tsuchiya
Background: Platelet concentrates (PC) are prepared by centrifugation of platelet-rich plasma (PRP) that is preparedby centrifugation of whole blood. The resuspension of the platelet pellet during PC preparation from dogs is diﬃcult
because of platelet activation induced by centrifugation.
Objectives: To investigate the eﬃcacy of adding prostaglandin E1 (PGE1) to prevent platelet activation during PC
preparation from dogs.
Animals: Fifteen healthy Beagle dogs.
Methods: Prospective, experimental trial: PGE1 was added to PRP before the high-speed centrifugation during PC
preparation. To estimate the eﬀect of this addition, we assessed the platelet aggregability before transfusion, the survivalof the platelets after transfusion, and the platelet reactivity after transfusion, which is estimated by the P-selectin expression of the platelets when stimulated by thrombin.
Results: The diﬃculty associated with platelet resuspension was resolved by PGE1. PGE1 strongly inhibited platelet
aggregation induced by collagen and ADP; however, it recovered after the platelets were resuspended in plasma without
PGE1 (mean aggregation ratio; collagen: 10.00–80.80%, ADP: 8.20–53.60%). Survival of the platelets after transfusion
was not aﬀected by PGE1 (mean 8.04 and 7.56 days, without and with PGE1), and thrombin-induced P-selectin expression
after transfusion in PGE1-treated PC was also well maintained (mean positive ratio 53.7 and 47.9%, before and 24 hours
after transfusion).
Conclusions and Clinical Importance: The addition of PGE1 in PRP before the centrifugation of PRP can improve thepreparation eﬃciency of PC from dogs, while maintaining the therapeutic eﬃcacy of the platelets.
Key words: Aggregability; P-selectin; Survival; Transfusion.

latelet transfusions are indicated when animals
have bleeding tendencies associated with thrombocytopenia, thrombocytopathy, or both. This isespecially important before surgery or until immunosuppressive drugs become eﬀective in the face oflife-threatening bleeding in immune-mediated thrombocytopenia.1–3 In the context of platelet transfusion,platelet concentrates (PC) have advantages over whole
blood transfusion because of the decreased risk ofhemolytic transfusion reactions, volume overload, andthe development of polycythemia.
Platelet concentrates from dogs are prepared byserial diﬀerential centrifugation of fresh whole
blood.4,5 or apheresis.6,7 Apheresis results in high quality PC, but it requires special equipment and generalanesthesia in veterinary practices. Consequently, PC
preparation by the serial diﬀerential centrifugation of
fresh whole blood is more practical in most veterinary
clinics.

P

Abbreviations:
PGE1
PRP
PC
PPP
ADP
PE
PBS
ANOVA
SD

prostaglandin E1
platelet-rich plasma
platelet concentrates
platelet-poor plasma
adenosine diphosphate
phycoerythrin
phosphate buﬀered physiological saline
analysis of variancestandard deviation

A protocol for the preparation of PC from dogs byusing serial diﬀerential centrifugation was ﬁrst reported
in 1993.4 In the serial diﬀerential centrifugation
method, PC are harvested by a high-speed centrifugation of platelet-rich plasma (PRP) after a low-speed
centrifugation of fresh whole blood. However, it is difﬁcult to resuspend platelet pellets following the highspeed centrifugation caused by platelet activation.
Platelet activation during PC preparation contributes
to the development of platelet storage lesions anddecreased platelet viability.8–10 It is therefore important
to develop a method to minimize platelet activation
during the preparation of PC.
Prostaglandin E1 (PGE1) is a reversible inhibitor ofplatelet activation because of its ability to increase cyclic AMP levels within the platelets.9,11 Although earlystudies showed that the addition of PGE1 to human
PRP facilitated the separation, concentration, andresuspension of platelets,9,12–14 there have been nostudies of the eﬀects of PGE1 on the preparation of
PC in dogs. The aim of this study was to investigate

From The Laboratory of Internal Medicine II, School of
Veterinary Medicine, Azabu University, Kanagawa, Japan. This
work was done in The Laboratory of Internal Medicine II, Schoolof Veterinary Medicine, Azabu University. Previously presented at
the 150th Meeting of the Japanese Society of Veterinary Science,2010.
Corresponding author: Ryo Tsuchiya, DVM, PhD, The Laboratory of Internal Medicine II, School of Veterinary Medicine,
Azabu University, 1-17-71 Fuchinobe, Chuou-ku, Sagamihara-shi,
Kanagawa 252-5201, Japan; e-mail: tsutiyar@azabu-u.ac.jp.

Submitted May 30, 2011; Revised November 20, 2011;
Accepted December 16, 2011.
Copyright © 2012 by the American College of Veterinary Internal
Medicine
10.1111/j.1939-1676.2011.00881.x

287

PGE1 and Platelets of Dogs

the eﬃcacy of adding PGE1 during PC preparation.
We estimated the ease of platelet resuspension after
high-speed centrifugation of PGE1-treated PC andthen conﬁrmed platelet function before and after transfusion, and also evaluated survival of the PGE1-treatedplatelets after transfusion.

Materials and Methods
Study Design
The platelet aggregability in PC before transfusion, the survival of the platelets after transfusion, and the platelet reactivity
after transfusion were assessed to evaluate the beneﬁts of modifying the PC preparation by adding PGE1. The platelet viability in
PC prepared with PGE1 was conﬁrmed by the comparing platelet
aggregability before and after PGE1 treatment (n = 5). The survival of the platelets after transfusion was assessed by ﬂow
cytometry using a biotin label. The platelets in PC prepared with
PGE1 and that in PRP without PGE1 (both n = 5) were labeledwith biotin and then were reinfused into the same individuals.
Blood samples were collected 1 hour after transfusion (Day 0)
and at the same time on Days 1 through 7. The survival time of
the transfused platelets were calculated by the changes in the biotin-positive ratio of the platelets. Subsequently, the biotinylated
PC prepared with PGE1 (n = 5) were reinfused and blood samples were collected 1 and 24 hours after reinfusion. The P-selectin
expression following thrombin stimulation in the biotin-labeledplatelets was analyzed by ﬂow cytometry using FITC-labeledanti-P-selectin antibody to estimate the reactivity after transfusion.

Experimental Animals
Research protocols were approved by the Animal ExperimentCommittee of Azabu University. Fifteen healthy Beagle dogs, 11
intact males and 4 intact females, 1–12 years of age and weighing
9–15 kg, were used for the experiments. Some dogs were rotatedand used for more than 1 experiment (the number of dogs usedin each experiment is described below).They were maintained in
the Institute of Life Sciences or in the Veterinary Clinical Center,
Azabu University, according to the Animal Experiment Guidelines of the university. They had no history of venipuncture ortreatment with any drug, including nonsteroidal anti-inﬂammatory agents, for at least 2 weeks before the study. They wereallowed free access to water but were fasted for at least 12 hours
before blood collection and were fed after the last sampling wascomplete on each experimental day.

PC Preparation
A quantity of 200 mL of whole blood was drawn into 200 mL
blood transfusion bagsa containing citrate-phosphate dextrose
adenine 1 (CPDA-1) at the standard mixing ratio (1 part CPDA1 to 6.5 parts blood) from the jugular vein through 17-gauge
needles. The bag was weighed during blood collection, and thecollection tubing was clamped oﬀ when it reached the intended
collection volume.
Platelet concentrates were prepared by serial diﬀerential
centrifugation of fresh whole blood.4,5 All blood components
containing platelets were handled gently at 20–24°C (roomtemperature) to minimize platelet activation. After resting for
30 minutes, the whole blood was centrifuged (soft spin) for
3.5 minutes at 1,000 9 g to prepare PRP. The PRP were thentransferred into a satellite bagb using a plasma extractor. Extraction was stopped when the red blood cell interface was 1 cm

371

from the top of the collecting bag, and the PRP was left undisturbed for 15 minutes. For PC that were to be treated with
PGE1, PGE1c (1 mmol/L in ethanol) was injected into the PRP(ﬁnal concentration, 1 lmol/L) through an outlet port, and thenmixed manually by rocking the bags back and forth. The PRP
was centrifuged (hard spin) for 10 minutes at 2,000 9 g to produce a platelet pellet. The platelet-poor plasma (PPP) was transferred to another satellite bag, leaving behind 20 mL of plasma
with the platelet pellet. The platelet pellet was left undisturbed
for 1 hour to promote platelet disaggregation, and then the platelets were resuspended by gentle manual back and forth rocking
and massaging of the bag.

Aggregability of Platelets in PC Prepared with PGE1
The aggregability of the platelets in PC treated with and without PGE1 was assessed. Four samples for platelet aggregometry
were collected from the transfusion bags at each step of PC preparation. First, a sample of the PRP was collected from the satellite bag when the PRP was produced, and the remainder was
divided in 2 satellite bags. Second, the PGE1 was added to oneof the separated PRP bags, and then a sample (PGE1+ PRP)
was collected immediately. Third, the PGE1+ PRP was centrifuged (hard spin), and then the supernatant was replaced with
PGE1-free autologous plasma. The platelet pellet was resuspended after a 1 hour incubation, and a sample of the PGE1removed PC was collected. Fourth, a control PC sample was alsocollected from the paired untreated PRP bag after high-speed
centrifugation, after a 1‐hour incubation, and resuspension
according to the reported method.4,5Aggregometry was performed as follows. First, the platelet
counts were measured using an automated cell counterd andadjusted to approximately 250,000/lL by adding autologous
PPP. After a 30 minute rest for the resuspended platelets, opticalplatelet aggregometry was performed by using an aggregometer.e
Collagenf and adenosine diphosphate (ADP)g were used as agonists. Because CPDA-1 is not an ideal anticoagulant for aggregometry,15,16 the stimulating conditions were modiﬁed with theaddition of CaCl2 to a ﬁnal concentration of 15 mmol/L to accelerate the platelet reaction, and the addition of low molecularweight heparinh at a ﬁnal concentration 0.6 IU anti-Xa/mL toprevent the plasma coagulation induced by adding the CaCl2.
The ﬁnal concentrations of collagen and ADP were 100 lg/mL
and 20 lmol/L, respectively.

Survival of Platelets after Transfusion
The survival of the platelets after transfusion in PC preparedwith PGE1 and that in PRP without PGE1 were investigatedaccording to Heilmann’s technique of biotinylation.7,16,17 The PC
and PRP were produced as described above. Next, the platelet
survival was assessed as follows: N-hydroxysuccinimido biotin(0.2 mg/mL PC or PRP)i dissolved at 20 mg/mL in dimethylsulphoxide was added into PC prepared with PGE1 or PRP,
and then mixed well. After a 10-minute incubation at room temperature, the biotin-treated platelets were infused to the same
individuals through a cephalic vein over 30 minutes. Quantitiesof 2.5 mL of blood samples were collected 1 hour after reinfusion (Day 0) and at the same time on Days 1 to 7 to assess thesurvival of the reinfused platelets. The samples were anticoagulated with ethylenediaminetetraacetic acid disodium salt and centrifuged for 1 minute at 2,000 9 g to separate the PRP. Then the
PRP were stored at 4°C until the ﬁnal sample was collected.
After collecting all the samples for the platelet survival study,
the platelet counts of each PRP sample were adjusted to approximately 10,000/lL using a phosphate buﬀered physiological saline

288

372

Segawa et alcontaining 3% bovine serum albumin at pH 7.2 (PBS). A quantity of 10 lL of phycoerythrin-streptavidin (PE-SA)j was added
to 100 lL of the platelet suspensions. The percentages of biotinylated platelets were measured by PE positive events using ﬂow
cytometry.
The samples were analyzed using an Epics XL ﬂow cytometerk
or a Cyﬂow SL instrument.l The light scatter and ﬂuorescencechannels were set on a logarithmic scale. DNA-check beadsm
were used to calibrate the ﬂuorescence. The forward scatter
threshold was set to exclude debris and machine noise. Platelets
were identiﬁed by their forward and side light scatter. A minimum of 2,000 platelets were analyzed. The background ﬂuorescence was evaluated by use of nonbiotinylated platelets, andgates were established so that approximately 95% of the events
had no ﬂuorescence.
The survival of the platelets after transfusion was calculatedusing a linear regression model.18,19

diﬀerences in the platelet reactivity between the positive control
and the values after transfusion. In addition, the paired t-test
was used to compare the platelet reactivity between samples collected at 1 and 24 hours after transfusion. Data were expressedas the means ± standard deviation (SD). Box plots were also
used as descriptive parameters within ﬁgures. Values of P < .05
were considered to be signiﬁcant.

Results
Platelet Resuspension after High-Speed
Centrifugation
Platelets were packed tightly to the bottom, irrespective of the presence of PGE1 (Fig 1A). The plateletpellets prepared with PGE1 achieved uniform resuspension after the 20‐minute agitation (Fig 1B). Because
the untreated platelet pellet was diﬃcult to disaggregate, there were more platelet aggregates in the bag
without PGE1 (Fig 1C). The platelet pellets without
PGE1 took 30 minutes or more to achieve uniform
resuspension.

Platelet Reactivity after Transfusion
To investigate the platelet reactivity after transfusion, thrombin-induced P-selectin expression was analyzed. The Heilmann
platelet biotinylation technique described above was also used todiscriminate the transfused platelets from the other platelets.7,16,17
Biotinylated PC prepared with PGE1 were reinfused as describedabove. A quantity of 2.5 mL of blood was collected into sodium
citrate at 1 and 24 hours after reinfusion to assess platelet P-selectin expression with thrombin stimulation.7,20,21
Because the P-selectin expression changed over time, the analyses of P-selectin expression were performed at 1 and 24 hours
after reinfusion. The PRP was separated from these samples as
described above. After a 15‐minute incubation of the PRP, PGE1
at ﬁnal concentration of 1 lmol/L was added to the PRP to stop
the activation of the platelets. Next, the PRP was centrifuged for
3 minutes at 2,000 9 g, and platelets were resuspended in PBS
at approximately 10,000/lL. A total of 100 lL of the platelet
suspension was incubated with 20 lL of FITC-labeled antihuman P-selectin mouse monoclonal antibody7,21,n and 10 lL of
PE-SA.
While they were incubated with the antibody, the platelets
were activated with thrombino at a ﬁnal concentration 0.25 U/
mL in the stimulus group. This was done to determine if thetransfused platelets maintained their reactivity to thrombin stimulation. After a 20‐minute incubation at room temperature and 2
washes, the platelets were resuspended in 150 lL of PBS andanalyzed immediately using ﬂow cytometry.
The positive and negative control data were also acquired
from preliminary studies where platelets were not transfused, butwhere they were thrombin stimulated or were left unstimulated.
The FITC-labeled mouse IgG1p was used as an isotypic control for the anti-P-selectin monoclonal antibody, and no nonspeciﬁc binding was observed (data not shown). Although the same
ﬂow cytometer settings as described above were used, the background ﬂuorescence was evaluated by use of nonstimulated platelets. Compensation was performed for all dual-labeled samples toensure adequate separation of events with and without ﬂuorescence.

Aggregability of Platelets in PC Prepared with PGE1
Although there were signiﬁcant diﬀerences in theaggregability between PRP and PGE1+ PRP (collagen:P < .001; n = 5, ADP: P < .001; n = 5, Fig 2A and
2B), there was no signiﬁcant diﬀerence in the aggregability between PRP and PGE1-removed PC using
either agonist. As a result of the replacement of PGE1containing plasma with intact PPP, the collagen or
ADP-induced platelet aggregation recovered similar to
the PRP.

A

B

C

Statistical Analysis
All data were analyzed using the same statistical software program.q Normality was conﬁrmed by the Kolmogorov-Smirnov
test. The maximal aggregation values were compared between the
PRP and other fractions using Dunnett’s posthoc test. The
unpaired t-test was used for the analysis of the platelet survival
after transfusion. Unpaired t-tests were performed to analyze theFig 1. The platelet resuspension after high-speed centrifugation
and a 1‐hour resting period with (B) and without (C) PGE1 indogs. Platelet pellets (A) were manipulated gently by hand for
20 minutes to achieve uniform resuspension. A lot of platelet
aggregates were still found in bag C.

289

PGE1 and Platelets of Dogs

373

A

B

Fig 3. A dot plot of the transfused platelets evaluated by ﬂow
cytometry in dogs. Biotin/phycoerythrin-streptavidin (SA) labeledplatelets were discriminated as PE positive events (upperscatters).

was 8.04 ± 0.49 days (n = 5) in the group that wastransfused with PRP without PGE1 and was
7.56 ± 0.81 days (n = 5) in the group that was transfused with PC prepared with PGE1 (Fig 4). No signiﬁcant diﬀerence was found between the groups in theplatelet survival (P = .3).

Platelet Reactivity after Transfusion

Fig 2. The aggregability of platelets in platelet concentrates(PC) prepared with prostaglandin E1 (PGE1) as analyzed byturbidimetric aggregometry in dogs. The samples of platelet-richplasma (PRP), control PC (without PGE1), PGE1+ PRP, andPGE1 removed PC were collected from transfusion bags at eachstep of PC preparation. The ﬁnal concentrations of collagen (A;n = 5) and ADP (B; n = 5) were 100 lg/mL and 20 lmol/L,
respectively. Dunnett’s posthoc test was used to compare the
PRP and other fractions. For box graphs, the line within the box
represents the median value, the limits of the box represent the25th and 75th percentile values, and the whiskers represent therange. NS, not signiﬁcant (same in Figs 4 and 6).

Survival of Platelets after Transfusion
Transfused platelets were discriminated as biotin/
PE-SA positive events (Fig 3). The platelet survival

Transfused platelets were completely discriminatedfrom other platelets as PE-positive events (Fig 5).
There were no signiﬁcant diﬀerences between PE-positive and PE-negative platelets with regard to the P-selectin expression that was evaluated as FITC positive
events. Thrombin-induced P-selectin expression eventswere compared between transfused PC and control
data (Fig 6). There were no signiﬁcant diﬀerences
between the positive control (P-selectin positive ratio:
53.7 ± 5.1%; n = 5) and transfused PC prepared with
PGE1 (1 hour after transfusion: 42.0 ± 13.0%; n = 5,
24 hour after transfusion: 47.9 ± 13.3%; n = 5) byunpaired t-tests. No signiﬁcant diﬀerence was foundbetween the values at 1 and 24 hours after transfusionby the paired t-test.

Discussion
This study suggests that the addition of PGE1
during preparation of PC by serial diﬀerential centrifugation is an eﬀective tool for decreasing plateletactivation.
It is often diﬃcult to resuspend platelet pellets following high-speed centrifugation of PRP according to
the reported protocol,4,5 and treatment with PGE1
made platelet resuspension easier and faster. Therefore,

290

374

Segawa et al% Platelets remaining

A

Days after reinfusion% Platelets remaining

B

Fig 5. A dot plot of the transfused platelet responses to thrombin as quantitated by the P-selectin expression in dogs. To detect
platelet surface P-selectin, an FITC-labeled anti-human P-selectinmonoclonal antibody was used. Transfused platelets werediscriminated as indicated in Figure 3.

Days after reinfusion

Fig 4. The survival of biotin-labeled autologous platelets indogs, reinfused as platelet-rich plasma (A; n = 5) or PC preparedwith prostaglandin E1 (B; n = 5). The percentage of biotinylated
platelets at various times was quantitated by ﬂow cytometry as
indicated in Figure 3. The percentage of biotin-positive platelets
on Day 0 in each dog was estimated as 100% remaining. According to the consensus opinion of the Biomedical Excellence for
Safer Transfusion Collaborative, only time points beyond
20 hours after infusion were used to analyze the survival of platelets after transfusion. No signiﬁcant diﬀerence in the platelet
survival was found between the groups according to the unpaired
t-test. For box graphs, the line within the box represents themedian value, the limits of the box represent the 25th and 75th
percentile values, and the whiskers represent the range.

Fig 6. The reactivity of the transfused platelets in dogs. The
platelet responses at 1 and 24 hours after transfusion were quantitated by thrombin-induced P-selectin expression as indicated in
Figure 5. The positive and negative controls were derived fromplatelets before transfusion with and without thrombin stimulation (n = 5). There were no signiﬁcant diﬀerences between thepositive control and transfused Platelet concentrates as determined by unpaired t-tests. There was also no signiﬁcant diﬀerence found between the values at 1 and 24 hours after
transfusion by the paired t-test. Neg. cont., negative control; Pos.
cont., positive control; after TX, after transfusion. For box
graphs, the line within the box represents the median value, thelimits of the box represent the 25th and 75th percentile values,
and the whiskers represent the range. NS, not signiﬁcant.

addition of PGE1 resolved the diﬃculties associatedwith platelet pellet resuspension in dogs.
To treat hemostatic abnormalities caused by thrombocytopenia, thrombocytopathy, or both, platelets in
PGE1-treated PC should act normally after transfusion. The inhibitory eﬀect of PGE1 on platelets is
considered to be reversible,9,11 but it was necessary to
conﬁrm the reversibility in vitro before a PGE1-treated PC transfusion is performed. In this study, thecollagen- or ADP-induced platelet aggregation was
clearly inhibited by PGE1 treatment. After replacement of the supernatant plasma containing PGE1
with autologous intact PPP, the inhibitory eﬀects of
PGE1 were lost. This reversibility of the eﬀect of
PGE1 suggested that platelets in PGE1-treated PC act

normally during hemostasis when they are diluted
with a larger amount of circulating plasma after
transfusion.

291

PGE1 and Platelets of Dogs

Whenever new platelet-rich products are produced,
an assessment of the in vivo platelet survival is essential to predict the platelet viability after transfusion.
We found that the platelet survival after transfusion of
PGE1-treated PC was equal to that of intact PRP.
The reversible eﬀect of PGE1 on platelets was
conﬁrmed in vitro using platelet aggregometry as
described above. However, the viability of platelets
after transfusion in PGE1-treated PC is not necessarily
guaranteed by evidence of their aggregation. Therefore, we investigated the platelet reactivity after transfusion. Although there are many kinds of laboratory
examinations that can be used to assess the quality ofPC, there are no in vivo methods to predict the clinicaleﬃcacy.22 For this study, we combined the thrombininduced P-selectin expression analysis with Heilmann’s
biotinylation technique7,16,17 which distinguishes transfused platelets from other intact platelets using ﬂow
cytometry. This original technique was considered tobe quite powerful to estimate the viability of the platelets after transfusion. Our results showed that thestrong inhibitory eﬀect of PGE1 on platelet function
had disappeared after PC transfusion, and that platelets in PGE1-treated PC maintain their reactivity for atleast 24 hours after transfusion.
Because the PGE1 solution is available commerciallyand is licensed for clinical use, the addition of PGE1
during preparation of PC is easily implemented andpractical. In human medicine, the addition of PGE1 tothe preparation of PC and 5 days of storage reduced
the survival of platelets after transfusion to
5.8 ± 1.6 days compared with 6.9 ± 1.4 days in control
PC prepared without PGE1.9 Although the addition of
PGE1 did not shorten the survival of platelets after
transfusion in this study, we did not assess the in vivo
survival of stored platelets treated with PGE1. Therefore, the addition of PGE1 should only be consideredwhen fresh PC is transfused immediately in dogs. Furthermore studies are needed to investigate the storage
stability of PC prepared with PGE1.

Footnotesa

Teruﬂex, BB-SCD207J01, Terumo Company, Tokyo, Japan
Transfer Bags, BB-T015CJ, Terumo Companyc
Prostaglandin E1, Sigma Chemical Company, Saint Louis, MOdSysmex F-820, Sysmex Corporation, Kobe, Japane
Chrono-log C550 dual-channel aggregometer, Chrono-log Corp,
Havertown, PA
fChrono-collagen, Chrono-log Corpg
Chrono-ADP, Chrono-log Corph
FRAGMINiv5,000, Pﬁzer Japan Inc, Tokyo, Japani
N-hydroxysuccinimido biotin, Sigma Chemical CompanyjPhycoerythrin-streptavidin, Sigma Chemical Companyk
EPICS XL, Beckman Coulter, Fullerton, CAl
CyFlow SL, Partec Gmbh, Munster, GermanymDNA-check Beads, Beckman Coulter, Fullerton, CA
nAnti-human CD62P-FITC, clone AC1.2, Becton Dickinson,
San Jose, CAoThrombin, Sigma Chemical Company
b

375

p

Mouse IgG1 isotype control-FITC, clone MOPC-21, Becton
Dickinson, San Jose, CAqPrism, Version 5.0, Graphpad Software, San Diego, CA

Acknowledgments
This study was partially supported by The Promotion and Mutual Aid Corporation for Private Schoolsof Japan, and a Grant-in-Aid for Matching Fund
Subsidy for Private Universities.

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ࣃࢡ㉁ࢥ࣮ࢻ㡿ᇦࡢ F'1$ ࢆࡶࡘⓎ⌧⣔࣋ࢡࢱ࣮ࢆస〇ࡋࡓࠋࡉࡽ࡟ࠊ/HI 㑇ఏᏊࡢࣉ࣮ࣟࣔࢱ࣮㡿ᇦࢆࡶࡘࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮࣋ࢡࢱ࣮ࡶస〇ࡋࡓࠋ⤖ᯝ࡜⪃ᐹ
'1$ ࣐࢖ࢡࣟ࢔ࣞ࢖࡟ࡼࡿ⥙⨶ⓗ㑇ఏᏊⓎ⌧ẚ㍑ゎᯒࡢ⤖ᯝࠊ࢔ࣟ࣌ࢩ࢔ ; ⑕ࡢ⓶⭵⤌⧊࡛೺ᖖ⓶⭵࡟ẚ࡭࡚᭷ព࡟Ⓨ⌧㔞ࡢῶᑡࡀぢࡽࢀࡓ」ᩘࡢ㑇ఏᏊ࡟ࡘ࠸࡚⑌ᝈཎ
ᅉೃ⿵㑇ఏᏊ࡜ࡋ࡚ὀ┠ࡋࡓࠋḟ࡟ࠊࡇࢀࡽࡢೃ⿵㑇ఏᏊ࡟ࡘ࠸࡚ࠊ࢔ࣟ࣌ࢩ࢔ ; ⑌ᝈ≟࡜೺ᖖ≟ཬࡧ &'$ ⑌ᝈ≟ࡢᩘከࡃࡢಶయࡢ⓶⭵࡟࠾ࡅࡿཝᐦ࡞ᐃ㔞ⓗⓎ⌧ゎᯒࢆ
UHDOWLPH573&5 ἲࢆ⏝࠸࡚ᐇ᪋ࡋࡓࠋࡑࡢ⤖ᯝࠊ᭱᭷ຊೃ⿵㑇ఏᏊ㸦࢔ࣟ࣌ࢩ࢔ ;ࡢࡳ࡛ῶᑡࡋ࡚࠸ࡿ㑇ఏᏊ㸧࡜ࡋ࡚ࠊ'O[ ཬࡧ /HI ࡀᚓࡽࢀࡓ㸦ᅗ 㸧ࠋ'O[ 㑇ఏᏊ⏘≀ࡣẟໟࡢศ໬ࡸẟࡢ࿘ᮇ࡟㛵୚ࡍࡿࢱࣥࣃࢡ㉁ࡢ  ࡘ࡛࠶ࡾࠊ/HI 㑇ఏᏊ⏘≀ࡣ'O[ 㑇ఏᏊⓎ⌧ࢆಁ㐍ࡍࡿ㌿෗ᅉᏊ࡛࠶ࡿࡇ࡜ࡀ࣐࢘ࢫ࡛▱ࡽࢀ࡚࠸ࡿࠋ
࢖ࢾ 'O[ 㑇ఏᏊࡢ᥎ᐃࣉ࣮ࣟࣔࢱ࣮㡿ᇦ㸦Ѹࠥ㸧࡟ࣝࢩࣇ࢙࣮ࣛࢮ㑇ఏᏊࢆ⤌ࡳ㎸ࢇࡔ᪂つࡢ࣏࣮ࣞࢱ࣮ࣉࣛࢫ࣑ࢻࠊཬࡧࡑࡢࢹ࣮ࣜࢩ࣑࣮ࣙࣥࣗࢱࣥࢺࡢ࣏࣮ࣞ
ࢱ࣮࣋ࢡࢱ࣮ࢆ⏝࠸࡚ࠊ&& ⣽⬊ෆ࡛Ⓨ⌧⣔࣋ࢡࢱ࣮ࢆ⏝࠸࡚࢖ࢾ /HI 㑇ఏᏊࡢࢱ
ࣥࣃࢡ㉁ࢆ୍㐣ᛶ࡟㐣๫Ⓨ⌧ࡉࡏࠊࣝࢩࣇ࢙࣮ࣛࢮάᛶࢆ

ᐃࡋࡓࠋࡇࡢホ౯⣔࡛ࠊ/HI㌿෗ᅉᏊࡢⓎ⌧ࡣࠊᮇᚅ㏻ࡾ࡟ 'O[ 㑇ఏᏊࡢⓎ⌧ࢆቑຍࡉࡏࡓ㸦ᅗ 㸧ࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊ࣐࢘ࢫ࡛ሗ࿌ࡉࢀ࡚࠸ࡿ /HI ㌿෗ᅉᏊࡀᛂ⟅ࡍࡿ⤖ྜ㒊఩㸦ѸࠥѸ㸧௨እ࡟
ࡶࠊ'O[ 㑇ఏᏊࡢⓎ⌧ࢆไᚚࡍࡿᛂ⟅࢚࣓ࣞࣥࢺࡀ 'O[ 㑇ఏᏊࡢࣉ࣮ࣟࣔࢱ࣮㡿ᇦ࡟Ꮡᅾࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓ㸦ᅗ 㸧ࠋࡉࡽ࡟ࠊ࢖ࢾ࢔ࣟ࣌ࢩ࢔ ; ࡟ᑐࡋ࡚๻ⓗ࡞Ⓨẟຠᯝࡀ࠶ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿᕷ㈍ࡢື≀⏝೺ᗣ⿵ຓ㣗ရ 5 8㸦⏕⌮άᛶ≀㉁ࢆྵࡴࣜ
ࢰ࣮ࣉࢫ⳦ UKL]RSXVRU\]DH ⏤᮶ᢳฟ≀㸧ࢆᮏホ౯⣔࡛࢔ࢵࢭ࢖ࡋࡓ࡜ࡇࢁࠊ5 8 ࡣ⏝㔞౫Ꮡⓗ࡟ 'O[ 㑇ఏᏊⓎ⌧ࢆಁ㐍ࡋࡓ㸦ᅗ 㸧ࠋ5 8 ࡢⓎẟ࣭⫱ẟຠᯝࢆࢧ࣏࣮ࢺࡍࡿ
୍ࡘࡢᶵᗎ࡛࠶ࡿ࠿ࡶࡋࢀ࡞࠸ࠋࡲࡓࠊᵓ⠏ࡋࡓ /HI 㑇ఏᏊࡢࣉ࣮ࣟࣔࢱ࣮㡿ᇦࢆࡶࡘࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮ࣉࣛࢫ࣑ࢻ࡟ࡼࡿ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖⣔ࢆ⏝࠸࡚ 5 8 ࢆホ౯ࡋࡓ࡜ࡇࢁࠊ5 8 ࡣ㌿෗ẁ㝵࡛ࡢ /HI 㑇ఏᏊࡢⓎ⌧ࡶಁ㐍ࡍࡿࡇ࡜ࡀࢃ࠿ࡗࡓ㸦ᅗ㸧ࠋࡇࢀࡽࡢ࣏࣮ࣞࢱ࣮ホ౯⣔ࢆ⏝࠸࡚ࠊ'O[ 㑇ఏᏊⓎ⌧ࡢไᚚᶵᵓ࡟㛵ࡍࡿศᏊ⏕≀Ꮫⓗゎᯒࢆࡍࡿ࡜࡜ࡶ࡟ࠊᙜヱ㑇ఏᏊⓎ⌧ࢆಁ㐍ࡍࡿ≀㉁ࡢ᥈⣴ࢆ⾜ࡗ࡚࠸ࡿࠋᮏ◊✲࡛ࡢ࣏࣮ࣞࢱ࣮ホ౯⣔࡛ࡣࠊ࠸ࡃࡘ࠿ࡢᇵ㣴⣽⬊ᰴࢆ᳨ウࡋࡓୖ࡛ࠊ&& ⣽⬊ᰴࢆ⏝࠸ࡓࡀࠊࡼࡾಙ㢗ᛶࡢ㧗࠸Ⓨẟ࣭⫱ẟຠᯝࢆホ౯ࡍࡿࡓࡵ࡟࢖ࢾẟங㢌ึ௦ᇵ㣴⣽⬊ᰴࢆ☜❧ࡋ⏝࠸ࡿࡢࡀᮃࡲࡋ࠸࡜ᛮࢃࢀࡿࠋ

295

ࡲࡓࠊ࢔ࣟ࣌ࢩ࢔ ; ⑌ᝈ≟࡜೺ᖖ≟࡟࠾ࡅࡿࣉ࣮ࣟࣔࢱ࣮㡿ᇦࢆྵࡴ 'O[ 㑇ఏᏊࡢሷᇶ㓄ิࢆỴᐃࡋẚ㍑ࡋࡓࡀࠊㄪ࡭ࡓ㡿ᇦ࡟࠾ࡅࡿ୧⪅ࡢሷᇶ㓄ิ࡟┦㐪ࡣㄆࡵࡽࢀ࡞
࠿ࡗࡓࠋ

296

297

Lef1 promoter-Luc⌧

ᅗ4䚷R&U䛻䜘䜛⃰ᗘ౫ᏑⓗLef1㑇ఏᏊⓎ䛾ಁ㐍㸯.ㄽᩥⓎ⾲

1)

Katakawa Y, Funaba M, Murakami M.: Smad8/9 is regulated through the BMP pathway. J.
Cell. Biochem. [Epub ahead of print] 2016.

2)

Nishino Y, Murakami M, Funaba M.: Expression and role of the TGF-β family in glial
cells infected with Borna disease virus. Microbes and Infection., 18, 128-36, 2016.

3)

Kida R, Yoshida H, Murakami M, Shirai M, Hashimoto O, Kawada T, Matsui T, Funaba
M.: Direct action of capsaicin in brown adipogenesis and activation of brown adipocytes.
Cell Biochem. Funct., 34, 34-41, 2016.

4)

Kasai T, Kato Y, Saegusa S, Murakami M.: Distribution of major staphylococcalca cassettechromosome mec types and exfoliative toxin genes in Staphylococcus pseudintermedius
strains from dogs with superficial pyoderma. J Azabu Uni., 27, 27-31, 2015.

5)

Yamada A, Kodo Y, Murakami M, Kuroda M, Aoki T, Fujimoto T, Arai K.: Hybrid originof gynogenetic clones and the introgression of their mitochondrial genome into sexual
diploids through meiotic hybridogenesis in the loach, Misgurnus anguillicuadatus. J Exp
Zool A Ecol Genet Physiol 323, 593-606, 2015.

6)

Shirai M, Nomura R, Kato Y, Murakami M, Kondo C, Takahashi S, Yamasaki Y,
Matsumoto-Nakano M, Arai N, Yasuda H, Nakano K, Asai F.: Short communication:
Distribution of Porphyromonas gulae fimA genotypes in oral specimens from dogs withmitral regurgitation. Res Vet Sci 102, 49-52, 2015.

298

7)

Kanamori Y, Murakami M, Matsui T, Funaba M.: Role of TPA-responsive element in
hepcidin transcription induced by the bone morphogenetic protein pathway.

Biochem

Biophys Res Commun 466, 162-6, 2015.
8)

Murakami M, Ohi M, Ishikawa S, Shirai M, Horiguchi H, Nishino Y, Funaba M.: Adaptive
expression of uncoupling protein 1 in the carp liver and kidney in response to changes inambient temperature. Comp Biochem Physiol Part A, 185, 142-9, 2015. ᰝㄞ᭷

9)Kanamori Y, Murakami M, Matsui T. and Funaba M.: The regulation of hepcidin
expression by serum treatment: requirements of the BMP response element and STAT- andAP-1-binding sites. Gene 551, 119-126, 2014.

10) Kanamori Y, Murakami M, Matsui T, Funaba M.:Hepcidin expression in liver cells:
evaluation of mRNA levels and transcriptional regulation.Gene 546, 50-5, 2014. ᰝㄞ᭷11) Yamaguchi S, Sano A, Hiruma M, Murata M, Kaneshima T, Murata Y, Takahashi H,
Takahashi S, Takahashi Y, Chibana H, Touyama H, Nguyen Thi Thanh Ha, Nakazato Y,
Uehara Y, Hirakawa M, Imura Y, Terashima Y, Kawamoto Y,Takahashi K,Sugiyama K,
Hiruma M, Murakami M, Hosokawa A, Uezato H.: Isolation of Dermatophytes and Related
Species from Domestic Fowl (Gallus gallus domesticus). Mycopathologia., 178, 135-43,
2014.
12) Nishita T, Yatsu J, Murakami M, Kamoshida S, Orito K, Ichihara N, Arishima K, Ochiai
H.: Isolation and sequencing of swine carbonic anhydrase VI, an enzyme expressed in theswine kidney. BMC Res Notes 7, 116, 2014.
13) Asai K, Funaba M, Murakami M.: Enhancement of RANKL-induced MITF-E expression
and osteoclastogenesis by TGF-β. Cell Biochem Funct 32, 401-9, 2014.
14) Murata M, Murakami M.: Two distinct mtDNA lineages among captive African penguins inJapan. J Vet Med Sci 76, 559-63, 2014.
15) Watanabe M, Tanaka K, Takizawa T, Segawa K, Neo S, Tsuchiya R, Murata M, Murakami
M, Hisasue M.: Characterization of a canine tetranucleotide microsatellite marker locatedin the first intron of the tumor necrosis factor alpha gene.

J Vet Med Sci 76, 119-22. 2014.

16) Shibuya E, Murakami M, Kondo M, Kamei Y, Tomonaga S, Matsui T, Funaba M.:
Downregulation of Pgc-1α expression by tea leaves and their by-products. Cell Biochem
Funct 32, 236-40, 2013.
17) Suenaga M, Kurosawa N, Asano H, Kanamori Y, Umemoto T, Yoshida H, Murakami M,
Kawachi H, Matsui T, Funaba M.: Bmp4 expressed in preadipocytes is required for the
onset of adipocyte differentiation. Cytokine 64, 138-45, 2013.
18) Dong J, Murakami M, Fujimoto T, Yamaha E, Arai K.: Genetic characterization of theprogeny of a pair of the tetraploid silver crucian carp Carassius auratus langsdorfii. Fish Sci
79, 935-41, 2013.

299

 Iida N, Fukushima H, Hiroi M, Yagi M, Kanda T, Murakami M, Sugiyama K.:Development of duplex SYBR green real-time PCR for rapid and simultaneous detection of
16 specific genes of 16 major foodborne bacteria. Jpn J Food Microbiol (᪥ᮏ㣗ရᚤ⏕≀Ꮫ఍㞧ㄅ), 30, 160-4, 2013.
 Yoshida H, Kanamori Y, Asano H, Hashimoto O, Murakami M, Kawada T, Matsui T,
Funaba M.: Regulation of brown adipogenesis by the Tgf-β family: InvolvementofSrebp1c
in Tgf-β-and Activin-induced inhibition of adipogenesis. Biochim Biophys Acta1830,
5027–35, 2013.
 Ohnishi M, Okatani AT, Harada K, Sawada T, Marumo K, Murakami M, Sato R, Esaki H,
Shimura K, Kato H, Uchida N, Takahashi T.: Genetic characteristics of CTX-M-Type
extended-spectrum-β-lactamase (ESBL)-producing enterobacteriaceae involved in mastitis
cases on Japanese dairy farms, 2007 to 2011. J Clin Microbiol 51, 3117-22, 2013.
 Ohnishi M, Okatani AT, Esaki H, Harada K, Sawada T, Murakami M, Marumo K, Kato Y,
Sato R, Shimura K, Hatanaka N, Takahashi T.: Herd prevalence of Enterobacteriaceae
producing CTX-M-type and CMY-2 β-lactamases among Japanese dairy farms. J ApplMicrobiol 115, 282-9, 2013.
 Hirai N, Shirai M, Kato Y, Murakami M, Nomura R, Yamasaki Y, Takahashi S,
Matsumoto-Nakano M, Nakano K, Asai F.: Correlation of age with distribution of
periodontitis-related bacteria in Japanese dogs. J Vet Med Sci 75, 999-1001, 2013.
 Murakami M, Shirai M, Ooishi R, Tsuburaya A, Asai K, Hashimoto O, Ogawa K, Nishino
Y, Funaba M.: Expression of activin receptor-like kinase 7 in adipose tissues. Biochem
Genet 51, 202-10, 2013.
 Murata M, Takahashi H, Takahashi S, Takahashi Y, Chibana H, Murata Y, Sugiyama K,
Kaneshima T, Yamaguchi S, Miyasato H, Murakami M, Kano R, Hasegawa A, Uezato H,
Hosokawa A, Sano A.: Isolation of Microsporum gallinae from a fighting cock (Gallus
gallus domesticus) in Japan. Med. Mycol., 51, 144-9, 2013.
 Yamasaki Y, Nomura R, Nakanoa K, Inaba H, Kuboniwa M, Hirai N, Shirai M, Kato Y,
Murakami M, Naka S, Iwai S, Matsumoto-Nakano M, Ooshima T, Amano A, Asai F.:Distribution and molecular characterization of Porphyromonas gulae carrying a newfimA genotype. Vet Microbiol 161, 196-205, 2012.
 Haishima A, Murakami M, Ikeda T, Inoue K, Kamiie J, Shirota K.: Detection of Bcl-2
mRNA and its product in the glomerular podocytes of the normal rat kidney. Exp. Toxicol.
Pathol., 64, 633-7, 2012.
 Nomura R, Shirai M, Kato Y, Murakami M, Nakano K, Hirai N, Mizusawa T, Naka S,
Yamasaki Y, Matsumoto-Nakano M, Ooshima T, Asai F.: Diversity of fimbrillin amongPorphyromonas gulae clinical isolates from Japanese dogs. J Vet Med Sci 74, 885-91,300

2012.
 Tanaka M, Izawa T, Kuwamura M, Nakao T, Maezono Y, Ito S, Murata M, Murakami M.,,
Sano A,Yamate J.: Deep granulomatous dermatitis of the fin caused by fusarium solani in a
false killer whale (Pseudorca crassidens). J. Vet. Med. Sci., 74, 779-82, 2012.
 Nishikawa O, Arishima K, Kobayashi T, Shirai M, Murakami M, Sakaue M,Yamamoto M.:
Maternal exposure to low doses of DES altered mRNA expression of hepatic microsomal
enzymes in male rat offspring. J. Vet. Med. Sci., 74, 247-53, 2012.
 Ishii Y, Takizawa T, Iwasaki H, Fujita Y, Murakami M, Groppe JC, Tanaka K.: Nucleotide
polymorphisms inthe canine Noggin gene and their distribution among dog (Canis lupus
familiaris) breeds. Biochem Genet 50, 12-8, 2012.
 Ooishi R, Shirai M, Funaba M, Murakami M.: Microphthalmia-associated transcription
factor is required for mature myotube formation. Biochim Biophys Acta 1820, 76–83,
2012.
 Umemoto T, Furutani Y, Murakami M, Matsui T, Funaba M.: Endogenous Bmp4 in
myoblasts is required for myotube formation in C2C12 cells. Biochim. Biophys. Acta.,
1810, 1127-35, 2011.
 Morita H, Nakano A, Onoda H, Toh H, Oshima K, Takami H, Murakami M, Fukuda S,
Takizawa T, Kuwahara T, Ohno H, Tanabe S,Hattori M.:Bifidobacterium kashiwanohense
sp. nov., isolated from healthy infant faeces. Int. J. Syst. Evol. Microbiol., 61(Pt 11),
2610-5, 2011.
 Kato Y, Shirai M, Murakami M, Mizusawa T, Hagimoto A, Wada K, Nomura R, NakanoK,
Ooshima T, Asai F.: Molecular detection of human periodontal pathogens in oral swab
specimens from dogs in Japan. J Vet Dent 28, 84-89, 2011.

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Microbes and Infection 18 (2016) 128e136
www.elsevier.com/locate/micinf

Original article

Expression and role of the TGF-b family in glial cells infected with
Borna disease virus
Yoshii Nishino a,*, Masaru Murakami b, Masayuki Funaba c,*a

Department of Animal Medical Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan
Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara 252-5201, Japanc
Division of Applied Biosciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japanb

Received 24 June 2015; accepted 9 October 2015
Available online 19 October 2015

Abstract
A previous study revealed that the expression of the Borna disease virus (BDV)-encoding phosphoprotein in glial cells was sufﬁcient to
induce neurobehavioral abnormalities resembling Borna disease. To evaluate the involvement of the TGF-b family in BDV-induced changes incell responses by C6 glial cells, we examined the expression levels of the TGF-b family and effects of inhibiting the TGF-b family pathway in
BDV-infected C6 (C6BV) cells. The expression of activin bA and BMP7 was markedly increased in BDV-infected cells. Expression of Smad7, a
TGF-b family-inducible gene, was increased by BDV infection, and the expression was decreased by treatment with A-83-01 or LDN-193189,
inhibitors of the TGF-b/activin or BMP pathway, respectively. These results suggest autocrine effects of activin A and BMP7 in C6BV cells.
IGFBP-3 expression was also induced by BDV infection; it was below the detection limit in C6 cells. The expression level of IGFBP-3 wasdecreased by LDN-193189 in C6BV cells, suggesting that endogenous BMP activity is responsible for IGFBP-3 gene induction. Our results
reveal the regulatory expression of genes related to the TGF-b family, and the role of the enhanced BMP pathway in modulating cell responses in
BDV-infected glial cells.
© 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Borna disease virus; TGF-b family; Glial cells; BMP; IGFBP-3

1. Introduction
Borna disease virus (BDV) is a non-segmented negativestrand RNA virus belonging to the family Bornaviridae [1].
The genome of BDV encodes at least 6 proteins: a nucleoprotein (N), nonstructural protein (X), phosphoprotein (P),matrix protein (M), envelope protein (G), and RNA polymerase (L) [2]. BDV naturally infects a wide range of warmblooded hosts [3]; BDV infection induces severe signs ofneurological disease, including behavioral abnormalities [3].
Responses to BDV infection vary according to differences inthe species, animal strain, age of the host at the time of* Corresponding authors.
E-mail addresses: nishino@cc.kyoto-su.ac.jp (Y. Nishino), mfunaba@kais.
kyoto-u.ac.jp (M. Funaba).

infection, or viral strain, indicating that host-dependent factors
as well as virus-speciﬁc factors determine the onset of Borna
disease [4e6]. However, the precise mechanism underlying
the BDV-induced onset of behavioral disorders currently remains unclear.
Previous studies have attempted to identify the BDVderived molecule(s) responsible for the onset of Borna disease in BDV-infected animals [7,8], and found that the forced
expression of P in glial cells [7] but not N in neurons and glial
cells [8] induced behavioral abnormalities such as enhanced
intermale aggressiveness, hyperactivity, and spatial reference
memory deﬁcits resembling the neurobehavioral abnormalities
exhibited in BDV-infected animals. Furthermore, the ectopic
expression of BDV P did not affect neurodegenerative reactions [7]. Thus, functional modulations in glial cells havebeen implicated in these behavioral abnormalities [7].

http://dx.doi.org/10.1016/j.micinf.2015.10.001
1286-4579/© 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

303

Y. Nishino et al. / Microbes and Infection 18 (2016) 128e136

We previously demonstrated that the expression of transforming growth factor (TGF)-b family members and theirsignal components was altered in the brains of 3-week-old rats
infected with BDV [6]; the expression of TGF-b1, activin bE,
and GDF15 was increased in the brain in response to BDV
infection, whereas that of TGF-b2, inhibin a, and BMP2 wasdecreased [6]. Furthermore, the expression of TbRII and
ALK5, receptors for TGF-b [9,10], was shown to be increasedin BDV-infected brains, while that of Smads, signal mediatorsand regulators of the TGF-b family [9,10], was generally
increased by BDV infection [6]. Cell growth and differentiation are potently regulated by the TGF-b family in many typesof cell, and alteration of TGF-b family signal is implicated in
the pathogenesis of diseases through disturbances of cell
response [9,10]. In view of the (patho-)physiological role of
the TGF-b family, we hypothesized the involvement of the
TGF-b family in the BDV-induced functional modulation of
the central nervous system; previous ﬁndings on the Borna
disease-like phenotype resulting from the glial expression of
BDV P [7] and modulated expression of TGF-b family
signaling components [6] prompted us to hypothesize that
BDV infection in glial cells induces behavioral disturbances
through the modulation of TGF-b family signaling. In order tovalidate our hypothesis, the present study examined 1) the
regulatory expression of the signal components of the TGF-b
family in glial cells infected with BDV, and 2) the effects ofinhibiting the TGF-b family pathway on cell responses in
BDV-infected glial cells.
2. Materials and methods
2.1. Materials
The following reagents were purchased: TGF-b1 and
BMP7 were from R & D Systems (Minneapolis, MN, USA);LDN-193189, an inhibitor of BMP type I receptors [11,12],
was from Stemgent (San Diego, CA, USA); A-83-01, an inhibitor of TGF-b/activin type I receptors [13], was from
Calbiochem (La Jolla, CA, USA); rabbit polyclonal antibodies against phospho-Smad1 (Ser463/Ser465)/Smad5(Ser463/Ser465)/Smad8 (Ser465/Ser467) (#9511), phosphoSmad2 (Ser465/Ser467) (#3101), or phospho-ERK (Thr202/
Tyr204) (#9101) were from Cell Signaling Technology(Danvers, MA, USA); and a mouse monoclonal antibody
against a-tubulin (B-5-1-2) was from Abcam (Cambridge,
MA, USA).
2.2. Cell culture
C6 glioma cells [14] and C6 glioma cells infected with the
BDV He/80 strain (C6BV, [15]) were cultured in Dulbecco's
modiﬁed Eagle's medium (DMEM) supplemented with 10%
heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. In order to examine theeffects of inhibitors of the TGF-b family on gene expression,
C6 and C6BV cells were plated at the density of 1  104/mL.

304

129

Sixteen hours after plating, cells were treated with A-83-01(5 mM) or LDN-193189 (100 pM) or both for 72 h.
2.3. Western blot
C6 and C6BV cells were plated at the density of 2.5  105/mL to examine the phosphorylation of Smad and ERK. Twenty
hours after plating, cells were cultured in DMEM medium with
0.2% FBS for 4 h to eliminate the effects of serum on phosphorylation. Cells were pretreated with A-83-01 (5 mM) or
LDN-193189 (100 pM) for 15 min, followed by a treatment
with TGF-b1 (100 pM) or BMP7 (2 nM) for 1 h. Western blot
analyses were performed as described previously [16].
Immunoreactive proteins were visualized using the ECL SelectWestern blotting detection system (GE Healthcare, Buckinghamshire, UK), according to the manufacturer's protocol.
2.4. RNA isolation, cDNA synthesis, and real-timequantitative PCR
Total RNA was isolated from C6 and C6BV cells using the
RNeasy Mini Kit (Qiagen, Venlo, the Netherlands). The concentration of RNA was calculated from absorbance at 260 nm.
The ratio of absorbance at 260 nm to that at 280 nm wascomparable among samples. The cDNA was synthesized from
1 mg of total RNA by the ReverTra Ace qPCR Master Mix(Toyobo, Osaka, Japan), according to the manufacturers' protocols. The cDNA corresponding to 5 ng of total RNA, i.e.,
0.5% of the cDNA, was used as a template to evaluate
expression levels, except for IGFBP-3, for RT-quantitative
PCR (RT-qPCR); the cDNA corresponding to 20 ng of total
RNA (2% of the cDNA) was used to examine the expressionof IGFBP-3. qPCR was performed using Thunderbird qPCR
Mix (Toyobo) in Applide Biosystems StepOnePlus Real TimePCR Systems (Life Technologies, Carlsbad, CA, USA), according to the manufacturers' protocol. The qPCR proﬁle was
as follows: after denaturing for 20 s at 95  C, 40 cycles consisted of 15 s at 95  C and 60 s at 60  C. The oligonucleotide
primers for RT-qPCR and PCR efﬁciency [17] are shown in
Table 1. All the primer sets were designed to span anexoneexon junction to exclude the possibility of ampliﬁcationof genomic DNA. After 40 cycles of RT-qPCR, the dissociation (melting) curve of the products was examined by changes
in the ramp temperature from 60  C to 95  C. Each sample
showed a single peak, suggesting the expected PCR products.
The abundance of gene transcripts was calculated by thestandard curve method, and the value was normalized against
HPRT1, GAPDH or TBP. The expression level of genes in C6
cells treated without the inhibitor was set to 1, except for
IGFBP-3. Since the expression of IGFBP-3 in C6 cells was
below the detection limit, its level in C6BV cells treated
without the inhibitor was set at 1. The cell culture experiments, i.e., comparison of gene expression between C6 cells
and C6BV cells as well as effect of inhibitors for the TGF-b
family pathway on gene expression, were performed at least
two times, and showed similar results.

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Y. Nishino et al. / Microbes and Infection 18 (2016) 128e136

Table 1
Nucleotide sequences of primers used in RT-qPCR analyses, sizes of PCR products, and mean PCR efﬁciency.
Gene
TGF-b family:
TGF-b1
TGF-b2
TGF-b3
Inhibin a
Activin bA
Activin bB
Activin bC
Activin bE
BMP2
BMP4
BMP7
GDF15
Type I receptors:
ALK1
ALK2
ALK3
ALK4
ALK5
ALK6
Type II receptors:
TbRII
ActRIIA
ActRIIBBMPRII
Smad:
Smad1
Smad2
Smad3
Smad4
Smad5
Smad6
Smad7

PrimerSequence

GenBank accession number

Size (bp)

Efﬁciency (%)a

50
30
50
30
50
30
50
30
50
30
50
30
50
30
50
30
50
30
50
30
50
30
50
30

50 -cctggaaagggctcaacac-30
50 -ctgccgtacacagcagttct-30
50 -agtgggcagcttttgctc-30
50 -gtagaaagtgggcgggatg-30
50 -agtggctgttgcggagag-30
50 -gctgaaaggtatgacatggaca-30
50 -gcacaggacctctgaaccag-30
50 -agctgcctgatcctcacag-30
50 -atcatcacctttgccgagtc-30
50 -tcactgccttccttggaaat-30
50 -gatcatcagctttgcagagaca-30
50 -cataggggagcagtttcaggta-30
50 -ggaaaccctgttggagca-30
50 -agacgggtttggttgatgtt-30
50 -ccccaggcagcactgaccaga-30
50 -gcggtaggttgaagtggatt-30
50 -cggactgcggtctcctaa-30
50 -ggggaagcagcaacactaga-30
50 -cgggcttgagtaccctgag-30
50 -tgggatgttctccagatgttc-30
50 -cctgggcttacagctctcc-30
50 -tccatgccgtccaatcag-30
50 -cggatactcagtccagaggtg-30
50 -gcggtaggcttcggggaga-30

X52498100

97.5

NM_031131

73

120.9

NM_013174

72

102.9

NM_012590

88

94.5

NM_017128

71

94.2

XM_001053684

137

92.9

BC089799

90

104.4

AF140032

120

97.0

NM_017178

70

99.6

NM_012827

85

109.0

XM_001053727

82

101.1

NM_019216

99

86.6

50
30
50
30
50
30
50
30
50
30
50
30

50 -ctaaccgactggcagcagat-30
50 -ggtaccagcactctcgcatca-30
50 -tgggcctttggcctcgttctg-30
50 -ggtggcttgtaatcttccactatac-30
50 -ctgtattgtcgccatgatcg-30
50 -ggttgtaacgacctctgcttg-30
50 -tccttcttcccccttgttgtcct-30
50 -gcatgcacacagcagagcct-30
50 -ggccaaatattcccaacagat-30
50 -tctcataattttggccatcactc-30
50 -tccccaatcgatggagcagt-30
50 -cgcccagcactctgtcata-30

L3608866

117.0

BC167754

77

84.3

D38082

86

93.3

S7646684

96.1

BC087035

65

94.8

BC092609

68

100.2

50
30
50
30
50
30
50
30

50 -ctactctgtctgtggatgac-30
50 -ccaaattcatcctggattct-30
50 -cctaccctcctgtacttgttcc-30
50 -gcaatggcttcaaccctagtaa-30
50 -ggctcagctcatgaacgact-30
50 -ctctgccacgactgcttgt-30
50 -gagccctccctggacttg-30
50 -atatcgaccccgtccaatca-30

S6777093

86.1

S4819078

99.1

BC097358

69

108.8

NM_080407

60

97.0

50
30
50
30
50
30
50
30
50
30
50
30
50
30

50 -gcagcccttttcagatgccag-30
50 -ggctgagagccatcctgggc-30
50 -caggacgattagatgagcttg-30
50 -cgtatttgctgtactcagtcccc-30
50 -cctgccactgtctgcaagata-30
50 -gcaaattcctggttgttgaagat-30
50 -gaacactggatggacgactg-30
50 -acagacgggcatagatcaca-30
50 -cagcctatggacacaagcaa-30
50 -ggcaacaggctgaacatctc-30
50 -gttgcaacccctaccacttc-30
50 -ggaggagacagccgagaata-30
50 -acccccatcaccttagtcg-30
50 -aaatccatcgggtatctgga-30

BC061757

85

95.2

BC127497

113

96.0

U6647965

99.5

AF056002

69

103.0

AB010955

78

98.6

NM_001109002

76

93.3

AF15962673

94.2(continued on next page)305

Y. Nishino et al. / Microbes and Infection 18 (2016) 128e136131

Table 1 (continued )
Gene
Smad8
Target genes:
p21WAFIGFBP-3
Corrected gene:
Hprt1
GAPDH
TBP
a

PrimerSequence

GenBank accession number

Size (bp)

Efﬁciency (%)a503050 -accattaccgcagagtggaga-30
50 -tgagggttgtactcgctgtg-30

AF012347

73

91.2

50
30
50
30

50 -gcagtgcccgagttaagg-30
50 -ccaggatcggacatggtg-30
50 -ggaaagacgacgtgcattg-30
50 -gcgtatttgagctccacgtt-30

U2417491

92.8

NM_012588

78

101.5

50
30
50
30
50
30

50 -gaccggttctgtcatgtcg-30
50 -acctggttcatcatcactaatcac-30
50 -acaactttggcatcgtgga-30
50 -cttctgagtggcagtgatgg-30
50 -cccaccagcagttcagtagc-30
50 -caattctgggtttgatcattctg-30

NM_012583

61

82.2

NM_017008

62

92.9

NM_001004198

75

97.6

Mean value.

2.5. Statistical analyses
Data are expressed as the mean ± SE. Gene expression
levels were log-transformed to provide an approximation of anormal distribution before being analyzed. Regarding theexpression of genes related to the TGF-b family, differences
between C6 cells and C6BV cells were examined using an
unpaired t-test. Data for the expression of Smad7, p21WAF,
and IGFBP-3 were analyzed by ANOVA using the GLM
procedures of SAS [18]. Considered factors were cell type, A83-01, LDN-193189, interaction of cell type and A-83-01(cell  A-83-01), the interaction of cell type and LDN-193189(cell  LDN-193189), interaction of A-83-01 and LDN193189 (A-83-01  LDN-193189), and interaction of cell
type, A-83-01, and LDN-193189 (cell  A-83-01  LDN193189). The expression of IGFBP-3 was below the detection
limit in C6 cells irrespective of the treatment; therefore, thefactors were A-83-01, LDN-193189, and the interaction of A83-01 and LDN-193189 (A-83-01  LDN-193189). The effects of the inhibitor treatment in each cell type, and the effects of the cell type treated with the respective inhibitor (forthe expression of Smad7 and p21WAF) were examined byDunnett's test. Differences of P < 0.05 were consideredsigniﬁcant.

mediated by BMPRII, ActRIIA, and ActRIIB type II receptors, and ALK2/3/6 type I receptors. According to current
models, constitutively active type II receptor kinases transphosphorylate and activate type I receptors, which subsequently transmit signals downstream by phosphorylating
serines at the carboxyl terminus of receptor-regulated (R)Smads. There are two R-Smad subclasses: activin/TGF-b
pathway-speciﬁc R-Smads (Smad2 and Smad3) and BMP
pathway-speciﬁc R-Smads (Smad1, Smad5, and Smad8).
Once activated by phosphorylation, R-Smads form complexes
with Smad4, and accumulate in the nucleus where theyinteract with transcriptional regulators for target genes [9,10].
We ﬁrst explored the regulatory expression of 12 membersof the TGF-b family in C6 glioma cells in response to BDV
infection (Fig. 1). C6BV cells did not exhibit cytopathic

3. Results
3.1. Expression of the TGF-b family was modulated in
BDV-infected C6 glioma cells
TGF-b family consists of three major subfamilies, i.e.,
TGF-bs, activins and BMPs. TGF-b family transmits signals
through speciﬁc heteromeric complexes with type I receptors,
called activin-like kinases (ALKs), and type II receptors. Bothtype I and type II receptors have intracellular serine/threonine
kinase domains. In canonical signal transduction, TGF-bs
signal via the TbRII type II receptor and uses the ALK5 type I
receptor. Activins associates with ActRIIA and ActRIIB typeII receptors, and the ALK4 type I receptor. BMP signaling is306

Fig. 1. Expression of TGF-b family members in C6 glioma cells infected with
BDV. Total RNA was isolated from C6 and C6BV cells, and expression of the
TGF-b family was evaluated by RT-qPCR. Expression was normalized to thatof HPRT1, and expression in C6 cells was set to 1. Data are shown as themean ± SE (n ¼ 5). **: P < 0.01.

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Y. Nishino et al. / Microbes and Infection 18 (2016) 128e136

effects (data not shown), and the growth rate of C6BV cells
was similar to that of C6 cells (data not shown). The expression of TGF-b1, activin bA, and BMP7 was signiﬁcantly
increased in BDV-infected cells. The expression of activin bA
was clearly up-regulated; its expression level in C6BV cells
was 33-fold higher than that in C6 cells. In contrast, theexpression of activin bE and GDF15 was signiﬁcantly lower in
C6BV cells than in C6 cells; the expression of activin bE wasdown-regulated in C6BV cells (17-fold). The altered expression of the TGF-b family in BDV-infected cells did not result
from changes in the expression level of HPRT1, the reference
gene; the expression pattern was essentially unchanged by thecorrection by GAPDH or TBP (data not shown). Furthermore,
expression pattern of the other genes shown as below was also
comparable, irrespective of the reference gene (data notshown). Expression levels of inhibin a, activin bB, activin bC,
and BMP4 were below the detection limit (data not shown).
We next examined the expression levels of receptors for the
TGF-b family (Fig. 2). The expression levels of ALK3 and
ALK4 were signiﬁcantly decreased in C6BV cells (Fig. 2), andthe BDV infection also decreased the expression of ActRIIA(Fig. 2). In contrast, the expression levels of ActRIIB and
BMPRII were signiﬁcantly higher in C6BV cells than in C6
cells (Fig. 2). The expression of ALK1 and ALK6 was below
the detection limit (data not shown). We further evaluated theexpression levels of Smad (Fig. 3). The expression of Smad1
and Smad7 was signiﬁcantly lower and higher in C6BV cells
than in C6 cells, respectively.
3.2. BDV infection affected endogenous activities of
TGF-b family members in C6 glioma cells
In order to explore the role of the aberrant expression of
TGF-b family components, we used A-83-01 and LDN-

Fig. 2. Expression of receptors for the TGF-b family in C6 glioma cells
infected with BDV. Total RNA was isolated from C6 and C6BV cells, and theexpression of type I and type II receptors for the TGF-b family was evaluatedby RT-qPCR. Expression was normalized to that of HPRT1, and expression inC6 cells was set to 1. Data are shown as the mean ± SE (n ¼ 5). * and **:P < 0.05 and P < 0.01, respectively.

Fig. 3. Expression of Smads in C6 glioma cells infected with BDV. Total RNA
was isolated from C6 and C6BV cells, and the expression of Smad wasevaluated by RT-qPCR. Expression was normalized to that of HPRT1, andexpression in C6 cells was set to 1. Data are shown as the mean ± SE (n ¼ 5).
**: P < 0.01.

193189, inhibitors of the TGF-b/activin type I receptors [13]and BMP type I receptors [11,12], respectively. Inhibition of
the type I receptor serine/threonine kinase leads to block Smadphosphorylation and subsequent signaling [11e13]. As expected, the TGF-b1 treatment induced the phosphorylation of
Smad2 in C6 and C6BV cells; this phosphorylation was
blocked by the pretreatment with A-83-01, but not by that with
LDN-193189 (Fig. 4A, second line). In C6 cells, BMP7
potentiated the phosphorylation of Smad1/5/8, which was
blocked by the pretreatment with LDN-193189 (Fig. 4A, top).
The treatment with TGF-b only slightly induced the phosphorylation of Smad1/5/8, and this phosphorylation was
blocked by A-83-01, but not by LDN-193189. The phosphorylation of Smad1/5/8 was stronger in C6BV cells than in
C6 cells. Furthermore, BMP7 did not induce the phosphorylation of Smad1/5/8 in C6BV cells; this may have resulted
from the full activation of Smad1/5/8 in C6BV cells underbasal conditions.
Previous studies showed the modulation of the ERK MAP
kinase pathway in BDV-infected PC12 neuronal cells [19],primary cultured neurons [20], and oligodendroglial cells [21].
In addition, the functional interaction between the TGF-b
family pathway and the ERK pathway has been reported inmany biological systems [9,10]. Thus, we measured phosphorylated ERK levels (Fig. 4A, third line), because ERK isknown to be phosphorylated and activated [22]. The phosphorylation level of ERK was lower in C6BV cells than in C6
cells. In C6 cells, TGF-b1 and BMP7 decreased the phosphorylation of ERK, as did the treatment with A-83-01 or
LDN-193189, suggesting that the phosphorylation of ERK isregulated by the TGF-b family with dual effects, i.e., efﬁcient
ERK phosphorylation by the optimal activity of the TGF-b
family. In contrast, BMP7 increased phosphorylated ERK
levels in C6BV cells. Furthermore, A-83-01 increased thephosphorylation of ERK, irrespective of the treatment with307

Y. Nishino et al. / Microbes and Infection 18 (2016) 128e136

133

The expression of Smad7, which inhibits Smad-mediatedsignaling [9,10], is regulated by the TGF-b family; TGF-b,
activin A, and BMP were previously shown to increase Smad7
mRNA levels in various cell types [23e25]. Not only A-83-01,
but also LDN-193189 effectively decreased the expression of
Smad7 in C6 cells and C6BV cells, except for the treatment
with LDN-193189 alone in C6 cells (Table 2, Fig. 4B). These
results suggested that the endogenous activities of TGF-b/
activin and BMP regulated Smad7 expression in C6 cells and
C6BV cells. Consistent with the results in Fig. 3, the expression of Smad7 was signiﬁcantly higher in C6BV cells than in
C6 cells (Fig. 4B). In view of the marked up-regulation in theexpression of activin bA and lack of expression of inhibin a in
C6BV cells, activin A (a homodimer of activin bA), but notinhibin A (a heterodimer of inhibin a and activin bA) was
produced in C6BV cells, which may stimulate Smad7
expression in an autocrine manner. In contrast, BMP7 may be
the principle molecule responsible for enhanced BMP activity
in C6BV cells, as suggested by the higher level of Smad7(Fig. 4B) and phosphorylated Smad1/5/8 (Fig. 4A) in C6BV
cells.
3.3. Expression levels of p21WAF and IGFBP-3 weremodulated in C6 glioma cells infected with BDV

Fig. 4. Smad7 expression and phosphorylation of Smad and ERK regulated by
BDV infection and the TGF-b family. A: The phosphorylation of Smad and
ERK in C6 and C6BV cells. After being cultured in DMEM medium with
0.2% FBS for 4 h, C6 and C6BV cells were pretreated with vehicle (V), A-8301 (A: 5 mM) or LDN-193189 (LDN: 100 pM) for 15 min, followed by atreatment with TGF-b1 (T: 100 pM) or BMP7 (B: 2 nM) for 1 h. The phosphorylation of Smad and ERK was examined by Western blot analyses. B:
Smad7 expression in C6 and C6BV cells treated with inhibitors of the TGF-b
family. C6 and C6BV cells were treated with A-83-01 (5 mM) or LDN-193189(100 pM) or both for 72 h. Smad7 mRNA levels were normalized to those of
HPRT1 mRNA. Expression in wild-type C6 cells without the inhibitor treatment was set to 1. Data are shown as the mean ± SE (n ¼ 6). *: P < 0.05,
signiﬁcantly different from that in respective cells without the inhibitor
treatment, and y: P < 0.05, signiﬁcantly different from that in C6 cells treatedwith the respective inhibitor.

TGF-b1 or BMP7. These results suggested that the phosphorylation status of ERK was distinct between C6 cells and
C6BV cells, and that the involvement of TGF-b1 and BMP7 in
ERK phosphorylation was also dependent on the cell context.

A previous study showed that BDV P inhibited the p53induced expression of p21WAF, a cyclin-dependent kinase
inhibitor [26]. Furthermore, IGFBP-3 was identiﬁed by cDNA
microarray analyses as a gene that was more strongly
expressed in BDV P-expressing C6 cells than in C6 cells [27].
Therefore, we determined whether the expression of p21WAF
and IGFBP-3 was modulated in C6BV cells, and also if the
TGF-b/activin pathway or BMP pathway was involved in theirexpression. Consistent with previous ﬁndings [26,27], theexpression of p21WAF was decreased in BDV-infected cells(Fig. 5A). In addition, although the expression level of IGFBP3 was below the detection limit in C6 cells, its reproducible
expression was detected in C6BV cells, indicating that its
expression was up-regulated. An analysis of variance on theexpression of p21WAF indicated a signiﬁcant effect of cell
type and the interaction of cell type, A-83-01, and LDN193189 (Table 2); however, the effects of the inhibitors were
small (Fig. 5A). In contrast, LDN-193189 markedly decreased
the expression of IGFBP-3 in C6BV cells (Table 2, Fig. 5B).

Table 2
Effects of cell type, A-83-01, and LDN-193189 on gene expression.
Effects of
Cella
Smad7
p21WAFIGFBP-3

0.001
0.001
ee

Ab
0.001
NS
NS

LDNc
0.001
NS
0.001

Cell  A
0.009
NS
e

a

Cell type.
A-83-01.
c
LDN-193189.
dNon-signiﬁcant.
e
Not examined.
b

308

Cell  LDN
NS
NS
e

d

A  LDN

Cell  A  LDN

NS
NS
NS

NS
0.04
e

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Y. Nishino et al. / Microbes and Infection 18 (2016) 128e136

Fig. 5. Expression of p21WAF and IGFBP-3 in C6 glioma cells infected with
BDV. C6 and C6BV cells were treated with A-83-01 (5 mM) or LDN-193189(100 pM) or both for 72 h. The expression of p21WAF (A) and IGFBP-3 (B)was evaluated by RT-qPCR. Expression was normalized to that of HPRT1. The
expression of p21WAF in C6 cells without the inhibitor treatment was set to 1,
and the expression of IGFBP-3 in C6BV cells without the inhibitor treatment
was set to 1. Data are shown as the mean ± SE (n ¼ 6). *: P < 0.05,
signiﬁcantly different from that in respective cells without the inhibitor
treatment, and y: P < 0.05, signiﬁcantly different from that in C6 cells treatedwith the respective inhibitor.

4. Discussion
Previous studies reported that the forced expression of BDVP, but not N in glial cells induced neurobehavioral abnormalities resembling those in BDV-infected animals [7,8].
These ﬁndings suggested that the expression of BDV P in glial
cells was sufﬁcient for the onset of Borna disease. We previously revealed changes in the expression of genes related to
TGF-b family signaling in the brains of BDV-infected rats [6].
The TGF-b family has been shown to regulate diverse physiological processes including cell differentiation, cell-cycle
progression, reproductive function, development, bonemorphogenesis, immune surveillance, motility, adhesion,
neuronal growth, and wound healing [28e30]. Thus,

malfunctions in TGF-b family members have been implicatedin the pathogenesis of severe diseases [9,10]. We hypothesizethat alteration of the TGF-b family signaling is related to theonset of neurological disturbances induced by BDV infection.
As the ﬁrst step to elucidate the molecular mechanisms underlying behavioral disturbances induced by BDV infection,
the present study initially explored the regulatory expressionof the signal components of the TGF-b family in BDVinfected glial cells as well as the possible role of the TGF-b
family. We demonstrated that 1) the expression of the TGF-b
family, especially activin bA, activing bE, BMP7, and GDF15,
was markedly altered in C6 glioma cells infected with BDV, 2)
the endogenous activity of activin and BMP was stimulated by
BDV infection, possibly through the autocrine action of activin
A and BMP7, respectively, and 3) the BMP pathway was
involved in the BDV-induced expression of IGFBP-3. Honda
et al. [27] suggested the involvement of IGFBP-3 produced in
BDV-infected glial cells in onset of neurological disturbances.
Also, considering that IGFBP-3 has growth inhibitory andproapoptotic effects [31], the present results provide novel
information on the role of the BMP-mediated regulation ofcell responses in glial cells infected with BDV.
In addition to inhibiting the interaction between IGFs andtheir receptors, IGFBP-3 has been shown to function throughthe IGF/IGF receptor-independent pathway; this is now widely
recognized [32]. IGFBP-3 binds to the low-density lipoprotein
receptor-related protein-1/a2M receptor, which has also beenrecognized as a type V TGF-b receptor [33]. IGFBP-3mediated signaling has been reciprocally related to TGF-b/
activin signaling [34e36]; however, the precise relationshipbetween these two signaling pathways remains unclear. Futurestudies are warranted to identify the target cells and determine
the role of IGFBP-3 produced by BDV-infected glial cells.
Furthermore, the regulation of IGFBP-3 by the BMP pathway
needs to be clariﬁed.
The expression of activin bA was markedly increased in
C6BV cells, suggesting the up-regulated expression of activin
A. Previous studies revealed that the expression of activin A
was increased in T cells, B cells, macrophages, mast cells, andmicroglial cells in response to activation [37,38]. Furthermore,
serum concentrations of activin A were increased in patients
infected with inﬂuenza [39]. Therefore, the increased expression of activin A may be a general phenotype of activatedcells.
Previous studies demonstrated that activin A potentiated theup-regulated expression of p21WAF [40e42]. Since theexpression of activin A was markedly increased in C6BV cells(Fig. 1), we expected that of p21WAF to also be up-regulated
in C6BV cells. However, similar to previous ﬁndings [26], theexpression of p21WAF was signiﬁcantly lower in C6BV cells
than in C6 cells (Fig. 5A). The activin A-induced expressionof p21WAF is known to be dependent on the cell context [43];therefore, the enhanced expression of activin A in C6BV cells
regulates the expression of genes other than p21WAF. Alternatively, activin A produced in C6BV cells may act on neurons. Activin A has been identiﬁed as a neuron survival factor[44]. In addition, an activin A infusion into the dentate gyrus,309

Y. Nishino et al. / Microbes and Infection 18 (2016) 128e136

but not the CA1 pyramidal cell layer of the hippocampus
produced an antidepressant-like effect in a behavioral modelof depression [45].
We previously reported that the expression of TGF-b1 andactivin bE was up-regulated in the brains of rats infected with
BDV, whereas that of activin bA and BMP7 was unaffected by
the infection [6]. These results are inconsistent with the present
results: the expression of activin bA and BMP7 was markedly
higher in C6BV cells than in C6 cells, whereas that of activinbE and GDF15 was decreased. These controversial results may
be related to differences in the materials examined; in ourprevious study, 3-week-old rats were infected with BDV andinﬂammatory cells inﬁltrated the brain [6]. The expression of
TGF-b1 was generally up-regulated in activated immune cells[46,47]. Thus, the induction of brain TGF-b1 may be a resultof the inﬁltration of immune cells; however, its expression wasincreased in BDV-infected brains without the inﬁltration of
immune cells [48]. The expression of the TGF-b family may
also be affected in BDV-infected neurons. The modulation ofgene expression in glial cells and neurons and changes in thecell population of brains infected with BDV may result in aninconsistent expression pattern of the TGF-b family.
The present results suggest aberrant signaling of the ubiquitous pathway in glial cells infected with BDV. Endogenous
BMP activity was enhanced in C6BV cells, which may havebeen due to the enhanced expression of BMP7. Smad1/5/8
could not be further phosphorylated in response to BMP7
(Fig. 4A), suggesting the full activation of the BMP pathway
in C6BV cells. The phosphorylation of ERK was also modulated by BDV infection; phosphorylation levels of ERK werelower in C6BV cells than in C6 cells (Fig. 4A). There areinconsistent results regarding ERK phosphorylation in BDVinfected cells; ERK phosphorylation was decreased in primary cultured neurons infected with BDV [20]. In contrast,
BDV infection induced the constitutive phosphorylation of
ERK in PC12 neuronal cells and oligodendroglial cells,
whereas the nuclear translocation of ERK was inhibited by theinfection [19,21]. The present study also showed that ERK
phosphorylation was modulated by the TGF-b/activin and
BMP pathways; it was stimulated by the inhibition of the
TGF-b/activin pathway in C6BV cells, whereas endogenous
TGF-b/activin activity was required for ERK phosphorylationin C6 cells. Modulation of the ubiquitous signaling pathways
in glial cells infected with BDV may lead to dysregulation of
the central nervous system.
The present study extends basic information on BDVinduced cell responses; our results clearly indicate theinvolvement of the BMP pathway in IGFBP-3 gene induction
in C6BV cells. Previous studies revealed that BDV P negatively regulates p53-mediated signaling pathway as well astranscription of interferon-b gene through association with
Traf family member-associated NF-kB activator-binding kinase-1 [26,49]. Future studies are needed to evaluate theregulatory role of TGF-b family in these pathways, which may
be helpful in understanding the onset of Borna disease indetail.

310

135

Conﬂict of interest
The authors have no conﬂicting ﬁnancial or commercial
interests.
Acknowledgments
We thank Dr. K. Matsumoto for guidance of real-timequantitative PCR analyses. The present study was supported
by JSPS KAKENHI Grant Number 25450433 and Program tosupporting research activities of female researchers from theJapan Society for the Promotion of Science.
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Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/cbf.3162

Direct action of capsaicin in brown adipogenesis and activation ofbrown adipocytes
Ryosuke Kida1, Hirofumi Yoshida1, Masaru Murakami2, Mitsuyuki Shirai3, Osamu Hashimoto4, Teruo Kawada5,
Tohru Matsui1 and Masayuki Funaba1*1Division of Applied Biosciences, Kyoto University Graduate School of Agriculture, Kyoto, Japan
Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara, Japan
3
Laboratory of Veterinary Pharmacology, Azabu University School of Veterinary Medicine, Sagamihara, Japan
4
Laboratory of Experimental Animal Science, Kitasato University School of Veterinary Medicine, Towada, Japan
5
Division of Food Science and Biotechnology, Kyoto University Graduate School of Agriculture, Kyoto, Japan2

The ingestion of capsaicin, the principle pungent component of red and chili peppers, induces thermogenesis, in part, through the activationof brown adipocytes expressing genes related to mitochondrial biogenesis and uncoupling such as peroxisome proliferator-activated receptor(Ppar) γ coactivator-1α (Pgc-1α) and uncoupling protein 1 (Ucp1). Capsaicin has been suggested to induce the activation of brown adipocytes, which is mediated by the stimulation of sympathetic nerves. However, capsaicin may directly affect the differentiation of brown
preadipocytes, brown adipocyte function, or both, through its signiﬁcant absorption. We herein demonstrated that Trpv1, a capsaicin receptor,
is expressed in brown adipose tissue, and that its expression level is increased during the differentiation of HB2 brown preadipocytes. Furthermore, capsaicin induced calcium inﬂux in brown preadipocytes. A treatment with capsaicin in the early stage of brown adipogenesis did
not affect lipid accumulation or the expression levels of Fabp4 (a gene expressed in mature adipocytes), Pparγ2 (a master regulator of adipogenesis) or brown adipocyte-selective genes. In contrast, a treatment with capsaicin in the late stage of brown adipogenesis slightly increased the expression levels of Fabp4, Pparγ2 and Pgc-1α. Although capsaicin did not affect the basal expression level of Ucp1, Ucp1
induction by forskolin was partially inhibited by capsaicin, irrespective of the dose of capsaicin. The results of the present study suggest
the direct effects of capsaicin on brown adipocytes or in the late stage of brown adipogenesis. Copyright © 2015 John Wiley & Sons, Ltd.
key words—capsaicin; Trpv1; brown adipocytes; brown adipogenesis; Ucp1

INTRODUCTION

by the peripheral stimulation of transient receptor potential(Trp) channels in sensory neurons.2,3
The ingestion of capsaicin, the principle pungent component of red pepper,4 and non-pungent capsaicin analogs(capsinoids) has been shown to increase energy expenditureand brown adipocyte-mediated thermogenesis.3,5–7 Because
capsaicin stimulates Trpv1,8 the capsaicin-induced stimulation of brown adipocytes may be mediated through the activation of sympathetic nervous activity.3 Previous studies
reported that capsaicin and capsinoids stimulated sympathetic nerve activity,9,10 and capsinoid-induced energy expenditure was attenuated by denervation of the spinal andvagus nerves or in mice lacking the Trpv1 gene.11
However, the activation of brown adipocytes may also be
directly induced by capsaicin. Capsaicin is effectively
absorbed from the gastrointestinal tract.12 Trpv1 is predominantly expressed in sensory nerves, but is also expressed inother tissues including the brain, lung, liver, testis and white
adipocytes.13,14 These ﬁndings suggest that absorbed capsaicin directly acts on the other tissues expressing Trpv1.
During an investigation of Trpv1 expression in the whole
body, we detected its expression in brown adipose tissue

There are two kinds of thermogenesis, shivering and nonshivering. The latter mainly occurs in brown adipocytes;
non-shivering thermogenesis in brown adipocytes has been
attributed to the expression of a series of genes related tomitochondrial biogenesis and enhancements in cellularrespiration that is uncoupled from ATP synthesis.1 This
uncoupling occurs through the expression of uncoupling
protein 1 (Ucp1), a proton channel located at the innermitochondrial membrane.1 Non-shivering thermogenesisin brown adipocytes is induced by the dissipation ofchemical energy in the form of heat against cold exposure.1 The stimulatory effects of cold exposure on brown
adipocyte activation are known to be mediated throughthe activation of the sympathetic nervous system, initiated*Correspondence to: Masayuki Funaba, Division of Applied Biosciences,
Kyoto University Graduate School of Agriculture, Kitashirakawa Oiwakecho,
Kyoto, Japan.
E-mail: mfunaba@kais.kyoto-u.ac.jp

Received 3 November 2015
Revised 16 December 2015
Accepted 16 December 2015

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capsaicin and brown adipocytes(BAT). Capsaicin has been shown to induce calcium inﬂux
via Trpv1,8 and increases in the concentration of intracellular
calcium inhibit the differentiation of white preadipocytes.15,16 These ﬁndings prompted us to pursue thepossibility of the direct activation of brown adipocytes aswell as brown adipogenesis by capsaicin without the mediation of sympathetic nerve activation. In the present study, we
employed a cell culture system that avoids the effects ofsympathetic nerve activation in order to evaluate the directrole of capsaicin.

least three independent experiments were performed, andrepresentative results are shown.
RNA isolation, RT-PCR and RT-quantitative PCRInterscapular BAT was obtained from adult female
C57BL/6 mice (Japan SLC, Hamamatsu, Japan); mice werecared for under a protocol approved by the Azabu
University Animal Experiment Committee. Total RNA isolation and RT-PCR were performed to examine Trpv expression as previously described.23 The cDNA synthesized from
20 ng of total RNA was used as the template for RT-PCR.
Thermal cycling parameters consisted of 40 cycles for Trpv
or 35 cycles for Tbp (TATA-binding protein) of denaturation at 95 °C for 20 s, annealing at 58 °C for 20 s andextension at 72 °C for 20 s. The PCR products were electrophoresed in agarose gels, and subsequently stained withethidium bromide.
The expression levels of Trpv and genes related to brown
adipogenesis in HB2 brown (pre)adipocytes were examinedby RT-real-time quantitative PCR (RT-qPCR) as described
previously.24 The oligonucleotide primers used are shownin Table 1. The Ct value was determined, and the abundanceof gene transcripts was analysed using the ▵▵Ct methodwith Tbp as the reference gene.25

MATERIALS AND METHODS
Materials
The following reagents were purchased: capsaicin andforskolin, an activator of adenylate cyclase,17 were from
Wako (Osaka, Japan); iRTX (5′-iodoresiniferatoxin), an
antagonist of Trpv1,18 was from Alomone Labs (Jerusalem,
Israel); A23187, a calcium ionophore,19 was from Cayman(Ann Arbor, MI, USA) and Fluo-8-AM, a ﬂuorescencebased calcium indicator,20 was from AAT Bioquest(Sunnyvale, CA, USA).
Cell culture
HB2 brown preadipocytes,21 which were isolated frommouse interscapular BAT and were kindly provided by Dr.
M. Saito, Emeritus Professor of Hokkaido University, were
cultured in Dulbecco’s modiﬁed eagle medium with 10%
FBS and antibiotics as described previously;22 HB2 cells
were stimulated to differentiate into brown adipocytes byinsulin (20 nM) 2 days after conﬂuence (day 0). In order toexamine the effects of capsaicin on brown adipogenesis,
capsaicin with or without iRTX at the indicated concentration (0–10 μM) was added to the culture medium from day
0 to day 8. We also evaluated the ability of capsaicin to activate brown adipocytes by treating cells with capsaicinfrom day 8 to day 12, followed by a stimulation withforskolin (10 μM) for 4 h. Lipid accumulation was examinedby Oil Red O staining on day 8 or 12. Images were obtainedby scanning stained wells (GT-9400UF, EPSON, Tokyo,
Japan).

Statistical analyses
Data are expressed as the mean ± standard error (SE). Dataon gene expression were log-transformed to provide an approximation of a normal distribution before analyses. Differences between gene expression levels in cells were
examined using unpaired t-tests. Differences of p < 0.05
were considered signiﬁcant.

RESULTS
Regulatory expression of Trpv1 during brown adipogenesis
We examined the expression of Trpv family members inmouse BAT (Figure 1A). The signiﬁcant expression of
Trpv1, Trpv2, Trpv3 and Trpv4 was detected. Based onthese results, we next evaluated changes in the expressionof Trpv family members during brown adipogenesis using
HB2 brown preadipocytes (Figure 1B–E). The expressionof Trpv1 increased with time after day 4 of the inductionof differentiation, whereas that of Trpv2 and Trpv4 decreased after the induction of differentiation. The expressionof Trpv3 increased on day 6 after the induction of differentiation; however, this increase was not signiﬁcant becauseof large variations. The expression levels of TrpV5 andTrpv6 were lower than the detection limit throughout brown
adipogenesis (data not shown).

Calcium imaging
The calcium imaging reagent was freshly prepared asfollows: 5-μM Fluo-8 AM (Invitrogen, Paisley, UK) and
0.04% Pluronic F-127 (AnaSpec Inc., Fremont, CA, USA)
were suspended in phosphate buffered saline with
1.27 mM Ca2+ (PBS(Ca2+)), and treated with ultrasonicationfor 10 s. HB2 cells plated in 96-well plates were washedwith PBS(Ca2+) twice, and loaded with 100 μl of thecalcium imaging reagent for 20 min at 37 °C under a 5%
CO2/95% air. Cells were subsequently washed with
PBS(Ca2+) twice, and 50 μl of PBS(Ca2+) was added,
followed by a stimulation with or without capsaicin or
A23187. Fluo-8 ﬂuorescence was captured using a ﬂuorescence microscope (FSX100, Olympus, Tokyo, Japan). At

Capsaicin induces calcium inﬂux in brown preadipocytes
Capsaicin induces calcium inﬂux via the activation of
Trpv1; the overexpression of Trpv1, but not that of Trpv2
or Trpv4, was previously reported to induce calcium inﬂux
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Table 1.

r. kida ET AL.
Nucleotide sequences of primers used in RT-qPCR analyses

Gene

PrimerSequence

GenBank accession number

Size (bp)

5′
3′
5′
3′
5′
3′
5′
3′
5′
3′
5′
3′

5′-caacaagaaggggcttacacc-3′5′-tctggagaatgtaggccaagac-3′5′-caccatagttgcctaccacca-3′5′-gtcgcttttgatgagggaat-3′5′-tcgtctacaacaccaacattga-3′5′-tttcgtgtgtagcagcgtatg-3′5′-tcagaagatcctcttcaaagacct-3′5′-acggattcaggagggtgac-3′5′-atggaggagaccacctgaag-3′5′-cgcttccgtttctgcattag-3′
5′-gctcagagccgagacgag-3′5′-gaagaggtgattcccagatcc-3′

NM_001001445

77

NM_011706

63

NM_145099

77

NM_02201798

NM_001007572

124

NM_022413

61

5′
3′
5′
3′
5′
3′
5′
3′

5′-aaggtgaagagcatcataaccct-3′5′-tcacgcctttcataacacattcc-3′5′-tgtggaactctctggaactgc-3′
5′-gccttgaaagggttatcttgg-3′5′-tgctgttatgggtgaaactctg-3′5′-ctgtgtcaaccatggtaatttctt-3′5′-ctttgcctcactcaggattgg-3′5′-actgccacacctccagtcatt-3′

NM_024406

133

NM_008904

81

NM_011146

110

NM_009463

123

5′
3′

5′-ccaatgactcctatgaccccta-3′5′-cagccaagattcacggtagat-3′

NM_013684

104

Trpv family:
Trpv1
Trpv2
Trpv3
Trpv4
Trpv5
Trpv6
Adipocyte-related genes:
Fabp4
Pgc-1αPparγ2
Ucp1
Reference gene:Tbp

in response to capsaicin.8,26,27 Furthermore, capsaicin modulated membrane currents in cells expressing Trpv1,8 but
not in those expressing Trpv226 or Trpv3.28 Thus, Trpv1,
but not Trpv2, Trpv3 or Trpv4, mediates capsaicin-induced
cell responses. In order to evaluate the activation of Trpv1
by capsaicin, calcium inﬂux was examined using ﬂuorescentanalyses (Figure 2). The treatment of cells with capsaicin at
10 μM, but not 0.1 or 1 μM, increased ﬂuorescent intensity,
suggesting the induction of calcium inﬂux by a high dose of
capsaicin. Capsaicin-induced calcium inﬂux was detected
within 10 s, and continued for at least 120 s. A23187, acalcium ionophore, expectedly induced rapid and robust
calcium inﬂux within 10 s, which thereafter decreased.
These results are consistent with previous ﬁndings obtainedin white preadipocytes; capsaicin induced the prolonged inﬂux of calcium in 3T3-L1 preadipocytes,29 although theeffective concentration of capsaicin to induce calcium inﬂux
was lower (~0.01 μM). In addition, A23187-induced calcium inﬂux was found to be transient in human white
preadipocytes.16 We also attempted to examine calcium
inﬂux in mature brown adipocytes, but were unsuccessful
because of autoﬂuorescence (data not shown).

inhibition by iRTX affected lipid accumulation, as evaluatedby Oil Red O staining (Figure 3A). In addition, theexpression level of Fabp4, a marker of maturation in whiteand brown adipocytes,30,31 was unaffected by capsaicin(Figure 3B). Because peroxisome proliferator-activatedreceptor (Ppar) γ2 is a master regulator of white and brown
adipogenesis,32 we also measured the expression level of
Pparγ2 and showed that it was not affected by capsaicin(Figure 3C). Pparγ coactivator-1α (Pgc-1α) and Ucp1 are
genes that are more strongly expressed in brown fat thanin white fat33 and are the principle genes for thermogenesisin brown adipocytes.1 The treatment of cells with capsaicin
during brown adipogenesis did not affect the expression
levels of Pgc-1α (Figure 3D) or Ucp1 (Figure 3E). Because
the expression levels of these genes reﬂect the activity ofbrown adipocytes,1,34,35 these results suggest that capsaicin
does not signiﬁcantly affect the early stage of brown
adipogenesis.
Effects of capsaicin on activation of brown adipocytes
We next examined the role of capsaicin in the function ofbrown adipocytes and late stage of brown adipogenesis;
HB2 brown adipocytes were treated for 4 days with capsaicin, i.e. the treatment was performed during the period between day 8 and 12. The treatment of these cells withcapsaicin did not affect lipid accumulation, irrespective ofthe dose of capsaicin (Figure 4A). However, the expressionof Fabp4 was up-regulated by increases in the concentrationof capsaicin used; the expression level of Fabp4 in cells
treated with 10 μM of capsaicin was signiﬁcantly higher
than that in control cells (Figure 4B). The expression levels

Effects of capsaicin on brown adipogenesis
The result that Trpv1 is expressed in brown preadipocytes
and its expression is increased with differentiation suggests
the direct action of capsaicin in the early stage of brown adipogenesis. Thus, we treated cells with capsaicin or iRTX
for 8 days after the induction of differentiation in order toexamine their effects on the early stage of brown adipogenesis. Neither Trpv1 activation by capsaicin nor Trpv1

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capsaicin and brown adipocytes

37

modulates brown adipogenesis in the late stage rather thanbrown adipogenesis in the early stage. Furthermore, capsaicin modulates the activation of brown adipocytes; a treatment with capsaicin during the late stage of brown
adipogenesis increased the expression levels of Pparγ2 andPgc-1α, and blunted forskolin-induced Ucp1 expression.
Our results indicate the direct action of capsaicin in brown(pre)adipocytes.
Previous studies reported the expression of Trpv1 inwhite adipose tissue (WAT),36 and that the expression levelsof Trpv1 decreased with time during white adipogenesis.29,37 This is in contrast to the changes observed in theexpression levels of Trpv1 during brown adipogenesis,
which suggests the differential regulation of Trpv1 expression between brown and white (pre)adipocytes. Trpv1 transcription is known to be stimulated by the transcription
factors Sp1, Sp4, Runx1 and C/ebpβ in neuronal cells.38,39
Gene transcription is regulated in a cell context-dependent
manner,40 and the transcriptional regulation in adipogenic
cells currently remains unclear. Nevertheless, in view of
the regulation of adipogenesis by Sp1 and C/ebpβ,31 theabove described transcription factors may be involved inchanges in Trpv1 during brown adipogenesis.
Capsaicin at 0.1 μM or 10 μM increased the expression of
Pparγ2 and Pgc-1α when cells were treated during the latestage of brown adipogenesis. Pparγ2 promotes a transcriptional cascade involved in the function of adipocytes,31,32
while Pgc-1α stimulates mitochondrial biogenesis as wellas brown adipocyte function including the transcriptional
activation of Ucp1.1 Therefore, the present results suggestthat capsaicin stimulates the differentiation of brown
preadipocytes present in the late stage of brown adipogenesis, increases expression levels in brown adipocytes or both.
In contrast to the treatment with capsaicin during the latestage of brown adipogenesis, the up-regulated expression of
Pparγ2 and Pgc-1α was not detected when cells were treatedwith capsaicin during the early stage of brown adipogenesis.
This differential response to capsaicin may be related toincreases in the expression level of Trpv1 during brown
adipogenesis. Capsaicin-induced calcium inﬂux may begreater in brown adipocytes than in brown preadipocytes.
Previous studies demonstrated that increases in the concentration of intracellular calcium induced the up-regulated
expression of Pparγ2 in human white adipocytes,16 andthe calcium signalling pathway activated the transcriptionof the Pgc-1α gene.41 Alternatively, the cell response itself
is likely to change during brown adipogenesis.
Norepinephrine-induced increases in intracellular calcium
concentrations were found to be slower in brown
preadipocytes than in brown adipocytes.42 Shi et al.16 also
revealed differential white adipogenesis in response tocalcium signalling in a stage-dependent manner; increases
in intracellular calcium inhibited adipogenesis in the earlystages of differentiation, whereas they promoted adipogenesis in the late stage.
The cell stage-dependent effects of capsaicin might berelated to changes in expression level of Trpv1 during
brown adipogenesis. We explored calcium ﬂux in response

Figure 1. Expression of Trpv in mouse brown adipose tissues and HB2cells. (A) Total RNA was isolated from the brown adipose tissue of adult
C57BL/6 mice, and RT-PCR was performed in order to examine the expression of Trpv. PCR products were electrophoresed on agarose gels,followed by staining with ethidium bromide. A representative result isshown. (B–E) Two days after conﬂuence (day 0), HB2 brown
preadipocytes were differentiated into brown adipocytes by a treatment withinsulin. The expression of Trpv1 (B), Trpv2 (C), Trpv3 (D) and Trpv4 (E)was examined using RT-qPCR and expressed as ratios to Tbp, with thelevel being set to 1 in cells on day 4. Data shown are the mean ± SE
(n = 4). * and **: p < 0.05 and p < 0.01 signiﬁcantly different from expression levels on day 0, respectivelyof Pparγ2 and Pgc-1α were signiﬁcantly increased inbrown adipocytes treated with 0.1 μM or 10 μM of capsaicin (Figure 4C and D). Although capsaicin did not affectUcp1 expression under the unstimulated state, it partiallyinhibited Ucp1 expression induced by forskolin, irrespective of the dose used (Figure 4E). These results suggest thatbrown adipocytes are target cells of capsaicin or that
capsaicin affects the late stage of brown adipogenesis.

DISCUSSION
The present study showed that the capsaicin receptor Trpv1
is expressed in mouse BAT, and also that its expression
level is increased with brown adipogenesis. By taking theseresults together with the efﬁcient absorption of capsaicinfrom the gastrointestinal tract,12 we expected capsaicin to
directly modulate brown adipogenesis, the activation ofbrown adipocytes or both. We revealed that capsaicin

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r. kida ET AL.

Figure 2. Calcium inﬂux by capsaicin or A23187 in HB2 brown preadipocytes. HB2 brown preadipocytes loaded Fluo-8 AM were treated with the indicatedconcentration of capsaicin or A23187 (1 μM) for the indicated time. A representative ﬂuorescent image is shown

Figure 3. Effects of capsaicin and the Trpv1 antagonist on brown adipogenesis. Two days after conﬂuence (day 0), HB2 brown preadipocytes were differentiated into brown adipocytes by a treatment with insulin in the presence or absence of the indicated concentration of capsaicin or iRTX, a Trpv1 antagonist(200 nM). (A) Cells on day 8 were examined by Oil Red O staining. The expression of Fabp4 (B), Pparγ2 (C), Pgc-1α (D) and Ucp1 (E) was examined using
RT-qPCR and expressed as ratios to Tbp, with the level being set to 1 in cells treated without capsaicin and iRTX. Data shown are the mean ± SE (n = 4)

to capsaicin in brown preadipocytes but not in mature brown
adipocytes because of autoﬂuorescence as described above.
In view of higher concentration of capsaicin to induce

calcium inﬂux effectively in brown preadipocytes than in
3T3-L1 preadipocytes,29 capsaicin-induced calcium inﬂux
in brown preadipocytes may not be mediated by Trpv1.
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capsaicin and brown adipocytes

39

Figure 4. Effects of capsaicin on brown adipocyte function. HB2 brown preadipocytes were differentiated into brown adipocytes by a treatment with insulin.
Brown adipocytes were treated with or without the indicated concentration of capsaicin in the presence of insulin on days 8–12. (A) Cells on day 12 were examined by Oil Red O staining. The expression of Fabp4 (B), Pparγ2 (C) and Pgc-1α (D) was examined. In addition, on day 12, cells were treated with orwithout forskolin (10 μM) for 4 h, and the expression of Ucp1 (E) was examined using RT-qPCR and expressed as ratios to Tbp, with the level being set to
1 in cells treated without capsaicin and forskolin. Data shown are the mean ± SE (n = 4). * and **: p < 0.05 and p < 0.01 signiﬁcantly different from expression
levels in cells treated without capsaicin (and with forskolin (E)), respectively

effect of capsaicin is in contrast to the in vivo effects of capsaicin and capsinoids on brown adipocytes.7,49,50 In adulthumans, the chronic ingestion of capsinoids for 6 weeks
did not affect basal energy expenditure, but increased coldinduced thermogenesis;7 capsaicin or capsinoid-induced
thermogenesis in animals has been attributed to sympathetic
nerve-mediated events,3 whereas capsaicin intrinsically inhibits the activation of brown adipocytes.
The precise reason why capsaicin partially inhibited
forskolin-induced Ucp1 expression currently remains unclear.
Forskolin activates adenylate cyclase,17 which increases
cAMP concentrations and the subsequent stimulation of theprotein kinase A pathway, leading to the transcription of the
Ucp1 gene.1 Capsaicin-induced calcium inﬂux may stimulate
the activity of calmodulin-dependent phosphodiesterase,
which degrades cAMP.51 This enhancement in phosphodiesterase activity with increases in intracellular calcium concentrations has also been suggested in brown adipocytes.52
Chaiyasit et al.53 reported that peak concentration ofplasma concentration was 2.47 ng/ml (8.1 nM) in humans
ingested capsicum containing 26.6 mg of capsaicin. The
concentration is lower than that examined effect on brown
adipogenesis and brown adipocyte function in this study.
Therefore, it is not clear whether capsaicin at the concentration detected in plasma affects brown adipogenesis andbrown adipocyte function. Future studies are needed to clarify the effect of physiological concentration of capsaicin.

Molecules responsible for capsaicin-induced calcium inﬂux
at the higher concentration should be explored in future.
There are two kinds of thermogenic adipocytes, i.e.
brown and beige. In mice, BAT consists of brown adipocytes, whereas beige adipocytes relatively exhibit sparse distribution in WAT.43 Both adipocyte types express genes
involved in thermogenesis such as Ucp1 and Pgc-1α at similar levels,44 and forskolin has been shown to up-regulate
Ucp1 expression in both types of adipocytes.45 However,molecular signatures differ between brown and beige adipocytes,44–47 as do the cell lineages; brown adipocytes arederived from skeletal muscle cells,48 whereas beige adipocytes are from smooth muscle-derived cells.44 Baboota
et al.37 recently revealed that the treatment of cells with capsaicin during the early stage of 3T3-L1 white adipogenesis
induced the up-regulation of Pparγ2, Pgc-1α and Ucp1 expression, suggesting the promotion of differentiation tobeige adipocytes. Capsaicin has the potential to increase
the activity of brown/beige adipocytes. However, the action
point of capsaicin may differ between brown and beige(pre)adipocytes; the target of capsaicin is brown adipocytes
or brown preadipocytes present in the late stage of brown
adipogenesis, whereas capsaicin induces beige adipocytes
through the promotion of adipogenesis in the early stage.
Forskolin-induced Ucp1 expression was partiallyinhibited by capsaicin, which suggests the inhibitory effectof capsaicin on the activation of brown adipocytes. This

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CONFLICT OF INTEREST
The authors declare no conﬂict of interest.

ACKNOWLEDGEMENTS
The authors thank Prof. Dr. Masayuki Saito for providing
HB2 brown preadipocytes.

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ARTICLE

Journal of Cellular Biochemistry 9999:1–9 (2016)

Smad8/9 Is Regulated Through the BMP Pathway
Yuko Katakawa,1 Masayuki Funaba,2* and Masaru Murakami1**
1
2

Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara 252-5201, Japan
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

ABSTRACT
Members of the transforming growth factor-b (TGF-b) family function through Smad-dependent and Smad-independent pathways. The
Smad-dependent pathway is stimulated through the phosphorylation of receptor-regulated Smad (R-Smad) and inhibited through thedephosphorylation of R-Smad or the gene induction of inhibitory Smad (I-Smad). Little information is available on the regulation of R-Smadgene expression. BMP4 potentiated the up-regulation of Smad8/9 expression in C2C12, H9c2, 3T3-L1, HepG2, B16, and primary ﬁbroblasts.
BMP4-induced Smad8/9 expression was cycloheximide-insensitive and LDN-193189-sensitive, suggesting a direct event mediated throughBMP type I receptors. BMP4 transcriptionally stimulated the Smad8/9 gene, and BMP-responsive elements (BREs) spanning nt 121 to nt 44
are involved in the up-regulation of Smad8/9 expression in response to BMP4. Phosphorylated Smad1/5/8/9 speciﬁcally bound to the BREs of
Smad8/9 gene. The present study reveals that Smad8/9 is a unique R-Smad regulated through the BMP pathway at the mRNA level. J. Cell.
Biochem. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.

KEY WORDS: BMP; SMAD8/9; TRANSCRIPTION

T(I)-Smads (Smad6 and Smad7) is up-regulated through TGF-b1,
activin A, BMP2, and BMP7 [Nakao et al., 1997; Ishisaki et al., 1998;
Takase et al., 1998], and I-Smads directly associate with activatedtype I receptors to compete with R-Smads [Hayashi et al., 1997].
Alternatively, I-Smads interact with a Smad-interacting ubiquitin
ligase known as Smurf, leading to the ubiquitination andproteasomal degradation of these receptors [Ebisawa et al., 2001;
Murakami et al., 2003]. The signaling of the TGF-b family is further
negatively regulated through the de-phosphorylation of R-Smads[Duan et al., 2006; Knockaert et al., 2006; Lin et al., 2006].
In contrast to I-Smads, the activity of R-Smads is not generally
regulated at the expression level; the TGF-b family does not affectthe expression of R-Smad [Nakao et al., 1997; Poncelet et al., 1999;
Herrera et al., 2009; Murakami et al., 2009], although a few studies
have reported the modulation of Smad3 expression through TGF-b[Yanagisawa et al., 1998; Poncelet et al., 1999; Winbanks et al.,2011]. We previously showed that the expression of Smad8/9, but
not Smad1 and Smad5, is decreased in response to the differentiation
stimulation of myoblasts into myotubes [Furutani et al., 2011]; thedynamic changes in Smad8/9 expression paralleled changes in
endogenous BMP activity. The results suggested that unlike the other
R-Smads, Smad8/9 is a target gene for the BMP pathway. Here wehe transforming growth factor-b (TGF-b) family, comprising
three subfamilies, TGF-b, activin, and BMP groups [Miyazono
et al., 2010; Massague, 2012; Sakaki-Yumoto et al., 2013], plays
diverse physiological roles, including the control of cell division,
early embryonic patterning, cell differentiation, and cell determination [Derynck et al., 2008]. Compared with these diverse functions,the signal transduction pathways of these transcription factors,
collectively referred to as the Smad pathway, are relatively simple.
Members of the TGF-b family transmit signals through speciﬁcheteromeric complexes through type I and type II receptors, followedby the phosphorylation of the type I receptor via the type II
receptor serine/threonine kinase. The phosphorylation of the type I
receptor activates the serine/threonine kinase activity ofthis receptor, resulting in the phosphorylation of C-terminal serinesof receptor-regulated (R)-Smads (Smad1, 2, 3, 5, and 8 [or 9]: Smad1/
5/8/9 are regulated through BMP receptor complexes, and Smad2/3
are regulated via activin or TGF-b receptor complexes),
complex formation with common Smads (Smad4) and the transactivation of target genes [Miyazono et al., 2010; Massague, 2012;
Sakaki-Yumoto et al., 2013].
The negative regulation of TGF-b family signaling is also partly
mediated through the Smad proteins. The expression of inhibitory

Conﬂicts of interest: The authors declare that there are no conﬂicts of interest.
Grant sponsor: Azabu University; Grant number: 2013K08.
*Correspondence to: Dr. Masayuki Funaba, Ph.D., Division of Applied Biosciences, Graduate School of Agriculture,
Kyoto University, Kyoto 606-8502, Japan. E-mail: mfunaba@kais.kyoto-u.ac.jp **Correspondence to: Dr. Masaru
Murakami, Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara 252-5201,
Japan. E-mail: murakami@azabu-u.ac.jp
Manuscript Received: 21 May 2015; Manuscript Accepted: 4 January 2016
Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2015
DOI 10.1002/jcb.25478  © 2016 Wiley Periodicals, Inc.

320

1

showed that the BMP pathway up-regulates Smad8/9 expression in
multiple cell types, indicating the unique regulation of the BMPinduced Smad-dependent signaling pathway.

MATERIALS AND METHODS
MATERIALS
The following reagents were purchased: BMP2, BMP4, TGF-b1,
and activin A were from R & D Systems (Minneapolis, MN);
LDN-193189, an inhibitor for BMP type I receptor [Yu et al.,2008], was from Stemgent (San Diego, CA); a rabbit monoclonal
antibody against Smad5 (D4G2, #12534), and rabbit polyclonal
antibodies against phospho-Smad1 (Ser463/Ser465)/Smad5(Ser463/Ser465)/Smad8 (Ser465/Ser467) (#13820 for Western
blot and #11971 for chromatin immunoprecipitation [ChIP]) werefrom Cell Signaling Technology (Danvers, MA); a rabbit
monoclonal antibody against Smad1 (EP565Y, ab33902), agoat polyclonal antibody against Smad8/9 (ab48011), and amouse monoclonal antibody against a-tubulin (B-5-1-2,
ab11304) were from Abcam (Cambridge, MA).
CELL CULTURE
C2C12, H9c2, 3T3-L1, HepG2, and B16 cells were cultured in
Dulbecco0 s modiﬁed Eagle0 s medium (DMEM) supplemented with
10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillinand 100 mg/ml streptomycin. C2C12, 3T3-L1, and B16 are mouseoriginated cell lines. H9c2 and HepG2 are rat- and human-originatedcell lines, respectively. Rat primary ﬁbroblasts were prepared fromskin of Wistar rats [Messenger, 1984] and cultured in DMEM
supplemented with 10% FBS and antibiotics.
RNA ISOLATION AND QUANTITATIVE RT-PCR (QRT-PCR)
Total RNA isolation and qRT-PCR analyses were performed aspreviously described [Murakami et al., 2015]; total RNA was
isolated using the RealTime ready Cell Lysis Kit (Roche,
Mannheim, Germany), and cDNA was synthesized using Transcriptor Universal cDNA Master (Roche). The oligonucleotide
primers for qRT-PCR are shown in Table I. As described above,
because we used cell lines and primary cells originated fromseveral species, that is, C2C12 (mouse), H9c2 (rat), 3T3-L1(mouse), HepG2 (human), B16 (mouse), and primary ﬁbroblasts(rat), we designed PCR primers to detect human, rat, and mousegenes. The mRNA levels were expressed relative to b-actin or
GAPDH mRNA levels.

KARYOTYPE ANALYSESTo prepare the metaphase chromosomes, 3T3-L1 cells were grown to
80% conﬂuency in 100-mm dishes. Colchicine (Calbiochem, La
Jolla, CA) was added at a ﬁnal concentration of 0.1 mg/ml for 1 h tostop the cell cycle. The cells were collected and subsequently treatedwith 0.075 M potassium chloride for 30 min at 37°C. The cells were
ﬁxed in Carnoy0 s solution (methanol: acetic acid, 3:1) and spread
onto glass slides using the air-drying method. After staining
with a 5% Giemsa solution, the number of chromosomes with 23
metaphases was counted.

2

REGULATION OF SMAD8/9 EXPRESSION BY BMP

321

WESTERN BLOT
The cells were lysed using the EzRIPA Lysis Kit (Atto, Tokyo, Japan),according to the manufacturer0 s instructions. The protein concentration was determined using the Pierce BCA Protein Assay Kit(Thermo Fisher Scientiﬁc, Waltham, MA). After heating for 5 min
at 95°C, 10 mg of the protein samples was subjected to 10%
SDS–polyacrylamide gel electrophoresis, followed by blotting onto
iBlot Gel Transfer Stacks PVDF, Mini membranes (Life Technologies,
Grand Island, NY) using an iBlot Gel Transfer Device (LifeTechnologies). The membranes were blocked with EzBlock (Atto)for 2 h and reacted with primary antibody diluted 1:1,000–5,000 in
Can Get Signal solution (Toyobo, Tokyo, Japan) overnight at 4°C.
Subsequently, the membranes were reacted with the appropriate
secondary antibody conjugated with HRP for 1 h at room temperature. The immunoreactive proteins were visualized using the ECL
Select Western blotting detection system (GE Healthcare, Buckinghamshire, UK) according to the manufacturer0 s instructions.
PLASMIDSThe DNA sequence spanning nt 1000 to nt þ113 of the mouse
Smad8/9 promoter was ampliﬁed and cloned into a pGL4 basic
vector containing the ﬁreﬂy luciferase reporter (Smad8/9(1000)luc); nt þ1 was deﬁned as the transcriptional initiation site. Reporter
plasmids with deleted promoters or point mutations were preparedusing a PCR-based method. The plasmids were veriﬁed throughDNA sequencing.
REPORTER ASSAYS
C2C12 and 3T3-L1 cells were transiently transfected with the
Smad8/9 reporter construct and a plasmid expressing Renillaluciferase under the control of thymidine kinase promoter(pRL-tk) using Polyethylenimine Max reagent (Polysciences,
Warrington, PA). At 24 h post-transfection, the cells were cultured
with members of the TGF-b family in the presence of 0.2% FBS in
DMEM for 16 h. The ﬁreﬂy luciferase activity in the cell lysate wasnormalized to the Renilla luciferase activity.
ChIP
C2C12 cells were treated with or without BMP4 (4 nM) for 2 h, andChIP assays were performed by use of a commercial kit (Simple ChIP
Plus Enzymatic Chromatin IP (#9005), Cell Signaling Technology).
Binding of Smad1/5/8/9 to the region including BMP-responsive
elements (BREs) of Smad8/9 gene (nt 164 to nt þ21) was evaluatedby PCR. Nucleotide sequence of the used PCR primers is shown in
Table I. Because the GC-content of the region is extremely high(77%), we ampliﬁed the region by use of PrimeSTAR GXL DNA
polymerase (TaKaRa, Otsu, Japan), which is suitable for ampliﬁcation of the GC-rich template. According to the manufacturer0 s
protocol, the PCR proﬁle was as follows: after denaturing for 2 min
at 98°C, 30 cycles consisted of 10 sec at 98°C and 20 sec at 68°C,
followed by extension for 3 min at 68°C. We also ampliﬁed outside
region of the BREs of Smad8/9 (nt 2861 to nt 2702), theunrelated region to BMP signaling of hepcidin (nt 150 to nt 35),
and the region including BREs of Id2 gene (nt 2872 to nt 2673,
Nakahiro et al., 2010); hepcidin is a BMP-responsive gene,but the region spanning nt 150 to nt 35 is not involved in

JOURNAL OF CELLULAR BIOCHEMISTRYTABLE I. Oligonucleotide Sequences For qRT-PCR and ChIP Assay

Gene
Human
Smad1
Smad2
Smad3
Smad4
Smad5
Smad6
Smad7
Smad9
b-actin
Mouse
Smad1
Smad2
Smad3
Smad4
Smad5
Smad6
Smad7
Smad8/9b-actin
GAPDH
Rat
Smad1
Smad2
Smad3
Smad4
Smad5
Smad6
Smad7
Smad9
b-actin
ChIP
BRE of Smad8
Outside BRE of Smad8
Unrelated to responsiveness to BMP in hepcidin
BRE of Id2

50 -primer

30 -primer

50 -caacagccacccgtttcct-30
50 -tcatagcttggatttacagccag-30
50 -tggacgcaggttctccaaac-30
50 -gccaactttcccaacattcctg-30
50 -ctttccaccagcccaacaacac-30
50 -tctcctcgcgacgagtacaa-30
50 -acccgatggattttctcaaac-30
50 -atgtgatttactgctgcgtgt-30
50 -ccaaccgcgagaagatga-30

50 -gtttgggtaactgctattggga-30
50 -acaccaaaatgcaggttctgag-30
50 -gtgctggggacatcggattc-30
50 -tgctgctgtcctggctgag-30
50 -taggcaggaggaggcgtatcag-30
50 -ccggagcagtgatgagggagt-30
50 -gccagataattcgttccccc-30
50 -cggtagtggtaagggttaatgc-30
500 -ccagaggcgtacagggatag -30

U59423BC014840
BC0550743
BC002379
AF009678
NM_005585
AF015261
BC011559
NM_001101

50 -tgaaaacaccaggcgacata-30
50 -tgtgcagagccccaactgt-30
50 -ggaatgcagccgtggaact-30
50 -aagctgccctgttgtgactgt-30
50 -gcagtaacatgattcctcagacc-30
50 -gttgcaacccctaccacttc-30
50 -acccccatcaccttagtcg-30
50 -cggatgagctttgtgaagg-30
50 -ctaaggccaaccgtgaaaag-30
50 -cgtgttcctacccccaatgt-30

50 -tgaggcattccgcatacac-30
50 -gcctggtgggatcttacacact-30
50 -aagacctcccctccgatgtag-30
500 -ggagagttgacccaagcaaaag-30
50 -gcgacaggctgaacatctc-30
50 -ggaggagacagccgagaata-30
50 -gaaaatccattgggtatctgga-30
50 -gggtgctcgtgacatcct-30
50 -accagaggcatacagggaca-30
50 -tgtcatcatacttggcaggtttct-30

NM_008539
U60530AB008192
NM_008540
AF063006
AF010133
AF015260
AY145520
NM_007393
NM_008084

50 -gcagcccttttcagatgccag-30
50 -caggacgattagatgagcttg-30
50 -cctgccactgtctgcaagata-30
50 -gaacactggatggacgactg-30
50 -cagcctatggacacaagcaa-30
50 -gttgcaacccctaccacttc-30
50 -acccccatcaccttagtcg-30
50 -accattaccgcagagtggaga-30
50 -ctaaggccaaccgtgaaaag-30

50 -ggctgagagccatcctgggc-30
50 -cgtatttgctgtactcagtcccc-30
50 -gcaaattcctggttgttgaagat-30
50 -acagacgggcatagatcaca-30
50 -ggcaacaggctgaacatctc-30
50 -ggaggagacagccgagaata-30
500 -aaatccatcgggtatctgga-30
50 -tgagggttgtactcgctgtg-30
50 -accagaggcatacagggaca-30

NM_013130
NM_019191
NM_013095
NM_019275
NM_021692
NM_001109002
NM_030858
AF012347
NM_031144

50 -aaggctctctgcaggcatgatgtg-30
50 -gggtggtgccagtcaggtttgctt-30
50 -cactattttcttggaaatgagtcagagc-30
50 -gctgagtgacggcgcggttgctat-30

50 -tccagaggcgtcagtcaggctgga-30
50 -cacatcaatcataaactaagaaaa-30
50 -tgtgtggtggctgtctaggagcc-30
50 -gtgatccctgacaggtgatgaatt-30

NC_000069.6
NC_000069.6
NC_000073.6
NC_000078.6

RESULTSBMP-mediated transcriptional activation [Truksa et al., 2009].
Because the GC-content of the former two sequences was 43% and53%, respectively, we ampliﬁed the region by use of Ex-Taq DNA
polymerase (TaKaRa) with the PCR proﬁle of initial denaturationfor 3 min at 95°C, 30 cycles consisted of 20 sec at 95°C, 20 sec at
60°C and 20 sec at 72°C, followed by extension for 3 min at 72°C.
The region including BREs of Id2 gene is GC-rich (75%). Thus, weused PrimeSTAR GXL DNA polymerase; the two-step PCR proﬁleto detect the BREs of Smad8/9 gene; however, did not properly
amplify the Id2 promoter. Therefore, we performed three-step PCR
with the PCR proﬁle of initial denaturation for 2 min at 98°C,
30 cycles consisted of 10 sec at 98°C, 15 sec at 55°C and 20 sec at
68°C, followed by extension for 3 min at 68°C, according to the
manufacturer0 s protocol. The PCR products were electrophoresedin agarose gels, followed by ethidium bromide staining andvisualization under ultraviolet light. We also examined several
antibodies against Smad for ChIP assays in preliminary experiments; reproducible results were obtained, when antibody against
phosphorylated Smad1/5/8/9 was used. Thus, chromatin samples
were reacted with control IgG as a negative control or antiphosphorylated Smad1/5/8/9 antibody.

SMAD8/9 EXPRESSION IS UP-REGULATED THROUGH BMP4 IN
MULTIPLE CELL TYPESIn a previous study, we revealed that changes in Smad8/9 expression,
but not Smad1/5, paralleled endogenous BMP activity during
myogenesis [Furutani et al., 2011]. We explored whether Smad8/9
expression is up-regulated through exogenous BMP and determined
whether this regulation is detected in multiple cell types. Treatment
with BMP4 for 2 h increased Smad8/9 expression in all examined cells(Fig. 1); expectedly, the expression of I-Smads, Smad6 and Smad7
were increased as previously shown [Nakao et al., 1997; Takase et al.,1998]. In addition, the expression levels of R-Smads other than
Smad8/9 were unaffected after BMP4 treatment. The up-regulation of
Smad8/9 expression was also detected in cells treated with BMP2, aclosely related molecule to BMP4 (Fig. 2, data not shown).
Smad8 is closely related to Smad9; the nucleotide sequence ofmouse Smad9 (GenBank accession number: NM_019483) indicatesthat six nucleotides (50 -cgagtc-30 ) are inserted at nt þ961 of mouse
Smad8 (AF175408, Fig. 3A), leading to differences of two aminoacids between Smad8 and Smad9. To characterize Smad8/9 inthe cells examined in Figure 1, the nucleotide sequence of theregion spanning the six nucleotides was determined (Fig. 3B). The
insertion of the six nucleotides was detected in all cells
examined (Fig. 3B upper, data not shown), except for C2C12 cells(Fig. 3B middle), indicating that C2C12 cells express Smad8,
whereas the other cells express Smad9. Treatment with BMP4 did

STATISTICAL ANALYSESThe data are expressed as the means  standard error (SE).
Differences in the gene expression between cells were examinedusing unpaired t-tests. Differences of P < 0.05 were consideredsigniﬁcant.

JOURNAL OF CELLULAR BIOCHEMISTRY

GenBank accession number

322

REGULATION OF SMAD8/9 EXPRESSION BY BMP

3

Fig. 1. Smad expression in response to BMP4 in multiple cell types. Smad expression in response to treatment with BMP4 was examined in C2C12 cells (A), H9c2 cells (B),primary ﬁbroblasts (C), 3T3-L1 cells (D), HepG2 cells (E), and B16 cells (F). The cells were cultured in the presence or absence of BMP4 (4 nM) for 2 h. Expression of Smadwas examined using qRT-PCR and expressed as ratios to b-actin, with the level set to 1 in cells cultured in the absence of BMP4. The data are presented as the means  SE (n ¼ 4).
, : P < 0.05 and P < 0.01, respectively, compared with expression in cells treated without BMP4.

not induce expression of Smad9 in C2C12 cells, which was veriﬁed
by direct sequencing of the RT-PCR products (data not shown).
Notably, the nucleotide sequencing in 3T3-L1 cells reproducibly
indicated the expression of both Smad8 and Smad9 (Fig. 3B bottom).
We cloned Smad8/9 mRNA expressed in 3T3-L1 cells anddetermined the nucleotide in a total of 58 clones: 24 clones (41%)
of Smad8 and 34 clones (59%) of Smad9 (data not shown).
The karyotype analyses of a total of 23 cells showed 63  15

Fig. 2. Smad expression in response to BMP2 in 3T3-L1 cells. Smad expression
in response to treatment with BMP2 was examined in 3T3-L1 cells. The cells
were cultured in the presence or absence of BMP2 (4 nM) for 2 h. The expressionof Smad was examined using qRT-PCR and expressed as ratios to GAPDH, withthe level set to 1 in cells cultured in the absence of BMP2. The data arepresented as the means  SE (n ¼ 4). , : P < 0.05 and P < 0.01, respectively,
compared with expression in cells treated without BMP2.

4REGULATION OF SMAD8/9 EXPRESSION BY BMP

Fig. 3. Nucleotide sequence of Smad8/9 in multiple cell types. A: Nucleotide
sequence surrounding nt þ961 of mouse Smad9 compared with that of mouse
Smad8. B: Waveform of the nucleotide sequence of Smad8/9 in B16 (upper),C2C12 (middle), and 3T3-L1 cells (bottom).

323

JOURNAL OF CELLULAR BIOCHEMISTRY

chromosomes in 3T3-L1 cells. Considering the 40 chromosomes
observed in normal mice, the mixed and uneven ratio of Smad8 andSmad9 might reﬂect abnormal chromosomes.
We next evaluated the effects of the other members of the TGF-b
family, TGF-b1, and activin A, on Smad8/9 expression (Fig. 4).
As expected, TGF-b1 increased the expression of I-Smad in C2C12
cells (Fig. 4A) and 3T3-L1 cells (Fig. 4B). Activin A potentiated theup-regulation of I-Smad expression in 3T3-L1 cells. In contrast,
activin A did not up-regulate I-Smad expression in C2C12 cells,
although the effects of activin A were statistically signiﬁcant. Both
TGF-b1 and activin A did not substantially increase Smad8/9
expression. These results indicate that Smad8/9 is a unique R-Smadgene regulated at the mRNA level through BMPs.
BMP4-INDUCED SMAD8/9 EXPRESSION IS TRANSCRIPTIONALLYREGULATEDTo examine the molecular mechanism underlying the regulation of
Smad8/9 expression, the effects of cycloheximide, an inhibitor ofprotein synthesis, on BMP-induced Smad8/9 expression were
examined (Fig. 5). Smad8/9 expression in response to BMP4
treatment was up-regulated, irrespective of treatment with cycloheximide, suggesting that the Smad8/9 induction is a direct event.
Thus, we speculated that BMP-induced Smad8/9 expression istranscriptionally regulated. To this end, luciferase-based reporter
assays using the Smad8/9 promoter were performed.
Initially, we examined the BMP4-induced transcription of Smad8/
9 using a reporter containing 1,000 bp of the Smad8/9 gene promoter(Fig. 6A and B). BMP4 increased the expression of luciferase, andpretreatment with LDN-193189, an inhibitor of BMP type I receptors[Yu et al., 2008], suppressed the responsiveness to BMP4 in C2C12
and 3T3-L1 cells, indicating that BMP4-induced Smad8/9 expression is transcriptionally regulated through the activation of BMP
type I receptors. Considering that, BMP type I receptors phosphorylate and activate Smad1/5/8/9 [Miyazono et al., 2010; Massague,2012; Sakaki-Yumoto et al., 2013], the stimulated Smad8/9transcription is likely achieved through a Smad-dependent pathway.
Indeed, LDN-193189 inhibited the BMP4-induced phosphorylationof Smad1/5/8/9 (Fig. 6C).

Fig. 4. Smad expression in response to the TGF-b family in C2C12 and 3T3-L1
cells. Smad expression in response to treatment with TGF-b1, activin A, or
BMP4 was examined in C2C12 cells (A) and 3T3-L1 cells (B). The cells were
cultured in the presence or absence of TGF-b1 (100 pM), activin A (4 nM), or
BMP4 (4 nM) for 2 h. The expression of Smad was examined using qRT-PCR
and expressed as ratios to b-actin, with the level set to 1 in cells cultured
in the absence of ligand. The data are presented as the means  SE (n ¼ 4).
, : P < 0.05 and P < 0.01, respectively, compared with expression in cells
treated without ligand.

MULTIPLE BMP-RESPONSIVE ELEMENTS ARE REQUIRED FOR
BMP4-INDUCED SMAD8/9 TRANSCRIPTION
We next explored the potential region within the Smad8/9 promoter
responsible for transcriptional activation in response to BMP4
treatment using deleted mutants of the Smad8/9 reporter (Fig. 7).
The deletion of 330 bp within 1,000 bp upstream of the Smad8/9
gene decreased responsiveness to BMP4 in C2C12 cells (Fig. 7A);
nevertheless, signiﬁcant transcriptional activation through BMP4
was detected. Compared with Smad8/9(338)-luc, further deletionsof the Smad8/9 promoter resulted in gradual decrease of BMP4induced transcription. The responsiveness to BMP4 nearly disappeared when Smad8/9(43)-luc was used as a reporter. Similareffects of BMP4 were detected in 3T3-L1 cells, although the
responsiveness to BMP4 in Smad8/9(1000)-luc was not different
from that in Smad8/9(670)-luc (Fig. 7B). Similar to the response in
C2C12 cells, BMP4 did not induce transcriptional activation, when
Smad8/9(43)-luc was used in 3T3-L1 cells. These results suggestthat BREs are located at the distal region of nt 43 of the Smad8/9
promoter.
The BMP-induced phosphorylation of Smad1/5/8/9 stimulates
transcription through binding to a GCCG or GC-rich sequence
located in the promoter region of the target gene [Kusanagi et al.,
2000; L
opez-Rovira et al., 2002]. Examination of the nucleotide
sequence indicated ﬁve potential regions of GCCG (BRE-1, 2, 4,
and 5) or GC-rich sequence (BRE-3) within nt 121 of the Smad8/9
promoter (Fig. 8A). To examine the involvement of these BREs in
BMP4-induced transcription, reporter plasmids with individual or
combinational mutations of the BREs were prepared.
All ﬁve mutations of the BREs completely inhibited responsiveness to BMP4 (Fig. 8B). The mutations of individual BREs partiallydecreased the BMP response; speciﬁcally, BRE-3 mutations
markedly reduced luciferase activity in response to BMP4. BRE-1
and BRE-2 mutations did not have the additive effects of BRE-3

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REGULATION OF SMAD8/9 EXPRESSION BY BMP

5

Fig. 5. Modulation of BMP4-induced Smad8/9 expression through cycloheximide. The necessity of new protein synthesis was examined for BMP4-induced mRNA expression of
Smad8 in C2C12 cells (A) and 3T3-L1 cells (B). The cells were treated with or without BMP4 (4 nM) for 12 h after pre-treatment with or without cycloheximide (0.5 mg/ml) for
15 min. Smad8/9 expression was examined using qRT-PCR and expressed as ratios to b-actin, with the level set to 1 in cells cultured in the absence of BMP4 and cycloheximide.
The data are presented as the means  SE (n ¼ 4). The numbers on the bar indicate fold-induction.

Fig. 6. Modulation of BMP4-induced Smad8/9 transcription through LDN-193189. BMP type I receptor kinase activity was examined for the BMP4-induced transcription andphosphorylation of Smad8/9 in C2C12 cells (A and C) and 3T3-L1 cells (B and C). The cells were treated with or without BMP4 (4 nM) for 1 h (C) or 16 h (A and B) after pretreatment with or without LDN-193189 (100 nM) for 15 min. A, B: Smad8/9 transcription was evaluated as Smad8/9(1000)-luc expression. Fireﬂy, luciferase activity was
expressed as a ratio to Renilla luciferase activity, with the level set to 1 in cells cultured in the absence of BMP4 and LDN193189. The data are presented as the means  SE (n ¼ 6).
‡
P < 0.01, compared with the respective treatment with BMP4, but not with LDN-193189. C: The effects of LDN-193189 on the phosphorylation of Smad1/5/8/9 in C2C12 and
3T3-L1 cells were examined through western blot analyses. Expression level of total Smad1, Smad5, Smad8/9, and a-tubulin was also examined.

6

REGULATION OF SMAD8/9 EXPRESSION BY BMP

325

JOURNAL OF CELLULAR BIOCHEMISTRY

Fig. 7. Transcriptional regulation of Smad8/9 through BMP4. The BRE in Smad8/9 promoter was examined using a series of Smad8/9 reporters with deleted promoters in C2C12
cells (A) and 3T3-L1 cells (B). The cells were transiently transfected with the indicated reporter plasmid with Renilla-luc and treated with or without BMP4 (4 nM) for 16 h. Fireﬂy
luciferase activity was expressed as a ratio to Renilla luciferase activity, with the level set to 1 in cells transfected with Smad8/9(1000)-luc and cultured in the absence of BMP4.
The data are presented as the means  SE (n ¼ 4).

Fig. 8. Identiﬁcation of BRE in Smad8/9. A: Nucleotide sequence of BRE-1 to BRE-5 of Smad8/9. B: BRE in Smad8/9 promoter was explored through reporter assays using
Smad8/9 promoter with point mutations in C2C12 cells. The cells were transiently transfected with the indicated reporter plasmid with Renilla-luc and treated with or without
BMP4 (4 nM) for 16 h. Fireﬂy luciferase activity was expressed as a ratio to Renilla luciferase activity, with the level set to 1 in cells cultured in the absence of BMP4. The data arepresented as the means  SE (n ¼ 4). C: C2C12 cells were treated with (þBMP4) or without BMP4 (-BMP4, 4 nM) for 2 h. Chromatin extracts were immuneprecipitated with
antibody against phosphorylated Smad1/5/8/9 or control IgG, followed by ampliﬁcation of the region spanning BREs of Smad8 gene (Smad8 BRE), outside BREs of Smad8 gene
(Smad8 outBRE), unrelated to responsiveness to BMP in hepcidin gene (Hepc), and BREs of Id2 gene (Id2). PCR was also performed using distilled water as the negative control(NC). The PCR products were electrophoresed in agarose gels and stained by ethidium bromide. A representative result is shown.

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REGULATION OF SMAD8/9 EXPRESSION BY BMP

7

mutations. Similar results were obtained with BRE-4 and BRE-5
mutations; mutations of both BRE-4 and BRE-5 did not inhibit the
responsiveness to BMP4 induced through BRE-3 mutations. These
results suggest that all ﬁve BREs are necessary for BMP-induced
Smad8/9 transcription, although BRE-3 is the most critical region.
We further explored whether Smad1/5/8 binds to the region
including BREs of Smad8 gene, that is, nt 164 to nt þ21, in C2C12
cells by ChIP assay (Fig. 8C). When anti-phosphorylated Smad1/5/8
antibody but not control IgG was used for ChIP assay, the region
including the BREs of Smad8 was ampliﬁed; the band intensity
tended to increase in response to BMP4 treatment. Phosphorylated
Smad1/5/8 also bound to the region including the BREs of Id2 in a
BMP4-dependent manner. On the contrary, signiﬁcant band was notdetected, when the primer sets to detect the outside BREs of Smad8
gene or the unrelated region to BMP responsiveness in hepcidin gene
were used. These results suggest that phosphorylated Smad1/5/8
binds to the BREs of Smad8 gene, and that the binding is dependent
on BMP signaling in C2C12 cells.

DISCUSSION
BMPs elicit physiological effects through the transcriptional activation oftarget genes; BMPs induce phosphorylation of R-Smads and complex
formation with Smad4, leading to transcriptional activation of the target
genes. The signaling model of BMP indicates that the dephosphorylationof R-Smads and gene induction of I-Smads terminate the stimulated BMP
signaling [Miyazono et al., 2010; Massague, 2012; Sakaki-Yumoto et al.,2013]; the activity of R-Smads is not regulated through expression, butrather through the phosphorylation status [Miyazono et al., 2010]. The
present study revealed that Smad8/9 is an exceptional R-Smad positively
regulated via the BMP pathway at the mRNA level, and that geneinduction occurs in multiple cell types; recently, Tsukamoto et al.
[2014] reported similar results that BMP regulates expression of Smad9.
The present study extends their results [Tsukamoto et al., 2014]:
expression of Smad8 and Smad9 is cell-type dependent, Smad8/9 gene
expression is transcriptionally regulated by BMP, and several BREs are
involved in the transcriptional activation of Smad8/9.
Functional role of Smad8/9 is still controversial. As describedabove, Smad8/9 is categorized as R-Smad that is phosphorylated in
response to treatment with BMP [Miyazono et al., 2010; Massague,2012; Sakaki-Yumoto et al., 2013]. Similar to Smad1 and Smad5,
Smad8/9 mediates the transcription of a reporter with BREs derived
from the Id1 gene [Kawai et al., 2000; Tsukamoto et al., 2014],
suggesting that functions of Smad8/9 are compatible with thoseof Smad1 and Smad5. Indeed, Smad8/9 is required for brain
development [Hester et al., 2005] and dorso-ventral patterning
during embryogenesis [Wei et al., 2014]; these effects were similar to
those of Smad1. In contrast, Tsukamoto et al. [2014] recently
demonstrated the inhibition of the BMP pathway by Smad8/9; BMP
induced phosphorylation of Smad1 and Smad8/9, and phosphorylated Smad8/9 formed complex with Smad1 to inhibit Smad1mediated transcription. These results suggest a dominant-negativerole of Smad8/9 in transcriptional activation by Smad1. In thecase of similar role of Smad8/9 to Smad1 and Smad5, the present
results on BMP-induced up-regulation of Smad8/9 expression is

8

REGULATION OF SMAD8/9 EXPRESSION BY BMP

327

interpreted to accelerate the BMP signaling. Taken the role with wellcharacterized gene induction of I-Smad, that is, Smad6 and Smad7,
by the BMP pathway [Miyazono et al., 2010] together, net BMP
signaling may be ﬁnely tuned by simultaneous stimulation andinhibition of BMP activity. In contrast, in the case of inhibitory roleof Smad8/9 in BMP pathway, the present results reﬂect a negative
feedback of BMP signaling. The dominant-negative role of Smad8/9
suggested by Tsukamoto et al. [2014] has not been shown inbiological process yet. Further studies to explore the biological roleof Smad8/9 clarify the signiﬁcance of Smad8/9 gene induction.
There is an alternative splicing variant of Smad8/9 gene,designated Smad8B in humans, which lacks C-terminal serines
phosphorylated through receptor complexes [Nishita et al., 1999]. We
examined Smad8B expression in HepG2 cells, a human-derived cellline, using previously reported PCR primers [Nishita et al., 1999], but
could not detect signiﬁcant Smad8B expression, irrespective of the
BMP treatment (data not shown). We also explored the corresponding
sequence of murine Smad8/9, but did not detect Smad8B. Thus, weconcluded that the induction of Smad8/9 gene expression through
BMP4 in multiple cell types does not reﬂect the expression of Smad8/9
variant lacking region with C-terminal serines.
A search of the nucleotide database indicated mouse Smad8/9 variants
with inserted sequences between exons 1 and 2 (variant 1: GenBank
accession number XM_006501728) and the deletion of a part of exon 2(variant 2: XM_00651729). Furthermore, other human Smad8/9 variants
lacking lack exons 2 and 3, or exon 3 alone have been identiﬁed [Chenget al., 2004]. The expression of these Smad8/9 variants and effects of
BMP4 treatment were further examined using RT-PCR analyses withprimers to discriminate these variants in C2C12 cells. We detected Smad8/
9 variants 1 and 2, but not Smad8/9 variants lacking exons 2 and 3, orexon 3 alone as previously reported [Cheng et al., 2004] (data not shown).
BMP4 increased the gene transcript levels of all 3 Smad8/9, that is,wild-type Smad8/9, Smad8/9 variants 1 and 2 (data not shown). The
amino acid sequence of Smad8/9 variant 1 is identical to that of wild-type
Smad8/9. In addition, differences in the amino acid sequences between
wild-type Smad8/9 and Smad8/9 variant 2 only involves 13 amino acids;
the ﬁrst nine amino acids of wild-type Smad8/9 are deleted in Smad8/9
variant 2, and four amino acids within the next ﬁve amino acids aredifferent between wild-type Smad8/9 and Smad8/9 variant 2. Thus, thepresent results suggest that the stimulated BMP pathway substantially
increases activity of wild-type Smad8/9.
The R-Smads, except for Smad2, and Smad4 can bind to preferred
DNA sequences. However, the afﬁnity of Smads to the DNA sequence is
not sufﬁcient to support the association of Smads with regulatory
promoter sequences [Miyazono et al., 2010; Massague, 2012; SakakiYumoto et al., 2013]. Therefore, Smad complexes interact and cooperatewith the other transcription factors, leading to transcriptional
regulation of target genes with the help of transcriptional
coactivators and corepressors [Miyazono et al., 2010; Massague,2012; Sakaki-Yumoto et al., 2013]. Future studies are needed tocharacterize Smad complexes bound to the BREs of Smad8/9.

ACKNOWLEDGMENT
This work was supported by a research project grant awarded by the
Azabu University (2013K08).

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Sample collecting
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GPST BOEFYUFOTJPOBU$GPST XJUIBOBEEJUJPOBM

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 #BTTFU   $IJO   (FSNBO TIFQIFSE   1FLJOHFTF  

Results and Discussion

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JEFOUJGJFE BT TUBQIZMPDPDDJ CBTFE PO DPMPOZ NPSQIPMPHZ 

JOWFTUJHBUFE VTJOH TFWFSBM DPOWFOUJPOBM 1$3 NFUIPET BT

(SBNTUBJOBQQFBSBODFBOEUIFDBUBMBTFUFTUQSFWJPVTMZ EFTDSJCFE  "T TIPXO JO 5BCMF   .341
XJUINFD"BDDPVOUFEGPS  PGUIFTUSBJOT5IJTGSFRVFODZXBTWFSZIJHIDPNQBSFEUPQSFWJPVTTVSWFJMMBODF

Species identiﬁcation, determination of methicillin
resistance and SCC

EBUB GSPN PUIFS DPVOUSJFT  JO UIF 6,   JO

typing,PSFB  BOE  JO /PSUI $IJOB  BOE XBT TJNJMBS UP
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5P JEFOUJGZ NFUIJDJMMJO SFTJTUBODF  B 1$3 NFUIPE 

JOGFDUFE XJUI .341 BOE    PG  EPHT JOGFDUFE

GPS EFUFDUJPO PG UIF NFD" HFOF XBT VTFE 4VCTFRVFOUMZ 

XJUI .441 %PHT JOGFDUFE XJUI .341 UFOEFE UP IBWF

4$$NFD UZQJOH PG UIF .341 TUSBJOT JEFOUJGJFE XBTHSFBUFS FYQPTVSF UP BOUJCJPUJDT UIBO .441JOGFDUFE EPHT

330



(FOPUZQJDEJTUSJCVUJPOPG4QTFVEJOUFSNFEJVTGSPNQZPEFSNBJO+BQBO

Table 1

Distribution of methicillin-resistantstrains from canine pyoderma

Table 2

/PPGTUSBJOT 
4QTFVEJOUFSNFEJVT.FUIJDJMMJOTVTDFQUJCMF.FUIJDJMMJOSFTJTUBOU
4$$NFDUZQF***MJLF
4$$NFDUZQF7/POUZQFBCMF

   


 

Distribution of two exfoliative toxin genes among
strains

4QTFVEJOUFSNFEJVTO.FUIJDJMMJOTVTDFQUJCMF.FUIJDJMMJOSFTJTUBOU/PPGQPTJUJWFTUSBJOT 
FYQ"
FYQ#
   
 

Fig. 1 Multiplex PCR analysis ofgene complex (A) andgene complex(B).
Lane 1, SCC
type III-like MRSP; lane 2, SCC
type V MRSP;
lane 3, SCC
nontypeable MRSP. (A) Upper bands in lanes 1 and 2
represent types 3 and 5
genes, respectively. Lane 3 is a nontypeable strain that possesses
gene only. (B) Lanes 1 and 2 show
single bands speciﬁc to class A and C
gene complexes.

1SFWJPVT VTF PG BOUJNJDSPCJBMT NBZ CF BTTPDJBUFE XJUI
.341JOGFDUJPOJOEPHT'VSUIFSNPSF  NVMUJQMFY BOE EVQMFY 1$3 BTTBZT GPS
4$$NFD UZQJOH SFWFBMFE UIBU    PG  .341
TUSBJOT XFSF UZQF ***MJLF     CFMPOHFE UP UZQF
7  BOE POMZ POF XBT EFUFSNJOFE BT OPOUZQFBCMF 5IFSFQSFTFOUBUJWFFMFDUSPQIPSFUJDQBUUFSOTPG4$$NFDUZQF***
MJLF XJUI GSBHNFOUT PG CPUI UZQF  DDS BOE DMBTT" NFD 
BOE7 XJUIGSBHNFOUTPGCPUIUZQFDDSBOEDMBTT$NFD 
BSFTIPXOJO'JH
5P FYBNJOF UIF QSFTFODF PG &5 HFOFT  BNQMJàDBUJPO PG
UIF FYQ" BOE # HFOFT XBT DPOEVDUFE BT TIPXO JO 'JH 
SFQSFTFOUBUJWFEBUB 5BCMFTIPXTUIFGSFRVFODZPGFYQ"
BOE#JOUIFJTPMBUFE4QTFVEJOUFSNFEJVTTUSBJOT5IFFYQ"

331

Fig. 2

PCR analysis of exfoliative genes.
Lane 1, a MSSP isolate carrying thegene; lane 2, positive control strain for;lane 3, a MSSP carrying thegene;lane 4, positive control strain for.



麻布大学雑誌

第  巻 年

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Acknowledgements*OWFTU   

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332

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RESEARCH ARTICLE

Hybrid Origin of Gynogenetic
Clones and the Introgression of
Their Mitochondrial Genome Into
Sexual Diploids Through Meiotic
Hybridogenesis in the Loach,Misgurnus anguillicuadatus
AYA YAMADA1, YUKIHIRO KODO2,MASARU MURAKAMI2,
MASAMICHI KURODA1, TAKAO AOKI1,
TAKAFUMI FUJIMOTO1,
1
AND KATSUTOSHI ARAI *1

Faculty and Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido,Japan
2
Faculty of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan

ABSTRACT

J. Exp. Zool.
323A:593–606,
2015

In a few Japanese populations of the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae),clonal diploid lineages produce unreduced diploid eggs that normally undergo gynogenetic
reproduction; however the origin of these clones remains elusive. Here, we show the presence of
two diverse clades, A and B, within this loach species from sequence analyses of two nuclear genes
RAG1 (recombination activating gene 1) and IRBP2 (interphotoreceptor retinoid-binding protein,2) and then demonstrate heterozygous genotypes ﬁxed at the two loci as the evidence of thehybrid nature of clonal lineages. All the clonal individuals were identiﬁed by clone-speciﬁc
mitochondrial DNA haplotypes, microsatellite genotypes, and random ampliﬁed polymorphic DNA
ﬁngerprints; they commonly showed two alleles, one from clade A and another from clade B,
whereas other wild-type diploids possessed alleles from either clade A or B. However, we also
found wild-type diploids with clone-speciﬁc mitochondrial DNA and nuclear genes from clade B.
One possible explanation is an introgression of a clone-speciﬁc mitochondrial genome from clonal
to these wild-type loaches. These individuals likely arose by a cross between haploid sperm frombisexual B clade males and haploid eggs with clone-speciﬁc mtDNA and clade B nuclear genome,
produced by meiotic hybridogenesis (elimination of unmatched A genome followed by meiosis
after preferential pairing between two matched B genomes) in clone-origin triploid individual(ABB). J. Exp. Zool. 323A:593–606, 2015. © 2015 Wiley Periodicals, Inc.
How to cite this article: Yamada A, Kodo Y, Murakami M, Kuroda M, Aoki T, Fujimoto T, Arai K.
2015. Hybrid origin of gynogenetic clones and the introgression of their mitochondrial genome
into sexual diploids through meiotic hybridogenesis in the loach, Misgurnus anguillicuadatus.
J. Exp. Zool. 323A:593–606.

© 2015 WILEY PERIODICALS, INC.

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YAMADA ET AL.

The loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) is agonochoristic diploid with 2n ¼ 50 chromosomes and it reproduces sexually, but clonal diploid lineages have been found in thenorthern area of Hokkaido Prefecture and the Notojima Island of
Ishikawa Prefecture, Japan (Morishima et al., 2002, 2008a). Theseclonal diploid loaches are essentially all-female and generate
unreduced diploid eggs by premeiotic endomitosis, that is,chromosome doubling without cytokinesis (Itono et al., 2006).
Most clonal diploid eggs initiate gynogenetic development after
fertilization by sperm of sympatric sexually reproducing wildtype males, whereas some incorporate the sperm nucleus into theegg to develop as triploid animal (Itono et al., 2007). Genetics andatypical reproduction of clonal diploid and other polyploidMisgurnus loaches have been reviewed by Arai and Fujimoto(2013).
Interspeciﬁc hybridization is considered to be involved in theorigin of unisexual vertebrates, which atypically reproduce byparthenogenesis, gynogenesis (sperm-dependent parthenogenesis), or hybridogenesis, and this is often accompanied bypolyploidy (reviewed in Dawley, '89; Vrijenhoek et al., '89;
Vrijenhoek, '94), although nonhybrid origins were recently
reported in parthenogenetically reproducing lizard Lepidophyma
genus (Sinclair et al., 2010). Hybridization often causes a
disruption of regular meiosis because of unsuccessful pairing
between non-homologous chromosomes derived from different
species, and it often results in sterility, but it seldom induces
atypical gametogenesis and unusual modes of reproduction inﬁsh species (Dawley et al., '85; Fujimoto et al., 2008). Examples ofunisexual ﬁsh with an apparent hybrid origin are included in thegenera Poecilia, Poeciliopsis, Menidia, Phoxinus, Squalius,
Carassius, and Cobitis (reviewed in Dawley, '89; Vrijenhoek
et al., '89; Gui and Zhou, 2010; Collares-Pereira et al., 2013).
Recent large-scale multi-locus analyses revealed the hybrid
origin of asexuality, establishment of clones in different ploidy
levels, and effect of asexual complexes to the initiation of
clonality and polyploidy in Cobitis loach, different members of
the family Cobitidae (Janko et al., 2012). In the Misgurnus loach,however, a relationship between clonal reproduction in certain
diploids and hybridization is ambiguous, because M. anguillicaudatus has been identiﬁed as a single species entity (Saitoh,'89).

Grant sponsor: JSPS-KAKENHI; grant number: 21380114.
Correspondence to: Katsutoshi Arai, Faculty and Graduate School ofFisheries Sciences, Hokkaido University, 3-1-1, Minato, Hakodate,
Hokkaido 041-8611, Japan.
E-mail: araikt@fish.hokudai.ac.jp
Received 2 February 2015; Revised 9 May 2015; Accepted 29 May 2015
DOI: 10.1002/jez.1950
Published online 14 July 2015 in Wiley Online Library(wileyonlinelibrary.com).

There are two major groups of Japanese M. anguillicaudatus,
namely, A and B, which are further clustered into two sub-groups,
B-1 and B-2, based on highly differentiated sequences (averagesequence divergence, 13%) of the mitochondrial DNA control
region (mtDNA-CR) (Morishima et al., 2008a). Similar intergeneric equivalent diversity has also been observed in M.
anguillicaudatus (Koizumi et al., 2009), in which different mtDNA
regions, particularly cytochrome b sequences, were analyzed, anda large genetic divergence was observed (average sequence
divergence, 15–18%). However, recent molecular phylogenetic
studies strongly suggest that the entire mitochondrial genome ofgroup B Misgurnus must have originated from the ancestral loach
genus Cobitis through introgression; thus, the presence of two
different lineages diverging early in the speciation process within
M. anguillicaudatus is highly spurious (Slechtov
a et al., 2008;
Kitagawa et al., 2011). The hypotheses about the mitochondrial
introgression are supported by the recent analyses of thecomplete mitochondrial genomes of different polyploid M.
anguillicaudatus (Zhou et al., 2014). Although these results raise
the question of genealogy based on previous mtDNA analyses(Morishima et al., 2008a; Koizumi et al., 2009), the presence of
two different groups in the Japanese wild populations have also
been demonstrated by using allozymes (Khan and Arai, 2000) andmicrosatellite analyses (Arias-Rodriguez et al., 2006). Recent
molecular phylogenetic studies on Misgurnus from the Far
Eastern region of Russia also suggested the presence of different
species within M. anguillicaudatus (Perdices et al., 2012).
Disruption of normal meiosis and subsequent gametogenesis
was also reported in inter-populational M. anguillicaudatus
hybrids between group A females and group B males as in
atypical reproduction occurred in inter-speciﬁc hybrids: most
hybrids generated unreduced diploid and other types of unusual
gametes (Arias-Rodriguez et al., 2009, 2010). All these studies
support the hybrid-origin theory for the occurrence of gynogenetically reproducing clones in nature.
In natural triploid loaches arising from the spontaneous
incorporation of a haploid sperm nucleus from a wild-type
diploid into an unreduced diploid egg of clonal lineage, haploideggs are formed by meiotic hybridogenesis: one of the twodifferent chromosome-sets (haploid genome) of the clone
preferentially pairs with the genetically similar haploid set
from a sperm donor to form bivalents and then they produce
haploid gametes by regular meiosis, but another haploid set of theclone is eliminated before meiosis because of the low pairing
afﬁnity between the two haploid genomes of the clone(Morishima et al., 2008b). This unusual pattern of meiotic
hybridogenesis also suggests that the genomic constitution of theclonal loach might be heterozygous because of its presumptive
hybrid origin.
All the above-mentioned results support the involvement ofhybridization in the origin of clonal diploid loaches in nature, but
direct genetic evidence has not been obtained so far. In the

J. Exp. Zool.

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HYBRID ORIGIN OF CLONAL DIPLOID LOACH

595

present study, we analyzed sequences of the nuclear genes, RAG1
(recombination activating gene 1) and IRBP2 (interphotoreceptor
retinoid-binding protein 2), which have been utilized formolecular phylogenetic studies in Cypriniformes ﬁshes, includ
ing loach species (Slechtov
a et al., 2008; Saitoh et al., 2010;
Perdices et al., 2012), in putative clonal diploid loaches and wildtype diploid loaches from different localities. Here, we demonstrate that the sequences of the RAG1 and IRBP2 genes can beclassiﬁed into two different clades, which closely correspond to
two groups that were previously observed in mtDNA-CR
sequences. We then show that clonal diploid specimens withclone-speciﬁc mtDNA-CR sequences and microsatellite genotypes are heterozygotes possessing two different alleles fromdiverse clades at both RAG1 and IRBP2 loci, indicating the hybrid
origin of clonal lineage.

MATERIALS AND METHODS
EthicsAll experiments procedures were performed in accordance with
Institutional guidelines on animal experimentation and care, andwere approved by the Animal Research Committee of Hokkaido
University.
Fish Specimens
From 2000 to 2010, M. anguillicaudatus (n ¼ 54) were collectedfrom 12 Japanese localities (Table 1). The ploidy status of eachindividual was examined by ﬂow cytometry according toMorishima et al. (2002) and one triploid individual was detectedamong the specimens collected from site number 1. Clonal
individuals (#3 and #6) were previously identiﬁed byreproduction experiments including artiﬁcial fertilization
with normal sperm, hybridization with goldﬁsh sperm, andinduced gynogenesis with UV-irradiated sperm as well as
genetically identical genotypes in several microsatellite lociand multi-locus DNA ﬁngerprinting (Morishima et al., 2002).
Clone 1 strain was established by reproduction of the identiﬁed
female (#3: Morishima et al., 2002) and then maintained in the
Aquarium room of the Environment Control Experiment
Building of the Faculty of Fisheries Sciences, Hokkaido
University, Hakodate. Clone 1 strain was used as reference
for clone 1. The other clonal strains (2 to 4) were later identiﬁedby the presence of mtDNA-CR haplotype III as well as
absolutely identical proﬁles in random ampliﬁed polymorphic
DNA (RAPD)-PCR analyses using several random primers andmulti-locus DNA ﬁngerprinting using a tetranucleotide probe(Morishima et al., 2008a). Preserved specimens of clones 2 to 4
were also used as reference control samples to identify clonalstrains. The mud loach M. mizolepis specimen (n ¼ 1), obtained
from a commercial aquarium shop, was also used in theanalysis.
Mitochondrial DNA Analysis
DNA was extracted from ﬁn clips or muscle tissue by using thestandard phenol/chloroform protocol (Asahida et al., '96). The
mtDNA-CR was ampliﬁed and subjected to restriction fragment
length polymorphism (RFLP) analysis, followed by determinationof the partial sequences of the mtDNA-CR region (444–448 bp)

Table 1. Sampling sites, sample size, and year of sampling of specimens used in the present.
Locality
Site no.

Prefecture

City/town/village (former name)(Site no. in Morishima et al. [2008a])

1
2
3
4
5
6
7
8
9
10
11
12
Total

Hokkaido
Hokkaido
Aomori
Iwate
Yamagata
Miyagi
Niigata
Saitama
Aichi
Mie
Ishikawa
ShimaneOzora (Memanbetsu) [5]
Iwamizawa [8]Yomogita
Hanamaki [18]Tsuruoka [20]Osaki (Naruko) [22]Niigata (Maki) [30]
Hanyu [27]Inazawa [40]Yokkaichi [42]Nanao2 [33]Izumo [49]12NYear of sampling

12 (1)1
8
2
2
2
1
9
4
2
2
9
1
54

1998/2009
1996
2009
2004
2004
1998
1996
1998
2003
2004
2009
1997

Number of triploid individual is indicated in parentheses.
Hannoura region, Notojima island.

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YAMADA ET AL.

according to the procedures described in Morishima et al. (2008a).
Precise group identiﬁcation of each specimen was performed by
100 to 99.3% matching in the corresponding site between thedeposited 942–954 bp mtDNA-CR sequences (AB306717AB306793) clustered into different genetic groups (A, B-1, andB-2; Morishima et al., 2008a) and present partial 444–448 bp
sequences by using Bioedit (http://www.mbio.ncsu.edu/BioEdit/
bioedit.html).
Identiﬁcation of Clonal Diploid Individuals by DNA Marker Analyses
Since all the previously identiﬁed clonal individuals and cloneorigin triploid individuals had mtDNA-CR RFLP haplotype III,
that is, sequences III-1 or III-2 (Morishima et al., 2008a),
candidates of clones were predicted to have such a clone-speciﬁc
haplotype. Thus, samples taken from localities (site no, 1, 7, and11), where previously known clones were found, were ﬁrstly
screened to separate candidates of clones among diploid loaches
by presence or absence of the clone-speciﬁc mtDNA-CR
haplotype.
Next, selected candidates of clones were analyzed byindependent microsatellite loci (Mac 3, Mac37, Mac 404,
Mac458 and Mac477; Morishima et al., 2008b, c, 2012) in order
to ﬁnd out clonal individuals with genotypes identical to those of
the reference control samples of clones 1, 2, 3, and 4. These ﬁveloci were used to genotype progeny of clone 1 in Morishima et al.
(2008b, 2012).
Candidates of clones were further examined by RAPD (random
ampliﬁed polymorphic DNA) ﬁngerprinting using three different
random primers, Wako-01 (Arbitorary Primer set A-01, Wako,
Osaka, Japan), OPA-11 (Kit A-11, Operon Technologies, Alameda,
CA, USA) and OPA-12 (Kit A-12) according to the procedures
described by Itono et al. (2006, 2007) and Morishima et al.
(2008a). The other specimens without clone-speciﬁc mtDNA-CR
haplotype, clonal microsatellite genotypes and clonal RAPD
ﬁngerprints were conveniently categorized to wild-type diploids,
although the bisexual reproductive mode was not individuallyconﬁrmed by reproduction experiments.
For microsatellite genotyping, PCR was performed with a 10 mL
mixture containing 1.0 mL of the DNA sample, 6.45 mL of DDW,
0.8 mL of dNTP mixture (TaKaRa, Otsu, Shiga, Japan), 1.0 mL of
10PCR buffer (TaKaRa), 0.05 mL of r Taq DNA Polymerase(TaKaRa), 0.37 mL of the primer mix (Forward:Reverse ¼ 1:10),
and 0.33 mL of the M13-tailed forward primer (with ﬂuorescencelabeled 50 ends). The PCR conditions were as follows: initialdenaturation at 94° for 1 min, 35 cycles of 94° for 15 sec, 56° for
15 sec, and 72° for 1 min 30 sec. The reaction was completed by aﬁnal extension at 72° for 60 min. Electrophoresis was carried out
using ABI PRISM 3130xl (Applied Biosystems, Foster City, CA,
USA). The alleles were distinguished by differences in themolecular size using the Gene Scan 500LIZ (Applied Biosystems).
Genotyping was performed using the Gene Mapper Software v.3.7(Applied Biosystems).

The ampliﬁcation reactions were conducted for RAPD ﬁngerprinting in a mixture comprising DNA template (100 ng/mL),
DDW 16.0 mL, dNTP mixture (TaKaRa) 1.6 mL, 10 PCR buffer(TaKaRa) 2.0 mL, rTaq polymerase (TaKaRa) 0.2 mL and random
primer 0.2 mL in 0.2 mL microtube. Ampliﬁcation proﬁles were
follows: initial step of 3 min at 95°, subsequent 30 cycles of 30 sec
at 95°, 1 min at 36°, and 1 min at 72°, followed by ﬁnal primer
extension for 7 min at 72°. About 5 mL of PCR products were
electrophoresed on 1.5% agalose gel and stained with ethidium
bromide and photographed on a UV transilluminator using a gel
documentation system (UVP BioDoc-ItTM Imagining System,
Cambridge, UK).
Sequencing of the RAG1 Gene
RAG1 was ampliﬁed using the primer RAG-1F (50 -AGCTGTAGTCAGTAYCACAARATG-30 : Quenouille et al., 2004) and RAG-RV1
(50 -TCCTGRAAGATYTTGTAGAA-30 : Slechtov
a et al., 2007). PCR
was performed with a 20 mL mixture containing 1.0 mL of the
DNA sample, 13.3 mL of DDW, 1.6 mL of a dNTP mixture (TaKaRa),
2.0 mL of 10xEx Taq Buffer (TaKaRa), 0.1 mL of Ex Taq DNA
Polymerase (TaKaRa), 1.0 mL of 10 mM RAG-1F primer, and 1.0 mLof 10 mM RAG-RV1 primer. The PCR conditions were as follows:
initial denaturation at 95° for 5 min, touch-down proﬁle of 1 min
at 94°, 1 min 30 sec at 60–55° (1° /cycle), and 2 min at 72°
followed by 30 cycles with annealing temperature held at 54°. The
reaction was completed by a ﬁnal extension at 72° for 7 min. The
PCR products were puriﬁed using AMPure Kits (Beckman Coulter,
Brea, CA, USA) and the puriﬁed products were sequenced with
PCR primers using BigDye Terminator v3.1 Cycle Sequencing Kit(Applied Biosystems). The sequence products were puriﬁed using
Clean SEQ (Beckman Coulter) and resolved on an ABI PRISM
3130xl (Applied Biosystems). The sequences were aligned using
ClustalW in the program BioEdit (http://www.mbio.- ncsu.edu/
BioEdit/bioedit.html), and the alleles were identiﬁed when no or
only one double-peaked site was observed. When two or more
double-peaked sites were detected, the PCR product was cloned
again using the TA Cloning Kit (Invitrogen, Paisley, UK), and eachallele per clone was identiﬁed.
Sequencing of the IRBP-2 Gene
IRBP-2 was ampliﬁed using the primer IRBP 76F(50 -CTTRTTGTGGATATGGCAAAAAT-30 ) and IRBP 1162R(50 -TGGTGGWCTTYAGGCACTTGT-30 ) (Chen et al., 2008). PCR
was performed with a 20 mL mixture containing 1.0 mL of the
DNA sample, 13.3 mL of DDW, 1.6 mL of dNTP mixture (TaKaRa),
2.0 mL of 10 Ex Taq Buffer (TaKaRa), 0.1 mL of Ex Taq DNA
polymerase (TaKaRa), 1.0 mL of 10 mM IRBP 76F primer, and
1.0 mL of 10 mM IRBP 1162R primer. The PCR conditions were as
follows: initial denaturation at 95° for 4 min, 30 cycles of 95° for
40 sec, 58° for 40 sec, and 72° for 1 min 30 sec. The reaction wascompleted by a ﬁnal extension at 72° for 7 min. The PCR products
were puriﬁed using AMPure Kits (Beckman Coulter), and the

J. Exp. Zool.

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597

puriﬁed products were sequenced using PCR primers and the
BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequence products were puriﬁed using Clean SEQ(Beckman Coulter) and resolved on ABI PRISM 3130xl (Applied
Biosystems). The sequences were aligned using ClustalW in the
program BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.
html), and alleles were identiﬁed when no or only one doublepeaked site was observed. When two or more double-peaked sites
were detected, the PCR product was cloned using a TA Cloning Kit(Invitrogen), and alleles were identiﬁed in the clone.
Data Analysis
We conducted maximum likelihood (ML) phylogenetic analysis
by using MEGA 5 (Tamura et al., 2011). The best-ﬁt substitution
models indicated that the Kimura 2-parameterþ discrete gamma
model (Kimura, '80; Yang, '94) was the best ﬁt implemented in
MEGA 5. Bootstrap analysis was performed on 1,000 replicationsin the phylogenetic tree. We obtained Cobitis biwae (RAG-1:
AB531299, IRBP-2: AB531238), Cobitis striata (RAG-1:
AB531286, IRBP-2: AB531223), and Acantopsis choirorhynchos(RAG-1: AB531313, IRBP-2: 531254) as the outgroup from DDBJ/
GenBank/EMBL. The genetic distance between the groups was
also calculated using MEGA5 against the p-distance.

RESULTS
Table 2 shows the genetic groups inferred from mtDNA-CR RFLP
haplotypes and the partial sequences of all the loach specimens
examined. Clone-speciﬁc mtDNA-CR haplotypes (III-1 or III-2)
were detected in six out of 12 samples from site number 1, one outof 9 samples from site number 7, and six out of nine samples fromsite number 11. No clone-speciﬁc mtDNA-CR haplotypes weredetected in specimens from other sampling sites.
When microsatellite genotyping was performed at ﬁve different loci in above-mentioned samples, ﬁve candidates from site
number 1 were identiﬁed as clone 1, because they exhibited
microsatellite genotypes identical to the reference control ofclone 1 and other samples gave polymorphic variations (Table 3).
On the other hand, clone 2 was found in site number 7, and clones
3 and 4 were detected in site no. 11 (Table 3). Clonal individual
was not detected by microsatellite genotyping in other candidates
from site number 11, although they had clone-speciﬁc mtDNACR III-1 or III-2 haplotype (Tables 2 and 3). A naturally triploid
individual (ﬁsh no. 7 from site no. 1) harbored two microsatellite
alleles speciﬁc to clone 2, which may presumably be aspontaneous triploid progeny of clone 2 (Table 3).
RAPD-ﬁngerprinting with three different random primers(Wako-01, OPA-11, and OPA-12) veriﬁed genetically identicalcharacteristics of ﬁve candidates from site number 1 (clone 1), one
candidate from site number 7 (clone 2) and two candidates fromsite number 11 (clone 3 and 4), because they gave ﬁngerprints
identical to that of reference control samples of clone 1–4 (Fig. 1).
Triploid sample (no. 7 from site no. 1; lane 12 in left gels) gave DNA

ﬁngerprints comprising all fragments of clone 2 and additional
fragments (Fig. 1). Genetic variation was found by RAPD
ﬁngerprinting in other diploids from site number 1, 7, and 11(Fig. 1). Several samples that had clone-speciﬁc mtDNA-CR III-1 orIII-2 haplotype also gave polymorphic ﬁngerprints (Fig. 1).
The 527-bp RAG1 gene sequence was determined in 52
individuals of M. anguillicaudatus and one individual of M.
mizolepis. Consequently, 16 different sequences were distinguished among M. anguillicaudatus specimens examined and allthese sequences were deposited in DDBJ/GenBank/EMBL as
accession numbers AB698049 to AB698064, with allele names
serially designated as RAG1-01 to RAG1-16 in the present study.
Two sequences (AB704298, 704299) from a M.mizolepis specimen were also deposited in DDBJ/GenBank/EMBL and designated as RAG1-a and RAG1-b, respectively. ML phylogenetic
analysis resulted in the construction of a tree (Fig. 2), whichclustered in two major clades, A and B, with clade B further
divided in two sub-clades, B-1 and B-2. RAG1 sequences of M.
anguillicaudatus were clearly distinguished from those of M.
mizolepis and this difference indicated the presence of geneticallydiversiﬁed groups within the species. The genetic distance of
RAG1 by mean uncorrected p-distance was 4.3  0.8% between
Cobitis and Misgurnus, 3.1  0.6% between M. anguillicaudatus
and M. mizolepis, 2.6  0.6% between clade A and B, and2.4  0.6% between sub-clades B-1 and B-2.
Among M. anguillicaudatus and M. mizolepis specimens
examined, 20 genotypes were detected in the RAG1 gene locus(Table 2). In wild-type diploids of M. anguillicaudatus from site
numbers 2 to 5, 7, 9, 10, and 12, the individuals were either
homozygous (Rag1-01/Rag1-01, Rag1-02/Rag1-02) or heterozygous (Rag1-01/Rag1-02, Rag1-01/Rag1-11, Rag1-01/Rag112) for the RAG1 alleles of the sub-clade B-1. All these genotypes
had mtDNA-CR sequences classiﬁed in the B-1 group. Loaches
from site number 8 were either homozygous (Rag1-13/Rag1-13)
or heterozygous (Rag1-13/Rag1-14, Rag1-13/Rag1-16, Rag114/Rag1-15) for the RAG1 alleles of the sub-clade B-2. All thesegenotypes contained an mtDNA-CR classiﬁed as B-2. In 13
loaches from site number 1, ﬁve wild-type diploids were either
homozygous (Rag1-05/Rag1-05) or heterozygous (Rag1-03/
Rag1-04, Rag1-04/Rag1-05, Rag1-05/Rag1-06) for the RAG1
alleles of clade A. The wild-type diploids with RAG1 alleles of theclades A, B-1, and B-2 were categorized into genetic groups A, B1, and B-2, as inferred from mtDNA-CR, respectively (Table 2).
On the other hand, clonal diploid loaches from site numbers 1,
7, and 11 showed the characteristic RAG1 genotype: all clonal
individuals were heterozygous for clade A and clade B-1 (Rag101/Rag1-07, Rag1-07/Rag1-10), that is, hybrids between genetically different clades. In site number 11, however, wild-type
diploid loaches with homozygous (Rag1-10/Rag1-10) or heterozygous genotypes (Rag1-01/Rag1-09, Rag1-01/Rag1-10, Rag101/Rag1-11) within the same B-1 clade showed clone-speciﬁc
mtDNA-CR.
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1
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
1
2
1
2
1
2
1
1
2
3
4
5
6
7
8

1

J. Exp. Zool.

339

6
7

5

4

3

2

Fish
no.

Site
no.

III
I
III
III
III
IV
III
III
I
I
I
I
V
V
V
V
V
V
V
V
V
V
V
V
V
V
X
V
VII
V
VII
V
V
IX
V

RFLP

III-1, III-2
I-3, I-4, I-5
III-1, III-2
III-1, III-2
III-1, III-2
IV-1
III-1, III-2
III-1, III-2
I-3, I-4, I-5
I-7
I-7
I-7
V-1, V-4
V-3
V-1, V-4
V-1, V-4
V-1, V-4
V-1, V-4
V-1, V-4
V-1, V-4
V-5, V-6, V-24
V-5, V-6, V-24
V-2, V-21
V-27
V-5, V-6, V-24
V-5, V-6, V-24
n.d.
V-23
VII-8, VII-15
V-23
VII-8, VII-15
V-5, V-6, V-24
V-23
IX-1
V-23

Haplotype (444–448 bp)mtDNA-CR

A
A
A
A
A
A
A
A
A
A
A
A
B-1
B-1
B-1
B-1
B-1
B-1
B-1
B-1
B-1
B-1
B-1
B-1
B-1
B-1
A
B-1
B-1
B-1
B-1
B-1
B-1
B-1
B-1

Group1

Identification ofClone3
Clone 1

Clone 1
Clone 1
Clone 1

Triploid 4
Clone 1




























Clone-specific
Haplotype2
þ

þ
þ
þ

þ
þ



























7
45
7
7
7
55
7
7
34
55
56
.n.d.














88







n.d.

Clade A
allele

11
11
11
11
11
11
11

11
12
22
12
22
11
22
22
11
1 12
1 12
11
11
11

1

1
1
1

1
1




Clade B
alleleRAG1 Genotype5

9
11 12
9
9
9
11 12
.n.d.6
9
n.d.
12 13
10 12
10 12




n.d.

n.d.





n.d.

77

n.d.
n.d.
n.d.
n.d
n.d.
n.d.


Clade A
allele


66

66

22

23
12
12
24
24

12




11
12
22
12

6

6

6
6
6


Clade B
alleleIRBP2 Genotype5

Table 2. Clones and wild-type diploids inferred from mtDNA-CR haplotypes, microsatellite genotypes and RAPD ﬁngerprinting and clade A and B alleles of RAG1 and IRBP2 loci.

598
YAMADA ET AL.

340

RFLP

III
VII
VII
VII
VII
V
V
V
V
III
III
III
III
V
III
VII
V
III
VII

Fish
no.

9
1
2
3
4
1
2
1
2
1
2
3
4
5
6
7
8
9
1

III-1, III-2
VII-1, VII-12
VII-4, VII-9
VII-14VII-1, VII-12
V-19, V-26
V-23
V-20
V-20
III-1, III-2
III-1, III-2
III-1, III-2
III-1, III-2
V-7
III-1, III-2
VII-7
V-7
III-1, III-2
VII-11

Haplotype (444–448 bp)
A
B-2
B-2
B-2
B-2
B-1
B-1
B-1
B-1
A
A
A
A
B-1
A
B-1
B-1
A
B-1

Group1

Identification ofClone3
Clone 2








Clone 4







Clone 3


Clone-specific
Haplotype2
þ








þ
þ
þ
þ

þ


þ

7








7







7


Clade A
allele
1
14 15
13 13
13 14
13 16
11
11
1 11
1 11
10
19
10 10
19
1 10
1 10
19
1 10
1
11

Clade B
alleleRAG1 Genotype5

9
n.d.
n.d.
n.d.

n.d.
n.d.
d.d.
n.d
8







8
n.d.

Clade A
allele




5
66
66
56
56
66
56
66
5

14 15

6

Clade B
alleleIRBP2 Genotype5

2

Group inferred from mtDNA-CR haplotypes (RFLP, sequences) by Morishima et al. (2008a).
Presence (þ) or absence () of clone-speciﬁc mtDNA haplotypes based on Morishima et al. (2008a).
3
Clone-candidates were ﬁrst screened for presence of clone-speciﬁc mtDNA haplotypes and then identiﬁed as clone 1 to 4 based on identical microsatellite genotypes as shown in Table 3 and identical
RAPD ﬁngerprints as shown Figure 1. Symbol “” means non-clonal wild-type.
4
Triploid probably derived from clone 1 diploid by accidental sperm nucleus incorporation into diploid egg.
5
Symbol “” means absence of allele categorized to clade A or B.
6
Not determined.

1

12

11

10

9

8

Site
no.

mtDNA-CR

Table 2. (Continued)

HYBRID ORIGIN OF CLONAL DIPLOID LOACH
599

J. Exp. Zool.

clone 1
clone 2
clone 3
clone 4
1
2
3
4
5
6
7
8
7
8
9
1
2
3
4
5
6
7
8
9

Reference control

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341

Mac 3(12)103/145
103/145
103/161
103/161
103/145
113/113103/145
103/145
103/145
117/117103/113/145
103/145
123/151
109/163
103/145
103/161
137/143
135/135
135/151
103/145
105/135
137/151
103/143
103/161

Samples with clone-speciﬁc mtDNA haplotypes.

11

7

1

Fish No.

Site No.
101/107
101/105
101/107
101/107
101/107
99/105101/107
101/107
101/107
79/85
93/101/105
101/107
105/109
107/107
101/105
101/107
105/131105/107
105/107
105/107
105/131115/117105/107
101/107

Mac37 (10)207/211213/223203/211203/211207/211
195/199207/211207/211207/211181/189201/213/223
207/211193/243
193/201
213/223203/211203/219219/219203/219203/229203/205203/219203/219203/211

Mac404 (4)224/232232/232
228/234
216/238224/232
228/244
224/232
224/232
224/232
250/264232/260
224/232
234/264
212/260
232/232
216/238200/200
218/236202/216
218/234
200/200
202/228202/230
228/234

Mac458 (16)

Microsatellite locus (Linkage group)

124/192
124/202
124/196
124/196
124/192
126/126
124/192
124/192
124/192
126/126
124/126/202
124/192
136/140
138/202
124/202
124/196
178/194
196/216
178/214178/178
178/196
194/212178/216
124/196

Mac477 (11)clone 3

clone 2
clone 4clone 2 origin 3nclone 1clone 1clone 1clone 1clone 1clone 2
clone 3
clone 4clone 1

Identification of clone

Table 3. Identiﬁcation of clonal individuals by matching of genotypes of ﬁve independent microsatellite loci with those of reference control samples identiﬁed as clones 1–4 byMorishima et al. (2002, 2008a, b, 2012).

600
YAMADA ET AL.

HYBRID ORIGIN OF CLONAL DIPLOID LOACH

601

Figure 1. RAPD-PCR ﬁngerprints using random primers WAKO-01(A), OPA-11(B), and OPA-12(C). Left gels: Lanes 1–4 denote reference
control samples of clone 1, 2, 3, and 4, respectively. Lane 5, blank. Lanes 6–13 denote ﬁsh no. 1–8 from site number 1 (see Table 1 and 2).
Central gels: Lanes 1–3 denote ﬁsh no. 7, 8, and 9 from site number 7 (see Table 1 and 2). Right gels: Lanes 1–4 denote reference control
samples of clone 1, 2, 3, and 4, respectively. Lane 5, blank. Lanes 6–13 denote ﬁsh number 1–9 from site number 11 (see Table 1 and 2). M in agel indicates size marker. Left bars also indicate molecular size marker of 3.0, 1.5, 1.0, and 0.5 kbp.

The 575-bp IRBP2 gene sequence was determined in 35
individuals of M. anguillicaudatus and one individual of M.
mizolepis. Consequently, 15 different alleles were detected in M.
anguillicaudatus specimens and all these sequences were
deposited in DDBJ/GenBank/EMBL under accession numbers
AB702651 to AB702665, with allele names serially designatedasIRBP2-01 to IRBP2-15. Two sequences (AB704300 and
704301) from a M. mizolepis specimen were also deposited in
DDBJ/GenBank/EMBL and designated as IRBP2-a and IRBP2-b,
respectively. ML phylogenetic analysis resulted in the construction of a tree (Fig. 3), which clustered at two major clades, A and
B. The IRBP2 sequences of M. anguillicaudatus were clearly
distingusished from those of M. mizolepis and indicated thepresence of two genetically differentiated groups in M.
anguillicaudatus. Further sub-clade analysis was not possibleusing the phylogenetic tree. The genetic distance of IRBP2 bymean uncorrected p-distance was 5.3  0.7% between Cobitis

and Misgurnus, 3.8  0.6% between M. anguillicaudatus and M.
mizolepis, and 2.9  0.6% between clades A and B.
A total 15 genotypes were detected in the IRBP2 gene locus(Table 2). Wild-type diploid samples from site numbers 2 to 5, 7,
and 8 were either homozygous (IRBP2-01/ IRBP2-01, IRBP2-02/
IRBP2-02, IRBP2-06/ IRBP2-06) or heterozygous (IRBP2-01/
IRBP2-02, IRBP2-02/ IRBP2-04, IRBP2-05/ IRBP2-06, IRBP214/ IRBP2-15) for IRBP2 sequences of the clade B. All thesegenotypes harbored an mtDNA-CR that was classiﬁed as
belonging to either the B-1 or B-2 subgroup. In loaches fromsite number 1, ﬁve diploid included only the IRBP2 alleles ofclade A (IRBP2-11/ IRBP2-12, IRBP2-12/ IRBP2-13, IRBP2-10/
IRBP2-12). The wild-type loach individuals with IRBP2 alleles of
the clades A and B were categorized into genetic groups A and B,
as inferred from mtDNA-CR, respectively.
On the other hand, all the clonal diploid individuals from site
numbers 1, 7, and 11 exhibited heterozygosity between alleles
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YAMADA ET AL.

Figure 2. ML phylogenetic tree using RAG1 sequences. The numbers on the branches are bootstrap values.

from clades A and B of the IRBP2 gene (IRBP2-05/ IRBP2-08,
IRBP2-06/ IRBP2-09). However, in wild-type diploid loaches
from site number 11, four individuals showed IRBP2 genesequences exclusively from clade B (IRBP2-06/ IRBP2-06),
although these showed an mtDNA-CR belonging to group A(Table 2).

DISCUSSION
Although recent molecular phylogenetic studies using both
nuclear genes and mitochondrial DNA markers suggest that theentire mitochondrial genome of one of the two major groups inJapanese M. anguillicaudatus should have been replaced by thatof the genus Cobitis due to introgression after a hybridization
event that occurred approximately 7 to 10 million years ago(Slechtova et al., 2008; Kitagawa et al., 2011; Pardices et al.,2012), both RAG1 and IRBP2 sequences examined in this study

were clustered into two clades of the same species. Among wildtype diploid M. anguillicaudatus specimens within clade A ofboth RAG1 and IRBP2 gene sequences were categorized as groupA, as inferred from its mtDNA-CR, whereas those within clade Bof both gene sequences as that of group B. Specimens within the
subclade B-1 of RAG1 and those within the subclade B-2 also
correspond to the B-1 and B-2 groups, as inferred from the
mtDNA-CR, respectively. Thus, genetic results from mtDNA andnuclear gene sequence analyses strongly suggest the presence ofdifferent groups within M. anguillicaudatus.
The inter-clade genetic distance for RAG1 (2.6  0.6%)
estimated in the present study was slightly higher than theinter-speciﬁc genetic distance reported for several Misgurnus
species (2.1  0.8%; Perdices et al., 2012) and similar to that for
the Cobitis species (2.5  0.5%; Perdices et al., 2012). A similarinter-clade distance was also estimated in the present study for

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603

Figure 3. ML phylogenetic tree using IRBP2 sequences. The numbers on the branches are bootstrap values.

IRBP2 (2.9  0.6%). Molecular systematics of Asian and Far
Eastern Misgurnus are poorly understood (Perdices et al., 2012),
but present RAG1 and IRBP2 genetic distances between clades of
the Japanese M. anguillicaudatus suggest the presence ofgenetically diverse groups or cryptic species in loaches.
Wild-type loaches were homozygous or heterozygous for
RAG1 and IRBP2 gene alleles within the same clade. In contrast,
all the clonal diploid individuals that were identiﬁed by mtDNACR haplotype, microsatellite genotype analyses and RAPD
ﬁngerpinting showed heterozygous genotypes between clades
A and B alleles in both RAG1 and IRBP2 genes. These results
show that natural clonal diploid loaches may have occurred froman earlier hybridization event between the loaches from different
genetic groups (Fig. 4). Hybridization presumably caused a
disruption of normal meiosis and subsequent irregular gametogenesis because of the failure of pairing between chromosomes,
with each set originating from a different species or genetic

group. However, in the inter-populational (or inter-clade)Misgurnus hybrids, females might have generated genetically
identical unreduced eggs to recover fertility, and very few such
eggs might have acquired the developing mode of gynogenesis
through mutation for clonal reproduction. The unreduced diploidegg formation was reported in inter-populational M. anguillicaudatus hybrids between group A and B-1, as inferred from
mtDNA-CR sequences (Arias-Rodriguez et al., 2009). Recently in
the genus Cobitis spinous loaches, clonality and polyploidy wereartiﬁcially synthesized by hybridization: F1 hybrids between two
bisexual species produced unreduced eggs, a few of which
initiated gynogenesis in nature (Choleva et al., 2012). The
formation of unreduced diploid eggs has also been reported ininter-speciﬁc hybridizations in Salmo (Johnson and Wright, '86),
Oryzias (Shimizu et al., 2000), Lepomis (Dawley et al., '85) and in
the inter-generic hybridization between Carassius and Cyprinus(Liu et al., 2001). As a cytological mechanism responsible for
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YAMADA ET AL.

Figure 5. A scenario of introgression of clone-speciﬁc mtDNA
from clonal diploid loaches to sexual diploid loaches throughmeiotic hybridogenesis of triploid progeny with one group A
genome and two group B genomes, accidentally formed byincorporation of a group B sperm nucleus into a diploid unreduced
egg from a clonal diploid loach. III in ﬁgure means clone-speciﬁc
mitochondrial DNA control region haplotype transmitted from A
group female.

Figure 4. A scenario for the formation of natural clonal diploids
through the hybridization of a group A female and a group B malein Misgurnus loaches. III in ﬁgure means clone-speciﬁc
mitochondrial DNA control region haplotype transmitted from A
group female.

unreduced oogenesis, premeiotic endomitosis has been proposed
because natural clonal diploid loaches were shown to form
unreduced diploid eggs through this mechanism (Itono et al.,2006), probably occurring at an early germ cell stage (Yoshikawa
et al., 2009; Morishima et al., 2012).
In specimens from site number 11 (Hannoura, Notojima,
Nanao City, Ishikawa Prefecture, site no. 33 in Morishima et al.,2008a), wild-type diploid loaches with clone-speciﬁc mtDNACR haplotypes occurred sympatrically with clonal diploid
individuals, although these harbored RAG1 and IRBP2 genesequences exclusively from clade B. The mitochondrial genomeof clade B loaches may have thus been replaced by that fromthe natural clonal lineage. One possible explanation to thisoccurrence is an introgression of clone-speciﬁc mitochondrial
genomes from clones to sympatric wild-type diploid loaches.
However, introgression of mitochondrial genome by backcrossing of paternal species (group) to hybrids is not realistic,
because most hybrids from intergeneric crosses are often sterileor sometimes nonviable (reviewed in Chevassus, '83). Intergeneric hybrids between M. anguillicaudatus and Cobitis
species have been reported to produce viable progeny(Minamori, '53; Kusunoki et al., '94), although sterility inmost hybrids has also been reported by Minamori ('57). It is also

been shown that inter-populational Misgurnus hybrids derived
from genetically different groups are generally sterile orundergo unusual reproduction. Experimental Misgurnus hybrids from groups A and B-1 produce a small number of haploideggs, a large number of unreduced diploid eggs, and othergenetically abnormal eggs, including aneuploids and mosaicisms (Arias-Rodriguez et al., 2009). The inter-populational
male hybrids generated a very small number of fertile haploidor/and diploid spermatozoa (Arias-Rodriguez et al., 2010).
Similar results were also obtained in hybrids within the genus
Cobitis (Choleva et al., 2012). Production of functional gametes
by hybrids is a prerequisite for the introgression theory by
subsequent backcrossing of fertile hybrids with its original
species or population.
Wild-type diploid genotypes with clone-speciﬁc mtDNA-CR
are likely to occur through meiotic hybridogenesis (Morishima
et al., 2008b). Unreduced diploid eggs with a heterozygous
genotype (AB) are formed by a clone from a crossing between aclade A female with clone-speciﬁc mtDNA and a clade B male(Fig. 4) and these diploid eggs infrequently develop into triploidﬁsh with genotype ABB: a haploid sperm from a wild-type male

J. Exp. Zool.

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HYBRID ORIGIN OF CLONAL DIPLOID LOACH

605

from clade B was thus accidentally incorporated into an
unreduced diploid egg (genotype AB) of the clonal diploid(Fig. 5). These clone-derived triploid loaches (genotype ABB)
are known to produce haploid eggs by meiotic hybridogenesis(Morishima et al., 2008b), that is, elimination of an unmatchedchromosome set from clade A and pairing between two
chromosome sets from clade B, followed by quasinormal
meiosis (Fig. 5). Thus, the occurrence of wild-type diploidloaches with chromosome sets of clade B and a mtDNA-CR ofgroup A in site number 11 is well explained by this meiotic
hybridogenetic mechanism. Consequently, the formation of
triploids from clonal lineages and the subsequent reproduction
through meiotic hybridogenesis presumably produce bisexually
reproducing diploid loaches with clone-speciﬁc mtDNA. Therefore, introgression of clonal mtDNA through meiotic hybridogenesis of clone-origin triploids produces bisexually
reproducing loaches with clone-derived group A mitochondrial
genomes.
The present study concluded that the RAG1 and IRBP2
genotypes in natural clonal loaches, that generate unreduced
diploid eggs with identical genotypes and develop throughgynogenesis, arose through the hybridization of two geneticallydifferent groups of M. anguillicaudatus.

ACKNOWLEDGMENTS
We thank the staff of the Ishikawa Prefecture Fisheries ResearchCenter for sampling loaches in the Ishikawa Prefecture.

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Research in Veterinary Science 102 (2015) 49–52

Contents lists available at ScienceDirect

Research in Veterinary Sciencejournal homepage: www.elsevier.com/locate/yrvsc

Short communication: Distribution of Porphyromonas gulae ﬁmAgenotypes in oral specimens from dogs with mitral regurgitation
Mitsuyuki Shirai a,1, Ryota Nomura b,⁎,1, Yukio Kato c, Masaru Murakami d, Chihiro Kondo a, Soraaki Takahashi a,
Yoshie Yamasaki e, Michiyo Matsumoto-Nakano e, Nobuaki Arai f, Hidemi Yasuda f,
Kazuhiko Nakano b, Fumitoshi Asai aaDepartment of Pharmacology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252–5201, Japan
Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, Suita, Osaka 565–0871, Japanc
Department of Veterinary Public Health II, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252–5201, Japand
Department of Molecular Biology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252–5201, Japane
Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700–8556, Japanf
Yasuda Veterinary Clinic, Meguro, Tokyo 152–0034, Japanba r t i c l e

i n f oArticle history:
Received 18 November 2014
Received in revised form 8 July 2015
Accepted 13 July 2015
Keywords:
ﬁmbriaemitral regurgitation
periodontal disease
Porphyromonas gulaea b s t r a c t
Porphyromonas gulae, a suspected pathogen for periodontal disease in dogs, possesses approximately 41-kDa
ﬁmbriae (FimA) that are encoded by the ﬁmA gene. In the present study, the association of ﬁmA genotypes
with mitral regurgitation (MR) was investigated. Twenty-ﬁve dogs diagnosed with MR (age range 6–13 years
old, average 10.8 years) and 32 healthy dogs (8–15 years old, average 10.8 years) were selected at the participating clinics in a consecutive manner during the same time period. Oral swab specimens were collected from thedogs and bacterial DNA was extracted, then polymerase chain reaction analysis was performed using primers
speciﬁc for each ﬁmA genotype, with the dominant genotype determined. The rate for genotype C dominant
specimens was 48.0% in the MR group, which was signiﬁcantly higher than that in the control group (18.8%)(P b0.05). These results suggest that P. gulae ﬁmA genotype C is associated with MR.
© 2015 Elsevier Ltd. All rights reserved.

Periodontal disease, including gingivitis and periodontitis, iscommonly found in dogs, with prevalence rates ranging from 5070% (Harvey and Emily, 1993). Gingivitis involves inﬂammation of thegingiva, whereas periodontitis is a more progressive form of periodontal
disease that involves the loss of tooth supporting tissues (i.e., periodontal
ligament, cementum, alveolar bone). Proper periodontal therapy can reduce gingival bleeding, tooth mobility, and, in some cases, sophisticatedmethods can be utilized to regenerate lost tissues. The distribution ofbacterial species related to periodontal disease has been investigatedusing oral swab specimens taken from dogs, with Porphyromonas gulae,
a Gram-negative black-pigmented anaerobe, one of the species frequently detected (Kato et al., 2011). The cell surface component Fimbrillin(FimA), a 41-kDa ﬁmbriae subunit, of P. gulae has also been characterized
and shown to be a possible factor related to periodontal disease in dogs
⁎ Corresponding author at: Department of Pediatric Dentistry, Osaka University
Graduate School of Dentistry, 1–8 Yamada-oka, Suita, Osaka 565–0871, Japan.
E-mail addresses: shirai@azabu-u.ac.jp (M. Shirai), rnomura@dent.osaka-u.ac.jp(R. Nomura), katohy@azabu-u.ac.jp (Y. Kato), murakami@azabu-u.ac.jp (M. Murakami),
v09058@azabu-u.ac.jp (C. Kondo), v09083@azabu-u.ac.jp (S. Takahashi), yoca777@live.jp(Y. Yamasaki), mnakano@cc.okayama-u.ac.jp (M. Matsumoto-Nakano), narai@slj.co.jp(N. Arai), hidemi@yasuda-vet.org (H. Yasuda), nakano@dent.osaka-u.ac.jp (K. Nakano),
asai@azabu-u.ac.jp (F. Asai).
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.rvsc.2015.07.009
0034-5288/© 2015 Elsevier Ltd. All rights reserved.

348(Hamada et al., 2008). In our recent study, FimA of P. gulae was classiﬁed
into 3 major genotypes; A, B, and C, with type C shown to have a high virulence for periodontal disease, followed by B and A (Nomura et al., 2012;
Yamasaki et al., 2012).
The association of periodontal disease with systemic diseases has
recently been gaining attention (Pavlica et al., 2008; Rawlinson et al.,2011). Mitral regurgitation (MR) caused by myxomatous mitral valve
disease is one of the most common types of cardiovascular diseases occurring in dogs (Serfass et al., 2006; Atkins et al., 2009) and characterized by chronic progression, with the condition worsening from mild
to severe over time (Borgarelli et al., 2008). A few reports have shown
an association of periodontal disease with MR in dogs (Glickman et al.,2009). In the present study, oral specimens from dogs diagnosed with
MR were analyzed to investigate the association of the disease withthe presence of P. gulae and genotypes of ﬁmA encoding FimA.
Twenty-ﬁve dogs diagnosed with MR and treated at veterinary
clinics in Tokyo in 2012 and 2013 were consecutively enrolled (MRgroup). In addition, 36 dogs older than 6 years old and without periodontitis, MR, diabetes mellitus, or other relevant clinical history,
were selected as Control group. Among them, we removed the 4 larger
breed dogs that weighed more than 20 kilograms and used the remaining 32 as controls. The study protocol was approved by the Animal
Research Committee of Azabu University (No. 130307–3). Prior to50

M. Shirai et al. / Research in Veterinary Science 102 (2015) 49–52

sample collection, the owners were informed of the purpose of thestudy and gave approval for their participation. None of the dogs ortheir owners received antibiotic treatment within the previous 3
months prior to collection. Oral specimens were obtained from supraand sub-gingival areas of the left and/or right upper fourth premolar
and/or lower ﬁrst molar using Seed-SwabR g-1 or − 2 swabs (Eiken
Chemical, Tokyo, Japan), then bacterial DNA was extracted using amethod previously described (Kato et al., 2011).
According to the ACVIM (American College of Veterinary Internal
Medicine) functional classiﬁcation (Atkins et al., 2009), the 25 dogs in
the MR group (18 males, 7 females; 10.8 ± 0.3 year old) were diagnosed
based on physical examination, thoracic radiography, electrocardiography, and echocardiography ﬁndings, as follows; Stage A (n = 0), StageB1 (n = 4), Stage B2 (n = 10), Stage C (n = 7), and Stage D (n = 4).
In addition, the severity of MR has been reported to be associated withmurmur intensity and audibility (Ljungvall et al., 2009). Thus, thesedogs were also evaluated using the Levine classiﬁcation, which is a scoring system used to classify the intensity or loudness of a heart murmur(Freeman and Levine, 1933), as follows; I/VI (n = 0), II/VI (n = 8),
II-III/VI (n = 2), III/VI (n = 7), IV/VI (n = 7), V/VI (n = 0), and VI/VI(n = 1). Breeds represented in the MR group are listed in Supplementary Table 1. The 32 healthy dogs in the control group (17 males, 15 females, 10.8 ± 0.4 years old) had no abnormal cardiac conditions
shown in physical examination, thoracic radiography, electrocardiography, and echocardiography ﬁndings. Breeds represented in the control
group are listed in Supplementary Table 2.
Statistical analyses were performed using the computational
software packages StatView 5.0e and Prism 4f. Fisher’s protected leastsigniﬁcant difference test was utilized to compare the detection
frequencies of each ﬁmA genotype for each gingival condition. Odds
ratio (OR) and 95% conﬁdence interval (CI95) values were calculated
to determine the signiﬁcance of the association of each ﬁmA genotype
in the dogs with each periodontal condition. A P value of b 0.05 wasconsidered to be statistically signiﬁcant.
We evaluated calculus coverage, and gingival and periodontal scoresbased on methods previously described, with some modiﬁcations(Warrick and Gorrel, 1997; Harvey and Emily, 1993; Löe, 1967; Wolfet al., 2005). For each dog, 22 teeth were scored according to a previously described technique (Kate and Cecilia, 1999). Dental calculus

accumulation was scored visually, as follows; (3) more than half of
the area of the portion of the tooth from the gingival margin to the tipof the crown, (2) less than half of that area, (1) slight, only at gingival
margin, and (0) none. Gingival scores were evaluated visually as follows; (3) prominent inﬂammation (intensive redness with gingival
swelling) identiﬁed throughout entire area, (2) prominent inﬂammation (intensive redness with gingival swelling) identiﬁed in limitedarea, (1) slight inﬂammation (slight redness without gingival swelling)
identiﬁed only in gingival margin, and (0) no signiﬁcant ﬁndings. Periodontal scores were evaluated based on visual examination and palpation, as follows; (3) tooth mobility with severe attachment loss,(2) attachment loss identiﬁed in more than one quarter of the portionof the tooth but no tooth mobility, (1) attachment loss identiﬁed inless than one quarter of that portion, and (0) no signiﬁcant ﬁndings.
Table 1 summarizes the oral conditions in the MR and Control groups.
There were no signiﬁcant differences in regard to dental calculus andgingival scores. As for periodontal scoring, the MR group had higherscores than the control group, though there were no signiﬁcant differences between the groups.
Detection of P. gulae and ﬁmA genotypes in addition to Tannerella forsythia and Campylobacter rectus were performed by PCR using speciesspeciﬁc sets of primers (Kato et al., 2011; Nomura et al., 2012;
Yamasaki et al., 2012). PCR ampliﬁcation was performed in a total volume of 20 μl with 1 μl of bacterial DNA using Ex Taq polymerase (Takara
Bio, Shiga, Japan) with the following cycling parameters: initial denaturation at 95 °C for 4 minutes and then 30 cycles consisting of 95 °C for 30
seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds, with a ﬁnal extension at 72 °C for 7 minutes. Speciﬁcity with this method was shownin our previous study, with the minimum detection level reported to be
10–100 cells (Nomura et al., 2012; Yamasaki et al., 2012). The dominant
genotypes were determined using samples with multiple ﬁmA genotypes detected. PCR was performed with titrated DNA samples (1/10,
1/100, 1/1000) from each specimen. The dominant genotype was determined as that with the lowest detection limit. When the detection limits
were similar for at least two genotypes, the results were described as no
dominancy. Fig. 1A shows the results of detection using the PCR systemwith genomic DNA extracted from titrated cultures of representative
strains of each type. The lowest numbers of tested strains for identiﬁcation were the same for all 3 ﬁmA genotypes. Fig. 1B, C, and D show

Table 1
Oral conditions and distribution frequency of P. gulae and its ﬁmA genotypes in oral specimens in MR and Control groups.
MR (n = 25)

Control (n = 32)

Statistical analysis

10.8 ± 0.3 (6–13)

10.8 ± 0.4 (8–15)

NS b

5.7 ± 0.8 (1.6-14.6)

5.7 ± 0.7 (1.6-18.8)

NS b

Oral condition
Dental calculus score (mean ± SE a)
Gingival score (mean ± SE a)
Periodontal score(mean ± SE a)

1.7 ± 0.1
0.8 ± 0.2
0.6 ± 0.2

1.8 ± 0.2
0.8 ± 0.1
0.3 ± 0.1

NS b
NS b
NS b

PCR detection
P. gulae-positive
A-dominant

24 (96.0%)
5 (20.0%)30 (93.8%)
17 (53.1%)

B-dominant
C-dominant

3 (12.0%)
12 (48.0%)

4 (12.5%)
6 (18.8%)

A=C
A=B=C
Untypeable

2 (8.0%)0 (0%)
2 (8.0%)1 (3.1%)1 (3.1%)1 (3.1%)NS b
P = 0.0143
OR: 0.22(CI95: 0.07-0.73)
NS b
P = 0.0240
OR c: 4.00
(CI95 d: 1.22-13.08)
NS b
NS b
NS b

Age in years
Mean ± SE a (range)
Body weight in kg
Mean ± SE a (range)aSE, standard error.
NS, not signiﬁcant.
cOR, odds ratio.
dCI95, 95% conﬁdence interval.
b349

M. Shirai et al. / Research in Veterinary Science 102 (2015) 49–52

A M ATCC 51700 (Type A)
105

B M

103

102

10

Primer for Type A
1

C M

104

10-1 10-2 10-3

Primer for Type A
1

10-1 10-2 10-3

D077 (Type B)
105

104

103

102

10

Primer for Type B
1

10-1 10-2 10-3

Primer for Type B
1

10-1 10-2 10-3

D049 (Type C)
105

104

103

102

M

10-1 10-2 10-3

Primer for Type C
1such as type II and IV, indicated that they are highly virulent for periodontitis (Nakano et al., 2008). In addition, it is interesting that analysisof the evolutionary relationship revealed that type C ﬁmA of P. gulae isclose to type IV ﬁmA of P. gingivalis (Yamasaki et al., 2012). Taken
together, these results indicate the possibility of an association of
periodontal disease with MR.
In summary, the present results indicate a possible association
between the presence of a speciﬁc genotype of ﬁmA of P. gulae with
MR. In addition, our novel method used to specify the dominant
genotype of P. gulae in the specimens obtained may enable identiﬁcation of subjects at risk and in need of preventive approaches.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.rvsc.2015.07.009.

10

Primer for Type C
1

M

51

Conﬂicts of interestM

10-1 10-2 10-3

The authors declare that they have no competing interests.
Acknowledgements

D

M

Primer for Type A
1

10-1

10-2

10-3

Primer for Type B
1

10-1

10-2

10-3

Primer for Type C
1

10-1

10-2

This study was supported by Grants-in-Aid for Scientiﬁc Research for
Challenging Exploratory Research (No. 23658256 and 25670873) fromthe Japan Society for Promotion of Science, the Promotion and Mutual
Aid Corporation of Private Schools of Japan, and a Grant-in-Aid forMatching Fund Subsidy for Private Universities. We also thank Prof.
Howard K. Kuramitsu (State University of New York at Buffalo) for
editing the manuscript.

M

10-3

References
Fig. 1. Methodology designed to identify the dominant genotype ﬁmA of P. gulae, using polymerase chain reaction (PCR). A. Comparisons of PCR products using diluted genomic
DNA extracted from P. gulae strains with each ﬁmA genotype. M; molecular size marker(100-bp DNA ladder). B, C, D. Representative results obtained with clinical specimens
using PCR system constructed for the present study.

representative results obtained using titrated bacterial DNA samples
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Biochemical and Biophysical Research Communications 466 (2015) 162e166

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Role of a TPA-responsive element in hepcidin transcription induced by
the bone morphogenetic protein pathway
Yohei Kanamori a, Masaru Murakami b, Tohru Matsui a, Masayuki Funaba a, *abDivision of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara 252-5201, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 21 August 2015
Accepted 28 August 2015
Available online 3 September 2015

Systemic iron balance is governed by the liver-derived peptide hormone hepcidin. The transcription of
hepcidin is primarily regulated by the bone morphogenetic protein (BMP) and inﬂammatory cytokine
pathways through the BMP-response element (BMP-RE) and STAT-binding site, respectively. In addition
to these elements, we previously identiﬁed a TPA-responsive element (TRE) in the hepcidin promoter
and showed that it mediated the transcriptional activation of hepcidin through activator protein (AP)-1
induced by serum. In the present study, we examined the role of TRE in the BMP-induced transcription of
hepcidin in HepG2 liver cells. The serum treatment increased the basal transcription of hepcidin;
however, responsiveness to the expression of ALK3(QD), a constitutively active BMP type I receptor, wasunaffected. Consistent with these results, mutations in TRE in the hepcidin promoter decreased basaltranscription, whereas responsiveness to the expression of ALK3(QD) remained unchanged. HepG2 cells
signiﬁcantly expressed AP-1 components in the basal state, whereas BMP did not up-regulate theexpression of these components. The expression of c-fos enhanced the basal transcription of hepcidin as
well as ALK3(QD)-mediated hepcidin transcription, whereas that of dominant-negative c-fos decreased
hepcidin transcription. The results of the present study suggested that the cis-elements of the hepcidin
promoter, BMP-RE and TRE, individually transmitted BMP-mediated and AP-1-mediated signals,
respectively, whereas transcription was synergistically increased by the stimulation of BMP-RE and TRE.
© 2015 Elsevier Inc. All rights reserved.

Keywords:
Hepcidin
TPA-Responsive element
BMP
AP-1

1. Introduction
Hepcidin is a peptide hormone that regulates systemic ironbalance. Under iron overload conditions, hepcidin is mainly produced in the liver, and inhibits intestinal iron absorption and ironrelease from macrophages, thereby decreasing iron concentrationsin the blood [1,2]. The expression of hepcidin was also previously
shown to be induced by inﬂammation caused by infection; this has
been suggested as a host defense mechanism to reduce the availability of iron for pathogens [3,4]. The ability of hepcidin to
decrease systemic iron levels is primarily regulated at its expression level; hepcidin mRNA levels are primarily controlled by transcription [1,2].

Abbreviations: AP, activator protein; BMP, bone morphogenetic protein; BMPRE, BMP-response element; IL, interleukin; STAT-BS, STAT-binding site; TRE, TPAresponsive element.
* Corresponding author. Division of Applied Biosciences, Graduate School of
Agriculture, Kyoto University, Kitashirakawa Oiwakecho, Kyoto 606-8502, Japan.
E-mail address: mfunaba@kais.kyoto-u.ac.jp (M. Funaba).

Accumulating evidence has indicated that the transcription of
hepcidin is mainly regulated by the BMP-Smad and inﬂammatory
cytokine-STAT pathways [1,2]. Under iron overload conditions,
BMP6 produced in the liver binds to its receptors, consisting of type
I receptor and type II receptors, which phosphorylate and activate
Smad1/5/8 and subsequently stimulate the transcription of hepcidin via promoter elements, i.e., BMP-response element (BMP-RE) 1
located at nt 155 to nt 150 (nt þ 1 is deﬁned as the translational
initiation site of hepcidin) and BMP-RE2 (nt 1678 to nt 1673)[5e8]. In contrast, inﬂammatory cytokines such as interleukin (IL)6 and oncostatin M have been shown to induce the phosphorylation and activation of STAT3, which result in the transcriptional
activation of hepcidin through binding to the STAT-binding site
(STAT-BS, nt 143 to nt 134) [9,10].
We recently revealed that serum exhibited potency to stimulate
the transcription of hepcidin [11]. A TPA-responsive element (TRE,nt 133 to nt 127), which has not yet been characterized, wasfound to play a role in the serum-induced transcription of hepcidin;
mutations in TRE resulted in decreases in hepcidin promoter activity induced by serum. The inhibition of basal BMP activity or

http://dx.doi.org/10.1016/j.bbrc.2015.08.123
0006-291X/© 2015 Elsevier Inc. All rights reserved.

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Y. Kanamori et al. / Biochemical and Biophysical Research Communications 466 (2015) 162e166

Aluciferase activity0

serum
stimulation

mutations in BMP-REs also inhibited the serum-induced transcription of hepcidin, suggesting a functional interaction between
BMP-REs and TRE. However, the involvement of TRE in hepcidintranscription mediated by the BMP pathway currently remains
obscure. The aim of the present study was to determine the role of
TRE in the transcriptional activation of hepcidin by the BMP
pathway in luciferase-based reporter assays using the hepcidin
promoter.

163

10

20

+

B

30

40

16.6
12.1
luciferase activity
BMPRE2

2. Materials and methods

BMPTRE
RE1

0

10

20

wild-type2.1. Plasmids, cell culture, and reporter assays

16.6

X

mTRE

The hepcidin reporter construct, i.e., the hepcidin promoter
spanning nt 2018 to nt 35 inserted into the luciferase reporter
vector pGL4 (hepcidin(-2018)-luc), and the mutated plasmids werepreviously described [11]. The expression plasmid for ALK3(QD)[12], a constitutively active BMP type I receptor, was kindly provided by Dr. K. Miyazono. HepG2 human hepatoma cells were
cultured in DMEM supplemented with 10% heat-inactivated fetal
bovine serum (FBS) and antibiotics. HepG2 cells (6  104 cells perwell) seeded onto 24-well plates were transfected with hepcidin(2018)-luc or the indicated reporter plasmid (0.5 mg), indicatedexpression plasmid, or empty plasmid (0.5 mg) and b-galactosidase
expression plasmid under the control of a cytomegalovirus-derived
promoter (pCMV-bGal, 0.1 mg) using polyethylenimine Max reagent(Polysciences, Warrington, PA, USA). Cells were harvested 30 h after
transfection. In the ligand stimulation, transfected cells were
cultured in DMEM with 0.2% FBS for 4 h, followed by a culture withor without 4 nM of BMP2 (R & D Systems, Minneapolis, MN, USA) or
2.5 nM of IL-6 (R & D Systems) for 24 h. In order to examine theeffects of inhibiting the BMP pathway, transfected cells weretreated with or without LDN-193189 (100 nM, Stemgent, San Diego,
CA, USA), an inhibitor of the BMP type I receptor [13] for 24 h.
Luciferase activity was normalized to b-galactosidase activity as
previously described [14].
2.2. RNA isolation, RT-PCR, and RT-quantitative PCR
Total RNA isolation, cDNA synthesis, PCR, and real-time quantitative PCR (qPCR) were performed as described previously [15].
The following primers were used: 50 -ACTACCACTCACCCGCAGAC-30
and50 -CCAGGTCCGTGCAGAAGT-30
forc-fos,
50 -AGGATAGTGCGATGTTTCAGG-30 and 50 -GACTTCTCAGTGGGCTGTCC-30 for
c-jun, 50 -ATACACAGCTACGGGATACGG-30 and 50 -GCTCGGTTTCAGGAGTTTGT-30 for junB, 50 -CAGTTCCTCTACCCCAAGGTG-30 and 50 TTCTGCTTGTGTAAATCCTCCA-30 for junD, 50 -CAGAGACAAAATATTTCCCATTTGT-30 and 50 -CAGAGACAAAATATTTCCCATTTGT-30
for Fra1, 50 -TTCTCCGGCTCCTTTTCTAA-30 and 50 -ATTTCAGACAGGGCCAAAAA-30 for Fra2, and 50 -AGCCACATCGCTCAGACAC-30
and 50 -GCCCAATACGACCAAATCC-30 for GAPDH. The ▵▵Ct method
was used to normalize the levels of target transcripts to GAPDHlevels [16].
3. Results and discussion
In order to evaluate the role of TRE in hepcidin transcription
induced by the BMP pathway, the effects of serum and theexpression of ALK3(QD) were examined in luciferase-based reporter assays using the hepcidin promoter (Fig. 1A). Consistent withprevious ﬁndings [11,15], the treatment with serum or expressionof ALK3(QD) increased luciferase expression, indicating the stimulation of hepcidin transcription. The ALK3(QD)-induced transcription of hepcidin was enhanced in the presence of serum,
whereas the fold-induction of luciferase activity by the expression

353

C

15.1

luciferase activity0

1

2

STAT-BS TRE
wild-type

mSTAT

4

5

4.3

X

X
mSTAT, TRE X
X
mTRE

3

1.6
3.4

1.3

Fig. 1. TRE was required for the full activation of hepcidin transcription induced by the
BMP pathway and IL-6 pathway. (A) HepG2 cells were transfected with hepcidin(2018)-luc and pCMV-bGal with or without ALK3(QD). Cells were stimulated with orwithout 10% FBS for 24 h (B and C) HepG2 cells were transfected with the indicatedreporter and pCMV-bGal. Cells were also co-transfected with or without ALK3(QD) (B)or treated with or without IL-6 (2.5 nM) for 24 h (C). Luciferase activity was normalized
to b-galactosidase activity, and luciferase activity in cells in the absence of ALK3(QD)
and FBS (A) or in cells transfected with the wild-type reporter, but not ALK3(QD) (B) or
IL-6 (C) was set to 1. Results are shown as the mean ± SE (n ¼ 3). The number at theside of the bar indicates fold-induction.

of ALK3(QD) was similar between cells treated with (12.1-fold) andwithout serum (16.6-fold). Mutations in TRE decreased the basaltranscription of hepcidin, whereas responsiveness to ALK3(QD)
expression was unchanged, i.e., the fold-induction in response to
the stimulation of the BMP pathway was 16.6 for the wild-type
reporter and 15.1 for the reporter with TRE mutations (Fig. 1B).
These results indicated the synergistic effects of TRE on the
BMP-mediated transcription of hepcidin; although TRE wasrequired for the basal transcription of hepcidin, TRE-mediatedtranscription did not modulate the efﬁciency of transcriptional
activation by the BMP pathway.
We subsequently investigated the role of TRE in IL-6-mediatedhepcidin transcription (Fig. 1C). A previous study revealed that IL6 induced STAT3 phosphorylation and hepcidin transcription via
STAT-BS [9], whereas another study showing that mutations in
STAT-BS did not change responsiveness to IL-6 did not support
these ﬁndings [17]. Since STAT-BS was located adjacent to TRE, we
examined the role of TRE as well as STAT-BS in IL-6-induced hepcidin transcription (Fig. 1C). Mutations in STAT-BS blocked thetranscriptional activation of hepcidin by IL-6, whereas TRE mutations did not affect fold-induction in response to the IL-6 treatment
on hepcidin transcription. These results suggested that, similar to
the regulation of BMP-induced hepcidin transcription, TRE did notaffect the efﬁciency of IL-6-induced hepcidin transcription, but was
required for the maximal transcription of hepcidin.
Activator protein (AP)-1 has been shown to bind to TRE andstimulate the transcription of its target genes [18,19]. We examinedthe expression of AP-1 components in cells treated with or without
BMP2 (Fig. 2). All AP-1 components examined were signiﬁcantly

Y. Kanamori et al. / Biochemical and Biophysical Research Communications 466 (2015) 162e166

c-fos

+

relative mRNA level

B

-

c-jun

-

+

junB

+

-

junD

+

3

-BMP2
+BMP2

1
0
0

1

Fra1

-

+

C

c-fos

2

-

2

-BMP2
+BMP2
1

0

F
-BMP2
+BMP2

0
1

time (h)

-BMP2
+BMP2

1

2

0

time (h)

1

0

2

0
1

2

Fra1

-BMP2
+BMP2

2

1

0
0

1

2

time (h)Grelative mRNA level

2

junB

D

2

0

relative mRNA level

relative mRNA level

junD

-

c-jun

time (h)

E

Fra2

+

relative mRNA levelA

relative mRNA level

164

1

2

time (h)

Fra2
2

-BMP2
+BMP2

1

0
0

1

2

time (h)Fig. 2. BMP did not induce the expression of AP-1 components. (A) The basal expression of AP-1 components in HepG2 cells. Total RNA was isolated from basal HepG2 cells, and theexpression of the indicated gene was evaluated by RT-PCR. cDNA was synthesized by a treatment with reverse transcriptase, and was used as the template of PCR (RT(þ)). As thenegative control, an equal amount of RNA was treated without reverse transcriptase, followed by PCR (RT()). (BeG) Time-course changes in the expression of AP-1 components in
response to the BMP2 treatment. The expression of c-fos (B), c-jun (C), junB (D), junD (E), Fra1 (F), and Fra2 (G) was quantiﬁed and expression levels in cells before the treatment
with BMP2 were set to 1. Results are shown as the mean ± SE (n ¼ 4).

expressed under the basal condition (Fig. 2A), and BMP2 did notaffect the expression of these components (Fig. 2BeG). Provided
that TRE plays a role in the basal transcription of hepcidin, as shownin Fig. 1B and C, we suspected that at least one of the AP-1 components expressed in unstimulated cells was important for thebasal transcription of hepcidin. Thus, the effects of the forced
expression of c-fos, a representative AP-1 component, were
examined (Fig. 3A). The expression of c-fos increased the basaltranscription of hepcidin, which was blocked by the TRE mutations.
ALK3(QD)-induced hepcidin transcription was further increased by
the expression of c-fos, for which TRE in the promoter was
required; enhancements in ALK3(QD)-induced hepcidin transcription by the expression of c-fos were not detected with mutations in
TRE. The fold-induction of the transcription of the reporter with
TRE mutations in response to ALK3(QD) expression was unaffectedby c-fos expression, whereas that of the wild-type reporter was
decreased by the overexpression of c-fos; the precise reason for thedecrease observed in the fold-induction in the presence of overexpressed c-fos currently remains unknown, but may reﬂect thesaturation of transcriptional activation induced by ALK3(QD) and cfos. The involvement of c-fos in the basal transcription of hepcidin
was also examined by the expression of dominant-negative c-fos;
although dominant-negative c-fos inhibited the basal transcriptionof hepcidin, responsiveness to BMP2 remained unchanged (Fig. 3B).
In contrast to the effects of c-fos, the forced expression of c-jun
did not affect the basal transcription of hepcidin, but decreased
responsiveness to the stimulation of the BMP pathway, irrespectiveof TRE mutations (Fig. 3C). These results suggested that c-jun
negatively regulated the BMP-mediated transcription of hepcidin
through regions other than TRE in the hepcidin promoter. AP-1
components may regulate the BMP-mediated transcription of
hepcidin differently.
The role of endogenous BMP activity in the TRE-mediated basaltranscription of hepcidin was further evaluated using LDN-193189,
an inhibitor of the BMP pathway [13] (Fig. 4A). In this case, we used

reporter plasmids with the hepcidin promoter spanning nt 270 tont 35, which included both BMP-RE1 and TRE; BMP-RE1 rather
than BMP-RE2, which was previously shown to be involved inserum-induced hepcidin transcription [11]. The treatment with
LDN-193189 decreased the expression of the wild-type reporter
gene to one-ﬁfth; transcriptional activity in cells treated with LDN193189 was similar to that of the reporter gene with BMP-RE1
mutations in cells treated without LDN-193189. In addition, thetranscriptional activity of the BMP-RE1-mutated reporter wasinsensitive to LDN-193189. Consistent with the results shown in
Fig. 1B and C, the expression of the reporter with TRE mutations
was lower than that of the wild-type reporter, and the treatment
with LDN-193189 further decreased the transcription of the TREmutated reporter. The extent of this decrease was less than thatinduced by LDN-193189 on the transcription of the wild-type reporter; LDN-193189 decreased transcription by as much as onehalf. Luciferase expression of the reporter with mutations in
BMP-RE1 and TRE was similar to that of the TRE-mutated reporterin cells treated with LDN-193189, and the expression of the double
mutated reporter was LDN-193189-insensitive. These results suggested that not only AP-1 expression in the steady state, but also
endogenous BMP activity was required for the basal transcription of
hepcidin. Our previous ﬁndings also suggested the necessity ofendogenous BMP activity for the basal transcription of hepcidin[11]. The differential fold-inhibition in response to LDN-193189
between the wild-type reporter (0.21) and TRE-mutated reporter(0.47) may be explained by the regulation of molecules related to
BMP signaling by AP-1 (Fig. 4B). For example, Smurf1 has been
identiﬁed as a negative regulator of the BMP pathway through thedegradation of BMP receptors [20], and the transcriptional activation of Smurf1 is known to be stimulated by AP-1 [21]. Liberated
AP-1 resulting from TRE mutations may stimulate Smurf1 transcription, thereby decreasing endogenous BMP activity. As a result,the expression of the TRE-mutated reporter gene may be decreased
more than luciferase expression resulting from the loss of TRE-

354

Y. Kanamori et al. / Biochemical and Biophysical Research Communications 466 (2015) 162e166

AB
luciferase activity
BMPRE2

BMPTRE
RE1

wild-type

X

mTRE

c-fos

0

+
+

20

40

60

dn c-fos

6.6

C

5

10

8.8
9.5
-BMP2
+BMP2

vector
ALK3(QD)

27.5

luciferase activity
0vector

20.2

24.0

165

luciferase activity
0

BMPRE2

BMPTRE
RE1

wild-type

mTRE

X

5

10

15

c-jun

+
+

13.9
4.9

13.8

vector
ALK3(QD)

4.7

Fig. 3. AP-1 components regulated BMP-induced hepcidin transcription differently. (A and C) HepG2 cells were transfected with the indicated reporter and pCMV-bGal with orwithout c-fos (A) or c-jun (C). (B) HepG2 cells were transfected with hepcidin(-2018)-luc and pCMV-bGal with or without dominant-negative c-fos. Cells were stimulated with orwithout BMP2 (4 nM) for 24 h. Luciferase activity was normalized to b-galactosidase activity, and luciferase activity in cells transfected with the wild-type reporter, but not the AP-1
component (A and C) or in cells transfected without dominant-negative c-fos and BMP2 (B) was set to 1. Results are shown as the mean ± SE (n ¼ 3). The number at the side of thebar indicates fold-induction.

mediated transcription itself, ultimately reducing responsiveness to
LDN-193189. In any case, the results of the present study suggestthat the cis-elements of the hepcidin promoter, BMP-RE and TRE,
individually transmitted BMP-mediated and AP-1-mediated signals, respectively; however, transcription was synergisticallyincreased by the stimulation of BMP-RE and TRE, indicating that
BMP-RE and TRE are both required for the maximal transcription of
hepcidin.
Two relationships are likely to exist between TRE- and BMP-REmediated hepcidin transcription; they individually or interactively
regulate its transcription. When the transcription of hepcidin is
individually regulated through TRE or BMP-RE, TRE- and BMP-REmediated transcription may be additively or synergisticallyincreased. The ﬁrst case is that TRE-mediated transcription is independent of and additive to BMP-induced transcription; theextent of decreases in BMP-induced hepcidin transcription by TRE
mutations corresponds to those in basal transcription. The secondcase is that TRE-mediated transcription is independent of BMPinduced transcription, similar to the ﬁrst case, whereastranscription is synergistically increased. In this case, responsiveness to the stimulation of the BMP pathway, which is evaluated by
the fold-induction of hepcidin transcription, is not changed by TRE
mutations, whereas basal transcription is decreased. Furthermore,when hepcidin transcription is interactively regulated through TRE
and BMP-RE, BMP-induced hepcidin transcription may be blocked
by TRE mutations; not only basal transcription but also responsiveness to the stimulation of the BMP pathway is reduced (thethird case). The present study revealed that TRE is required for basaltranscription of hepcidin, but does not affect efﬁciency of BMPmediated transcription. We conclude that hepcidin transcription
via TRE and BMP-RE is regulated by the mode indicated by thesecond case.
Various relationships are known to exist between AP-1 and the
BMP pathway. BMP2 was previously found to block AP-1dependent gene transcription in monocytes, suggesting the negative regulation of AP-1 activity by the BMP pathway [22]. A previous
study demonstrated that BMP6 and AP-1 had reciprocal effects on
miRNA-21 expression; AP-1 stimulated the transcription of miRNA-

Fig. 4. Functional interaction between the BMP-responsive element and TRE in BMP-mediated hepcidin transcription. (A) HepG2 cells were transfected with the indicated reporterand pCMV-bGal. Cells were treated with or without LDN-193189 (100 nM) for 24 h. Luciferase activity was normalized to b-galactosidase activity, and luciferase activity in cells
transfected with the wild-type reporter and treated with vehicle was set to 1. Results are shown as the mean ± SE (n ¼ 3). The number at the side of the bar indicates fold-induction.
(B) A proposed model for the role of TRE in hepcidin transcription mediated by endogenous BMP activity.

355

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Y. Kanamori et al. / Biochemical and Biophysical Research Communications 466 (2015) 162e166

21, whereas BMP6 inhibited it by blocking the binding of AP-1 tothe promoter of the miRNA-21 gene [23]. In contrast, a cooperative
relationship has been reported between AP-1 and the BMP
pathway; BMP4 promoted hematopoiesis in concert with AP-1,
which was partly achieved through the up-regulation of junD
expression [24]. Furthermore, BMP2 has been shown to stimulate
GlcAT-1 gene transcription, which is mediated by AP-1 binding to
the AP-1 binding site of the GlcAT-1 gene promoter; mutations in
the AP-1 binding site completely blocked the BMP2-induced transcription of GlcAT-1 [25]. Similar to previous ﬁndings reported by
Lee et al. [24] and Hiyama et al. [25], the results of the present study
suggest a cooperative relationship between AP-1 and the BMP
pathway for transcriptional activation. However, BMP2 did not
induce AP-1 gene expression in the present study, and the basal
expression of c-fos was important for the transcription of hepcidin.
Furthermore, AP-1 did not affect the efﬁciency of transcriptional
activation by the BMP pathway. The interactive mode between AP1 and the BMP pathway depends on the regulated gene, whichleads to the diverse effects of BMP and AP-1 in various physiologicalprocesses.
Conﬂict of interest
All authors have no conﬂict of interest.
Acknowledgments
We thank Dr. K. Miyazono for providing the plasmid for
ALK3(QD). This work was partly supported by JSPS KAKENHI(15J02394 to Y.K.), research project grants from the Azabu University (2014-K-6 to M.M.) and from Tokyo Institute of Technology (toY.K.).
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Comparative Biochemistry and Physiology, Part A 185 (2015) 142–149

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part A
journal homepage: www.elsevier.com/locate/cbpa

Adaptive expression of uncoupling protein 1 in the carp liver and kidney
in response to changes in ambient temperature
Masaru Murakami a,⁎, Masahiro Ohi a, Shoko Ishikawa a, Mitsuyuki Shirai b, Hiroki Horiguchi a,
Yoshii Nishino c, Masayuki Funaba d,⁎aLaboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara 252-5201, Japan
Laboratory of Veterinary Pharmacology, Azabu University School of Veterinary Medicine, Sagamihara 252-5201, Japanc
Department of Animal Medical Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japand
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japanba r t i c l e

i n f oArticle history:
Received 17 May 2014
Received in revised form 16 October 2014
Accepted 2 April 2015
Available online 10 April 2015
Keywords:
UCP1
PPAR
PGC-1
RXR
Ambient temperatureCarps

a b s t r a c t
The expression of uncoupling protein (UCP1) is up-regulated in mammalian brown adipocytes during cold exposure. However, a previous study revealed that UCP1 was highly expressed in the liver of common carps, and thatthe hepatic expression of UCP1 was down-regulated during cold exposure. The present study examined the effects of temperature on the recovery of UCP1 expression levels and the expression of genes involved in UCP1
transcription in the livers and kidneys of common carps. The hepatic and renal expressions of UCP1 weredecreased by acclimation from 22 °C to 8 °C, and a subsequent increase in the water temperature from 8 °C to
28 °C recovered the renal, but not hepatic expression of UCP1. Changes in the expression of peroxisome
proliferator-activator receptor (PPAR) γ, retinoid X receptor (RXR) α and PPARγ co-activator (PGC)-1α, genesthat are involved in the expression of UCP1 in mammals, with ambient temperature indicated that the expressions of PPARγ and RXRα, but not expression of PGC-1α was decreased in response to cold exposure; the hepaticand renal expressions of PPARγ and RXRα recovered to basal levels with the cessation of cold exposure, although
this was not complete for hepatic expression of PPARγ. The results of the present study indicate that a uniqueregulatory mechanism is responsible for the hepatic and renal expressions of carp UCP1 during cold exposureand subsequent reacclimation, and is distinct from that in murine brown adipocytes.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction
Uncoupling protein (UCP) 1 increases proton leakage from the mitochondrial inner membrane, resulting in a decreased proton motive
force, which leads to increased oxygen consumption and heat generation without the concomitant generation of ATP. The restricted expression of UCP1 in mammalian brown adipocytes located in brown andwhite fat renders these cells responsible for non-shivering heat production (Cannon and Nedergaard, 2004).
The expression of UCP1 and its orthologs has been detected not onlyin placental mammals, but also in ectothermic vertebrates (Gesta et al.,2007). UCP1 was identiﬁed in the common carp (Cyprinus carpio), an
ectothermic vertebrate, based on the conserved synteny within the
mammalian lineage (Jastroch et al., 2005). It was found to be highly
expressed in the liver and to a lesser extent in the kidney. The⁎ Corresponding authors.
E-mail addresses: murakami@azabu-u.ac.jp (M. Murakami),
mfunaba@kais.kyoto-u.ac.jp (M. Funaba).

expression of UCP1 could not be detected in carp adipose tissues,
which suggested that the role of UCP1 in energy expenditure in ﬁsh differed from that in placental mammals (Jastroch et al., 2005).
The expression of UCP1 in murine brown fat was previously shown
to be up-regulated in response to cold exposure (Puigserver et al.,
1998; Barbatelli et al., 2010), whereas hepatic UCP1 transcript levels
were lower in the common carp acclimated at 8 °C for 4 weeks than inthose kept at 20 °C (Jastroch et al., 2005). Similar responses wereobserved in gilthead sea bream (Sparus aurata); expression levels of
hepatic UCP1 were lower in winter than in summer and fall (BermejoNogales et al., 2010). The mechanism underlying the regulation of ﬁsh
UCP1 expression currently remains unknown. In addition, changes in
UCP1 expression upon recovery from cold exposure have not experimentally been examined. Furthermore, studies are needed to elucidate
the relationship between the expression of UCP1 and non-shivering
heat production in ectothermic vertebrates.
In murine brown fat, the transcription of UCP1 is regulated by several key molecules including the tissue-restricted transcription factors
peroxisome proliferator-activator receptor (PPAR) α, β/δ and γ, thyroid
hormone receptor (TR) α, and the transcriptional co-activator PPARγ
co-activator (PGC)-1α (Puigserver et al., 1998; Barbera et al., 2001;

http://dx.doi.org/10.1016/j.cbpa.2015.04.003
1095-6433/© 2015 Elsevier Inc. All rights reserved.

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Cannon and Nedergaard, 2004; Cao et al., 2004; Komatsu et al., 2010;
Seebacher and Glanville, 2010). In addition, PGC-1β, a molecule
structurally related to PGC-1α, is known to be highly expressed inmurine brown fat (Lin et al., 2002; Seale et al., 2007). PPAR and
TR heterodimerize with retinoid X receptor (RXR), and the formed
complex regulates transcription of target genes including UCP1 and
PGC-1 (Cannon and Nedergaard, 2004; Evans and Mangelsdorf, 2014).
Furthermore, a transcription factor, cAMP responsive element binding
protein (CREB) is phosphorylated and activated by protein kinase
A, which leads to stimulation of UCP1 transcription (Cannon andNedergaard, 2004). The expression levels of nuclear respiratory factor
1 (NRF1), which stimulates mitochondrial biogenesis by interactingwith PGC-1α (Scarpulla, 2008), have been closely related to those of
UCP1, PPARδ, and PGC-1α during cold exposure in murine brown fat(Seebacher and Glanville, 2010).
We hypothesized that regulation of carp UCP1 expression is similar
to that established in murine UCP1 expression, although response tocold exposure is different between common carps and mice. As theﬁrst step to clarify regulation of ﬁsh UCP1 expression, we isolated not
only 5′ ﬂanking region of carp UCP1 gene but also coding region ofcarp mRNA for several molecules involved in regulation of murine
UCP1 expression partially, because the information has not yet been
available yet. By use of the information, we examined the effects ofambient temperature on expression levels of UCP1 and the candidates
to regulate UCP1 expression in common carps.
2. Materials and methods
2.1. Animals
A total of 30 common carps aged 7 months were used. These carps
were fed with commercial pellets ad libitum with a 12-h light/12-hdark cycle. They were kept in a temperature-controlled recirculatingwater system maintained at 22 °C for at least 2 weeks (day 0). The
water temperature was gradually lowered to 8 °C over 3 days. After
14 days at 8 °C (days 3–17), the water temperature was gradually
returned to 28 °C over 3–5 days and kept at 28 °C for 14 days (days
21–35). Carps were sacriﬁced on days 0, 17, and 35, and the livers andkidneys were removed.
2.2. RNA isolation and RT-quantitative real-time PCR
RNA isolation and reverse transcription (RT)-quantitative real-time
PCR (qPCR) analyses were performed as described previously(Murakami et al., 2008; Asai et al., 2014). Total RNA was isolated fromthe livers and kidneys of carps using QuickGene RNA tissue kit S(Wako, Osaka, Japan) in QuickGene-810 (Wako, Osaka, Japan), an automatic nucleic acid extraction system, according to the manufacturer's
protocol. The concentration of RNA was determined from absorbanceat 260 nm. In RT-qPCR analyses, cDNA was synthesized using the highcapacity cDNA reverse transcription kit with an RNase inhibitor (Life143

Technologies, Carlsbad, CA, USA), according to the manufacturer's
protocol. The cDNA corresponding to 5 ng of total RNA was used as atemplate of qPCR; the qPCR was performed using KAPA SYBR FAST Universal qPCR Master Mix (Kapa Biosystems, Boston, MA, USA) in Thermal
Cycler Dice Real Time System TP800 (Takara, Otsu, Japan), according to
the manufacturer's protocol. The qPCR proﬁle was as follows: after denaturing for 30 s at 95 °C, 40 cycles consisted of 5 s at 95 °C and 30 s
at 60 °C. The oligonucleotide primers for qPCR are shown in Table 1.
After 40 cycles of RT-qPCR, the dissociation (melting) curve of the products was examined by changes in the ramp temperature from 60 °C to
95 °C. Each sample showed a single peak, suggesting the expected PCR
products. The mRNA levels were expressed relative to EF1α mRNAlevels, and the expression level in the liver of control carps was set at100.

2.3. Isolation of 5′ ﬂanking region of UCP1 gene from carps
The 5′ ﬂanking region of carp UCP1 gene was isolated by two roundsof PCR; we designed 4 PCR primers. Because of lack of information on 5′
ﬂanking region of carp UCP1 gene, forward primers were designed on
the basis of information of zebraﬁsh UCP1 gene; 5′-gttgtagttttgg
tttattacacaagg-3′ named as primer A that corresponds to nt −4500 tont − 4474 of zebraﬁsh UCP1 gene (GenBank accession number
NC_007112) and 5′-taaagtcctgctgcagtggaaacag-3′ as primer B thatis nt − 3000 to nt − 2976 of zebraﬁsh UCP1 gene. The reverse
primers were designed based on the carp UCP1 gene (AY461434),
i.e., 5′-gatggagtgacaggatgatgcctcgtg-3′ (nt + 45 to nt + 19) and 5′ctgacagctgtgattgagttcctctg-3′ (nt + 74 to nt + 49) as primer C andprimer D, respectively. The ﬁrst round PCR was performed using genomic DNA, which was isolated from carp muscle cells, as a template DNA as well as primers A and D; PCR was conducted in a
total volume of 50 μL by use of PrimeSTAR GXL DNA Polymerasethat is a high ﬁdelity polymerase (TaKaRa). The PCR proﬁleconsisted of 35 cycles of denature for 10 s at 98 °C, annealing for
15 s at 55 °C and extension for 4 min at 68 °C. The second round of
PCR re-ampliﬁed using one-ﬁftieth of the PCR products as the template, and primers B and C with the same PCR proﬁle. The productsof the nested-PCR were electrophoresed in agarose gels, followed byethidium bromide staining and visualization under ultraviolet light.
A signiﬁcant band was detected at ~ 2800 bp, which was within therange of expected size. The band was excised from the gels, and thenucleotide sequence was determined by direct sequencing anddeposited in GenBank (LC003596). To examine the validity ofsequence, PCR was performed by use of genomic DNA as a template
DNA and PCR primers spanning nt − 434 to nt − 412 (5′-tttctgagctc
ctttaatgcatc-3′) and nt + 930 to nt + 908 (5′-gcggtgtccagaggg
aaggtgac-3′), and the nucleotide sequence of PCR product was conﬁrmed by direct sequencing. The 5′ ﬂanking region of carp, human,
mouse, rat and zebraﬁsh UCP gene was compared by use of Pairwise
Sequence Alignment of EMBOSS (http://emboss.open-bio.org/).

Table 1
Nucleotide sequence of primers used in RT-qPCR analyses.
Gene

Forward primer

Reverse primer

GenBank accession number

CREB
EF1α
NRF1
PGC-1αPGC-1βPPARα
PPARβ/δ
PPARγ
RXRα
TRα
UCP1

5′-ctcagcagattgccaccttgg-3′5′-atgcggtggaatcgacaa-3′
5′-aagccctgaggactattgtt-3′5′-tgcctgagcttgacctctct-3′5′-tggggaagaggaggtctgc-3′
5′-gggaaagagcagcacgagtcc-3′5′-tggctttgtggatctcttcc-3′5′-aggcaactctacgagtcctatct-3′5′-cacccaatgatccagtcacaaaca-3′5′-aatcacccgcaaccagtgccag-3′5′-cgccttctacaaaggtttcg-3′5′-gggcagctgaactaaggtcac-3′5′-cagagagcaatgtcaatggtg-3′5′-gctcctgtgccaacctgtat-3′5′-cgtcttcatccactgggatac-3′
5′-ccgtccaggctgtctgtg-3′5′-ggtaggcttcatgcatctgtc-3′
5′-gatctcgccgaaaggtttgc-3′
5′-agttgatcatctgctcgccttcc-3′5′-agctcattccatcctgctcgtaga-3′
5′-tcgatcagacgcctcttggcc-3′5′-cgaatgacacgaacatcacc-3′

LC0006801
AF485331
AB9246411
AB7673021
AB7673031
FJ849065
LC0006831
FJ849064
LC0006821
LC0006811
AY461434

1

Nucleotide sequence was determined in this study.

358

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M. Murakami et al. / Comparative Biochemistry and Physiology, Part A 185 (2015) 142–149

2.4. Isolation of PGC-1α, PGC-1β, PPARβ/δ, TRα, RXRα, CREB, and NRF1mRNA from carp livers

2.5. Statistical analyses
Data are expressed as the mean ± SE. Data were log-transformed toprovide an approximation of a normal distribution before analysis.
Differences between groups in each tissue were analyzed by Tukey's
multiple comparison test. P b 0.05 was considered signiﬁcant.
3. Results
The expression of UCP1 was evaluated in the livers and kidneys ofcommon carps acclimatized at various temperatures (Fig. 1). A decreasein the water temperature from 22 °C to 8 °C signiﬁcantly reduced the expression of UCP1 in the liver and kidney within 14 days. Although increasing the water temperature from 8 °C to 28 °C did not change thehepatic expression of UCP1, the renal expression of UCP1 returned tobasal levels. Consistent with previous ﬁndings (Jastroch et al., 2005),
the expression level of UCP1 was higher in the liver than in the kidney.
Expression of mammalian UCP1 gene is transcriptionally regulated(Cannon and Nedergaard, 2004). We determined nucleotide sequenceof 5′ ﬂanking region of carp UCP1 gene, because it has not yet been
available. Whole genome sequence of common carps is not known,
and, therefore, we isolated 2634 bases upstream sequence of the150

relative mRNA level

To isolate PGC-1α, PGC-1β, PPARβ/δ, TRα, RXRα, CREB, and NRF1
cDNA from carp livers, 1 μg of the recovered RNA was treated with
RNase-free DNase I (Invitrogen) to remove residual DNA. RNA was subsequently reverse-transcribed in a 21 μl volume reaction using theoligo(dT) primer and Superscript III First-Strand Synthesis System for
RT-PCR (Invitrogen) to generate ﬁrst-strand cDNA (Murakami et al.,2008).
Based on the nucleotide sequence of the PGC-1α mRNAs of goldﬁsh(Carassius auratus, FJ710611 and EU426842), zebraﬁsh (Danio rerio,
FJ710604), grass carp (Ctenopharyngodon idella, JN195739), and goldenshiner (Notemigonus crysoleucas, FJ710606), the following PCR primers
were designed to isolate PGC-1α cDNA: 5′-atggcgtgggacaggtgtaatc-3′
and 5′-tcaaaggaagtggcaagatggt-3′. To isolate PGC-1β mRNA, the following
PCR primers were designed based on the nucleotide sequence of zebraﬁsh
PGC-1β (XM_009291061): 5′-atggcggactgcgcttcactgttagatg-3′ and 5′agacccccgactccgtcctcaacaat-3′. To isolate PPARβ/δ mRNA, the PCRprimers, 5′-cagtgcaccacagtggaaactg-3′ and 5′-gaatgccatcctgaatctgctc-3′,
were designed based on the nucleotide sequence of zebraﬁsh PPARδb(NM_131468 and XM_683192). The mRNA of TRα was isolated on thebases of nucleotide sequences of goldﬁsh TRα1 (AY973629) andTRα2 (DQ172902) and zebraﬁsh TRαa (NM_131396) by use of 5′gcatcacatgtgagggctgcaa-3′ and 5′-tctccatacagcagcctttcag-3′ as PCRprimers. The mRNA of RXRα was isolated on the bases of nucleotide
sequences of goldﬁsh RXRα (AY197562) and zebraﬁsh RXRαa(NM_001161551 and XM_001923838) by use of 5′-cagaagtgtttgg
ccatgggc-3′ and 5′-gcaaaccttccaggttgttcag-3′ as PCR primers. The
mRNA of CREB was isolated on the bases of nucleotide sequences of
goldﬁsh CREB1 (AM886438) and zebraﬁsh CREB1 (NM_001161551
and XM_001923838) by use of 5′-caggagcagatgtccagcagg-3′ and 5′gcatactgcaggatggtggtg-3′ as PCR primers. As for NRF1 mRNA, the following PCR primers were designed based on the nucleotide sequenceof zebraﬁsh NRF1 (XM_005164709) and Japanese pufferﬁsh (Takifugu
rubripes) NRF1-like isoform 1 (XM_003972802): 5′-caggcccagctccg
agcgttcat-3′ and 5′-ggccacagcctgcgtggcctct-3′. Using these primers,
cDNA was isolated from common carp livers, and agarose gel electrophoresis of the PCR products indicated bands with an expected size of~ 920 bp for PGC-1α, ~ 350 bp for PGC-1β, ~ 270 bp for PPARβ/δ,~ 440 bp for TRα, ~ 450 bp for RXRα, ~ 440 bp for CREB, and ~ 560 bp
for NRF1. The nucleotide sequence of the PCR product was determinedby direct sequencing.

UCP1
control
8°C-14 d
28°C-14 d

A
100

50

B B
a

a
b
0

liver

kidneyFig. 1. UCP1 expression in the livers and kidneys of common carps acclimated to various
temperatures. Common carps acclimated at 22 °C were subjected to cold exposure for
14 days at 8 °C. They were then acclimated at 28 °C for 14 days. The gene transcript levelsof UCP1 were determined by RT-qPCR and are expressed relative to the expression ofEF1α, with the hepatic level determined on day 0 (control) set to 100. Data shown are
the mean ± SE (n = 10). A, B and a, b: P b 0.05.

putative transcriptional initiation site of carp UCP1 through thenested-PCR strategy in reference to the nucleotide sequence of 5′
ﬂanking region of zebraﬁsh UCP1 gene (Fig. 2A). When genomic DNA
from three carps was individually used as the template DNA, comparable results were obtained (data not shown). The validity of the isolated
nucleotide sequence was also determined by the expected ampliﬁcationof genomic DNA using PCR primers designed within the region of determined nucleotide (data not shown).
The nucleotide sequence indicated the lack of the ﬁrst 18 bases (5′tggatccaaagaattcgg-3′) within 5′ untranslated region of carp UCP1mRNA in GenBank accession number AY461434, and three nucleotide
substitutions, T, C, and T instead of A, G, and G of the 20th, 22nd, and25th nucleotides, respectively. We performed RT-PCR using liver cDNA
as the template DNA and the ﬁrst 22 bases of AY461434 (primer E: 5′tggatccaaagaattcggcacg-3′) as the forward primer and primer for the
RT-qPCR as the reverse primer, and found no band (Fig. 2B, lanes 1–3). In contrast, when the ﬁrst 22 bases overlapping to AY461434 determined in this study (primer F: 5′-ctccagtcatcatcctgtcact-3′), i.e., corresponding region spanning the 19th nucleotide to the 40th nucleotide
of AY461434, were used as the forward primer, we detected the PCR
product with the expected size (Fig. 2B, lanes 5–7). Therefore, we
conclude that the 19th nucleotide of DNA sequence recorded in
AY461434 is the putative transcriptional initiation site of UCP1 gene incommon carps used in this study; the 19th nucleotide is designated as
nt + 1. The nucleotide sequence near 5’′ untranslated region of carp
UCP mRNA may be different between the strains. The nucleotide sequence has limited homology with that of 5′ ﬂanking region ofhuman, mouse, and rat UCP1 genes (Fig. 2C). The less homology of thenucleotide sequence was also detected between carps and zebraﬁsh.
Previous studies identiﬁed and characterized the brown adipose
tissue (BAT)-speciﬁc enhancer region (~220 bp) around nt −2500 upstream of murine UCP1 transcriptional initiation site (Kozak et al., 1994;
Rim and Kozak, 2002). The region plays a critical role in the regulation of
UCP1 that includes responsive element to PPAR, TR, and CREB (Cannon
and Nedergaard, 2004). Homology search indicated no similarity between the murine BAT-speciﬁc enhancer and the carp UCP1 promoter(data not shown). In addition, there was no consensus responsive element to PPAR, TR, and CREB within the carp UCP1 promoter, i.e., direct
repeats (DR) 1 and 2 (core sequence 5′-agg(a/t)ca-3′ separated by one
and two base-pairs, respectively) for PPAR (Evans and Mangelsdorf,

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145

Carp

Zebrafish

Rat

Primer F
K L L
S S H

Mouse

Forward primer: Primer E
Tissue: K L L
RT: S S H

C
Human

B

DNA ladderA

Human 100 45 44 39 36
100 70 39 41
Mouse
100 41 40
Rat
100 46
Zebrafish100
Carp

1 2 3 4 5 6 7 8
Fig. 2. Nucleotide sequence of the 5′ ﬂanking region of the carp UCP1 gene. (A) Nucleotide sequence of the 5′ ﬂanking region of the carp UCP1 gene. The nt +1 is deﬁned as putative transcriptional-initiation site. Possible CRE, BRE-1, and NF-E2 recognition sequences are shown with underline, box, and double underline, respectively. (B) RT-PCR analyses were performed
using cDNA prepared from kidney (K: lanes 1 and 5) and liver (L: lanes 2, 3, 6 and 7) as the template. The cDNA was prepared by Superscript III First-Strand Synthesis System (S) to isolate
full-length of carp UCP1 mRNA (lanes 1, 2, 5 and 6) or by high capacity cDNA reverse transcription kit (H: lanes 3 and 7). Lanes 1–3 and 5–7 indicate PCR products using primer E and thereverse primer for RT-qPCR, and primer F and the reverse primer for RT-qPCR, respectively. Lanes 4 and 8: negative control and 100-bp DNA ladder, respectively. (C) Homology between
nucleotide sequences spanning nt −2634 to nt −1 of UCP1 gene of various animal species.

2014), DR-4 for TR (Evans and Mangelsdorf, 2014), and cAMP response
element (5′-tgacgtca-3′) for CREB (Montminy, 1997). We also searched
speciﬁc sequence motifs present in the murine BAT-speciﬁc enhancer
region, and found two half-site consensus sequence of CRE (5′-cgtca3′ and its complementary sequence 5′-tgacg-3′, Fig. 2A, underline),
two contiguous TTCC motifs within brown adipocyte-regulatory360

element 1 (BRE-1, Fig. 2A, box), and two possible NF-E2 sites (Fig. 2A,
double underline).
The expression of genes involved in the expression of UCP1 in mammals (Puigserver et al., 1998; Barbera et al., 2001; Cannon andNedergaard, 2004; Cao et al., 2004; Komatsu et al., 2010; Seebacher
and Glanville, 2010) was examined next. Sequence information for146

M. Murakami et al. / Comparative Biochemistry and Physiology, Part A 185 (2015) 142–149

common carp PGC-1α, PGC-1β, PPARβ/δ, TRα, RXRα, CREB, and NRF1
cDNAs was unavailable; thus, we isolated gene transcripts from theliver of the common carp. The partial amino acid sequences deduced

from the nucleotide sequences exhibited sequence similarity to ﬁsh
PGC-1α, PGC-1β, PPARβ/δ, TRα, RXRα, CREB, and NRF1, respectively(Fig. 3), which suggested that these are orthologs of respective genes

Fig. 3. Comparison of the amino acid sequence deduced from the nucleotide sequence of PGC-1, PPARβ/δ, TRα, RXRα, CREB, and NRF1. The partial gene transcripts of PGC-1α, PGC-1β,
PPARβ/δ, TRα, RXRα, CREB, and NRF1 were isolated and sequenced. The deduced amino acid sequences of PGC-1α (A), PGC-1β (B), PPARβ/δ (C), TRα (D), RXRα (E), CREB (F), andNRF1 (G) were compared to that of ﬁsh PGC-1α, PGC-1β, PPARβ/δ, TRα, RXRα, CREB, and NRF1, respectively. The dot stands for the same amino acid as that in Cca. Cca: Cyprinus carpio,
Cau: Carassius auratus, Dre: Danio rerio, Cid: Ctenopharyngodon idella, Spr: Schizothorax prenanti, Ncr: Notemigus crysoleucas, Ssa: Salmo salar, Caca: Carassius carassius, Omy: Oncorhynchus
mykiss, Pma: Pagrus major, Ola: Oryzias latipes. β/δ, β, and β2 and δb: PPARβ/δ, PPARβ, PPARβ2, and PPARδb, respectively. α1, α2, and αA: TRα1, TRα2, and TRαA, respectively.

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in common carps. Since ﬁsh PGC-1α, PGC-1β, PPARβ/δ, TRα, RXRα,
CREB, and NRF1 consist of ~ 878, ~ 495, ~ 517, ~ 421, ~ 343, ~ 318, and~ 514 amino acids, respectively, the gene transcripts clariﬁed in thepresent study are expected to correspond to approximately one-ﬁfth
of PGC-1β and PPARβ/δ, one-third of PGC-1α, TRα, and NRF1, and halfof RXRα and CREB of the coding regions.
Unlike the hepatic and renal expressions of UCP1, the expression
level of PGC-1α did not decrease with a reduction in the water temperature from 22 °C to 8 °C; the renal expression of PGC-1α was increased
by cold exposure (Fig. 4A). The hepatic expression of PGC-1α remained
unchanged by increases in the water temperature from 8 °C to 28 °C,
whereas the cold-induced expression of renal PGC-1α returned tobasal levels with increases in the water temperature from 8 °C to
28 °C. Decreasing the water temperature from 22 °C to 8 °C decreased
the expression of PGC-1β in the kidney, but not signiﬁcantly in theliver (Fig. 4B). The renal expression of PGC-1β tended to return tobasal levels with increases in the water temperature from 8 °C to 28 °C.
The hepatic expression of PPARα was unaffected by cold exposure,but decreased with increases in the water temperature from 8 °C to
28 °C (Fig. 5A). In contrast, changes in the water temperature did not affect the renal expression of PPARα. Expression of hepatic and renal
PPARβ/δ was increased by cold exposure, and the increase in water temperature from 8 °C to 28 °C decreased the expressions; hepatic expression levels at 28 °C were lower than those at 22 °C (Fig. 5B). Both thehepatic and renal expressions of PPARγ were markedly reduced withdecreasing water temperature from 22 °C to 8 °C, and the cessation ofcold exposure returned to these expression levels back to basal levels;
however, hepatic expression was not completely recovered (Fig. 5C).
Expression of renal TRα was decreased during the cold exposure, andthe cessation of cold exposure further decreased the expression; hepatic
expression of TRα was also decreased by the increase in water temperature from 8 °C to 28 °C (Fig. 5D). Similar to the regulatory expression of
PPARγ, RXRα expressions in the liver and kidney were signiﬁcantly
decreased during cold exposure, and the re-warming resulted in returnto the basal expression levels (Fig. 5E).
Cold exposure did not affect expression levels of hepatic and renal
expressions of CREB, but the increase in water temperature from 8 °C
to 28 °C signiﬁcantly decreased renal CREB expression (Fig. 6A). The
cold exposure increased the hepatic and renal expression levels of
NRF1, and an increase in the water temperature from 8 °C to 28 °C
decreased expression levels; expression levels at 28 °C were lower
than those at 22 °C (Fig. 6B).

down-regulated by cold exposure, 2) expression levels in the kidney,
but not in the liver recovered with the cessation of cold exposure,3) water temperature-related changes in the renal expression of UCP1
paralleled those in the renal expression of PPARγ and RXRα, 4) the
cold-induced down-regulation of hepatic PPARγ partially recovered
with the cessation of cold exposure, 5) contrary to hepatic PPARγ andRXRα expressions, expressions of hepatic and renal PPARβ/δ and
NRF1 were up-regulated during cold exposure, and the cessation ofcold exposure returned the expression, and 6) the expression of hepaticand renal PGC-1α was not decreased by cold exposure. Of these, thedown-regulation observed in the hepatic expression of UCP1 duringcold exposure was consistent with previous ﬁndings (Jastroch et al.,2005); considering that the common carp is an ectothermic animal,
down-regulation of UCP1, a drive engine of non-shivering thermogenesis, during cold exposure is reasonable. The other 5 points were notevaluated in the study by Jastroch et al. (2005). Our results conﬁrm
that regulatory expression of carp UCP1 during cold exposure and subsequent recovery from cold exposure is distinct from that of mammalian UCP1 expression, but suggests that expression level of carp UCP1 ispartly but not completely related to that of molecules involved in regulation of mammalian UCP1 expression. The present study has provided anovel insight into the regulatory expression of carp UCP1.
Although expression levels of UCP1 were closely related to those ofmolecules regulating UCP1 expression in murine brown adipocytes during the changes in ambient temperature in common carps, the detailed
regulation was distinct between murine brown adipocytes and carp
tissues such as liver and kidney. In murine brown adipocytes, thecold-induced stimulation of the sympathetic nervous system and accompanying β3 adrenergic receptor activation induce the transcriptionof PGC-1α, which in turn stimulates UCP1 transcription through complex formation with PPARγ (Oberkoﬂer et al., 2002; Cao et al., 2004).
The expression level of PGC-1α, but not PPARγ was shown to increaseduring cold exposure in murine brown fat (Seale et al., 2007;
Karamitri et al., 2009; Komatsu et al., 2010). The present study indicatesthat renal expression of UCP1 is closely related to that of PPARγ rather
than PGC-1α; water temperature-related changes in the renal expression of UCP1 paralleled those in the expression of PPARγ, but not in
PGC-1α. Cold exposure also decreased hepatic expressions of UCP1
and PPARγ, but the expression level of hepatic PGC-1α was unaffected.
Consistent with previous ﬁndings on the effects of cold on theexpression of PPARα in murine brown fat (Komatsu et al., 2010), the hepatic and renal expression levels of PPARα in common carps were unaffected by cold exposure. In addition, hepatic and renal expressions of
PPARβ/δ were rather increased during cold exposure. Taking theexpression level of PPARs and PGC-1α together, the down-regulationof PPARγ expression during cold exposure may be responsible for the
down-regulation of UCP1 expression in common carps. Changes in
expression levels of RXRα, a PPARγ partner (Evans and Mangelsdorf,

4. Discussion
This study revealed six aspects of the regulation of the hepatic andrenal expressions of UCP1 in carps in response to changes in ambient
temperature 1) both the hepatic and renal expressions of UCP1 wereA

B

PGC-1αPGC-1 β

a

300
b

200

b

100

relative mRNA level

relative mRNA level

control
8°C-14 d
28°C-14 d

300

400

147

a
ab

200

b
100

0

0

liver

kidneyliver

kidneyFig. 4. PGC-1 expression in the livers and kidneys of common carps acclimated to various temperatures. Common carps acclimated at 22 °C were subjected to cold exposure for 14 days at
8 °C. They were then acclimated at 28 °C for 14 days. Transcript levels of PGC-1α (A) and PGC-1β (B) were determined by RT-qPCR and are expressed relative to the expression of EF1α,with the hepatic level determined on day 0 (control) set to 100. Data shown are the mean ± SE (n = 10). a, b: P b 0.05.

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M. Murakami et al. / Comparative Biochemistry and Physiology, Part A 185 (2015) 142–149

A

B

PPAR α800

relative mRNA level

relative mRNA level

150
A A

100

50

B

0

150

a

600
400

A

200

b

B

PPAR γbCDkidney

liver

E

TR α

kidneyRXR αrelative mRNA level

A

100
a

A

50

b

B

c

B

A

a

a

50
C

b

liver

kidneycontrol
8°C-14 d
28°C-14 d

150

150

A

100

0

0

liver

relative mRNA level

C

PPAR β /δrelative mRNA level

148

A

100
a

a

50

B
b

0

0

liver

kidneyliver

kidneyFig. 5. Expression of PPAR, TRα, and RXRα in the livers and kidneys of common carps acclimated to various temperatures. Common carps acclimated at 22 °C were subjected to cold exposure for 14 days at 8 °C. They were then acclimated at 28 °C for 14 days. Transcript levels of PPARα (A), PPARβ/δ (B), PPARγ (C), TRα (D), and RXRα (E) were determined by RT-qPCR and
are expressed relative to the expression of EF1α, with the hepatic level determined on day 0 (control) set to 100. Data shown are the mean ± SE (n = 10). A, B, C and a, b, c: P b 0.05.

2014), basically paralleled to those of PPARγ. Therefore, expression
levels of RXRα may be also involved in regulation of UCP1 expression
by PPARγ during cold exposure.
However, the regulatory expressions of PPARγ/RXRα do not explain
UCP1 expression in liver of common carps during the water temperature from 8 °C to 28 °C; the cessation of cold exposure increased hepatic
expressions of PPARγ and RXRα but not UCP1. It is possible that inhibitory factor(s) on UCP1 expression is induced in carp livers during therecovery period from cold exposure.
We isolated 2634 bases upstream to putative transcription initiationsite of UCP1 gene. The nucleotide sequence has low homology to corresponding region of humans, mice, rats, and even zebraﬁsh, althoughsome sequence motifs in the murine BAT-speciﬁc enhancer are present
scattered widely. The enhancer region of human UCP1 gene, which

A

B

CREB

NRF1
400

a a
300

b

200
A
AB B

0

relative mRNA level

relative mRNA level

400

100

includes responsive elements to PPAR, TR, and CREB, is located aroundnt −3800 (Rim and Kozak, 2002; Shore et al., 2013). It is possible thatthe transcriptional enhancer is located in a more distal region of UCP1
promoter. In addition, the sequence motifs in ﬁsh may not match tothose suggested in mammals. These should be evaluated in futurestudies.
We also evaluated the expression of NRF1, because NRF1 has beenshown to regulate energy metabolism in concert with PGC-1α in mammals (Scarpulla, 2008). A previous study revealed that changes in NRF1
expression paralleled those in UCP1, PGC-1α, and PPARδ expressions
during cold exposure and physical activity in murine brown fat(Seebacher and Glanville, 2010). Cold exposure increased the renal
expression of PGC-1α, PPARβ/δ, and NRF1, and the cessation of cold
exposure decreased the expression of these genes; similar changes

control
8°C-14 d
28°C-14 d

a

300
200
100

A

b

B

c
C

0

liver

kidneyliver

kidneyFig. 6. Expression of CREB and NRF1 in the livers and kidneys of common carps acclimated to various temperatures. Common carps acclimated at 22 °C were subjected to cold exposure for
14 days at 8 °C. They were then acclimated at 28 °C for 14 days. Transcript levels of CREB (A) and NRF1 (B) were determined by RT-qPCR and are expressed relative to the expression ofEF1α, with the hepatic level determined on day 0 (control) set to 100. Data shown are the mean ± SE (n = 10). A, B, C and a, b, c: P b 0.05.

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M. Murakami et al. / Comparative Biochemistry and Physiology, Part A 185 (2015) 142–149

were also detected in hepatic expressions of PPARβ/δ and NRF1.
However, water temperature-related changes in the expression patternof PGC-1α, PPARβ/δ, and NRF1 differed from those in UCP1 expression.
PGC-1α, PPARβ/δ, and NRF1 may cooperatively function through their
up-regulation during cold exposure, and this is distinct from the regulation of energy metabolism through the modulation of UCP1 expression.
A previous study showed that the hepatic expressions of PGC-1α,
PGC-1β, and PPARβ and NRF1 were higher in goldﬁsh acclimated at
4 °C for 3 weeks than in those acclimated at 20 °C, and PPARα expression was lower during cold exposure (LeMoine et al., 2008). Bremer
et al. (2012) observed that cold exposure signiﬁcantly increased expression of PGC-1β and NRF1, decreased expression of PGC-1α, PPARα, andTRα, and did not affect expression of PPARβ/δ and RXRα in white muscle of goldﬁsh. The temperature-related changes in gene expression
were partly but not completely consistent with the previous results.
At present, it is difﬁcult to understand relationships among expression
levels of these genes comprehensively; effects of cold exposure arealso different among tissues in gold ﬁsh (LeMoine et al., 2008), andthere may be species differences on cold-related changes in gene
expression.
Although carp UCP1 may be a functional uncoupling protein, similar
to its mammalian counterpart (Jastroch et al., 2007), a correlation between the expression of UCP1 and non-shivering heat production has
yet to be established in ﬁsh. Future studies are needed to determine
the role of UCP1 expression, the physiological signiﬁcance of regulatorychanges in the hepatic and renal expressions of UCP1, and the molecularmechanisms underlying altered expression levels of genes during cold
exposure and subsequent recovery from cold exposure in common
carps. Also, aging, diet, feed restriction, and hypoxia are known to affectexpression levels of UCP2 and UCP3, structurally related molecules to
UCP1, in gilthead sea bream (Bermejo-Nogales et al., 2010, 2014). To
understand uncoupling-related regulation of energy metabolism inectothermic animals fully, it is needed to explore regulatory expressionsof UCP2 and UCP3 in addition to UCP1.
Acknowledgments
We thank Dr. Masaaki Hasegawa for providing the common carps.
Also, we are grateful to anonymous reviewer's constructive comments.
This work was supported by a research project grant awarded by the
Azabu University (2013K08).
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Gene 551 (2014) 119–126

Contents lists available at ScienceDirectGenejournal homepage: www.elsevier.com/locate/gene

The regulation of hepcidin expression by serum treatment:
Requirements of the BMP response element and STAT- andAP-1-binding sites
Yohei Kanamori a, Masaru Murakami b, Tohru Matsui a, Masayuki Funaba a,⁎ab

Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara 252-5201, Japana r t i c l e

i n f oArticle history:
Received 9 April 2014
Received in revised form 26 June 2014
Accepted 20 August 2014
Available online 21 August 2014
Keywords:
Hepcidin
Serum
AP-1
BMP
Liver

a b s t r a c t
Expression of hepcidin, a central regulator of systemic iron metabolism, is transcriptionally regulated by the bonemorphogenetic protein (BMP) pathway. However, the factors other than the BMP pathway also participate in theregulation of hepcidin expression. In the present study, we show that serum treatment increased hepcidin expression and transcription without inducing the phosphorylation of Smad1/5/8 in primary hepatocytes, HepG2
cells or Hepa1-6 cells. Co-treatment with LDN-193189, an inhibitor of the BMP type I receptor, abrogated thishepcidin induction. Reporter assays using mutated reporters revealed the involvement of the BMP response
element-1 (BMP-RE1) and signal transducers and activator of transcription (STAT)- and activator protein (AP)1-binding sites in serum-induced hepcidin transcription in HepG2 cells. Serum treatment induced the expressionof the AP-1 components c-fos and junB in primary hepatocytes and HepG2 cells. Forced expression of c-fos or
junB enhanced the response of hepcidin transcription to serum treatment. By contrast, the expression of dominant negative (dn)-c-fos and dn-junB decreased hepcidin transcription. The present study reveals that serum
contains factors stimulating hepcidin transcription. Basal BMP activity is essential for the serum-induced
hepcidin transcription, although serum treatment does not stimulate the BMP pathway. The induction of c-fos
and junB by serum treatment stimulates hepcidin transcription, through possibly cooperation with BMPmediated signaling. Considering that AP-1 is induced by various stimuli, the present results suggest that hepcidin
expression is regulated by more diverse factors than had been previously considered.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
Iron is essential for fundamental metabolic processes in cells and organisms. Iron homeostasis is strictly maintained through cellular andsystemic systems. Cellular iron metabolism is regulated through ironregulatory proteins that bind iron-responsive elements in regulated
mRNAs. By contrast, systemic iron metabolism is mainly governed by
the synthesis and secretion of the iron-regulating hormone hepcidin.
Hepcidin is produced by liver parenchymal cells and orchestrates

Abbreviations: BMP, bone morphogenetic proteins; TGF-β, transforming growth
factor-β; IL, interleukin; AP, activator protein; STAT, signal transducers and activator oftranscription; BMP-RE, BMP response element; CMF, calcium and magnesium-free;
HBSS, Hank's buffered salt solution; DMEM, Dulbecco's modiﬁed Eagle's medium; FBS,
fetal bovine serum; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; pCMV-βGal, plasmid expressing β-galactosidase under the control cytomegalovirus promoter; SE, standard error;
TRE, TPA-responsive element.
⁎ Corresponding author at: Division of Applied Biosciences, Graduate School of Agriculture,
Kyoto University, Kitashirakawa Oiwakecho, Kyoto 606-8502, Japan.
E-mail address: mfunaba@kais.kyoto-u.ac.jp (M. Funaba).

http://dx.doi.org/10.1016/j.gene.2014.08.037
0378-1119/© 2014 Elsevier B.V. All rights reserved.

365

systemic iron ﬂuxes and decreases plasma iron levels by binding toand degrading the iron exporter ferroportin on the surface of ironreleasing cells, especially on the basolateral membrane of enterocytes(Anderson et al., 2012; Hentze et al., 2010; Lee and Beutler, 2009).
Hepcidin expression is transcriptionally regulated; bone morphogenetic protein (BMP), a member of the transforming growth factor-β(TGF-β) family, potently stimulates the process (Finberg, 2013; Hentze
et al., 2010; Lee and Beutler, 2009; Muckenthaler, 2008). Members of
the TGF-β family, including BMP, TGF-β, and activin, elicit their activities
through similar but distinct signal transduction pathways. Upon ligand
binding, the receptor complexes phosphorylate carboxyl-terminal serinesof receptor-regulated (R-) Smad; BMP induces the phosphorylation of
Smad1/5/8, whereas TGF-β/activin does Smad2/3 phosphorylation. Phosphorylated R-Smad forms complexes with the common Smad, Smad4,
and these translocate into the nucleus where they participate in the regulation of gene transcription (Massagué, 2012; Miyazono et al., 2010;
Sakaki-Yumoto et al., 2013).
In addition to the BMP pathway, several other molecules, such as
interleukin (IL)-6, IL-22, oncostatin M and testosterone, also regulate
hepcidin transcription (Chung et al., 2010; Guo et al., 2013; Kanda

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Y. Kanamori et al. / Gene 551 (2014) 119–126

et al., 2009; Nemeth et al., 2003; Smith et al., 2013); other factors andmechanisms that regulate hepcidin expression have not yet been fully
elucidated.
During the exploration of BMP-induced hepcidin expression, we noticed that serum starvation down-regulates hepcidin expression in hepatocytes. This observation prompted us to pursue the role of serum
as a positive regulator in hepcidin expression. Here we show that
serum stimulates hepcidin transcription via the following: an activator
protein (AP)-1 binding site within the hepcidin promoter, whichspans nt −133 to nt −127 and was identiﬁed previously but was notcharacterized (Truksa et al., 2007); the signal transducers and activatorof transcription (STAT)-binding site (STAT-BS), which spans nt −143 tont − 134; and the BMP response element 1 (BMP-RE1), which spansnt − 155 to nt − 150. Although serum treatment does not stimulate
the BMP pathway, the basal BMP activity is indispensable for seruminduced hepcidin transcription. In addition, the AP-1 components
c-fos and junB induced by serum treatment would be involved in thetranscriptional activation. The present study extends our understandingof hepcidin expression. AP-1 is induced by various stimuli, such as
stress, infections and inﬂammations, and certain stimuli that are mediated by cytokines and growth factors (Hess et al., 2004). Thus, thepresent results suggest that hepcidin is regulated by more factors thanwere previously considered.
2. Materials & methods
2.1. Materials
The following reagents were purchased and used: BMP2 was fromR&D Systems (Minneapolis, MN, USA); BMP6 was from GeneTex (Irvine,
CA, USA); LDN-193189, an inhibitor for BMP type I receptor (Yu et al.,2008a) was from Stemgent (San Diego, CA, USA); rabbit polyclonal antibodies against phospho-Smad1 (Ser463/Ser465)/Smad5 (Ser463/
Ser465)/Smad8 (Ser426/Ser428) (#9511) and p38 (#9212) werefrom Cell Signaling Technology (Danvers, MA, USA); a mouse monoclonal antibody against Smad1 (A-4, sc-7965) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and a rabbit monoclonal antibody
against Smad1 (EP565Y, ab33902) and a mouse monoclonal antibody
against β-actin (AC-15, ab6276) were from Abcam (Cambridge, MA,USA).
2.2. Cell culture
Animal experiments were approved by the Kyoto University Animal
Experiment Committee. Primary rat hepatocytes were isolated by collagenase digestion of livers from male Sprague–Dawley rats aged 4 weeks.
The livers were perfused from the portal vein to the incised inferior
vena cava with calcium and magnesium-free (CMF) buffer (40 mM
Hepes, pH 7.4, 120 mM NaCl, 5.4 mM KCl, 5.0 mM NaHCO3 and 5.6 mM
glucose supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin
and 250 ng/mL amphotericin B) for 10 min at a rate of ~12 mL/minfollowed by perfusion with CMF buffer containing 0.05% collagenase(Wako, Osaka, Japan) for 10 min. Subsequently, hepatocytes were liberated into Hank's buffered salt solution (HBSS), i.e., 140 mM NaCl, 5.4 mM
KCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 0.81 mM MgSO4·7H2O,
1.3 mM CaCl2, 4.2 mM NaHCO3 and 5.6 mM glucose supplemented
with 100 U/mL penicillin, 100 μg/mL streptomycin and 250 ng/mL
amphotericin B. After cell recovery by centrifugation at 50 ×g for
2 min, the cells were washed with HBSS three times and were then resuspended in Dulbecco's modiﬁed Eagle's medium (DMEM) with 10%
heat-inactivated fetal bovine serum (FBS) and antibiotics. Cells (N90%
hepatocytes by microscopy) were seeded on collagen-coated plates at
1.5 × 105 cells per well in 12-well plates. Attached cells were subsequently used. HepG2 hepatoma cells and Hepa1-6 hepatoma cells
were cultured in DMEM with 10% heat-inactivated FBS and antibiotics.

2.3. RNA isolation and RT-quantitative PCR
Total RNA was isolated from rat primary hepatocytes by using TRIZOL(Invitrogen, Grand Island, NY, USA) and cDNA was synthesized by the ABI
high capacity cDNA reverse transcription kit (Applied Biosystems, Foster
City, CA, USA), according to the manufacturers' protocols. The cDNA thatwas reverse-transcribed from 5 ng of total RNA was used as a templatefor RT-quantitative PCR (RT-qPCR) as previously described (Asano et al.,2013). The oligonucleotide primers for RT-qPCR are as follows: 5′gatggcactcagcactgga-3′ and 5′-gctgcagctctgtagtctgtct-3′ for hepcidin, 5′gaacactggatggacgactg-3′ and 5′-acagacgggcatagatcaca-3′ for Smad4,
and 5′-cgtgttcctacccccaatgt-3′ and 5′-tgtcatcatacttggcaggtttc-3′ forglyceraldehyde-3-phosphate dehydrogenase (Gapdh). The Ct value
was determined, and the abundance of gene transcripts was analyzed
using the ΔΔCt method and by normalizing against the Gapdh gene(Duran et al., 2005).
2.4. Western blot
Western blot analyses were performed as previously described(Funaba and Murakami, 2008). The immunoreactive proteins werevisualized using the ECL Select Western blotting detection system (GEHealthcare, Buckinghamshire, UK) according to the manufacturer's
protocol.
2.5. Plasmids
The DNA sequence that spans nt − 2018 to nt − 35 of the mouse
hepcidin (Hamp1) promoter was ampliﬁed and cloned into a pGL4
basic vector that contained the ﬁreﬂy luciferase reporter (Hepcidin(− 2018)-luc); nt + 1 was deﬁned as the translation initiation site.
Reporter plasmids with deleted promoters or point mutations wereprepared by a PCR-based method. The dominant negative (dn)-c-fos,
which lacks region encoding amino acid 133 to amino acid 159(Ransone et al., 1990), was also prepared by a PCR-based method.
The plasmids were veriﬁed by DNA sequencing. Expression vectors
for c-fos, junB and dn-jun B were kindly provided by Dr. M. Hibi.
2.6. Reporter assays
HepG2 and Hepa1-6 hepatoma cells were transiently transfectedwith the described expression vectors, reporter construct and a plasmid expressing β-galactosidase (pCMV-βGal) with the use of thepolyethylenimine Max reagent (Polysciences, Warrington, PA, USA) according to the manufacturer's protocol. DNA transfection was performed in the absence of heat-inactivated FBS. Equal amounts of DNA,adjusted with empty vector, were transfected in each experiment. At
4 h post-transfection, cells were cultured with medium with or without
heat-inactivated FBS for 24 h. Luciferase activity in the cell lysate wasnormalized to β-galactosidase activity.
2.7. Statistical analyses
Data are expressed as the mean ± standard error (SE). Data on gene
expression were log-transformed to provide an approximation of a normal distribution before analysis. Differences between gene expressions
in the cells were examined using unpaired t-tests. Differences of P b 0.05
were considered signiﬁcant.
3. Results and discussion
3.1. Serum stimulates hepcidin transcription in liver cells
When effects of exogenous ligand are examined, cells are typicallycultured with a reduced concentration of heat-inactivated FBS before
and during ligand stimulation. During the analysis of BMP-induced

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hepcidin expression in rat primary hepatocytes, we reproducibly observed that serum starvation itself down-regulates hepcidin expression
signiﬁcantly (P b 0.05, Fig. 1A). The decreased expression levels of
hepcidin were not caused by a non-speciﬁc down-regulation of gene expression, because serum starvation did not affect Smad4 expression.
These results suggest that serum has a stimulatory effect on hepcidin
expression. Thus, we aimed to verify the up-regulation of hepcidin
expression levels in response to serum treatment. Attached rat primary
hepatocytes were cultured in the absence of heat-inactivated FBS
followed by treatment with or without 10% FBS. Consistent withthe results on the effects of serum starvation (Fig. 1A), the serum treatment clearly increased the expression of hepcidin but not Smad4 in primary hepatocytes (P b 0.01, Fig. 1B).
Previous studies revealed that hepcidin expression is primarily increased by the activation of the BMP pathway (Finberg, 2013; Hentze
et al., 2010; Lee and Beutler, 2009). Especially, BMP6 principally regulates hepcidin transcription in livers; mice lacking BMP6 exhibited
inappropriately low level of hepcidin and massive iron overload(Andriopoulos et al., 2009; Meynard et al., 2009). BMP2 is relatively
heat-stable, and heat-inactivation of FBS for 30 min at 56 °C did not decrease the bioactivity of BMP2 (Ohta et al., 2005), although the stabilityof BMP6 in response to the heat treatment is unknown. Thus, it is possible that BMP or the related molecule(s) in heat-inactivated FBS isA

121

responsible for serum-induced hepcidin expression. To this end, we
evaluated phosphorylated Smad1/5/8 in response to serum stimulationin primary hepatocytes because BMPs elicit their activities through thephosphorylation of carboxyl-terminal serines of Smad1/5/8 (Massagué,
2012; Miyazono et al., 2010; Sakaki-Yumoto et al., 2013).
Serum treatment did not increase the phosphorylation levels of
Smad1/5/8 in primary hepatocytes; rather, phosphorylated Smad1/5/8
levels tended to decrease with serum treatment, implying the presenceof inhibitor(s) for the BMP pathway in serum (Fig. 1C, lanes 2–9). As expected, Smad1/5/8 was clearly phosphorylated within 1 h after BMP2
treatment in primary hepatocytes (Fig. 1C, lanes 10 and 11). Pretreatment with LDN-193189 decreased phosphorylation levels of
Smad1/5/8 (Fig. 1D) and abrogated serum-induced hepcidin expression (Fig. 1E).
To explore serum-induced hepcidin expression in detail, experiments were next performed in HepG2 cells and Hepa1-6 cells. Consistent with the results from primary hepatocytes, serum treatment did
not induce the phosphorylation of Smad1/5/8 in either HepG2 cells or
Hepa1-6 cells (Fig. 2A). We next examined hepcidin transcription by
luciferase-based reporter assays in HepG2 cells and Hepa1-6 cells;
these cell lines are suitable to examine hepcidin transcription but not
hepcidin expression at the mRNA level (Kanamori et al., 2014). Serum
treatment increased the expression levels of hepcidin (−2018)-luc in

Brelative mRNA level01

hepcidinrelative mRNA level
2

0

5

10

hepcidin***□ -serum■ +serum

Smad4

Smad4
□ 10% FBS
■ FBS starvation

C
time (h): 0 0.5
124
serum: - - + - + - + - +pSmad1/5/8
Smad1
p38
1 2 3 4D
LDN-193189: - - + +serum: - + - +pSmad1/5/8
Smad1
p38
1 2 3 4time (h):1
BMP2: - +pSmad1/5/8
Smad1
p38

5 6 7 8 9

10 11

E

relative mRNA level
0

5

10

vehicleLDN-193189***‡□ -serum■ +serumFig. 1. Modulation of hepcidin expression levels by serum concentrations in rat primary hepatocytes. (A) Attached rat primary hepatocytes were cultured in the presence or absence ofheat-inactivated FBS for 12 h. Hepcidin expression was examined by RT-qPCR and expressed as ratios to Gapdh, with the level set to 1 in cells cultured in the presence of heatinactivated FBS. Data shown are the mean ± SE (n = 3). *: P b 0.05, vs. cells treated with 10% FBS. (B) Attached rat primary hepatocytes were cultured in the absence of heatinactivated FBS for 4 h, followed by culturing with or without 10% heat-inactivated FBS for 12 h. Hepcidin expression was examined by RT-qPCR and is expressed as ratios to Gapdh,
with the level set to 1 in cells treated without FBS. Data shown are the mean ± SE (n = 4). **: P b 0.01, vs. cells treated without serum. (C) Attached rat primary hepatocytes were cultured
in the presence or absence of heat-inactivated FBS for the indicated time. Phosphorylated Smad1/5/8, Smad1 and p38 were examined by Western blot analysis. As a positive control, rat
primary hepatocytes were also treated with BMP2 (4 nM) for 1 h. (D and E). Attached rat primary hepatocytes were treated with or without LDN-193189 for 20 min followed by treatment
with or without 10% heat-inactivated FBS for 1 h (D) or 12 h (E). (D) Phosphorylated Smad1/5/8, Smad1 and p38 were examined by Western blot analysis. (E) Hepcidin expression was
examined by RT-qPCR and is expressed as ratios to Gapdh, with the level set to 1 in cells treated without LDN-193189 and FBS. Data shown are the mean ± SE (n = 4). **: P b 0.01, vs. cells
treated with the respective reagent (vehicle or LDN-193189) but without serum. ‡: P b 0.01, vs. cells treated with vehicle and serum.

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B

A

1

2 3 4

C

- - + +
- + - +

D

3 4 5

6 7

heated BMP6
BMP6 (4 nM)

5

10

0

1

2

3

vehicleHepa1-6-□ -serum■ +serum

HepG2□ -serum■ +serumHepa1-6

8 9 10 11 12 13 14

luciferase activity
02

5 6 7 8

HepG2

2

1

LDN-193189

heated BMP6: pSmad1/5/8
Smad1β-actin
1

0

BMP6(4 nM)

LDN-193189: - - + +serum: - + - +pSmad1/5/8
Smad1β-actinluciferase activity

luciferase activity

Hepa1-6

BMP6(4 nM)

HepG2

luciferase activity
15 0

1

2

HepG2

3

Hepa1-6

Fig. 2. Role of endogenous BMP activity in serum-induced hepcidin expression. (A and C) HepG2 cells and Hepa1-6 cells were cultured in the absence of heat-inactivated FBS for 4 h.
(A) After pre-treatment with or without LDN-193189 for 20 min, cells were cultured with or without 10% heat-inactivated FBS for 1 h. (C) Cells were treated with or without various concentrations of BMP6, which was heated for 30 min at 56 °C, for 1 h. Concentration of the treated BMP6 is as follows; lanes 2 and 9: 0.24 pM, lanes 3 and 10: 1.2 pM, lanes 4 and 11: 6 pM,
lanes 5 and 12: 30 pM, and lanes 6 and 13: 150 pM. As a positive control, cells were treated with 4 nM BMP6 (lanes 7 and 14). (A and C) Phosphorylated Smad1/5/8, Smad1 and β-actin
were examined by Western blot analysis. (B and D) After transfection with hepcidin(−2018)-luc and CMV-βGal, HepG2 cells and Hepa1–6 cells were cultured in the absence of heatinactivated FBS for 4 h. (B) After pre-treatment with or without LDN-193189 for 20 min, cells were cultured with or without 10% heat-inactivated FBS for 24 h. (D) Cells were treatedwith or without 30 pM BMP6, which was heated for 30 min at 56 °C, for 24 h. As a positive control, cells were treated with 4 nM BMP6. (B and D) Luciferase activity was normalized
to β-galactosidase activity, and the luciferase activity in cells cultured in the absence of LDN-193189 and FBS was set to 1. Data were expressed as the mean ± SE from a representative
experiment (n = 3).

HepG2 cells and Hepa1-6 cells (Fig. 2B), which indicated that seruminduced hepcidin expression is regulated at the level of transcription.
In addition, pre-treatment with LDN-193189 decreased the luciferase
activity and blocked serum-induced hepcidin transcription (Fig. 2B).
These results indicate that the BMP activity is not further enhanced byserum treatment but that the endogenous BMP activity is required forserum-induced hepcidin expression and transcription in liver cells.
We also evaluated the effects of BMP6 in heat-inactivated FBS on
Smad1/5/8 phosphorylation and hepcidin transcription in HepG2 cells
and Hepa1-6 cells. Herrera and Inman (2009) reported that FBS
contains ~ 300 pM of BMP6, which was determined by the transcriptional activity of BMP-responsive reporter. Thus, it is estimatedthat the culture medium, i.e., DMEM with 10% FBS, contains ~ 30 pM
of BMP6 that was heated for 30 min for 56 °C. Signiﬁcant induction of
Smad1/5/8 phosphorylation and hepcidin transcription could not be
detected in HepG2 cells and Hepa1-6 cells treated with below 30 pMof the heat-treated BMP6 (Fig. 2C and D). Taken failure of Smad1/5/8
phosphorylation in response to serum treatment in primary hepatocytes (Fig. 1C and D) with the inability to phosphorylate Smad1/5/8
and transactivate hepcidin by 30 pM of the heat-treated BMP6 in live
cell lines together, BMP in the FBS is not likely to explain the seruminduced up-regulation of hepcidin expression. Previous studies also
revealed that a BMP concentration greater than 100 pM is required toinduce hepcidin expression (Maes et al., 2010; Truksa et al., 2006; Wu
et al., 2012).
3.2. Serum-induced hepcidin transcription is mediated via BMP-RE1, TRE
and STAT-BS
Previous studies revealed that the hepcidin promoter has two BMP
responsive elements: BMP-RE1 (spanning nt − 155 to nt − 150) and
BMP-RE2 (spanning nt − 1678 to nt − 1673) (Casanovas et al., 2009;
Truksa et al., 2009). We performed reporter assays using a series of deleted reporters of mouse hepcidin promoter. The responsiveness toserum treatment was still detected by a deletion of BMP-RE2, althoughbasal transcription was reduced and the extent of the increase inluciferase expression in response to serum treatment was decreased(Fig. 3A). The deletion of BMP-RE1 further decreased the basal transcription and inhibited the responsiveness to serum treatment (Fig. 3A),
which suggests an essential role of BMP-RE1 in serum-induced hepcidintranscription. Consistent with these results, reporter assays that used reporters with point mutations in BMP-RE1, BMP-RE2 or both revealed
that serum-induced hepcidin transcription was inhibited in reporters
containing mutations in BMP-RE1 but not BMP-RE2 (Fig. 3B).
Serum treatment is known to induce the expression of AP-1 components, which stimulate transcription by binding to the TPA-responsive
element (TRE) located in the transcriptional regulatory region of thegene (Hess et al., 2004; Shaulian and Karin, 2002). The TRE consensus
sequence is TGAG/CTCA (Hess et al., 2004; Shaulian and Karin, 2002),
but the closely related sequence TGAGACA also binds to AP-1 andconfers AP-1-mediated transcription (Kim et al., 1990). A search forthe hepcidin gene identiﬁed a possible TRE sequence from nt −133 tont −127 (Fig. 4A).
Truksa et al. (2007) reported the existence of the TRE sequence in the
hepcidin gene and explored its function in IL-6 and BMP9-induced
hepcidin transcription by using a reporter plasmid containing the
hepcidin promoter with TRE mutations. However, to create the “TRE mutated reporter” they mutated sequence outside of the core TRE sequence(TGAG/CTCA) (Truksa et al., 2007), and therefore the role of TRE was notstrictly evaluated in their study. Additionally, adjacent to the TRE sequence, a STAT-BS sequence spans nt − 143 to nt − 134 (Fig. 4A). Although the STAT-BS sequence has been reported to function in IL-6induced transcription (Wrighting and Andrews, 2006), mutations in the
STAT-BS in mouse hepcidin (Hamp1) did not affect the responsivenessto IL-6 (Truksa et al., 2007). In the present study, we prepared reporter
plasmids with mutated core TRE sequence (mTRE), STAT-BS sequence(mSTAT) or both (mSTAT, TRE) by introducing point mutations at distal
sites from the mutual elements (Fig. 4B). In this case, we used a reporter
plasmid with a shorter hepcidin promoter, hepcidin(− 270)-luc, as awild-type plasmid to evaluate serum responsiveness (Fig. 3A).
Serum-induced hepcidin transcriptional activation was decreased
when the TRE or STAT-BS was mutated (Fig. 4C), and the double

368

Y. Kanamori et al. / Gene 551 (2014) 119–126

A

BMPRE2123

luciferase activity

BMP0
RE1

.5

4

3

1

-2018
-1203

□ -serum■ +serum

-581
-270
-219

luciferase activity
0

-150
-109

B

0.05

-219
-150
-109

BMPRE20.1

0.15□ -serum■ +serum

luciferase activity

BMP- 0
RE1

.4 1

.2

2

3

wild-typemBMP-RE2
mBMP-RE1□ -serum■ +serummBMP-RE1,2

Fig. 3. Role of BMP-RE in serum-induced hepcidin expression. After transfection with the indicated reporter plasmid and CMV-βGal, HepG2 cells were cultured in the absence of heatinactivated FBS for 4 h. After pre-treatment with or without LDN-193189 for 20 min, cells were cultured with or without 10% heat-inactivated FBS for 24 h. Luciferase activity was normalized to β-galactosidase activity, and the luciferase activity in cells that were cultured in the absence of FBS and were transfected with hepcidin(−2018)-luc was set to 1. Data wereexpressed as the mean ± SE from a representative experiment (n = 3). Hepcidin transcription was examined by the use of reporter genes with series of deletions (A) or a reporter
gene with point mutations in BMP-RE1 and -RE2 (B). BMP-RE1 (GGCGCC) was mutated to aGaaCC, and BMP-RE2 (GGCGCC) was mutated to tcaGCC; the mutated nucleotides areshown in small characters.

A

BMP-RE1
STAT-BSTRE
H. sapiens: TTCGGCGCCACCACCTTCTTGGAAATGAGACAGAG

M. musculus (Hamp1): TTTGGCGCCACTATTTTCTTGGAAATGAGTCAGAG
R. norvegicus: TTTGGCGCCACTATCTTCTTGGAAATGAGTCAGGG

B

STAT-BSTRE

wild-type: TTCTTGGAAATGAGTCA
mSTAT: ggaTTGGAAATGAGTCAmTRE: TTCTTGGAAATGAGTtg
mSTAT,TRE: ggaTTGGAAATGAGTtg

C

BMPRE1

STAT-BS

luciferase activity
TRE

0

2

4

6

8

wild-type
mSTAT
mTRE

X
X

mSTAT,TRE□ -serum■ +serumFig. 4. TRE in the hepcidin promoter. (A) Nucleotide sequence of the hepcidin promoter around TRE. Possible TRE sequence is indicated by a box, and BMP-RE1 and STAT-BS are indicatedby a double underline and a dotted underline, respectively. (B) Mutations in STAT-BS and TRE. TRE and STAT-BS are indicated by a box and a dotted underline, respectively. The mutatednucleotides are shown in small characters. (C) The role of TRE and STAT-BS in serum-induced hepcidin transcription. After transfection with the indicated reporter plasmid and CMV-βGal,
HepG2 cells were cultured in the absence of heat-inactivated FBS for 4 h followed by culture with or without 10% heat-inactivated FBS for 24 h. Luciferase activity was normalized
to β-galactosidase activity, and the luciferase activity in cells that were cultured in the absence of FBS and were transfected with hepcidin(− 270)-luc was set to 1. Data wereexpressed as the mean ± SE from a representative experiment (n = 3).

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mutations completely abrogated serum-induced hepcidin transcription.
In addition, mutations in the TRE decreased basal transcription levels of
hepcidin. These results suggest that both the TRE and STAT-BS are required for full activation of hepcidin transcription induced by serum; especially, TRE is essential for the basal transcription of hepcidin.
Verga Falzacappa et al. (2007) identiﬁed a possible AP-1 bindingsite but not the TRE in the human hepcidin gene, which extends fromnt −313 to nt −304, by the use of the nucleotide database on putativetranscription factor binding sites. The nucleotide database did notindicate the corresponding region of mouse hepcidin gene (Hamp1),
nt −292 to nt −283, as a putative AP-1 binding site (Fig. 5A). Nevertheless, we examined the role of the region spanning from nt − 292 tont − 283 by the use of hepcidin(− 581)-luc as a wild-type plasmid.
The responsiveness to serum was not changed by the mutations of theregion (Fig. 5B). The results are consistent with those that deletion of
nt −581 to nt 271 did not affect hepcidin transcription in response toserum (Fig. 3A). Thus, we conclude that the putative AP-1 site suggested
by Verga Falzacappa et al. (2007) is not functional for serum-induced
transcription of mouse hepcidin.

3.3. Serum induces c-fos and junB expressions in liver cells, which areresponsible for hepcidin transcription via TRE and STAT-BS
We examined the induction of AP-1 in response to serum treatmentin primary hepatocytes (Fig. 6A) and HepG2 cells (Fig. 6B). The timecourse changes that occurred during serum stimulation were similar between the two cell types but were different among the genes analyzed;
c-fos expression was transiently induced within 30 min after serum
treatment, and c-fos expression levels returned to basal levels at 1.5 hafter serum treatment in primary hepatocytes and at 2 h in HepG2
cells. Expression levels of junB were signiﬁcantly higher in cells treatedwith serum at 1 h of serum treatment in primary hepatocytes and from
0.5 h to 4 h in HepG2 cells. In contrast, serum treatment did not significantly affect c-jun expression in primary hepatocytes, although itslightly but signiﬁcantly increased from 0.5 to 4 h in HepG2 cells.
Considering that serum treatment stimulated hepcidin expression andtranscription in primary hepatocytes and HepG2 cells, the expression ofc-fos and junB but not c-jun may be involved in serum-induced hepcidintranscription. Thus, we explored that the effects of the forced expression

A

-313

-304

-292

-283

-289

-280

H. sapiens: AATGACATCG

M. musculus (Hamp1): TGTGACATCA

R. norvegicus: GGTGACATCA

B

luciferase activity
AP1-like

TRE

0

2

4

6

wild-typemAP1-likeX□ -serum■ +serumFig. 5. Role of a putative AP-1 binding site in serum-induced hepcidin expression.
(A) Nucleotide sequence of human putative AP-1 binding site and the corresponding region in mouse and rat hepcidin promoter. (B) The role of the putative AP-1 binding site
in serum-induced hepcidin transcription. After transfection with the indicated reporter
plasmid and CMV-βGal, HepG2 cells were cultured in the absence of heat-inactivated
FBS for 4 h followed by culture with or without 10% heat-inactivated FBS for 24 h. Luciferase activity was normalized to β-galactosidase activity, and the luciferase activity in cells
that were cultured in the absence of FBS and were transfected with hepcidin(−581)-luc
was set to 1. Data were expressed as the mean ± SE from a representative experiment(n = 3). The corresponding region to human putative AP-1 binding site (TGTGACATCA)
was mutated to TGTGttATCA; the mutated nucleotides are shown in small characters.

of c-fos and junB were examined (Fig. 7). The expression of c-fos alone increased the transcription of hepcidin, and it also increased serum-induced
hepcidin transcription. The responsiveness to serum also increased
with junB expression, although junB expression alone did not stimulate
hepcidin transcription. Mutations of either the STAT-BS or TRE sequences
decreased hepcidin transcription, irrespective of serum stimulation. The
effects of the TRE mutations were more remarkable, but the reporters
still responded to serum treatment. Double mutations in both the STATBS and TRE sequences completely inhibited hepcidin transcription.
We further examined the effects of the expression of dn-c-fos or dnjunB on hepcidin transcription (Fig. 8). Basal transcription of hepcidin
was decreased by the expression of dn-c-fos or dn-junB. In addition,
the extent of the increase in luciferase activity in response to serum
treatment was smaller in cells expressing dn-c-fos or dn-junB than inthose expressing empty vector.
Here, we showed that the withdrawal of heat-inactivated FBS decreased the expression of hepcidin in primary hepatocytes and that
serum treatment increased hepcidin expression and transcription(and vice versa) in primary hepatocytes, HepG2 cells and Hepa1-6
cells. We also demonstrated that serum treatment does not stimulate
the BMP pathway but the basal BMP activity is required for hepcidin induction through the BMP-RE1 region of its promoter. The serum induced
expression of c-fos and junB and both the TRE and STAT-BS sequences
within the hepcidin promoter are essential for serum-induced transcription. AP-1 enhances the transcription of the α2-macroglobulin genethrough complex formation with STAT3 (Ginsberg et al., 2007). The requirement of adjacent STAT-BS and TRE sequences suggests the cooperative role of AP-1 and STAT3 in AP-1-mediated hepcidin transcription,
similar to that for the α2-macroglobulin gene.
Hepcidin transcription, via both STAT-BS and TRE regulation, may beelicited by some cytokines. For example, oncostatin M, a stimulator of
hepcidin expression (Chung et al., 2010; Kanda et al., 2009), can induce
the expression of AP-1 components (Botelho et al., 1998). Not only JAK inhibition but also MEK1/2 inhibition blocked oncostatin M-mediatedhepcidin expression (Chung et al., 2010; Kanda et al., 2009); JAK phosphorylates and activates STAT (Ihle, 2001), and MEK1/2 is a MAPK kinase
and an upstream molecule that induces AP-1 components (Shaulian andKarin, 2002).
The present study reveals the requirements of BMP-RE1, TRE and
STAT-BS for the full induction of hepcidin transcription by serum treatment; BMP-RE1 is essential for the responsiveness to serum, although
the BMP activity is not enhanced by serum. In view of a central role of
the BMP pathway in hepcidin transcription, AP-1 possibly accelerates
hepcidin transcription through enhancement of the basal BMP activity. Previous studies revealed that AP-1 cooperatively enhanced
TGF-β-mediated transcription through activated Smad3 and Smad4(Busnadiego et al., 2013; Funaba et al., 2006; Wong et al., 1999; Zhang
et al., 1998), but at present, no direct evidence is available on the roleof AP-1 in BMP-mediated signaling. Members of the Jun family physically interact with Smad4 (Liberati et al., 1999), a common Smad thattransmits both TGF-β and BMP signals (Massagué, 2012; Miyazono
et al., 2010; Sakaki-Yumoto et al., 2013). In addition, a liver-speciﬁc disruption of Smad4 decreased the expression of hepcidin in mice, and theoverexpression of Smad4 increased the transcription of hepcidin in
Hepa1-6 cells (Wang et al., 2005), which indicates the involvement of
Smad4 in hepcidin expression. To clarify the role of AP-1 in BMP signaling, the effects of exogenous BMP should be examined in future studies.
During the preparation of this manuscript, the effect of serum
treatment on hepcidin transcription was reported (Shanmugam andCherayil, 2013). These researchers showed that heat-inactivated FBS
treatment increased the phosphorylation levels of Smad1/5/8 andhepcidin mRNA levels in HepG2 cells, which were blocked by cotreatment with dorsomorphin, an inhibitor of the BMP type I receptor,
as well as AMPK (Yu et al., 2008b; Zhou et al., 2001). These authors concluded that BMP or BMP-related proteins in heat-inactivated FBS are responsible for the serum-induced hepcidin mRNA changes, and these370

Y. Kanamori et al. / Gene 551 (2014) 119–126

*
*

4
2

8

relative mRNA level

0

6
4
2
0

0

1

2

B

1

relative mRNA level

6

*
4
2

0

time (h)junB
8□ -serum■ +serum

6
4

*

**

**

2

*

0

0
0

2

4

2

2

c-jun

□ -serum■ +serum

1

4

2

8

*

□ -serum■ +serum6

time (h)

c-fos
8

**

0
0

time (h)relative mRNA level8□ -serum■ +serum

relative mRNA level

relative mRNA level□ -serum■ +serum

6

junB

c-jun

c-fos
8

relative mRNA levelA

125□ -serum■ +serum

6

**
4

**

**

**

2
0

0

time (h)

2

0

4

2

4

time (h)time (h)Fig. 6. The induction of AP-1 components in response to serum stimulation. Primary hepatocytes (A) and HepG2 cells (B) were cultured in the absence of heat-inactivated FBS for 4 hfollowed by culturing with or without 10% heat-inactivated FBS for the indicated time. The expression of AP-1 components such as c-fos, c-jun and junB was examined by RT-qPCR
and expressed as ratios to Gapdh, with the level in cells at time = 0 set to 1. Data shown are the mean ± SE (n = 4). * and **: P b 0.05 and P b 0.01, respectively, vs. cells treated without
serum at the same time point.

luciferase activity
BMPRE1

STAT
-BS

TRE

wild-type

mSTAT

mTRE

mSTAT,TRE

0

X
X
X X

4

8

vectorc-fos
junB
vectorc-fos
junB
vectorc-fos
junB
vector
c-fos
junB

12

□ -serum■ +serum

luciferase activity
0

mTRE

X

mSTAT,TRE

X X

vectorc-fos
junB
vector
c-fos
junB

0.2

0.4

0.6

0.8

□ -serum■ +serumFig. 7. Enhancement of hepcidin transcription by AP-1 via TRE. The role of AP-1 components in serum-induced hepcidin transcription. After transfection with the indicated AP-1 component expression vector and reporter plasmid, and CMV-βGal, HepG2 cells were cultured in the absence of heat-inactivated FBS for 4 h followed by culturing with or without 10% heatinactivated FBS for 24 h. Luciferase activity was normalized to β-galactosidase activity, and the luciferase activity was set to 1 in cells that were cultured in the absence of exogenous
AP-1 components and FBS and were transfected with hepcidin(−270)-luc. Data were expressed as the mean ± SE from a representative experiment (n = 3).

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luciferase activity
0

1

2

3

4

vectordn-c-fos
dn-junB□ -serum■ +serumFig. 8. Inhibition of hepcidin transcription by dn-c-fos and dn-junB. After transfection with
the indicated dominant negative form of the AP-1 component expression vector and reporter plasmid, and CMV-βGal, HepG2 cells were cultured in the absence of heatinactivated FBS for 4 h followed by culturing with or without 10% heat-inactivated FBS
for 24 h. Luciferase activity was normalized to β-galactosidase activity, and the luciferase
activity was set to 1 in cells that were cultured in the absence of exogenous AP-1 components and FBS and were transfected with hepcidin(−270)-luc. Data were expressed as themean ± SE from a representative experiment (n = 3).

researchers' results on serum-induced hepcidin expression are consistent
with those of the present study. However, the underlying mechanism
clearly contrasts with our results; the reason for this inconsistency is currently unknown.
Acknowledgments
We thank Dr. M. Hibi for providing plasmids. This work was partly
supported by a research project grant that was awarded by the Azabu
University (2013K08) and a grant awarded by the Tokyo Institute ofTechnology.
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Gene 546 (2014) 50–55

Contents lists available at ScienceDirectGenejournal homepage: www.elsevier.com/locate/gene

Hepcidin expression in liver cells: evaluation of mRNA levels andtranscriptional regulation
Yohei Kanamori a, Masaru Murakami b, Tohru Matsui a, Masayuki Funaba a,⁎ab

Division of Applied Biosciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan
Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara 252-5201, Japana r t i c l e

i n f oArticle history:
Received 3 February 2014
Received in revised form 7 May 2014
Accepted 19 May 2014
Available online 20 May 2014
Keywords:
HepcidinmRNA
Transcription
BMP
Liver cells

a b s t r a c t
Hepcidin produced in the liver negatively regulates intestinal iron absorption, and the bone morphogenetic protein (BMP) pathway is well-known to stimulate hepcidin expression. However, the regulation of hepcidin
expression has not been fully elucidated. In this study, we evaluate different systems that can be used to determine how hepcidin expression is regulated. The basal expression of hepcidin in liver cell lines, such as HepG2
cells and Hepa1-6 cells, was lower than that in the liver and primary hepatocytes; the expression levels of
hepcidin in the cell lines were near the limit of detection for RT-PCR and RT-qPCR analyses. Treatment withtrichostatin A, RNAlater, or MG-132 enhanced the expression of hepcidin in HepG2 cells, suggesting that histone
deacetylation, instability of mRNA, or proteosomal degradation of the protein(s) that positively regulate hepcidin
expression may be responsible for the decreased expression of hepcidin in HepG2 cells. In luciferase-based reporter assays, BMP induced the transcription of a reporter, hepcidin(− 2018)-luc, that contains nt − 2018
through nt −35 of the hepcidin promoter in HepG2 cells and Hepa1-6 cells. However, BRE-luc, a representative
reporter used to evaluate BMP signaling, was unresponsive to BMP in HepG2 cells. These results suggest that
hepcidin transcription can be best evaluated in liver cell lines and that the hepcidin promoter senses BMP signaling with high sensitivity. The present study demonstrates that studies regarding the regulation of hepcidin expression at the mRNA level should be evaluated in primary hepatocytes, and liver cell lines are well-suited forstudies examining the transcriptional regulation of hepcidin.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
The iron uptake of the body is tightly regulated through the modulation of its absorption from the intestine (Lee and Beutler, 2009). Ferriciron is reduced at the luminal site by intestinal cytochrome b, and ferrous iron is transferred into the enterocyte via the transmembrane protein divalent metal transporter-1. On the basolateral side, ferrous iron isexported from enterocytes to the circulatory system via ferroportin, andafter being oxidized by the membrane bound ferroxidase hephaestin,iron is incorporated into transferrin (Ganz and Nemeth, 2012).

Abbreviations: BMP, bone morphogenetic protein; CMF, calcium and magnesium-free;
HBSS, Hank's buffered salt solution; DMEM, Dulbecco's modiﬁed Eagle's medium; FBS,
fetal bovine serum; ALK, activin receptor-like kinase; RT-PCR, reverse transcriptionpolymerase chain reaction; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; pCMV-βGal, plasmid expressing β-galactosidase under the control cytomegalovirus promoter; SEM, standard error of
the mean; Id1, inhibitor of differentiation 1; Hprt1, hypoxanthine phosphoribosyltransferase
1.
⁎ Corresponding author at: Division of Applied Biosciences, Kyoto University Graduate
School of Agriculture, Kitashirakawa Oiwakecho, Kyoto 606-8502, Japan.
E-mail address: mfunaba@kais.kyoto-u.ac.jp (M. Funaba).

Hepcidin is a 25-amino acid antimicrobial peptide that serves as thecentral regulator of intestinal iron absorption (Lee and Beutler, 2009).
Hepcidin binds to the iron exporter ferroportin and induces its endocytosis and proteolysis, thus preventing intestinal absorption of iron (DeDomenico et al., 2007; Nemeth et al., 2004). Previous studies suggested
that hepcidin activity is mainly regulated at the gene transcript level(Ganz and Nemeth, 2012). The ratio of intestinal iron absorption toiron intake decreases as dietary iron levels increase (Laftah et al.,2004), and this is achieved by the up-regulation of hepatic hepcidin expression (Corradini et al., 2009; Kautz et al., 2008). The current model
suggests that hepcidin expression is transcriptionally regulated by thepathway induced by bone morphogenetic proteins (BMPs) such as
BMP2, BMP4, BMP6 and BMP9 (Babitt et al., 2006, 2007; Lee andBeutler, 2009; Muckenthaler, 2008; Truksa et al., 2006). Increased levelsof iron have been shown to increase the hepatic expression of BMP6
(Kautz et al., 2008), and the disruption of BMP6 resulted in decreased
hepatic hepcidin expression and the accumulation of iron in the liver(Andriopoulos et al., 2009; Meynard et al., 2009). However, factors
other than the BMPs are also involved in modulating hepcidin expression (Goodnough et al., 2012; Lee and Beutler, 2009; Muckenthaler,2008), indicating that the mechanisms governing hepcidin expression
have not yet been fully elucidated.

http://dx.doi.org/10.1016/j.gene.2014.05.040
0378-1119/© 2014 Elsevier B.V. All rights reserved.

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Y. Kanamori et al. / Gene 546 (2014) 50–55

While exploring the factors that affect hepcidin expression, we noticed that hepcidin expression in liver cell lines is extremely low compared to that in the liver and primary hepatocytes, and this low levelof expression makes it difﬁcult to evaluate the regulation of hepcidin
at the mRNA level. The main objective of this study is to clarify the differences of the hepcidin expression level among the livers, primary hepatocytes and liver cell lines. The present study provides information on
cultured cell systems suitable for evaluating hepcidin expression.
2. Materials & methods
2.1. Animals and cell culture
Animal experiments were approved by the Kyoto University Animal
Experiment Committee or the Azabu University Animal ExperimentCommittee. Mouse and rat livers were obtained from normal adult
C57BL/6 mice and Sprague–Dawley rats, respectively. Primary rat hepatocytes were isolated by collagenase digestion of livers from male Wistar
rats weighing 200 to 300 g. Livers were perfused from the portal vein to
the incised inferior vena cava with calcium and magnesium-free (CMF)
buffer consisting of 40 mM Hepes, pH 7.4, 120 mM NaCl, 5.4 mM KCl,
5.0 mM NaHCO3 and 5.6 mM glucose supplemented with 100 U/mL
penicillin, 100 μg/mL streptomycin and 250 ng/mL amphotericin B for
10 min at a rate of ~12 mL/min followed by perfusion with CMF buffer
containing 0.05% collagenase (Wako, Tokyo, Japan) for 10 min. Subsequently, hepatocytes were liberated into Hank's buffered salt solution (HBSS), i.e., 140 mM NaCl, 5.4 mM KCl, 0.34 mM Na 2 HPO 4 ,0.44 mM KH2 PO 4, 0.81 mM MgSO 4 ·7H2O, 1.3 mM CaCl2 , 4.2 mM
NaHCO3 and 5.6 mM glucose supplemented with 100 U/mL penicillin,
100 μg/mL streptomycin and 250 ng/mL amphotericin B. After cell recovery by centrifugation at 50 ×g for 2 min, the cells were washedwith HBSS three times and resuspended in Dulbecco's modiﬁed Eagle's
medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics.
Cells (N 90% hepatocytes by microscopy) were seeded on collagencoated plates at 1.5 × 105 cells per well in 12-well plates. After attachment, the medium was replaced with the same medium, and the cells
were incubated for 24 h. Then, the cells were treated with or without
BMP2 (4 nM; R&D Systems, Minneapolis, MN, USA) in the presence orabsence of cycloheximide (0.5 μg/mL) for 12 h.
HepG2 hepatoma cells, Hepa1-6 hepatoma cells and 3T3-L1
preadipocytes were cultured in DMEM with 10% FBS and antibiotics.
For the luciferase-based reporter assays, these cells were transiently
transfected using PolyFect transfection reagent (Qiagen, Valencia, CA,

51USA) or polyethylenimine Max reagent (Polysciences, Warrington,
PA, USA), according to the manufacturers' protocols. At 24 h posttransfection, the cells were pre-treated with or without LDN-193189(100 nM), an inhibitor of the BMP type I receptor (Cuny et al., 2008),
for 15 min before BMP2 (4 nM) treatment for 16 h. To evaluate the expression of constitutively active activin receptor-like kinase (ALK) 3(ALK3(QD)), cells were harvested at 36 h post-transfection. To evaluate
the levels of hepcidin mRNA, HepG2 cells and Hepa1-6 cells were treated for 24 h with or without trichostatin A (TSA: 0.1 μg/mL; Wako,
Tokyo, Japan), an inhibitor of class I and II histone deacetylases (Gräff
and Tsai, 2013), RNAlater (0.1 or 1%; Life Technologies, Carlsbad, CA,
USA) to denature RNase at a controlled pH (Zaitoun et al., 2010) or
MG-132 (20 μM; EMD Millipore, Billerica, MA, USA), a proteasome
inhibitor (Tsubuki et al., 1993). In Hepa1-6 cells, treatment with MG132 at 20 μM caused detachment of the cells from culture dish, and,
therefore, we also treated with MG-132 at 10 μM.
2.2. RNA isolation, RT-PCR and RT-quantitative PCR
Total RNA was isolated from the livers and cells, and cDNA was synthesized using TRIZOL (Invitrogen, Grand Island, NY, USA) and ReverTra
Ace qPCR RT kit (Toyobo, Osaka, Japan), respectively, according to the
manufacturers' protocols. The cDNA reverse-transcribed from 5 or
20 ng of total RNA was used as a template for conventional reverse
transcription-polymerase chain reaction (RT-PCR) or RT-quantitative
PCR (RT-qPCR) using SYBR Green I (Thunderbird SYBR qPCR mix,
Toyobo, Osaka, Japan) as described previously (Asano et al., 2013). The
oligonucleotide primers for the conventional RT-PCR and RT-qPCR arepresented in Table 1. The Ct value was determined, and the abundance of gene transcripts was analyzed using the ΔΔ Ct method
using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as thenormalization gene (Duran et al., 2005).
2.3. Plasmids and reporter assays
Plasmids were obtained as follows: BRE-luc reporter plasmids(Korchynskyi and ten Dijke, 2002) was from Dr. P. ten Dijke, andALK3(QD) (Imamura et al., 1997) was obtained from Dr. K. Miyazono.
The DNA fragment spanning from −2018 bp to −35 bp of the mouse
hepcidin (Hamp1) promoter was ampliﬁed and cloned into the basic
vector pGL4 containing the ﬁreﬂy luciferase reporter (Hepcidin(−2018)-luc) with nt +1 as the translation initiation site. The productwas veriﬁed through nucleotide sequencing. Luciferase-based reporter

Table 1
Oligonucleotide PCR primers for RT-PCR and RT-qPCR.
Oligonucleotide

GenBank accession number

5′-primer

3′-primer

5′-TTCATTGACCTCAACTACATGGT-3′5′-GCTAAGCAGTTGGTGGTGCAGGA-3′

Hprt1

5′-ATGGGAGGCCATCACATTG-3′5′-CTTTCCAGTTAAAGTTGAGAGATCA-3′

mhHepcidina

5′-TGCCTCCTGCTCCTCCTCCT-3′5′-GCAGAAAATGCAGATGGGGAAGT-3′

mrHepcidinb

5′-GCTGCCTGTCTCCTGCTT-3′5′-TTACAGCATTTACAGCAGAAGAGG-3′

RT-PCR:
Tmprss65′-CACTGTGACTGTGGCCTCCAGG-3′5′-CGTCGTAGTCATGGCTGTCCTC-3′

NM_153609 (human)
NM_027902 (mouse)
NM_001130556 (rat)RT-qPCR:
rGapdhrHepcidin5′-ACAACTTTGGCATCGTGGA-3′5′-GCTGCCTGTCTCCTGCTT-3′5′-CTTCTGAGTGGCAGTGATGG-3′5′-AGCCGTAGTCTGTCTCGTCT-3′

NM_017008 (rat)NM_053469 (rat)

RT-PCR and qPCR:
Gapdh

a
b

PCR primers common to mouse and human hepcidin.
PCR primers common to mouse and rat hepcidin.

374

NM_002046 (human)
NM_008084 (mouse)
NM_017008 (rat)NM_000194 (human)
NM_013556 (mouse)
NM_012583 (rat)
NM_021175 (human)
NM_032541 (mouse)
NM_053469 (rat)
NM_032541 (mouse)

Y. Kanamori et al. / Gene 546 (2014) 50–55

2.4. Statistical analyses
Data are expressed as the mean ± standard error of the mean
(SEM). Data on gene expression were log-transformed to provide anapproximation of a normal distribution before analysis. Differences between control cells and BMP2-treated cells, between cycloheximidetreated and untreated cells, and between LDN-193189-treated anduntreated cells were examined using unpaired t-tests. Differencesof P b 0.05 were considered signiﬁcant.
3. Results and discussion
3.1. Hepcidin expression is lower in cell lines than in liver and primary
hepatocytes

Mouse liver
Rat liverHepcidin expression was examined in mouse and rat livers, rat primary hepatocytes and the human HepG2 and mouse Hepa1-6 liver
cell lines. To compare the hepcidin expression levels, we designed PCRprimers to amplify both mouse hepcidin (Hamp1) and rat hepcidin(Fig. 1A), and both human hepcidin and mouse hepcidin (Fig. 1B). Signiﬁcant hepcidin expression was detected in livers and primary hepatocytes, but the levels of hepcidin expression in HepG2 cells and Hepa1-6
cells was lower or below the limit of detection. Gapdh and hypoxanthine phosphoribosyltransferase 1 (Hprt1), house-keeping genes,
were individually ampliﬁed by use of common PCR primers applicableto human, mouse and rat Gapdh and Hprt1. The Gapdh levels tended
to be higher in HepG2 cells and Hepa1-6 cells than in the livers and primary hepatocytes, and the Hprt1 levels were higher in primary hepatocytes and liver cell lines (Fig. 1C).
We also examined hepcidin expression by RT-qPCR analyses (Fig. 2).
Consistent with the RT-PCR analyses (Fig. 1), hepcidin expression levelin primary hepatocytes was as high as that in the livers (Fig. 2A), whereas hepcidin expression level was below detection limit in the liver celllines, including HepG2 cells and Hepa1-6 cells (Fig. 2B). In some samples, we could detect signiﬁcant ﬂuorescence in qPCR more than 35 cycles in HepG2 cells, but it was not reproducible (data not shown).
Hepcidin mRNA level has been clearly detected in liver cell lines including HepG2 cells (Ravasi et al., 2012); the discrepancy could be due to thedifferences of the sensitivity of RT-qPCR analyses. The RT-qPCR analyses
for Gapdh and Hprt1 indicated clear expression in all samples (Fig. 2C
and D); similar to the RT-PCR analyses (Fig. 1), expression level in theliver samples tended to be lower than that in the primary hepatocytes
and liver cell lines. All these results suggest that hepcidin expression
levels in liver cell lines are lower than those in the livers and primary hepatocytes, and that the lower expression of hepcidin in the liver celllines is not due to a non-speciﬁc decrease in gene transcript levels.
There are several possible explanations for the lower hepcidin mRNAlevels in the liver cell lines. Hepcidin mRNA is negatively regulated byTmprss6, a transmembrane matriptase-2 (Du et al., 2008); the increasein Tmprss6 mRNA levels in response to BMP treatment is delayed compared to the up-regulation of hepcidin expression, indicating a negative
feedback loop to avoid excess hepcidin expression (Meynard et al.,2011). Because the lower expression of hepcidin in the liver cell lines likely results from higher expression of Tmprss6, we examined the Tmprss6
expression level in liver cell lines. However, Tmprss6 expression was undetectable in the liver cell lines (Fig. 1D). Thus, the low level of hepcidin
expression in the liver cell lines is not due to high levels of Tmprss6.
We also examined the potential roles of histone deacetylase activation and mRNA instability in the low expression of hepcidin in liver

A

Water

assays were conducted as described previously (Murakami et al., 2009).
Cells were transiently transfected with the indicated expression vectors,
reporter construct and a plasmid expressing β-galactosidase under thecontrol cytomegalovirus promoter (pCMV-βGal). Equal amounts of
DNA were transfected in each experiment and adjusted with empty
vector. Luciferase activity was normalized to the β-galactosidase activity, and the luciferase activity in the cell lysate transfected without BMP
treatment or with the empty vector was set at 1.

Rat primary

52

Hepcidin

Water

B

Hepa1-6

Mouse liver
HepG2

1 2 3 4

Hepcidin

Gapdh

Water

Hepa1-6

HepG2

Rat primary

Rat liverHprt1
1 2 3 4 5 6

Water

Hepa1-6

HepG2

Rat liverRat primary

Mouse liver

1 2 3 4 5 6D

Mouse liver

Water

Hepa1-6

HepG2

C

Rat liverRat primary

Mouse liver

1 2 3 4

Tmprss6
1 2 3 4 5 6
Fig. 1. Hepcidin expression in the liver, primary hepatocytes and liver cell lines. The cDNA was prepared from total RNA from mouse and rat livers, rat primary hepatocytes and the liver celllines HepG2 and Hepa1-6. RT-PCR was performed using primers to detect (A) mouse and rat hepcidin using mrHepcidin primer set, (B) human and mouse hepcidin using mhHepcidin
primer set, (C) human, mouse and rat Gapdh and Hprt1 and (D) human, mouse and rat Tmprss6. The PCR products were electrophoresed on 2% agarose gels and stained with ethidium
bromide. A representative result is shown.

375

Y. Kanamori et al. / Gene 546 (2014) 50–55

B

HepcidinMouse liver
Rat liverRat primary

10-1

Fluorescence

Fluorescence

A

10-2

Hepcidin

5

10

15

20

25

30

35

Mouse liver
HepG2
Hepa1-6

10-1

10-2

0
0

53

0

40

0

5

10

15

cycle

D

Gapdh

Fluorescence

FluorescenceC
10-1

Mouse liver
Rat liverRat primary
HepG2
Hepa1-6

10-2

20
25
cycle

30

35

40

30

35

40

Hprt1
Mouse liver
Rat liverRat primary
HepG2
Hepa1-6

10-1

10-2

0

0
0

5

10

15

20
25
cycle

30

35

40

0

5

10

15

20
25
cycle

Fig. 2. Lower hepcidin expression in liver cell lines than in the liver and primary hepatocytes. The cDNA was prepared from total RNA from mouse and rat livers, rat primary hepatocytes
and the liver cell lines HepG2 and Hepa1-6. RT-qPCR was performed using primers to detect (A) mouse and rat hepcidin using mrHepcidin primer set, (B) human and mouse hepcidinusing mhHepcidin primer set, (C) human, mouse and rat Gapdh and (D) human, mouse and rat Hprt1. The ampliﬁcation plots are shown. Note that cDNA from HepG2 cells andHepa1-6 cells was not ampliﬁed (B).

TSA:

-

+

on hepcidin expression. Treatment with MG-132 increased hepcidintranscription in HepG2 cells but not in Hepa1-6 cells (Fig. 3C). These results suggest the different reasons underlying the lower expression
level of hepcidin between HepG2 cells and Hepa1-6 cells. Future studies
are needed to clarify the reason of the lower expression of hepcidin in
Hepa1-6 cells. Additionally, histone acetylation within the hepcidin promoter and hepcidin mRNA instability should be also evaluated to clarify
the relation to the silencing of hepcidin in HepG2 cells. Furthermore, the

-

liver
water

A

liver
water

cell lines. Treatment with TSA robustly increased the expression of
hepcidin in HepG2 cells but not in Hepa1-6 cells, although the expression level in cells without TSA treatment was near the limit of detection(Fig. 3A). Hepcidin expression was also induced by treatment with an
RNase inhibitor in HepG2 cells but not in Hepa1-6 cells (Fig. 3B). Unlike
Hepa1-6 cells, transcriptional repression due to histone deacetylation,
mRNA instability or both may be responsible for the low levels of
hepcidin in HepG2 cells. We further examined the effect of MG-132+

Hepcidin
Gapdh

-

++

+

-

+

++

water

RNase inh:

liver

B

water

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
HepG2
Hepa1-6

Hepcidin
Gapdh

-

++

-

+

++

water

MG-132:

liver

C

water

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Hepa1-6
HepG2

HepcidinGapdh
1 2 3 4 5 6 7 8 9
HepG2

10 11 12 13 14 15 16 17 18 19 20 21 22 23
Hepa1-6

Fig. 3. Altered expression of hepcidin by histone deacetylases, RNase and the proteasome in HepG2 cells but not in Hepa1-6 cells. HepG2 cells were treated with TSA (A), RNase inhibitor(B) or MG-132 (C) as described in the Materials and methods section. The treatments were performed in quadruplicate. The hepcidin (upper) and Gapdh (lower) expression levels were
examined by RT-PCR. The PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. A representative result is shown.

376

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Y. Kanamori et al. / Gene 546 (2014) 50–55

CHX:

The BMP-regulated expression of hepcidin has been studied in various systems, including the liver cell lines such as HepG2, Hep3B andHuh7 cells (Babitt et al., 2006, 2007; Mleczko-Sanecka et al., 2010;
Truksa et al., 2007, 2009). The Lin group has examined hepcidinmRNA by RT-qPCR analyses, and their studies revealed that treatment
with BMP increased the expression of hepcidin by 50 to 1100-fold and
12 to 25-fold in Hep3B cells and HepG2 cells, respectively. An approximately 8-fold induction was detected in mouse and rat primary hepatocytes, and intraperitoneal injection of BMP resulted in only a
1.3 to 2-fold increase in hepatic hepcidin expression (Andriopoulos
et al., 2009; Babitt et al., 2006, 2007; Corradini et al., 2009, 2010;
Theurl et al., 2011). One explanation for the larger effect BMP treatment on hepcidin expression in the liver cell lines may be related
to a lower basal expression level; the magnitude of the increase in
the hepcidin mRNA copy number in response to BMP treatment relative
to the total hepcidin mRNA is larger when the basal expression is lower.
Examining hepcidin mRNA in liver cell lines can allow one to overlook
factor(s) that positively and negatively affect hepcidin expression.

+-

Fig. 4. Hepcidin expression and transcription in response to BMP2 in liver cells. Rat primary hepatocytes were treated with or without BMP2 in the presence (+) or absence (−) of
cycloheximide (CHX). Hepcidin expression examined by RT-qPCR using rHepcidin primer
set was normalized to Gapdh expression, which was quantiﬁed by use of rGapdh primer
set. The expression level in cells treated without BMP2 or cycloheximide was set at 1. **:P b 0.01 vs. cells treated without BMP2. ‡: P b 0.01 vs. cells treated with BMP2 and without
cycloheximide.

3.2. Hepcidin transcription can be examined in cell lines
molecule(s) that positively regulate hepcidin, which is degraded by proteasome, should be identiﬁed in HepG2 cells.
As described above, the activation of hepcidin expression by the BMP
pathway is well-known (Babitt et al., 2006, 2007; Lee and Beutler, 2009;
Muckenthaler, 2008). We also evaluated hepcidin expression in response
to BMP treatment and the effect of cycloheximine, a protein synthesis inhibitor, in rat primary hepatocytes. BMP increased hepcidin expression
4.5-fold, and cycloheximide partially blocked the up-regulation of
hepcidin expression (Fig. 4). The results indicate that BMP-induced
hepcidin expression is partially a direct event, and that it is also enhanced
through de novo protein synthesis; the results clearly disagree with a previous study using HepG2 cells in which cycloheximide did not affect BMPinduced hepcidin expression (Babitt et al., 2006). The contradicting conclusions are possibly due to the different cell types used in the studies.

A

LDN-193189:

Primary hepatocytes are suitable for examining hepcidin mRNA levels,
but they are inappropriate for studying the transcriptional regulation of
hepcidin using a reporter gene; it is generally difﬁcult to transfect primarycells with plasmid DNA, although plasmid DNA can be highly transfectedin primary cells by use of a special apparatus (Gresch and Altrogge, 2012).
In contrast, cell lines can be easily transfected with plasmid DNA, andthe transcriptional regulation of hepcidin has been examined byluciferase-based reporter assays in many studies (Goodnough et al.,2012; Mleczko-Sanecka et al., 2010; Pandur et al., 2013; Truksa
et al., 2009). Consistent with the results of previous studies, the expression of hepcidin(− 2018)-luc was signiﬁcantly increased by
BMP treatment in HepG2 cells (P b 0.01) and was inhibited by pretreatment with LDN-193189; both basal expression of the luciferase

B

-

+

C

-

LDN-193189:

D

Reporter:
Cell:+

Cell: HepG2

Hepa1-6E

BRE

Hepcidin

HepG2

BRE

HepcidinReporter:

BRE

Hepcidin

3T3-L1

Fig. 5. Hepcidin expression and transcription in response to BMP2 in liver cells. HepG2 cells (A) and Hepa1-6 cells (B) were transiently transfected with Hepcidin(−2018)-luc and pCMVβGal and treated with or without BMP2 in the presence (+) or absence (−) of LDN-193189. (C) Hepcidin(−2018)-luc, ALK3(QD) and pCMV-βGal were transfected into the indicatedcells. (D) The indicated reporter gene and pCMV-βGal were transfected in HepG2 and 3T3-L1 cells, and the cells were treated with or without BMP2. (E) HepG2 cells were transfectedwith the indicated reporter, ALK3(QD) and pCMV-βGal. Luciferase activity was normalized to β-galactosidase activity, and luciferase activity in the cell lysates in the absence of BMP2,
ALK3(QD) and LDN-193189 was set at 1. Data are expressed as the mean ± S.E. (n = 3). **: P b 0.01 vs. cells treated without BMP2. ‡: P b 0.01 vs. cells treated without LDN-193189.

377

Y. Kanamori et al. / Gene 546 (2014) 50–55

and the induced expression in response to BMP treatment were signiﬁcantly decreased (P b 0.01, Fig. 5A). Similar results were obtainedin Hepa1-6 cells (Fig. 5B). Furthermore, the enhanced transcriptionof hepcidin(− 2018)-luc was signiﬁcantly elicited by the forced expression of ALK3(QD), a constitutively active BMP type I receptor(Imamura et al., 1997) (P b 0.01, Fig. 5C). These results conﬁrm previous results that showed that BMP-induced hepcidin transcription
is evaluated in liver cell lines.
BRE-luc, which is derived from the promoter region of inhibitor of differentiation 1 (Id1), a BMP target gene, is a representative reporter thattransmits canonical BMP signaling (Korchynskyi and ten Dijke, 2002).
Provided that hepcidin transcription is stimulated by the BMP pathway
in liver cells (Fig. 5A–C), we expected that not only hepcidin(−2018)luc but also BRE-luc is transcriptionally stimulated by the BMP pathway.
However, the BMP-induced transcriptional activation of BRE-luc was notdetected in HepG2 cells. 3T3-L1 cells and C2C12 cells are non-liver cells
that can transmit canonical BMP signaling (Korchynskyi and ten Dijke,
2002; Rebbapragada et al., 2003); in these cells, BMP treatment increased
expression of BRE-luc and hepcidin(−2018)-luc (P b 0.01, Fig. 5D, data
not shown). The higher response of hepcidin(−2018)-luc than BRE-luc
was also detected when the BMP signal was transmitted by ALK3(QD)
expression, although ALK3(QD) expression signiﬁcantly increased expression of hepcidin(−2018)-luc (P b 0.01, Fig. 5E). The results suggestthat it is difﬁcult to evaluate BMP signaling by BRE-luc and that the
hepcidin promoter senses exogenous BMP with high sensitivity in liver
cell lines, thus extending the current understanding of how hepcidin
expression is regulated by the BMP pathway.
4. Conclusions
The present study revealed that 1) compared with expression in thelivers and primary hepatocytes, hepcidin expression levels are lower inliver cell lines, 2) the activation of histone deacetylase and the accelerated degradation of mRNA or protein(s) may be involved in the lower
expression levels of hepcidin in HepG2 cells, and 3) the transcriptional
regulation of hepcidin, which is highly responsive to BMP, can be evaluated in liver cell lines. We conclude that primary hepatocytes should be
used to examine the regulation of hepcidin expression at the mRNAlevel. In addition, liver cell lines such as HepG2 cells and Hepa1-6 cells
should be limited to studies that aim to evaluate the transcriptional regulation of hepcidin.
Conﬂict of interest
The authors have declared that there is no conﬂict of interest.
Acknowledgments
We thank Drs. K. Miyazono and P. ten Dijke for providing plasmids.
This work was partly supported by a research project grant awarded
by the Azabu University (2013K08).
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Mycopathologia (2014) 178:135–143
DOI 10.1007/s11046-014-9758-0

Isolation of Dermatophytes and Related Species
from Domestic Fowl (Gallus gallus domesticus)
Sayaka Yamaguchi • Ayako Sano • Midori Hiruma • Michiko Murata •Takashi Kaneshima • Yoshiteru Murata • Hideo Takahashi • Sana Takahashi •Yoko Takahashi • Hiroji Chibana • Hidemi Touyama • Nguyen Thi Thanh Ha •Yasutomo Nakazato • You Uehara • Morihiko Hirakawa • Yoshimi Imura •Yoshie Terashima • Yasuhiro Kawamoto • Keji Takahashi • Kazutoshi Sugiyama •Masataro Hiruma • Masaru Murakami • Atsushi Hosokawa • Hiroshi Uezato
Received: 25 January 2014 / Accepted: 9 May 2014 / Published online: 22 June 2014
 Springer Science+Business Media Dordrecht 2014

Abstract We investigated 793 bird combs [645chickens and 148 ﬁghting cocks (Shamo)] to determine the prevalence of dermatophytes and theirrelated fungal species. The targeted fungal species
were recovered from 195 of the 793 examined birds(24.6 %). Prevalence ratios were compared in temperate (the mainland) and subtropical (Nansei Islands)
areas, genders, strains, breeding scale (individual andfarm), and housing system (in cage and free ranging).
The frequency of the fungal species in the mainland,males, ﬁghting cocks, breeding scale by individual

nursing, and free-range housing system exhibited
signiﬁcantly higher positive ratios than that in theother groups. A total of 224 dermatophytes and related
species were isolated, including 101 Arthroderma(Ar.) multiﬁdum, 83 Aphanoascus (Ap.) terreus, ﬁve
Uncinocarpus queenslandicus, two U. reesii, two Ap.
pinarensis, one Amauroascus kuehnii, one Ar. simii,
one Gymnoascus petalosporus, one Microsporum
gallinae, and 28 Chrysosporium-like (Chrysosporiumspp.) isolates, which were identiﬁed using internaltranscribed spacer regions of ribosomal RNA gene

S. Yamaguchi H. Uezato
Department of Dermatology, Faculty of Medicine,
University of the Ryukyus, Okinawa, JapanY. Murata
Murata Animal Hospital, Chiba, JapanY. Murata H. Takahashi
Committee of Infectious Diseases, Chiba Veterinary
Medical Association, Chiba, Japan

A. Sano (&) H. Touyama N. T. T. Ha
Y. Nakazato Y. Uehara M. Hirakawa
Y. Imura Y. Kawamoto
Faculty of Agriculture, University of the Ryukyus,
1 Sembaru, Nishihara-cho, Nakagusuku,
Okinawa 903-0213, Japane-mail: aya_grimalkin@yahoo.co.jp

H. Takahashi
A-Land Oyumino Animal Hospital, Chiba, Japan
Y. Takahashi H. Chibana
Medical Mycology Research Center, Chiba University,
Chiba, Japan

M. Hiruma M. Hiruma
Department of Dermatology, Faculty of Medicine,
Juntendo University, Tokyo, Japan

Y. Terashima
Tropical Bioresources Research Center, University of the
Ryukyus, Okinawa, Japan

M. Murata S. Takahashi M. Murakami
Faculty of Veterinary Medicine, Azabu University,
Kanagawa, Japan

K. Takahashi
Chiba Prefectural Government, Chiba, Japan

T. Kaneshima
Ryukyu Animal Clinic Center, Okinawa, Japan

123
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Mycopathologia (2014) 178:135–143

sequences. The predominant fungal species in themainland was Ap. terreus and that in the Nansei
Islands was Ar. multiﬁdum. Pathogenic fungal species
to humans and animals were limited to M. gallinae andAr. simii, which corresponded to 0.025 % of theisolates in this study.

Materials and Methods
Animals
Seven hundred ninety-three chickens nursing in Japan,
including 293 from the mainland and 500 from the
Nansei Island areas, were investigated. Prefectures are
not mentioned to comply with the privacy policy. The
survey was carried out from December 2008 to March
2013. The details of the chickens are given in Table 1.
Approval was obtained from the ethics committee foranimal welfare of the University of the Ryukyus (No.
2011-5271 and 2012-5437).

Keywords Dermatophyte Ketanophilic
Gallus gallus domesticus Internal transcribedspacer (ITS) rDNA

Introduction
Chickens (Gallus gallus domesticus) and their nursing
environments contain dermatophytes and related species. The predominant dermatophyte species related tochickens are Microsporum gallinae and Arthroderma(Ar.) simii; these are known causative agents ofmycotic zoonoses [1, 2]. The former fungal species
was isolated from a human [3] and from a ﬁghting
cock [4] in Japan. The latter fungal species caused
ringworm in an imported chimpanzee [5].
According to Chermette et al., human Ar. simii
infections are rare [2]; however, the fungal species isspread worldwide [6]. Furthermore, dermatophyterelated species, mainly keratinophilic fungi represented
by Chrysosporium-like species, have also been isolated
from chickens and their raising habitats in Egypt [7, 8],
India [9, 11], Nigeria [12], Iran [13], Germany [14],Bahrain [15], and Saint Kitts and Nevis [16].
We previously reported the isolation of chicken
dermatophytes; one human case of M. gallinae
infection was found in a subtropical area of Japan[3], and one case from a ﬁghting cock was reported in atemperate area [4]. However, no other dermatophytesor species related to chicken have been investigated inJapan. The present study, as a representative study in
the Far East, aims to investigate the prevalence ratio of
dermatophytes and their related fungal species inchickens and ﬁghting cocks (Shamo) with comparisons between temperate and subtropical areas ofJapan.

Isolation
Transparent plastic tape 18 mm in width (Scotch
Sumitomo 3A, Tokyo, Japan) was cut into strips
approximately 10 cm in length. Both 1-cm ends were
folded for handling. The adhesive surface (approximately 6 cm in length) was placed on the comb andgently rubbed with both the thumb and index ﬁnger tocollect scales. The tape was incubated at 42 C for 4 h
to remove the mites. Afterward, the tape was stampedon BBLTM MycoselTM Agar (Becton, Dickinson andCompany (BD), Sparks, MD, USA) plates in duplicate
and cultured at 35 C for up to 28 days.
White to slightly beige colored, cottony or powderycolonies, which are characteristics of dermatophytes
and/or related species, were picked and transferred
onto potato dextrose agar (DifcoTM Potato Dextrose
Agar; PDA, BD) slants and incubated at 25 C for
further molecular biological analysis. In addition, weplaced the transparent tape on the ﬁrst colonies toappear and observed them by light microscopy afterstaining with lactophenol cotton blue to detect roundor pyriform conidia attached to the right angle of thehyaline septate hyphae.
Following molecular biological identiﬁcation, plural fungal sprouts from one comb sample withdifferent genotypes of identical species were treatedas individual isolates.
Molecular Biological Identiﬁcation

K. Sugiyama
Sugiyama Veterinary Clinic, Shizuoka, Japan

Sequences of the internal transcribed spacer (ITS)
1-5.8S-ITS 2 region of the rRNA gene (ITS rDNA)
were obtained by routine methods [17]. Brieﬂy, DNA
was extracted with a DEXPAT kit (TaKaRa, Ohtsu,

A. Hosokawa
AM Dermatological Clinic, Okinawa, Japan

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Mycopathologia (2014) 178:135–143

137

Table 1 Number of positive samples from a total number of samples classiﬁed based on areas, sex, strains, breeding scale, and form
Area

Sex
FemaleMainland

42/136

StrainMale

47/147

Chicken

Shamo

FemaleMale

FemaleMale

28/115

28/113

14/21

19/44

Breeding scale

Housing system

Total

Individual

Farm

Cage

Free range

28/78

61/215

8/19

81/27489/293293(30.9)(40.0)(24.3)(24.8)(66.6)(43.2)(37.8)(28.4)(42.1)(29.6)(30.4)

Nansei Islands

50/33756/16346/321

40/96

4/16

16/67

54/18552/31547/34859/152

106/500500(14.8)(34.4)(14.3)(41.7)(25.0)(23.9)(29.2)(16.5)(21.2)(49.2)(21.2)

Total

92/473

103/32074/43668/20918/37

35/111

82/263

113/530

55/367140/426

195/793793(19.5)(32.2)(17.0)(32.5)(48.6)(31.5)(31.7)(21.3)(24.6)(32.9)(24.6)The data are shown as the number of dermatophyte-positive/total number of chickens with the percentage in parentheses

labeled samples were directly sequenced on an ABIPRISM 3100 sequencer (Applied Biosystems) using
the primers ITS-5, ITS-4, ITS-2 (50 -GCTGCGTTCTTCATCGATGC-30 ), and ITS3 (50 -GCATCGATGAAGAACGCAGC-30 ) [18]. The DNA sequenceswere aligned using GENETEX-MAC genetic information processing software (Software Development
Co., Ltd., Tokyo, Japan). Sequences were analyzed bya basic local alignment search tool (BLAST) (http://
www.ncbi.nlm.nih.gov/BLAST/Blast.cgi), and closely related sequences were obtained. The conﬁrmation of fungal species was based on[99 % identity to
the known fungal species, and those of fungal genera
was \98 %.

Japan) using a modiﬁed procedure. Approximately
100 lL fungal cells from the cultures that wereincubated at 25 C for 1 month on PDA slants wasplaced in a sterile microtube (1.5 mL); subsequently,
0.5 mL DEXPAT solution was added, and the
mixture was homogenized with a plastic pestle. The
tube was incubated at 100 C for 10 min and thencentrifuged at 13,2009g for 10 min, and the supernatants were used as DNA samples. Although this type of
kit is designed to extract DNA from parafﬁn-embedded tissue samples, we routinely use it to isolate genes
from DNA from fungal cultures because of its
convenience [17].
We mixed 2.5 lL DNA extract with Ready-To-Go
polymerase chain reaction (PCR) beads (Amersham
Pharmacia Tokyo, Japan), 2.5 lL 10 pM primers ITS5 (50 -GGAAGTAAAAGTCGTAACAAGG-30 ) and
ITS-4 (50 -TCCTCCGCTTATTGATATGC-30 ) [18],and 17.5 lL distilled water. The reaction mixture
was subjected to one denaturation cycle at 95 C for
4 min; 30 ampliﬁcation cycles at 94 C for 1 min,
50 C for 1 min, and 72 C for 2 min; and a ﬁnal
extension cycle at 72 C for 10 min in a PCR Thermal
Cycler MP (TaKaRa).
The PCR products were visualized by electrophoresis on 1.0 % agarose gels in 19 TBE buffer [0.04 M
Tris-boric acid, and 0.001 M ethylenediaminetetraacetic acid (EDTA, pH 8.0)] followed by ethidium
bromide staining. The PCR samples were puriﬁedusing a PCR puriﬁcation kit (QIAquick, Qiagen Co.
Ltd., Tokyo, Japan) and labeled with BigDye Terminator ver. 1.1 (Applied Biosystems, Foster City, CA,
USA) following the manufacturer’s protocol. The

Statistical Analysis
The comparison of the prevalence ratios of thechickens between the mainland and Nansei Islands,
the females and males, the chickens and ﬁghting cocks(Shamo strain), breeding scale (e.g., individual habits
and commercial or institutional farms), and housing
system (in cage including battery and individual cage
and free range) were evaluated using Pearson’s chisquare method with odds ratios and two-tailed
Fischer’s exact probability test via the website http://
vassarstats.net/odds2x2.html and the text http://
vassarstats.net/textbook/index.html. P values \0.05
were considered statistically signiﬁcant. The areadependent appearance of the fungal species of Ar.
multiﬁdum and Aphanoascus (Ap.) terreus was also
compared by the same method mentioned above.

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Mycopathologia (2014) 178:135–143

Table 2 Statistic comparisons of the prevalence ratio of dermatophyte and their related species in the poultries
Data
Number of dermatophyte-positive/
total number of birds (%)

SigniﬁcanceOdds ratio (range)

89/293 (30.4) versus 106/500 (21.2)

P \ 0.001

1.6216 (1.1674–2.2525)

Males versus females in totalbirds

217/320 (67.8) versus 92/473 (19.5)

P \ 0.0001

1.9657 (1.4176–2.7256)

Males versus females inmainland

50/158 (31.6) versus 39/135 (28.9)

Not signiﬁcant–Males versus females inNansei Islands

50/163 (30.7) versus 56/337 (16.6)

P \ 0.001

2.2203 (1.3463–3.4455)

Chickens versus ﬁghting
cocks in total birds

142/503 (28.2) versus 53/148 (35.8)

P \ 0.001

1.9762 (1.3458–2.9019)

Chickens versus ﬁghting
cocks in males

68/209 (32.5) versus 35/111 (31.5)

Not signiﬁcant–

Chickens versus ﬁghting
cocks in Females

74/436 (17.0) versus 18/37 (48.6)

P \ 0.001

4.6344 (2.3211–9.2534)

Chickens versus ﬁghting
cocks in Mainland

56/228 (24.6) versus 33/65 (50.8)

P \ 0.001

3.1674 (1.7873–5.6131)

Chickens versus ﬁghting
cocks in Nansei Islands

86/417 (20.6) versus 20/96 (20.8)

Not signiﬁcant–Individual versus Farm in
total birds

82/263 (31.2) versus 113/530 (21.3)

P \ 0.01

1.6718 (1.1976–2.3338)

Individual versus farm inmalesa58/166 (34.9) versus 48/155 (31.0)

Not signiﬁcant–Individual versus farm infemalesa

24/97 (24.7) versus 65/375 (17.3)

Not signiﬁcant–Individual versus farm inmainland

28/78 (35.9) versus 61/215 (28.4)

Not signiﬁcant–Individual versus farm in
Nansei Islands

54/185 (29.2) versus 52/315 (16.5)

P \ 0.001

2.0849 (1.3499–3.2200)

Cage versus free range intotal birds

55/367 (24.6) versus 140/426 (32.9)

P \ 0.001

2.7769 (2.0429–5.6727)

Cage versus free range inmalesaCage versus free range infemalesa

29/115 (25.2) versus 32/205 (15.6)

Not signiﬁcant–

26/252 (10.4) versus 66/221 (29.9)

P \ 0.001

3.7012 (2.3485–8.6923)

Cage versus free range inmainlanda

8/19 (42.1) versus 81/274 (29.6)

Not signiﬁcant–Cage versus free range inNansei Islandsa

47/348 (13.5) versus 59/152 (38.8)

P \ 0.001

4.0629 (1.6152–6.3607)

Comparisons

Area
Mainlands versus Nansei
Islands
GenderStrainBreeding scale

Housing systemaData were not shown in Table 1

Signiﬁcantly higher data were indicated in bold letters

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139

Results

according to the housing system of the mainland
samples, while a signiﬁcantly higher prevalence wasobserved in the free-range housing system in the
Nansei Islands (P \ 0.001; odds ratio 4.0629).
Two hundred twenty-four dermatophytes andrelated species were isolated in this survey. The
number of positive birds and the number of isolates did
not match because of duplicate or more isolates and/or
genotypes from ten females and 13 males. Isolated
species were identiﬁed based on ITS rDNA genesequences with more than 99 % identity to the known
isolates in the GenBank database and included 101 Ar.
multiﬁdum, 83 Ap. terreus, ﬁve Uncinocarpus queenslandicus, two U. reesii, one Ap. pinarensis, one
Amauroascus (Am.) kuehnii, one Ar. simii, one
Gymnoascus petalosporus, and one M. gallinae
isolates [4]. In addition, 28 Chrysosporium spp.
isolates were identiﬁed based on the gene sequenceswith \99 % homology to the known species(Table 3). The ITS rDNA gene sequences of theisolates were deposited in the GenBank database
under the accession numbers AB861650–AB861872
and AB667976 for M. gallinae [4].
Ar. multiﬁdum, Ap. terreus, and Chrysosporium
spp. were isolated from all areas, genders, strains,
breeding scales, and housing systems. The backgroundof the M. gallinae isolate from a male Shamo has been
described in a previous report [4]. U. queenslandicus
isolates were recorded from the mainland in both sexesof chicken strains with individual breeding andhousing in cages. The remaining species were limited
to the Nansei Islands. U. reesii was isolated frommales and females, breeding scales, and housing
systems in the Shamo strain. Ap. pinarensis was
isolated from a female chicken, Am. kuehnii was
isolated from a male chicken, and Ar. simii was
isolated from a male Shamo with individual breeding
scale and housing in cages, whereas G. petalosporus
was isolated from a female chicken in farm housing ina battery cage (Table 3).
Ar. multiﬁdum had a signiﬁcantly higher dermatophyte prevalence ratio in the Nansei Islands than in themainland (P \ 0.001; odds ratio 7.4622). The prevalence ratio of Ar. multiﬁdum in chickens was higher
than that in Shamo (P \ 0.05; odds ratio 1.9518). The
prevalence ratio of dermatophytes and related fungi in
Ap. terreus was signiﬁcantly higher in the mainland
than in the Nansei Islands (P \ 0.001; odds ratio
4.7866). Both Ar. multiﬁdum and Ap. terreus exhibited

Of the 793 birds that were examined, dermatophytes orrelated species were recovered from the combs of 195(24.6 %). The prevalence ratios based on the gender,
strain, breeding scale, and housing system are shownin Table 1. In addition, a marked clinical change was
detected in a ﬁghting cock that was positive for M.
gallinae [4].
The results of the comparisons of area, gender,
strain, breeding scale, and housing system are shownin Table 2. The prevalence ratio of the birds from themainland was signiﬁcantly higher than that of the
Nansei Islands (P \ 0.001; odds ratio 1.6216).
The prevalence ratio of the males was higher thanthat of the females (P \ 0.001; odds ratio 1.9657). No
signiﬁcant difference was noted between the ratios ofmales and females from the mainland. However, theprevalence ratio of males from the Nansei Islands was
signiﬁcantly higher than that of the females(P \ 0.001; odds ratio 2.2203).
The prevalence of dermatophyte-related fungi in
the Shamo strain was higher than that in chickens ofboth sexes (P \ 0.001; odds ratio 1.9762). The
comparison between female chickens and female
Shamo strain birds showed a signiﬁcant difference(P \ 0.001; odds ratio 4.6344). The signiﬁcance wasalso detected in the comparison of females from themainland and those from the Nansei Islands(P \ 0.001; odds ratio 3.1674). The comparisons inmales were not signiﬁcantly different.
The breeding scale by individual habit exhibited asigniﬁcantly higher positive ratio than that of commercial or institutional nursing farms (P \ 0.01; odds
ratio 1.6718). There was no signiﬁcant difference inthe prevalence of dermatophyte-related fungi according to the breeding scale of birds in the mainland andbirds grouped by gender, while a signiﬁcant difference
was detected in the individual breeding scale in the
Nansei Islands (P \ 0.001; odds ratio 2.0849).
The free-range housing system had a signiﬁcantly
higher positive ratio than that of birds in cages(P \ 0.001; odds ratio 2.7769). No signiﬁcant difference was noted in the housing system of the male sex,
but a signiﬁcant difference was observed in theprevalence of dermatophyte-related fungi according
to the housing system of females (P \ 0.001; odds
ratio 3.7012). No signiﬁcant difference was observedin the prevalence of dermatophyte-related fungi

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Mycopathologia (2014) 178:135–143

Table 3 Details of 224 isolates
Species or genus(number of isolates)

Area

Sex

Strain of bird

ML

NI

F

M

C

S

Breedingscale
I

Housing system

Arthroderma multiﬁdum (101)

17 (18.7)

84 (63.2)

44 (42.3)

57 (47.5)

81 (49.4)

20 (33.3)

61 (59.2)

40 (33.1)

40 (60.6)

Aphanoascus terreus (83)53 (58.2)

30 (22.6)

43 (41.3)

40 (33.3)

55 (33.5)

28 (46.7)

21 (20.4)

62 (51.2)

13 (19.7)

70 (44.3)

Chrysosporium sp. (28)

17 (18.7)

11 (8.3)

10 (9.6)

18 (15.0)

20 (12.2)

8 (13.3)

11 (10.7)

17 (14.0)

2 (3.0)26 (16.5)

Uncinocarpus queenslandicum(5)

5 (5.5)0 (0)4 (3.8)1 (0.8)5 (3.0)0 (0)5 (4.9)0 (0)5 (7.6)0 (0)Uncinocarpus reesii (2)0 (0)2 (1.5)

1 (1.0)1 (0.8)0 (0)2 (3.3)

1 (1.0)1 (0.8)1 (1.5)1 (0.6)

Aphanoascus pinarensis (1)0 (0)1 (0.8)1 (1.0)0 (0)1 (0.6)0 (0)1 (1.0)0 (0)1 (1.5)0 (0)Amauroascus kuehnii (1)0 (0)1 (0.8)0 (0)1 (0.8)1 (0.6)0 (0)1 (1.0)0 (0)1 (1.5)0 (0)Arthroderma simii (1)0 (0)1 (0.8)0 (0)1 (0.8)0 (0)1 (1.7)

1 (1.0)0 (0)1 (1.5)0 (0)Gymnoascus petalosporus (1)0 (0)1 (0.8)1 (1.0)0 (0)1 (0.6)

0 (0)

0 (0)

1 (0.8)1 (1.5)

0 (0)

FA

CA

FR
61 (38.6)

Microsporum gallinae (1)1 (1.1)

0 (0)

0 (0)

1 (0.8)0 (0)1 (1.7)

1 (1.0)0 (0)1 (1.5)0 (0)Total numbers of isolates (224)

93

131

104

120

164

60

103

121

66

158

The percentage was calculated from the number of isolate(s) divided by the total number of isolates times 100
ML mainland, NI Nansei Islands, F female, M male, C chicken, S ﬁghting cock (Shamo); I individual, FA farm, CA battery and individual cage, FR indoor andoutdoor free-range housing

target fungal species, because the housing management was out of political control until October 1, 2011.
Direct contact with soil by the free-range housing
system of the birds may increase the prevalence ratioof the fungal species. We supposed that the prevalence
ratio might reﬂect soil and environmental fungal ﬂora.
There were only two pathogenic dermatophyte
isolates: one Ar. simii isolate and one M. gallinae
isolate. The ratio of pathogenic species was extremely
low: two of 793 chickens, corresponding to 0.025 %.
Furthermore, these fungal species rarely caused zoonotic infection. Human records of Ar. simii infections
worldwide were reviewed by Beguin et al. [6].
However, both the rare zoonotic dermatophyte species
derived from the Shamo strain seemed to be endemic
to Japan. To our knowledge, the isolation of Ar. simii
from autochthonous animals is the ﬁrst record inJapan, although a previous study showed the isolationof the fungal species from an imported chimpanzee[5].
Interestingly, there were regional differences in theprevalence ratio of the major two fungal species, Ar.
multiﬁdum and Ap. terreus. The former specie was the
predominant species in the Nansei Islands and thelatter one in the mainlands. These results suggest thatthe differences are climate and environmental based.
The above two major species may not become
pathogenic. According to Chanasee et al. [19],signiﬁcantly higher prevalence ratios in individual
breeding scales than in farms (P \ 0.001; odds ratios
1.9861 and 4.1033, respectively). On the other hand,
Ar. multiﬁdum showed a signiﬁcantly higher ratio inbirds housed in cages (P \ 0.001; odds ratio 2.4464),
while Ap. terreus exhibited a signiﬁcantly higher ratioin free-range housing birds (P \ 0.001; odds ratio
3.2430) (Table 4).

Discussion
The average temperature in Tokyo, as a representative
area of the mainland, is 16.8 C and that of Naha of the
Nansei Islands is 23.1 C based on the data by theJapan Meteorological Agency. Chickens in the temperate regions (mainland) showed signiﬁcantly higher
prevalence ratio of dermatophyte and related species
than chickens in the subtropical area (Nansei Islands),
indicating that the prevalence of dermatophyte andrelated species was not correlated with the meanannual temperature.
The prevalence ratio was higher in males than infemales and in Shamo strain than in chickens, which
seems to reﬂect the vigorous action of males andShamo strain. Breeding scales also inﬂuenced theprevalence ratio. Sloppy management of the individual housing of the birds could increase the number of

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141

Table 4 Statistical comparisons of the prevalence ratio of Ar. multiﬁdum and Ap. terreus in poultry
Data
Number of dermatophyte-positive/
total number of isolates (%)

SigniﬁcanceOdds ratio (range)

Mainland versus Nansei
Islands

17/91 (18.7) versus 84/133 (63.2)

P \ 0.001

7.4622 (3.9582–14.068)

Males versus females

44/104 (42.3) versus 57/120 (47.5)

Not signiﬁcant–

Chickens versus ﬁghting
cocks

81/164 (49.4) versus 20/60(33.3)

P\ 0.05

1.9518 (1.0521–3.6207)

Individual versus farm

51/103 (49.5) versus 40/121 (33.1)

P\ 0.05

1.9861 (1.1562–3.4115)

Cage versus free range

40/66 (60.6) versus 61/158 (38.6)

P\ 0.001

2.4464 (1.3581–4.4068)

Mainland versus Nansei
Islands

53/91 (58.2) versus 30/133 (22.6)

P\ 0.001

4.7866 (2.6753–8.5712)

Males versus females

43/104 (41.3) versus 40/120 (33.3)

Not signiﬁcant–

Chickens versus ﬁghting
cocks

55/164 (33.5) versus 28/60 (46.7)

Not signiﬁcant–Individual versus farm

21/103 (20.4) versus 62/121 (51.2)

P\ 0.001

4.1033 (2.2579–7.4571)

Cage versus free range

13/66 (19.7) versus 70/158 (44.3)

P\ 0.001

3.2430 (1.6380–6.4207)

Comparisons

Arthroderma multiﬁdum

Aphanoascus terreus

Signiﬁcantly higher data were indicated in bold letters

Am. kuehnii was isolated from crater soil in India[31]. However, identiﬁcation based on ITS rDNA genesequences was insufﬁcient because of more than 10 %of diversities among the species tree via BLAST
search. The identity of the present Am. kuehnii to the
ex-type strain: CBS 539.72 was 98 %; however, thehomologies to the other Am. kuehnii sequences weremore than 99 % located at the same subcluster at the‘‘Distance tree of results’’ via BLAST search. Then,
we conﬁrm the isolate as Am. kuehnii.
G. petalosporus was isolated from a bird sanctuary[32], suggesting the isolate is related to a chickenbreeding environment.
Identiﬁcation of Chrysosporium-like isolates at the
species level requires further investigation. Chrysosporium spp. are known causative agents for mycotic
disease in poultry [9, 10] and are often isolated fromfeathers [15, 16, 22] and animal-related environments[7–15, 19, 23, 26]. Human cases of onychomycosis[33] and systemic infection in a patient with acutelymphocytic leukemia caused by Chrysosporium sp.
[34] have been reported. However, the present isolates
were not matched to pathogenic Chrysosporium spp.
based on the ITS rDNA sequences.
In conclusion, the predominant fungal species in
the Main Lands was Ap. terreus and that in the Nansei

pathogenicity of Ar. multiﬁdum to guinea pig skin andthe peritoneal cavity of mice were negative. Therefore,
we suspect that there is currently no need for concern
regarding to Ar. multiﬁdum infection. Ap. terreus is akeratinophilic fungus and has been isolated from soil
and birds [20–23], however, this fungal species has norecord of pathogenicity.
Uncinocarpus queenslandicus (Chrysosporium
queenslandicus anamorph) has been isolated fromindoor samples from India [24], soil from Egypt [25]and Kuwait [26]. This species also caused infections insnakes [27]. The present isolates are correspondent tothe ﬁrst isolates of this species in Japan.
U. reesii is related to highly pathogenic fungi, forexample, Coccidioides spp. and Paracoccidioides spp.
[28]. Isolation of this fungal species has been recorded
in India [29] and Egypt [25]. The present isolates arealso the ﬁrst report in Japan.
The identiﬁcation of the present Ap. pinarensis
isolate was based on ITS rDNA gene sequences(AB861811) exhibiting 99 % identity to AJ439433
derived from Ap. pinarensis isolated in Cuba. However, we did not detect ascospores with an irregularly
reticulate wall and two prominent and delicate equatorial rims under microscopy described by Cano et al.
[30].

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Islands was Ar. multiﬁdum. In addition, pathogenic
fungal species for humans and animals were limited to
M. gallinae [4] and Ar. simii corresponding to 0.025 %in the present study.
Acknowledgments This study was supported in part by theSpecial Research Fund for Emerging and Re-emerging
Infections of the Ministry of Health, Welfare and Labour(Grant No. H-21-Shinkou-004) and by a Grant-in-Aid for
Scientiﬁc Research (c) by the Japan Society for the Promotion of
Science (JSPS, No. 23580419). We express our gratitude to Drs.
Takako Murano (Chiba Prefecture), Tadao Ikeda (Association
Pasteur Japan), Eichiro Matayoshi and Taketoshi Kawamitsu(Okinawa Veterinary Medical Association), Takako Okubo andMasami Sudo (Ibaraki Prefecture), Tomo Inomata (Azabu
University), and Shigeyuki Matsui and Moriji Iketani (Shizuoka
Prefecture). We also thank Ms. Kyoko Yarita, Akiko Takayama,
and Yuki Hanami (Medical Mycology Research Center, Chiba
University) for technical support.
Conﬂict of interest The authors report no conﬂict of interest.
The authors alone are responsible for the content and writing of
the paper.

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http://www.biomedcentral.com/1756-0500/7/116

RESEARCH ARTICLE

Open Access

Isolation and sequencing of swine carbonic
anhydrase VI, an enzyme expressed in the swine
kidneyToshiho Nishita1*, Juro Yatsu2, Masaru Murakami3, Shino Kamoshida3, Kensuke Orito4, Nobutune Ichihara5,
Kazuyoshi Arishima6 and Hideharu Ochiai7*

Abstract
Background: Carbonic anhydrase VI (CA-VI) is produced by the salivary gland and is secreted into the saliva.
Although CA-VI is found in the epithelial cells of distal straight tubule of swine kidneys, the exact function of CA-VI
in the kidneys remains unclear.
Results: CA-VI was located in the epithelial cells of distal straight tubule of swine kidneys.
A full-length cDNA clone of CA-VI was generated from the swine parotid gland by reverse transcription polymerase
chain reaction, using degenerate primers designed based on conserved regions of the same locus in human andbovine tissues.
The cDNA sequence was 1348 base pairs long and was predicted to encode a 317 amino acid polypeptide with aputative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI was most similar(77.4%) to that of human CA-VI. CA-VI expression was confirmed in both normal and nephritic kidneys, as well as
parotid. As the primers used in this study spanned two exons, the influence of genomic DNA was not detected.
The expression of CA-VI was demonstrated in both normal and nephritic kidneys, and mRNA of CA-VI in the normal
kidneys which was the normalised to an endogenous β–actin was 0.098 ± 0.047, while it was significantly lower inthe diseased kidneys (0.012 ± 0.007). The level of CA-VI mRNA in normal kidneys was 19-fold lower than that of theparotid gland (1.887).
Conclusions: The localisation of CA-VI indicates that it may play a specialised role in the kidney.
Keywords: Carbonic anhydrase VI, cDNA, Swine kidney, mRNA, RT-PCR, Kidney disease

Background
Carbonic anhydrase (CA; EC 4.2.1.1) is a well-characterised enzyme that catalyses the reversible hydrationof CO2 to form HCO3− and protons according to thefollowing reaction: CO2 + H2O ↔ H2CO3 ↔ HCO3− +H+. The first reaction is catalysed by CA and the second
reaction occurs instantaneously. The mammalian α-CA
gene family includes at least 15 enzymatically active isoforms with different structural and catalytic properties. Sixof the active CA isozymes are cytosolic (CA-I, −II, −III,* Correspondence: nishida@azabu-u.ac.jp; ochiaih@azabu-u.ac.jp
1
Laboratory of Physiology I, School of Veterinary Medicine, Azabu University,
1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201, Japan
7
Research Institute of Biosciences, School of Veterinary Medicine, Azabu
University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201, Japan
Full list of author information is available at the end of the article−VII, −VIII, and -XIII), 4 are membrane-associated (CAIV, −IX, −XII, and -XIV), 2 are mitochondrial (CA-VA
and CA-VB), and 1 is secretory form (CA-VI), while 2
CA-related proteins (CA-X and XI) are inactive variants[1-3]. The physiological function of carbonic anhydrase is
to maintain the acid–base balance in various tissues andbiological fluids [4].
CA-VI has been previously purified from the saliva
and parotid glands of sheep [5] humans [6], cattle [7],pigs [8], and dogs [9]. The enzyme is localised in the serous acinar and demilune cells of the parotid and submandibular glands [7,10], from which it is secreted into
saliva. CA-VI may participate in the regulation of salivary pH and buffer capacity, and protect the mouth andupper alimentary canal against excess acidity [11]. On© 2014 Nishita et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.

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the other hand, Hooper et al. [12] suggested that theunique oligosaccharide structures on bovine CA-VI might
have an antibacterial function. Karhumaa et al. [13] alsosuggested that the glycoproteins on CA-VI confer multifunctionality on the enzyme.
To date, human, bovine, mouse, canine, and equine
CA-VI cDNAs have been cloned successfully [14-18].
CA-VI was previously reported to be present in the parotid gland, saliva, bile, and serum of pigs [19]. However,
the exact physiological and clinical significance of swine
CA-VI has not been established. Here, we demonstrated
immunohistochemical localization of CA-VI in the swine
kidney and deduced the nucleotide sequence of swine
CA-VI. Furthermore, we demonstrate expression of CAVI in both normal and diseased kidneys of pigs. These
data provide an initial step toward exploring the physiological and pathological roles of CA-VI.

Methods
Tissue samples

Samples from normal kidneys (n = 9), parotid gland (n = 1),
and diseased kidneys (n = 19) of domestic pigs were takenfrom a slaughterhouse in Miyagi prefecture (Japan).
Macroscopically, diseased swine kidneys showed signsof necrosis (n = 7), nephritis (n = 7), pyelectasis (n = 3),
and cystic kidney (n = 2).
All samples were obtained in accordance with theguidelines of the Laboratory Animal Care Committee of
Azabu University, Japan, and programs accredited by the
Office of Laboratory Animal Welfare (OLAW) USA(#A5393-01) were used.
The samples were immediately fixed in neutralised 10%
formalin and Bouin’s solution, dehydrated with a graded
series of alcohols, cleared with xylene, and then embedded in paraffin wax blocks that were cut into 4-μmthick histological sections. To observe the morphologicchanges, renal tissue samples were stained with hematoxylin
and eosin. Microscopic inspection of diseased kidney
revealed predominantly renal tubule necrosis and stromal
cell permeation.
Immunohistochemical staining of CA-VI in kidney

Biopsies of pig kidneys were performed. The samples
were immediately fixed in neutralized 10% formalin and
Bouin’s solution, dehydrated with a graded series of alcohols, cleared with xylene and then embedded in paraffin
wax blocks that were cut into 4 μm-thick histologicalsections.
Endogenous peroxidase activity was blocked in deparaffinized and rehydrated sections using 0.3% H2O2 inmethanol, and immersion in normal goat serum (2% inPBS) for 20 min blocked fragment crystallizable receptors. Monospecific primary antisera (diluted 1:2000)
against swine CA-VI produced in our laboratory [8] wasused to detect the respective isozymes during a 1-h incubation. Antibody binding was visualized using the Vectastain Elite avidin-biotin-peroxidase complex kit (ABCPOD reagent kit; Vector) and diaminobenzidine (DAB)
according to the manufacturer’s protocol.
The kidney sections were stained with hematoxylin,dehydrated through a graded alcohol series, and mounted
on coverslips.
Samples were observed and photographed under alight microscope.
cDNA sequence of pig CA-VI

Total RNA was isolated from the parotid gland of ahealthy pig by using RNA extraction solution (Isogen;Nippon Gene, Japan). Degenerate primers used for theamplification of a central region of swine CA-VI cDNA
were designed based on the conserved sequences in human [14] and bovine [15] cDNAs. Reverse transcriptionpolymerase chain reaction (RT-PCR) amplification wasthen performed using the SuperScript One-Step RT-PCR
system (Life Technologies, MS, USA), according to the
manufacturer’s instructions. The RT-PCR products wereverified to be single bands on agarose gel electrophoresis
and were then purified using Ultrafree-DA (Millipore,
MA, USA) and Microcon YM-100 (Millipore). The purified DNA was cloned into a pGEM-T Easy cloning vector (Promega, WI, USA) and sequenced using a Thermo
Sequenase Fluorescent Labeled Primer Cycle sequencing
kit (Amersham Biosciences, NJ, USA) and a DSQ2000L
DNA sequencer (Shimadzu, Japan). In order to minimise
PCR errors, sequences from several clones were analysed. The consensus nucleotide sequence showed a highsimilarity of approximately 80% to bovine CA-VI cDNA
sequences. In order to amplify the 3′ and 5′ regions of
pig CA-VI cDNA, 3′- and 5′ -rapid amplification of
cDNA ends (3′- and 5′-RACE) methods were employed.
3′-RACE was performed as previously described [20]and 5′-RACE was carried out using a SMART RACE
cDNA amplification kit (Clontech, CA, USA), according
to the manufacturer’s protocol.
Each RACE product was cloned into the pGEM-T
Easy vector and sequenced by employing an AmpliTaq
Dye Terminator Cycle Sequencing FS Ready Reaction kit(Applied Biosystems, CA, USA) on a 373A DNA sequencer (Applied Biosystems).
RNA extraction from FFPE tissue

A total of 19 diseased and 9 healthy kidney samples informalin-fixed, paraffin-embedded (FFPE) were used inthis study. The 4 pieces of 10-μm-thick FFPE sections
were cut from each paraffin block and collected in a 1.5mL tube. A NucleoSpin FFPE RNA isolation kit for FFPE
Tissues (Takara, Kyoto, Japan) was then used according
to the manufacturer’s protocol. Briefly, 1 mL of xylene389

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was added into the 4 pieces of 20-μm-thick FFPE sections to remove traces of paraffin. The tissues weredigested with proteinase K at 60°C for 3 h and treatedwith DNase I. After washing, total RNA, including asmall miRNA fraction, was eluted with distilled waterand stored at −80°C until use.
cDNA synthesis and PCR evaluation of pig CA-VI

Reverse transcription was performed using a SuperScript
III First-Strand Synthesis System (Invitrogen, Carlsbad,
CA) according to the manufacturer protocol, and theresulting cDNA was used as a template for RT-PCR. Primer set used was purchased from Takara (Kyoto, Japan).
Primer sequences for the CA-VI genes were following;
forward: 5′-AGAATGTCCACTGGTTTGTGCTTG-3′;
reverse: 5′-GGATGGTCTTGTTCTGGTCATTCA-3′.
The expected product size of the PCR using this primer
set is 102 bp. PCR reaction was performed using Takara
Ex Taq™ Hot Start version (Takara, Kyoto, Japan). Amplification was conducted using the following protocol: initialdenaturation phase at 95°C for 30 s, and then 40 cycles at
95°C for 15 s for denaturation, then at 60°C for 30 s for annealing and extension step. PCR products were loaded in
3% agarose gel.
Real-time PCR evaluation of pig CA-VI

Real-time quantitative PCR was performed using a Thermal Cycler Dice® Real Time System II (Takara). Samples(final volume of 25 μL) were run in duplicate and contained the following: X1 SYBR® Premix Ex Taq™ II(Takara) 1 μL 10 mM of each primer and 2 μL cDNA
template. Amplification conditions were carried out as
manufacturer’s protocol. The primer set used in CA-VI
amplification was the same as described above. The
housekeeping gene β-actin was used as a reference gene(forward: 5′-TCTGGCACCACACCTTCT-3′, reverse; 5′TGATCTGGGTCATCTTCTCAC-3′; DDBJ accession
number AY550069). The Real-Time RT-PCR results arepresented as the gene expression of the target gene (CAVI) relative to that of the housekeeping gene (β-actin),and CA-VI gene expression levels are achieved using the2-ΔΔCT method of quantification [21].

in Figure 1. CA-VI was located in the epithelial cells of
distal straight tubule of swine kidneys.

Nucleotide sequence of swine CA-VI cDNA

A 1348-bp nucleotide sequence corresponding to fulllength swine CA-VI cDNA was obtained (accession
number AB333806), consisting of a 951-bp open reading frame encoding swine CA-VI of 317 amino acids(Figure 2A). A typical polyadenylation signal was foundin the 3′ untranslated region. The deduced 317 aminoacids included a signal peptide (17 amino acids) typicalof most secreted proteins where the region was enriched with hydrophobic residues [22]; thus, the predictedmature protein consisted of 300 amino acids. To determine the genomic structure, the UCSC genome browser
site (http://genome.ucsc.edu/) was used to align canine
genomic sequences and the cDNA sequence of CA-VI(Figure 2B).
The amino acid sequence of the deduced swine mature
CA-VI was 3 residues shorter than that of canine CAVI, and 9 residues (in the carboxy-terminal region) longer than that of mouse and human CA-VI, respectively.
The sequence of swine CA-VI showed approximately
77.4% identity to human CA-VI (Figure 3). Two cysteine
residues (amino acid positions 25 and 207), which are
known to form intra-molecular disulphide bonds insheep CA-VI [23], are conserved, and 3 histidine residues (amino acid positions 94, 96, and 121) responsible
for zinc binding were also found. In addition, 2 potential
N-glycosylation sites (Asn-X-Thr/Ser) were detected; 1of these (amino acid positions 239–241, Asn-Lys-Thr) is
known to be glycosylated in sheep CA-VI [23].

Statistical analysis

To compare differences in the relative levels of CA-VI
mRNA between normal and nephritic kidneys, statisticalanalysis was carried out using an unpaired t-test. Valuesof P < 0.01 were considered to be statistically significant.

Results
Immunohistochemical study

The results of immunohistochemical localization of CAVI in the kidneys of clinically normal pigs were shown390

Figure 1 Immunohistochemical localization of CA-VI in theswine kidney. CA-VI was found in the epithelial cells of the distal
straight tubule of swine kidneys. Scale bar: 50 μm.

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Figure 2 Nucleotide and deduced amino acid sequences of swine CA-VI cDNA. The DDBJ accession number is AB333806. The termination
codon is asterisked and the polyadenylation signal is boxed. The putative signal peptides consisting of 17 amino acid is underlined. Arrows
indicate the positions of introns (A). Alignment of the cDNA sequence with swine genome chromosome 1 predicts 8 exons of 70–447 base pairs.
Exon ■: intron ▬(B).

Figure 3 Comparison of the amino acid sequences of mature CA-VI in pigs, dogs, humans, sheep, and cattle. Multiple sequencealignments were performed using the Genetyx program (version 12). Asterisks and dots indicate identical residues and conservative substitution,
respectively. Cys residues are indicated by diamonds, and His residues, by triangles. These are likely to form an intra-molecular disulphide bondand to bind to zinc, respectively. The potential glycosylation sites are indicated by circles.

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The levels of CA-VI mRNA in the kidney

Expression levels of CA-VI mRNA were measured by
qRT-PCR in FFPE samples of normal and diseased kidneys (Figure 4). The relative level of CA-VI mRNA in thenormal kidneys was 0.098 ± 0.047, while it was significantly
lower in the diseased kidneys (0.012 ± 0.007; p = 2.71 × 10−8,
Figure 5). The level of CA-VI mRNA in normal kidneys was 19-fold lower than that of the parotid gland(1.887).

Figure 4 RT-PCR analysis of CA-VI expression from FFPE
samples. m; 100-bp ladder, 1; parotid, 2; normal kidney, 3;
nephritic kidney.

Expression of swine CA-VI mRNA in kidney

Figure 4 shows the RT-PCR analysis of CA-VI expression from FFPE samples. CA-VI expression was confirmed in both normal and nephritic kidneys, as well as
parotid. As the primers used in this study spanned two
exons, the influence of genomic DNA was not detected.

Figure 5 Comparison of CA-VI expression in normal or diseased
kidneys from FFPE samples as shown by quantitative RT-PCR.
CA-VI levels were normalised to an endogenous β-actin mRNA. The
values represent the mean ± SD. Asterisks indicate a significant
difference between the normal and diseased kidneys (P < 0.05,
unpaired t-test).

Discussion
Fernley et al., [24] reported that CA-VI was absent fromthe sublingual salivary gland, kidney, lung, adrenal, brain,
skeletal muscle, liver, heart, pancreas, small intestine, andcerebrospinal fluid of sheep. However, CA-VI was foundin the lung, skeletal muscle, liver, heart, and pancreas ofpigs by using ELISA [19]. In the present study, we show
for the first time that CA-VI is expressed in the epithelial
cells of distal straight tubule of swine kidneys.
The expression of bovine CA-VI mRNA has previously
been detected in the parotid gland, liver, and mammary
gland of cow [25,26]. Canine CA-VI mRNA signals werestrong in the major salivary glands and weaker in theminor salivary glands and esophagus, and were absent in
the pancreas, liver, and almost all parts of the digestive
tract, except the esophagus [17]. In the horse, CA-VI
mRNA was detected in the digestive tract, salivary glands,
testis, thyroid gland, and liver, but not in nerve tissue,
skeletal muscle, spleen, or lymph node [18].
To our knowledge, there have been no previous studies on CA-VI mRNA expression in normal and diseased
kidneys. Although the levels of CA-VI mRNA werelower in diseased kidneys, further studies are necessary
to determine whether CA-VI is a suitable biomarker forkidney disorders.
In the kidney, cytosolic CA-II accounts for >95% of all
CA activity. In humans, rabbits, and bovine species,
most of the remaining ~ 5% of renal CA is membraneassociated and consists of CA-IV and CA-XII [27] CA-II
is expressed in the renal proximal tubule; thin descending limb; thick ascending limb; and intercalated cells of
the cortical collecting duct, outer medullary collecting
duct, and inner medullary collecting duct. Schwartz [28]described that the function of CA-II in renal H+/HCO3−
transport is perhaps best understood by examining CAII interactions with specific transporters.
Räisänen et al., [29] suggested that CA-III is an oxyradical scavenger that protects cells from oxidative damage. Using HK-2 cells, which represent an establishedmodel for normal human proximal tubule cells, Gailly
et al. [30] reported that exposure to 1 mM H2O2 induced a significant increase in CA-III mRNA expression.
This suggests that CA-III may be a multifunctional392

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enzyme, and that 1 of the functions is to protect cells
from oxidative damage.
Recently, Pertovaara et al. [31] reported that the levelsof anti-CA-VI antibody were significantly higher in patients with primary Sjogren’s syndrome (pSS). The
amount of antibody correlated significantly with urinary
pH, and inversely with serum sodium concentrations.
Anti-CA-VI antibody seems to be associated with renal
acidification capacity in patients with pSS. However, therole of CA-VI autoantibodies in modulating urinary pH
in the kidney remained perplexing, since the presence of
CA-VI has never been demonstrated in the human kidneys. Pertovaara et al. [31] speculated that anti-CA-VI
antibodies might exhibit cross-reactivity with CA-XIII
expressed in the kidneys. However, the molecular weightof CA-XIII is 30 kDa [2] and the subunit molecularweight of swine CA-VI is 37 kDa [8]. Furthermore, theamino acid sequence homology between human CA-VI
and CA-XIII is only 35%, which is also the degree ofhomology between CA-VI and CA-II [2]. We feel it isunlikely that cross-reactivity explains the results above.
In support of this, despite a 62% amino acid sequencehomology between equine CA-I and CA-II, sera raised
against each of these isoforms do not exhibit crossreactivity with the other [32]. These results indicate thatanti human CA-VI serum does not cross-react with bothhuman CA-XIII and CA-II.
The exact function of CA-VI in the kidneys remains unclear at this stage. However, based on our preliminary
data, additional studies should be conducted to determine
whether measuring the CA-VI concentration in the urineof pigs with kidney disorders is of clinical utility.

Conclusions
CA-VI was located in the epithelial cells of distal straighttubule of swine kidneys.
The cDNA sequence was 1348 base pairs long andwas predicted to encode a 317 amino acid polypeptide
with a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI was mostsimilar (77.4%) to that of human CA-VI. The expressionof CA-VI was demonstrated in both normal and nephritic kidneys, and the relative levels of CA-VI mRNA in
the nephritic kidneys were significantly lower than innormal kidneys. The level of CA-VI mRNA in normal
kidneys was 19-fold lower than that of the parotid gland.
Abbreviations
CA-VI: Carbonic anhydrase VI; RT-PCR: Reverse transcription- polymerase
chain reaction.

Competing interests
The authors declare that they have no competing interests.

Authors’ contributions
TN, JY and HO were responsible for the study conception and design,development of the questionnaire, carried out the data collection, performed
descriptive statistical analyses and drafted and revised the manuscript. KO
helped in the descriptive statistical analyses. MM and KA helped in thedesign of the study and coordination. SK and NI performed the drafting of
the manuscript. All authors read and approved the final manuscript.
Acknowledgement
The authors declare that no funding support was obtained for this study.
Author details
1
Laboratory of Physiology I, School of Veterinary Medicine, Azabu University,
1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201, Japan. 2Miyagi
Prefectural Meat Sanitation Inspection Station, 314 Imaizumi, Sakuraoka,
Yoneyamacho, Tome-city, Miyagi 987-0031, Japan. 3Laboratory of Molecular
Biology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe,
Sagamihara, Kanagawa 252-5201, Japan. 4Laboratory of Physiology II, Schoolof Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara,
Kanagawa 252-5201, Japan. 5Laboratory of Anatomy I, School of Veterinary
Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa
252-5201, Japan. 6Laboratory of Anatomy II, School of Veterinary Medicine,
Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201,
Japan. 7Research Institute of Biosciences, School of Veterinary Medicine,
Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201,
Japan.
Received: 17 October 2013 Accepted: 24 February 2014
Published: 28 February 2014
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1
2

Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara, Japan
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

Microphthalmia-associated transcription factor (MITF) is a transcription factor that is expressed in limited types of cells, including
osteoclasts, but the expression and role of MITF during osteoclastogenesis have not been fully elucidated. The expression of the MITF-E
isoform but not that of the MITF-A isoform was induced in response to differentiation stimulation towards osteoclasts by receptor activatorof NF-κB ligand (RANKL) in both RAW264.7 cells and primary bone marrow cells. The RANKL-induced formation of tartrate-resistant acidphosphatase (TRAP)-positive multinucleated cells was inhibited in RAW264.7 cells expressing siRNA for MITF-E. Transforming growth
factor-β (TGF-β) enhanced RANKL-induced MITF-E expression and -TRAP positive multinucleated cell formation. In particular, TGF-βpotentiated the formation of larger osteoclasts. The expression levels of NFATc1, TRAP and CtsK, genes related to osteoclast development
and activity, were concurrently enhanced by TGF-β in the presence of RANKL. Furthermore, the expression of dendritic cell-speciﬁctransmembrane protein (DC-STAMP), Itgav, Itga2, Itga5, Itgb1, Itgb3 and Itgb5, genes related to cell adhesion and fusion, were up-regulated
by co-treatment with TGF-β. In particular, the regulatory expression of Itgav and Itgb5 in response to RANKL with or without TGF-β resembled
that of MITF-E. Because MITF is involved in cell fusion in some cell systems, these results imply a role for MITF-E as an enhancer ofosteoclastogenesis and that RANKL-induced levels of both MITF-E mRNA and of MITF-dependent gene expression are enhanced by treatment
with TGF-β. Copyright © 2014 John Wiley & Sons, Ltd.
key words—osteoclastogenesis; MITF; TGF-β; cell fusion; integrins

INTRODUCTION

differentiation depend on the cell culture system and the stageof osteoclast differentiation,7,8 TGF-β consistently stimulates
RANKL-induced osteoclastogenesis in differentiation models
using RAW264.7 cells.9–11 The detailed mechanism underlying the enhanced osteoclastogenesis by TGF-β in RAW264.7
cells is not, as of yet, fully characterized.
The regulation of osteoclast differentiation and maturation is
not determined only by secreted proteins from osteoblasts andstromal cells. Microphthalmia-associated transcription factor(MITF) is a transcription factor expressed in a tissue-speciﬁcmanner.12,13 MITF is also expressed in monocyte–macrophagelineage cells and osteoclasts.14 The Mitfmi allele produces an
impaired bone phenotype. The bones of Mitfmi/Mitfmi micepresent signs of osteopetrosis due to a lack of functional osteoclasts.14,15 MITF regulates osteoclastogenesis in at least two
steps. First, MITF up-regulates the expression of genes related
to osteoclast activity such as TRAP,16 chloride channel 7,17
cathepsin K (CtsK)18 and v-ATPase d2.19 Second, MITF
governs osteoclast development through the regulation of cellfusion. Dendritic cell-speciﬁc transmembrane protein (DCSTAMP) is required for efﬁcient cell–cell fusion of
osteoclasts,20 and transcription of DC-STAMP is positively
regulated by MITF.21 The activity of MITF in stimulating cellfusion has also been shown in different cell types. MITF expression in myoblasts and nascent myotubes is essential for

Osteoclasts, which degrade bone matrix through thesecretion of acid and various enzymes,1–3 are tartrateresistant acid phosphatase (TRAP)-positive multinucleatedcells formed by the fusion of precursor cells of monocyte–
macrophage lineage.4,5 Osteoclast differentiation is
supported by osteoblasts and stromal cells, which produce
factors affecting the number and activity of osteoclasts.
Macrophage-colony-stimulating factor (M-CSF) and receptor
activator of NF-κB ligand (RANKL) principally regulate
osteoclastogenesis. M-CSF is required for the survival and proliferation of osteoclast precursor cells.4,5 In contrast,
RANKLstimulates osteoclast differentiation through regulatory
expression of osteoclastogenesis-related transcription factors
including nuclear factor of activated T-cells c1 (NFATc1).4,5
In addition to these molecules, various cytokines, including transforming growth factor-β (TGF-β), are involved inosteoclastogenesis.3,6,7 TGF-β is a pluripotent growth factor
that modulates cell differentiation and maturation in diverse
types of cells.7,8 Although the effects of TGF-β on osteoclast*Correspondence to: Masayuki Funaba, Division of Applied Biosciences,
Graduate School of Agriculture, Kyoto University.E-mail: mfunaba@kais.
kyoto-u.ac.jp; Masaru Murakami, Laboratory of Molecular Biology, Azabu
University School of Veterinary Medicine. E-mail: murakami@azabu-u.ac.
jp

Received 11 November 2013
Revised 14 January 2014
Accepted 15 January 2014

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k. asai ET AL.
Table 1.

Oligonucleotide PCR primers for RT-qPCR
Oligonucleotide
3′-primer

GenBank
accession
number5′-GCTGGCGTAGCAAGATGCGTGA-3′5′-GCTGGCGTAGCAAGATGCGTGA-3′

AB_009397
AF_465624

5′-primer
MITF
MITF-A
5′-GAGGAGTTTCACGAAGAACC-3′
MITF-E
5′-CCAGATACACAGACAGTCACAG-3′
Osteoclast-related genes
NFATc15′-TCCAAAGTCATTTTCGTGGA-3′
TRAP
5′-GCCAAAGAGATCGCCAGAAC-3′
CtsK
5′-TGGACTGTGTGACTGAGAATTATGG-3′
Cell fusion-related genes
DC-STAMP
5′-CGAAGCTCCTTGAGAAACGA-3′
Itgav
5′-GGTGTGGATCGAGCTGTCTT-3′Itga25′-ACTTCCGGCATACGAAAGAAT-3′
Itga55′-CACCATTCAATTTGACAGCAA-3′
Itgb15′-ATGCAGGTTGCGGTTTGT-3′5′-GTGGGAGGGCAGTCCTCTA-3′
Itgb3
Itgb5
5′-TGCCACCTGCCAAGATGGCATA-3′ADAM85′-AAAGGCTCCGAGACAAATCC-3′
ADAM125′-CAGAGCATCCCAGCCAAG-3′5′-CTTTGCTTCCATCTCCCAGA-3′5′-GAAGTAGAAATTGTCCCCCAGAGA-3′5′-CCGTTCTGCTGCACGTATTG-3′

NM_016791
NM_007388
NM_007802

5′-GGACTGGAAACCAGAAATGAA-3′
5′-CAAGGCCAGCATTTACAGTG-3′
5′-TCAGCCAGCAGGTGATGTTA-3′
5′-TCCTCTCCCTTGGCACTGTA-3′5′-CATCCGTGGAAAACACCAG-3′5′-CAGGATATCAGGACCCTTGG-3′5′-CACGGACACTTCAAAGGATG-3′5′-TTGGAGAGCCCCGAGATAG-3′5′-CAGGCTGAGGATCAGGTCTC-3′

AB_109560
NM_008402
NM_008396
NM_010577
NM_010578
NM_016780
NM_010580
NM_007403
NM_007400

Housekeeping gene
G3PDH

5′-TGTCATCATACTTGGCAGGTTTCT-3′

NM_008084

5′-CGTGTTCCTACCCCCAATGT-3′

RT-qPCR, reverse transcription quantitative polymerase chain reaction; MITF, microphthalmia-associated transcription factor; NFATc1, Nuclear factor of activated T-cells c1; TRAP, tartrate-resistant acid phosphatase; CtsK, cathepsin K; DC-STAMP, dendritic cell-speciﬁc transmembrane protein; Itg, integrin;
ADAM, a disintegrin and metalloirotease domein; G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

Cell culture

the maturation of myotubes via the up-regulation of integrin
α9 (Itga9) expression.13
Microphthalmia-associated transcription factor is expressed
as a series of isoforms differing in their ﬁrst exons andpromoters.22–24 For most isoforms, the initial exon, which isisoform-speciﬁc exon, is spliced onto the later part of exon
1B and then to the common exons 2–922–24; at present, nine
MITF isoforms have been identiﬁed in mice, i.e. MITF-A,-B, -C, -D, -E, -H, -J, -M and -mc. MITF isoforms are
expressed in a cell type-speciﬁc manner.25–28 Transcriptional activities of MITF are overlapped but distinct among
MITF isoforms depending on the target gene.29–33
Here, we explored the role of MITF duringosteoclastogenesis and the relationship between MITF-E
isoform and the TGF-β pathway. Our results indicate that
the MITF-E isoform is induced in differentiating
osteoclasts, and the expression is required for efﬁcientosteoclast formation. In addition, TGF-β enhances theexpression level of MITF-E induced by RANKL andpotentiates the formation of larger osteoclasts, possibly
through the up-regulation of integrins.

RAW264.7 cells were cultured in alpha minimal essential
medium (α-MEM) with 6% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin
at 37 °C under a humidiﬁed 5% CO2 atmosphere. Primaryosteoclasts were basically differentiated as described by
Sankar et al.34 Bone marrow cells were obtained from thefemurs of adult C57BL/6 mice and cultured for 3 days in basal
medium, i.e. Dulbecco’s modiﬁed Eagle’s medium (DMEM)
with 10% heat-inactivated FBS, 100 U/ml penicillin and100 μg/ml streptomycin, supplemented with M-CSF(50 ng/ml). Subsequently, the cells were cultured in thebasal medium supplemented with M-CSF (25 ng/ml) in
the presence or absence of recombinant soluble RANKL(50 ng/ml). The animal experiments were approved by the
Animal Care and Use Committees of Azabu University.
Histochemical detection of TRAP
Tartrate-resistant acid phosphatase was detected by staining ofﬁxed cells by the use of an acid phosphatase, leukocyte kit(Sigma, St. Louis, MO, USA) according to the manufacturer’s
protocol. The cells were counter-stained with hematoxylin,
and the number of cells with more than three nuclei wascounted. In addition, the area of TRAP-positive cells wasmeasured by the use of Image J software, and the number of
TRAP-positive cells was counted for every cell size.

MATERIALS AND METHODS
Materials
The following reagents were purchased, recombinantmurine M-CSF and recombinant murine soluble
RANKL from Peprotech (Rocky Hill, NJ, USA) andrecombinant human TGF-β1 from R&D Systems(Minneapolis, MN, USA).

Osteoclast function assay
Osteoclast functionality was examined via a pit formation
assay using 96-well Corning Osteo Assay plates (Corning
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osteoclastogenesis by mitf-e and tgf-β
810 (Wako, Osaka, Japan), an automatic nucleic acid extraction system, according to the manufacturer’s protocol. The
concentration of RNA was determined from the absorbanceat 260 nm. The cDNA was synthesized by the use of highcapacity cDNA reverse transcription kit with rnase inhibitor(Life Technologies, Carlsbad, CA, USA), according to the
manufacturer’s protocol. The cDNA corresponding to 5 ngof total RNA was used as a template of real-time qPCR;
qPCR was performed by the use of SYBR Premix Ex-taq
II (Takara, Otsu, Japan) in Thermal Cycler Dice Real Time
System TP800 (Takara, Otsu, Japan), according to the
manufacturer’s protocol. The proﬁle of qPCR is as follows:
after denature for 30 s at 95 °C, 40 cycles consisting of 5 s at
95 °C and 30 s at 60 °C. Subsequently, melting curve analyses were performed by increasing temperature from 60 °C to
95 °C to verify that the PCR products are not primer dimers
but single products. Gene transcript level in each sample
was determined by standard curve method; the standard
DNA was prepared as described in the succeeding
texts. The PCR primers used are presented in Table 1. The
5′-primers for MITF-A and -E were selected from therespective isoform-speciﬁc region; the speciﬁcity of the PCRprimers was also veriﬁed by agarose gel electrophoresis of
the PCR product and subsequent staining with ethidium
bromide. The relative mRNA level was expressed as a ratiowith the G3PDH mRNA level. When the gene expression
level was below detection limit, it was assumed to express atthe detection limit.

Figure 1. MITF-E is induced during osteoclastogenesis. Gene transcriptlevels of the MITF-A isoform (A and B) and MITF-E isoform (C and D)
were quantiﬁed by RT-qPCR during osteoclastogenesis in RAW264.7 macrophage cells (A and C) and primary cultures of bone marrow cells (B and
D). (A and C) RAW264.7 cells were treated with or without RANKL(100 ng/ml) for 3 days. (B and D) Bone marrow cells were treated with
M-CSF (50 ng/ml) for 3 days. Non-adherent cells were subsequently treatedwith M-CSF (50 ng/ml) with or without RANKL (100 ng/ml) for an additional 3 days. The expression of the MITF isoforms was normalized to
G3PDH expression (n = 3). **p < 0.01

Preparation of standard DNA
To examine time-course changes in the expression of MITF
isoforms, the number of cDNA molecules was precisely
quantiﬁed as described previously.35 Brieﬂy, the cDNA for
MITF-A, MITF-E and G3PDH was individually ampliﬁed
by PCR. The PCR product by the use of Wizard SV Gel
and PCR Clean-Up System (Promega, Madison, WI,
USA). The amount of the PCR product was precisely
quantiﬁed by the use of a DNA 1000 Lab Chip Kit (Agilent
Technologies, Santa Clara, CA, USA) in an Agilent2100
bioanalyzer (Agilent Technologies, Santa Clara, CA,
USA). The molecular number of the PCR product wascalculated from the DNA mass and the molecular weight.
Several levels of the PCR product were prepared by serial
dilution and used as standard solutions of qPCR.

Inc., Corning, NY, USA) according to the manufacturer’s
protocol. The pits were observed via light microscopy. The
pit area was measured using Image J software, and the total
pit area per well was calculated.
siRNA transfection
The siRNA for MITF-E were prepared (Bonac Inc., Kurume,
Japan). The nucleotide sequences used were 5′-GGUUACG
UAUCUUGUCCACAG-3′ and 5′- GUGGACAAGAUACGUAACCUC-3’. As a control, siRNA for green ﬂuorescentprotein (GFP) was used.35 For reverse transcription-quantitative
polymerase chain reaction (RT-qPCR) analyses, 20 pmol ofsiRNA was transfected into RAW264.7 cells seeded at a density of 2.5 × 104 cells in 24-well plates at 24 h before transfection. For morphological evaluation, 4 pmol of siRNA wastransfected in cells seeded at a density of 4 × 103 cells in
96-well plates. The transfection of siRNA was conductedby the use of Lipofectamine RNAiMAX (Life Technologies,
Carlsbad, CA, USA) according to the manufacturer’s
protocol. At 8 h after transfection, the cells were treated withRANKL (50 ng/ml) for 24 h.

Statistical analysis
All the experiments were basically performed two or moretimes; each experiment was performed in triplicate. The dataof a representative experiment are presented as the mean ±SE (n = 3). Comparisons between the cells treated withRANKL alone and the other cells and comparisons between
cells transfected with siRNA for green ﬂuorescent protein
and those with MITF-E siRNA were conducted using
student’s t-test. The results were considered statisticallysigniﬁcant at p < 0.05.

RNA isolation and RT-quantitative PCR
Total RNA was isolated by the use of QuickGene RNA
cultured cell kit S (Wako, Osaka, Japan) in QuickGene-

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k. asai ET AL.
RESULTS AND DISCUSSIONof the other MITF isoforms was not detected (data not shown).
Lu et al. showed the up-regulation of MITF-E expression in
response to RANKL treatment by semi-quantitative RTPCR analyses.37 In the present study, we quantitatively
reveal the speciﬁc induction of MITF-E mRNA duringosteoclastogenesis.
The role of induced MITF-E in the formation of osteoclastic cells was evaluated next. RAW264.7 cells weretransfected with MITF-E siRNA prior to RANKL treatment.
MITF-E siRNA potentiated a decrease in the number of
TRAP-positive multinucleated cells (Figure 2A and 2B),
suggesting a positive role for the induced MITF-E inosteoclastogenesis. The MITF-E gene transcript level was
reproducibly decreased by transfection with the siRNA(Figure 2C, data not shown). MITF-E isoform-speciﬁcregion is only 139 bp. We designed possible 2 siRNAs for
MITF-E; the knockdown efﬁciency and reproducibility of
the knockdown is better for the siRNA used in this study(data not shown), although nevertheless the inhibition of
MITF-E expression is limited. The knockdown of MITF-E
gene did not affect the expression level of MITF-A(Figure 2D). Transfection of siRNA for MITF-E inhibitedRANKL-induced expression of TRAP and CtsK, genes
related to osteoclast activity,16,18 on day 3 (Figure 2E
and 2F). Lu et al. also showed the blockage of MITF-E gene

The changes in gene expression of MITF isoforms duringosteoclastogenesis were examined (Figure 1). Because
RAW264.7 cells synthesize sufﬁcient M-CSF,36 only exogenous RANKL is required for the formation of TRAP-positive
multinucleated cells. In contrast, both M-CSF and RANKL
must be exogenously supplied in primary bone marrow cell
culture. The MITF-A isoform was expressed in both
RAW264.7 cells (Figure 1A) and bone marrow cells(Figure 1B). The expression level was relatively constantduring osteoclastogenesis in both RAW264.7 cells and bonemarrow cells, except for the higher expression of MITF-A inbone marrow cells on day 3 without RANKL (Figure 1B).
In contrast, the gene transcript levels of MITF-E wereincreased within 1 day in response to treatment with RANKLin RAW264.7 cells (ninefold, Figure 1C) and in bone marrowcells (1000-fold, Figure 1D). RANKL-induced MITF-E
expression was gradually decreased with incubation days in
RAW264.7 cells, and the effect of RANKL treatment was
not signiﬁcant on day 3. In contrast, the up-regulation of
MITF-E with treatment with M-CSF and RANKL wasmaintained for at least 3 days in bone marrow cells. Treatment
with M-CSF alone had no effects on MITF-E expression inbone marrow cells (data not shown). Signiﬁcant expression

Figure 2. MITF-E expression is required for osteoclastogenesis. RAW264.7 cells were transfected with MITF-E siRNA or GFP or treated with RNA transfection reagent only (mock). At 8 h after transfection, the cells were treated with or without RANKL (100 ng/ml) for 3 days. (A) The cells were stained for
TRAP and counter-stained with hematoxylin. A representative result is shown. *, TRAP-positive multinucleated cell. The bar in each photograph indicates
100 μm. (B) The number of TRAP-positive multinucleated cells in a well of 96-well plate was counted (n = 3). (C–F) The gene transcript level of MITF-E(C) and MITF-A (D) on day 1 and that of TRAP (E) and CtsK (F) on day 3 were quantiﬁed by RT-qPCR, and the expression was normalized to G3PDH
expression. The expression in cells treated with siRNA for GFP was set to 1 (n = 3). * and **, p < 0.05 and p < 0.01, respectively
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osteoclastogenesis by mitf-e and tgf-β
of cells with areas >1 × 104 and <2 × 104 μm2 was signiﬁcantly increased by TGF-β treatment, whereas the proportion of cells with areas <5 × 103 μm2 tended to be
decreased, indicating that TGF-β accelerates the formationof larger TRAP-positive multinucleated cells. TGF-β also
increased bone resorption activity, and RANKL-induced pit
formation was enhanced by the co-treatment with TGF-β(Figure 3D and 3E). The present results suggest that TGF-β
acts not only as an enhancer of osteoclast formation induced
by RANKL but also as an accelerator of mature osteoclast
formation through cell fusion of nascent osteoclasts.
The molecular basis of enhanced osteoclastogenesis by
TGF-β was then explored. The expression of NFATc1,
which is involved in the onset of osteoclastogenesis,38,39
was increased within 1 day after treatment with RANKL,
and TGF-β enhanced RANKL-induced NFATc1 expression (Figure 4A). Similarly, RANKL-induced MITF-E

induction results in the impaired formation of osteoclasts37;
we also observed the similar results. All these results
suggest that transient induction of MITF-E by RANKL isessential for efﬁcient osteoclastogenesis.37
Next, the effects of TGF-β on osteoclastogenesis were
examined in RAW264.7 cells. Treatment with TGF-β alone
did not induce formation of TRAP-positive multinucleatedcells, but TGF-β increased the number of TRAP-positive
multinucleated cells induced by RANKL (Figure 3A and
3B), which was consistent with previous results.9–11 We
also noticed that larger TRAP-positive multinucleated cells
were formed by co-treatment with TGF-β compared withthe treatment with RANKL alone. Chin et al.11 described
the TGF-β-induced formation of giant osteoclasts but did
not quantitatively analyze them. To characterize the cell sizein detail, the number of TRAP-positive multinucleated cells
was counted for every cell size (Figure 3C). The proportion

Figure 3. TGF-β enhances RANKL-induced osteoclastogenesis. RAW264.7 cells were pre-treated with or without TGF-β1 (100 pM) for 12 h, followed by
the co-treatment with or without RANKL (100 ng/ml) for 3 days. (A) Cells were stained for TRAP and counter-stained with hematoxylin. A representative result is shown. *, TRAP-positive multinucleated cell. The bar in each photograph indicates 100 μm. (B) The total number of TRAP-positive multinucleated cells
in a well of 96-well plate was counted (n = 3). (C) The number of TRAP-positive multinucleated cells induced by RANKL alone or RANKL plus TGF-β wascounted for every cell size. The percentage of the respective TRAP-positive multinucleated cell number to the total TRAP-positive multinucleated cell number
was calculated (n = 3). (D) Osteoclast functionality was evaluated by pit formation assays (n = 3). A representative result is shown. The bar in each photographindicates 100 μm. (E) The total area of pit in a well of 96-well plate was measured (n = 3). **, p < 0.01
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k. asai ET AL.

Figure 4. TGF-β enhances the expression of MITF-E and genes related to osteoclastogenesis. RAW264.7 cells were pre-treated with or without TGF-β1(100 pM) for 12 h, followed by the co-treatment with RANKL (100 ng/ml) for 3 days. The gene transcript levels of NFATc1 (A), MITF-E (B) and MITF-A(C) on day 1 and TRAP (D) and CtsK (E) on day 3 were quantiﬁed by RT-qPCR. The expression was normalized to G3PDH expression, and the expressionin cells treated without RANKL and TGF-β1 was set to 1 (n = 3). **, p < 0.01

expression was further up-regulated by TGF-β (Figure 4B).
The TGF-β treatment also signiﬁcantly increased genetranscript level of MITF-A, but the extent of increase in
the expression was relatively small (~twofold, Figure 4C).
In accordance with the enhancement of expression related
to the triggering of osteoclastogenesis, TGF-β further
increased the expression of TRAP and CtsK on day 3(Figure 4D and 4E).
The expression of the genes involved in cell fusion was
further examined. DC-STAMP is a master regulator of cellfusion in osteoclast formation.40,41 The expression of DCSTAMP was induced by RANKL treatment, and TGF-βslightly but signiﬁcantly enhanced the RANKL-induced
expression (Figure 5A).
We evaluated the gene transcript levels of integrins and adisintegrin and metalloprotease (ADAM). Integrins and
ADAMs are also involved in the adhesion and fusion of
monocyte/macrophage-lineage cells.41,42 Several integrin proteins, such as integrin αV (Itgav), α2(Itga2), α5(Itga5), β1(Itgb1)
and β5 (Itgb5), are expressed in human osteoclasts.43 The
forced expression of Itgav stimulated the formation of
TRAP-negative multinucleated cells in RAW264.7 cells, andRANKL changed these cells to TRAP-positive cells.11 AntiItga9 antibody partly inhibited M-CSF and RANKL-induced
osteoclast formation.44 Bone marrow cells prepared from
integrin β3 (Itgb3)-null mice are less differentiated into
osteoclasts in vitro.45 Furthermore, the down-regulationof ADAM8 and ADAM12 expression inhibits osteoclast
formation as well as cell fusion of macrophages.46,47

The expression levels of Itgav, Itga2, Itga5, Itgb1, Itgb3
and Itgb5 were signiﬁcantly higher in cells treated withRANKL and TGF-β than in cells treated with RANKL
alone (Figure 5B–5G). In contrast, the expression of
ADAM8 and ADAM12 was not increased by TGF-βtreatment (Figure 5H and 5I). RANKL treatment signiﬁcantly increased the expression of Itgav, Itgb5 and
ADAM12 (Figure 5B, 5G and 5I). The changes in theexpression level of Itgav and Itgb5 in response to treatment with RANKL with or without TGF-β resembled
those of MITF-E expression; RANKL increased theexpression (Figure 4B), but the extent of the increased
gene expression was much smaller than that of theincrease following co-treatment with TGF-β.
In the present study, DC-STAMP expression was increased
by RANKL treatment, which coincides with the induction of
MITF-E but not MITF-A. Previous studies showed that thetranscriptional activation of DC-STAMP, a master regulatorof cell fusion during osteoclastogenesis, is regulated by
MITF,21,48 although isoform of MITF responsible for the cellfusion has not been identiﬁed. It is possible that one of theroles of MITF-E induces transcriptional activation of DCSTAMP. However, the role of MITF-E expression in responseto treatment with RANKL and TGF-β is likely to be distinct
from that with RANKL alone; the extent of further increasein DC-STAMP expression in response to TGF-β was
relatively smaller than that in MITF-E expression. Considering that TGF-β formed larger osteoclasts with more nuclei,
the further increased MITF-E by TGF-β might regulate the
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osteoclastogenesis by mitf-e and tgf-β

Figure 5. TGF-β increases the expression of genes related to cell adhesion and fusion. RAW264.7 cells were pre-treated with or without TGF-β1 (100 pM) for
12 h, followed by the co-treatment with RANKL (100 ng/ml) for 3 days. The gene transcript levels of DC-STAMP (A), ItgaV (B), Itga2 (C), Itga5 (D), Itgb1(E), Itgb3 (F), Itgb5 (G), ADAM8 (H) and ADAM12 (I) on day 3 were quantiﬁed by RT-qPCR. The expression was normalized to G3PDH expression, and theexpression in cells treated without RANKL and TGF-β1 was set to 1 (n = 3). * and **, p < 0.05 and p < 0.01, respectivelytranscription of gene(s) other than DC-STAMP, leading to theacceleration of cell fusion.
Integrins are involved in cell fusion as described earlier.11,13
In addition, integrin α4 (Itga4) expression is induced by MITF
in hematopoietic stem/progenitor cells,49 and the regulatory
expression of Itga4 depends on the isoform of MITF.33 Thus,
we speculate that TGF-β-mediated up-regulation of MITF-E
expression is responsible for the formation of larger osteoclasts
through increased expression of integrins; especially, theregulatory expression of Itgav and Itgb5 may be important,because changes in expression of MITF-E paralleled to thoseof Itgav and Itgb5 in response to treatment with RANKL withor without TGF-β. The present results suggest the critical roleof DC-STAMP and integrins in osteoclastogenesis induced
by RANKL and in osteoclast maturation by TGF-β, respectively; MITF-E may regulate both processes.

The present study extends the knowledge of the expression
and role of MITF-E expression in developing osteoclasts.
We previously demonstrated the functional interaction
between MITF and the TGF-β pathway. TGF-β enhances
MITF-E-mediated transcription,30,31 whereas the MITF-M
isoform negatively regulates TGF-β-mediated signalling.50
In the present study, additional regulation of MITF activity
by the TGF-β pathway, i.e. enhancement of MITF expression
by TGF-β, was clariﬁed. Recently, it was shown that TGF-β
represses the transcription of MITF-M in melanoma cells.51
Taken the present results with the results of a previous study51
together, MITF expression may be regulated by TGF-β in an
isoform-dependent or cell type-dependent manner or both.
Balancing bone resorption and bone formation is important for bone and mineral homeostasis. Differentiation into
multinucleated TRAP-positive cells resulting from the
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k. asai ET AL.
16. Luchin A, Purdom G, Murphy K, et al. The microphthalmia
transcription factor regulates expression of the tartrate-resistant acidphosphatase gene during terminal differentiation of osteoclasts. J Bone
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family. Proc Natl Acad Sci U S A 2001; 98: 5798–5803.
19. Feng H, Cheng T, Steer JH, et al. Myocyte enhancer factor 2 andmicrophthalmia-associated transcription factor cooperate with
NFATc1 to transactivate the V-ATPase d2 promoter during RANKLinduced osteoclastogenesis. J Biol Chem 2009; 284: 14667–14676.
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2005; 202: 345–351.
21. Courtial N, Smink JJ, Kuvardina ON, Leutz A, Göthert JR, Lausen J.
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CONFLICT OF INTEREST
The authors have declared that there is no conﬂict of interest.

ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid for ScientiﬁcResearch (24580437) from The Japan Society for the
Promotion of Science and a research project grant awardedby Azabu University.

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NOTE

Wildlife Science

Two Distinct mtDNA Lineages among Captive African Penguins in Japan
Michiko MURATA1) and Masaru MURAKAMI1)*
1)Laboratory of Molecular Biology, School of Veterinary Medicine, Azabu University, 1–17–71 Fuchinobe, Chuo-ku, Sagamihara,

Kanagawa 252–5201, Japan(Received 25 July 2013/Accepted 25 November 2013/Published online in J-STAGE 9 December 2013)
The African penguin (Spheniscus demersus) is one of the world’s most endangered seabirds. In Japan, although the number ofAfrican penguins in captivity continues to increase, genetic data have not been collected for either wild or captive populations. To reveal
genetic diversity and characterization in captive African penguins, we analyzed the nucleotide sequences of mitochondrial DNA (mtDNA)
from a sample of 236 African penguins. Analysis of 433 bp of the control region and 1,140 bp of cytochrome b sequences revealed theexistence of two mtDNA clades. Control region haplotypes were much more divergent (d=3.39%) between the two clades than within each
FODGH7KHGLYHUJHQFHRIWKHVHFODGHVPD\UHÀHFWGLIIHUHQFHVDWWKHVXEVSHFLHVRUJHRJUDSKLFDOSRSXODWLRQOHYHOLQ$IULFDQSHQJXLQV7KHVH
¿QGLQJVVXJJHVWWKDWDWOHDVWWZRGLVWLQFWPDWHUQDOOLQHDJHVH[LVWLQWKHZLOGSRSXODWLRQVRIWKH$IULFDQSHQJXLQKEY WORDS: African penguin, control region, genetic diversity, mitochondrial DNA.
ABSTRACT.

doi: 10.1292/jvms.13-0377; J. Vet. Med. Sci. 76(4): 559–563, 2014

The African penguin (Spheniscus demersus), which isendemic to southern Africa, inhabits 31 islands and four
mainland sites in Namibia and South Africa, ranging fromsouthern Angola in the north to Nelson Mandela Bay inthe east [4]. Wild African penguin populations markedly
decreased in the 20th century. Approximately 56,000 pairs
estimated in 2001 have declined to 21,000 pairs in 2009[5]. This population decline is thought to be primarily due
WRIRRGVFDUFLW\UHVXOWLQJIURPRYHU¿VKLQJRYHUH[SORLWDWLRQ
DQGHQYLURQPHQWDOÀXFWXDWLRQVVXFKDVLQFUHDVHGVHDVXUIDFH
temperature linked to climate change. In addition, oil pollution has become a major factor in African penguin mortality.
Consequently, the African penguin is listed in Appendix II of
the Convention on International Trade in Endangered SpeFLHVDQGFODVVL¿HGDV(QGDQJHUHGE\WKH,QWHUQDWLRQDOUnion for Conservation of Nature.
While wild African penguin populations continue to decrease, properly maintained captive populations are increasing year on year. According to the 2011 Japanese regional
studbook for the African penguin, their original introduction
to Japan was in 1935, and from 1973 to 2011, 156 additionalfounders were introduced: 92 from South Africa, 30 fromoverseas zoos and aquariums and the remaining 34 fromunknown locations. The time and number of introductions
varied. The captive African penguin populations in Japan
contain 485 individuals, comprising an estimated 87 different founder lineages. In order to avoid close inbreeding andto maintain genetic diversity, the Japanese Association of*CORRESPONDENCE TO: MURAKAMI, M., Laboratory of Molecular
Biology, School of Veterinary Medicine, Azabu University, 1–17–
71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252–5201, Japan.
e-mail: murakami@azabu-u.ac.jp©2014 The Japanese Society of Veterinary Science
This is an open-access article distributed under the terms of the Creative
Commons Attribution Non-Commercial No Derivatives (by-nc-nd)
License <http://creativecommons.org/licenses/by-nc-nd/3.0/>.

404

Zoos and Aquariums (JAZA) keeps studbooks which it uses
to promote longer-term breeding plans. However, no genetic
data on the foundering population of African penguins introduced to Japan are described in the studbook, and the genetic
relationship of captive African penguins in Japan is unclear.
Genetic data have not been collected on either wildor captive African penguins in Japan so far. In this study,
we examined the genetic diversity of the captive Japanese
populations of African penguins by analyzing the control
region and cytochrome b sequences of mitochondrial DNA(mtDNA), since mtDNA sequences have higher rates ofnucleotide substitution than nuclear DNA sequences [2].
Analysis of mtDNA sequences can reveal genetic relationships among closely related species. This molecular information may prove useful to JAZA for future management
and implementation of breeding programs.
Blood or feather samples from African penguins (n=236)
were collected from 20 Japanese zoos and aquariums. Blood
samples were also collected from captive populations of
Humboldt penguins (Spheniscus humboldti) (n=20) and
Magellanic penguins (Spheniscus magellanicus) (n=2).
Genomic DNA was extracted using Dr. GenTLETM (TaKaRa Bio, Otsu, Japan) or Get pureDNA Kit-Cell, Tissue(Dojindo Molecular Technologies, Kumamoto, Japan). Two
mtDNA fragments of 653 bp (control region) and 1,140 bp
F\WRFKURPH E  ZHUH DPSOL¿HG E\ SRO\PHUDVH FKDLQ UHDFtion (PCR). The control region fragments and the entire
F\WRFKURPH E JHQH ZHUH DPSOL¿HG XVLQJ WKH SULPHU SDLU
L-tRNA Glu ƍ&&7*&77**&7777<7&&$$*$&& 
DQG +'ER[ ƍ&7*$&&*$**$$&&$*$**&*& >@ DQG WKH SULPHU SDLU %&/ ƍ$**&&7$&&7$*GATCCTTCGCCCT) and BCH1 (GTCTTTGGTTAATTACAAGACCAATGTTT) [8], respectively. The H-Dbox,
BCL1 and BCH1 primers were also used as sequencing
SULPHUV$PSOL¿FDWLRQUHDFWLRQVZHUHFDUULHGRXWLQDμl
solution containing 10–100 ng genomic DNA, 1 × buffer (20mM Tris-HCl, pH8.0, 100 mM KCL and 2 mM MgCl2), 200

560

M. MURATA AND M. MURAKAMIμM each of deoxyribonucleotides, 1.25 units of Ex Taq® HS
DNA polymerase (TaKaRa Bio) and 0.2 μM of each primer.
Reaction mixtures were incubated in a PCR thermal cycler(TaKaRa PCR Thermal Cycler DiceTM; TaKaRa Bio) with
LQLWLDO GHQDWXUDWLRQ RI  PLQ DW & ZLWK D W\SLFDO SUR¿OH
of 32 cycles, each consisting of 30 sec at 95°C, 30 sec at
60°C (for the control region) or 55°C (for cytochrome b)
and 30 sec (control region) or 1 min (cytochrome b) at 72°C
ZLWK¿QDOH[WHQVLRQRIPLQDW&3&5SURGXFWVZHUH
checked by electrophoresis on 1.5% agarose gel with TAE
buffer, and the gel was stained with ethidium bromide. PCR
SURGXFWV ZHUH SXUL¿HG E\ H[RQXFOHDVH , :DNR 2VDND
Japan) and shrimp alkaline phosphatase (TaKaRa Bio) andwere sequenced directly using an ABI PRISM 3130 Genetic
Analyzer and BigDye® Terminator v3.1 Cycle Sequencing
Kit (Applied Biosystems, Foster, CA, U.S.A.). The sequences of 433 nucleotides of the control region and 1,140
nucleotides of cytochrome b were determined. Phylogenetic
analyses were conducted using MEGA version 5 [16]. Multiple sequence alignment was performed by using Clustal W[17]. Genetic distances between haplotypes were obtainedusing the Tamura-Nei model [15]. Phylogenetic trees wereconstructed by the neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods. The
reliability of tree topology was assessed by 1,000 bootstrap
replications [7]. Lastly, Neighbor-Net analysis [3] wasperformed to construct a phylogenetic network using SplitsTree4 software [9] (http://www.splitstree.org/).
DNA sequences of the mitochondrial control region of
236 African penguin individuals were determined. Multiple
sequence alignments of the 433 bp constituting the partial
control region showed 39 polymorphic sites, generating a
total of 30 different haplotypes (Table 1). All substitutions
were transitions. Our nucleotide sequencing data are available under the accession numbers AB775475-775504 fromthe DDBJ/EMBL/GenBank databases. The available 45 of
the total 64 South African founder populations were also
examined, resulting in 27 haplotypes. This indicated that thefounder populations were derived from 27 maternal ancestors. These maternal genetic results are consistent with traceable records of captive breeding detailed in the studbook.
We also investigated the Humboldt penguin and Magellanic
penguin, which belong to Spheniscus, the same genus as theAfrican penguin. Eight different haplotypes were obtained
from these species (accession numbers AB775505-775512).
NJ phylogenetic tree analysis using mitochondrial DNA
control region sequences revealed that the captive African
penguin populations kept in a total of 20 Japanese zoos andaquariums clustered into two clades (A and B) as supported
by high bootstrap values (Fig. 1). Clades A and B contained
26 and 4 haplotypes, respectively. The 27 haplotypes fromSouth Africa described above belonged to both clades A andB, suggesting 2 maternal lineages derived from South Africa. Five substitutions at nucleotide numbers 110, 166, 217,
225 and 285 in the control region were characteristic of clade
B (Table 1). The mean genetic distance of the control region
sequences between the 2 clades was 3.39%, and the genetic
distances within clades A and B were 0.93% and 1.18%, re-405

spectively. Both MP and ML analyses also showed a similar
topology with the captive African penguins arranged in 2
different clades (data not shown). A network analysis further
supported the division of the maternal lineages of the captive
African penguins into these 2 different clades (Fig. 2).
The complete 1,140 bp sequences (accession numbers
AB776002-776009) in the cytochrome b gene were obtained
from 54 captive African penguins in Japan. There were 8
KDSORW\SHV GH¿QHG E\  YDULDEOH VLWHV 1LQH QXFOHRWLGH
substitutions were synonymous, and the remaining 2 substitutions were non-synonymous. The same two clades as thoseof the control region were separated by 1 non-synonymous
substitution (data not shown).
The mtDNA diversities of rockhopper penguin (Eudyptesspp.) and blue penguin (Eudyptula minor) samples from wild
populations have been previously reported. The rockhopper
penguin, which had been considered a single species with two
subspecies [6], was suggested to be two distinct species basedon the average genetic distance (6.1%) of the control region
sequences between its northern and southern clades [10]. The
blue penguin, a single species comprising 6 morphologicallydetermined subspecies [11], can be divided into an Australian-Otago clade and a New Zealand clade from analyses of
3 mtDNA sequences [1]. The control region sequences between these two clades have been found to differ by 11.8%.
Moreover, examination of the control region sequences ofindividuals collected from 7 southern Australian E. m. novaehollandiae colonies [11] showed no geographic clustering ofclosely related genetic variations among colonies [12]. The
average genetic distance among colonies of this subspecies
was 1.0%, based on our calculations made from sequence
data for EU043384-043403. Considering these molecular
data found in studies of the rockhopper penguin and blue
penguin, the divergence of African penguin clades A andB (d   VHHQ LQ WKH SUHVHQW VWXG\ PD\ UHÀHFW HLWKHU D
difference in geographical populations or the existence of
XQGH¿QHG VXEVSHFLHV RI$IULFDQ SHQJXLQV DOWKRXJK LW PXVW
be noted that our data focused on captive-bred individuals.
7KLVLVWKH¿UVWUHSRUWRIPROHFXODUGDWDREWDLQHGE\PW'NA analyses of captive African penguins. Here, we demonstrated the existence of two divergent clades of captive African penguins with moderate genetic distance (d=3.39%).
Although we currently have no descriptive information on
the founders of the captive African penguin population andgenetic data from wild African penguin populations mustalso be examined, our data imply that captive African penguins in Japan are derived from two distinct maternal lines.
We are presently analyzing nuclear DNA markers, including microsatellite diversity and repetitive DNA sequences,to elucidate further the genetic characterization of African
penguin populations.
In other endangered species, such as the Oriental white
stork (Ciconia boyciana), mtDNA control region analysis
has been utilized to develop an effective breeding plan inJapan [18, 19]. Likewise, careful genetic management isneeded to maintain genetic variability in the African penguin
and to use captive breeding projects to save this endangered
species from extinction.

561

mtDNA HAPLOTYPES OF AFRICAN PENGUINS

Table 1. Haplotypes and variable sites of the mitochondrial control region found in Japanese captive populations of African penguins (Spheniscus
demersus)
Nucleotide position
Haplotype1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3
1 3 3 0 0 1 1 2 2 3 3 3 4 6 6 6 7 7 8 9 1 2 2 3 3 4 7 8 8 8 9 9 9 9 0 0 0 2
3 1 0 1 7 8 0 7 1 2 4 5 9 1 1 5 6 0 7 4 8 7 0 5 7 9 3 5 5 6 8 2 3 4 5 5 6 9 6CladeA type1 A G T T T T A A A G A T C T G A A G G C G G G G A T A G A T G G G T T A A T T
type2 G . . . . . . G . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . C
type3 G . . . C . . G . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . .
type4 . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C
type5 G . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . C
type6 . . . . C . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C
type8 G . . . . . . G . . . . . . . . . A . . . . . . . . . . . . . . . . C . . . C
type9 G . . . . . . G . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . C
type10 G . . . . . . G . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . C
type11 . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C
type12 G . . . . . . G . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . C
type13 G . . . . . . G G . . . . . . . . A . . . . . . . . . . . . . . . . . . . . C
type14 G . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C
type15 G . . . . . . G . . G C . C . . . . . . . . . . . C . . . . A . . . . . . . C
type17 G . . . . . . G . . . . . . . . . . . T . . . . . C . . . . . . . . . . . . C
type18 G . . . . C . G . . . . . . . . . . . . A . . . . . . . . . A . . . . . . . .
type19 G . . . C . . G . A . . . . . . . A . . . . . . . . . . . . . . . . . . . . .
type20 G . . . . . . G . . . . . . . . . . . T . . . . . . . . . . A . A . . . . . C
type21 G . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . A C . . . . C
type22 G . . . . C . G . . . . . . . . . . . . A . . . . . . . . . A . . . . . . C .
type23 G . . . . . . G . . . . . . . . . . . . A . . . . . . . . . . A . . . . . . C
type25 G . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . C
type26 G . . . . . . G . . . . . . A . . . . . . . . . . C . . . . A . . . . . . . C
type27 G . . . . . . G G . . . T . . . . A . . . . . . . . . . . . . . . . . . . . C
type29 G . . . . . . G G . . . . . . . . . . . . . . . . . . . . . A . . . . . . . C
type30 . . . . . . . G . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . C
CladeB type7
type16
type24
type28. A C . C
. . . C C
. . . C C
. A C . C

.
.
.
.

G G
G G
G G
G G

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

A
A
A
A

.
.
.
.

G
G
G
G

. R .
. . .
. . .
. . .

.
.
.
.

A
A
A
A

.
.
.
.

A .
A G
A G
A .

. . A G . A. G A G C A. G . G . A. . A G . A

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

Numberof individuals
22
3
9
20
28
7
25
27
9
3
3
8
18
8
3
1
1
4
2
1
1
1
3
11
2
3

. G . C. . . C. . . C
. G . C

7
1
1
4

total

236

evolution of animal mitochondrial DNA. Proc. Natl. Acad. Sci.
U. S. A. 76: 1967–1971. [Medline] [CrossRef]
3. Bryant, D. and Moulton, V. 2004. Neighbor-net: an agglomerative method for the construction of phylogenetic networks. Mol.
Biol. Evol. 21: 255–265. [Medline] [CrossRef]
4. Crawford, R. J. M., Underhill, L. G., Upfold, L. and Dyer, B. M.
2007. An altered carrying capacity of the Benguela upwelling
ecosystem for African penguins (Spheniscus demersus). ICES J.
Mar. Sci. 64: 570–576. [CrossRef]
5. Crawford, R. J. M., Altwegg, R., Barham, B. J., Barham, P. J.,
Durant, J. M., Dyer, B. M., Geldenhuys, D., Makhado, A. B.,
Pichegru, L., Ryan, P. G., Underhill, L. G., Upfold, L., Visagie,J., Waller, L. J. and Whittington, P. A. 2011. Collapse of South
Africa’s penguins in the early 21st century. Afr. J. Mar. Sci. 33:
139–156. [CrossRef]
6. del Hoyo, J., Elliott, A. and Sargatal, J., editors. 1992. Spheniscidae (Penguin) pp. 140–160 In: Handbook of the bird of theworld, vol.1, Lynx Edicions, Barcelona.
  )HOVHQVWHLQ -  &RQ¿GHQFH OLPLWV RQ SK\ORJHQLHV DQ DSproach using the bootstrap. Evolution 39: 783–791. [CrossRef]
8. Hitosugi, S., Tsuda, K., Okabayashi, H. and Tanabe, Y. 2007.

ACKNOWLEDGMENTS. We thank Sunshine International
Aquarium, Ueno Zoological Gardens, Awashima Marine
Park, Nagasaki Penguin Aquarium, Yokohama Hakkeijima
Sea Paradise Aqua Museum, Kakegawa Kachoen, Matsue
Vogel Park, EPSON Shinagawa Aqua Stadium, Futami Sea
Paradise, Kobe Kachoen, Marine World Uminonakamichi,
Tohoku Safari Park, Nasu Safari Park, Kurume City Bird
Center, Yodel Forest, Izu Mito Sea Paradise, Noboribetsu
Marine Park, Marinepia Matsushima Aquarium, Miyazaki
City Phoenix Zoo and Kyoto Aquarium for their contributionof samples used in this study. We particularly thank Mr. M.
7RPL\DPDIRUKLVDVVLVWDQFHZLWK¿HOGFROOHFWLRQVREFERENCES
1. Banks, J. C., Mitchell, A. D., Waas, J. R. and Paterson, A. M.
2002. An unexpected pattern of molecular divergence within theblue penguin (Eudyptula minor) complex. Notornis 49: 29–38.
2. Brown, W. M., George, M. Jr. and Wilson, A. C. 1979. Rapid

406

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Fig. 1. Neighbor-joining tree of mitochondrial DNA control region haplotypes of African penguins. The tree
was constructed as described by Saitou and Nei [14] and was rooted using Humboldt penguins (Spheniscus
humboldti) as an outgroup. Phylogenetic analyses were conducted with MEGA5. Only bootstrap values greaterthan 70% are shown.

Phylogenetic Relationships of mitochondrial DNA cytochromeb gene in East Asian ducks. J. Poult. Sci. 44: 141–145. [CrossRef]
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[Medline] [CrossRef]
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rockhopper penguin Eudyptes moseleyi being a sibling species.
Mol. Ecol. 15: 3413–3423. [Medline] [CrossRef] .LQVN\ ) & DQG )DOOD 5$ $ VXEVSHFL¿F UHYLVLRQ RI
the Australasian Blue Penguin (Eudyptula minor) in the New
Zealand region. National Museum of New Zealand Records 1:
105–126.
12. Overeem, R. L., Peucker, A. J., Austin, C. M., Dann, P. andBurridge, C. P. 2008. Contrasting genetic structuring between
colonies of the world’s smallest penguin, Eudyptula minor(aves: Spheniscidae). Conserv. Genet. 9: 893–905. [CrossRef]
13. Roeder, A. D., Ritchie, P. A. and Lambert, D. M. 2002. New DNA
markers for penguins. Conserv. Genet. 3: 341–344. [CrossRef]
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new method for reconstructing phylogenetic trees. Mol. Biol.
Evol. 4: 406–425. [Medline]
15. Tamura, K. and Nei, M. 1993. Estimation of the number ofnucleotide substitutions in the control region of mitochondrial
DNA in humans and chimpanzees. Mol. Biol. Evol. 10: 512–526.
[Medline]
16. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M.
and Kumar, S. 2011. MEGA5: Molecular evolutionary genetics
analysis using maximum likelihood, evolutionary distance, andFig. 2. Neighbor-Net diagram of captive African penguin
populations. A network of relationships among the 30
haplotypes detected in 236 individuals of the captive
African penguin was generated by Neighbor-Net analysisof polymorphisms of the control region. Digits represent
the haplotype number.

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maximum parsimony methods. Mol. Biol. Evol. 28: 2731–2739.
[Medline] [CrossRef]
17. Thompson, J. D., Higgins, D. G. and Gibson, T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple seTXHQFHDOLJQPHQWWKURXJKVHTXHQFHZHLJKWLQJSRVLWLRQVSHFL¿F
gap penalties and weight matrix choice. Nucleic Acids Res. 22:
4673–4680. [Medline] [CrossRef]
18. Yamamoto, Y. 2011. New haplotypes in the mitochondrial con-

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and Furuyama, J. 2000. Determination of the complete nucleotide sequence and haplotypes in the D-loop region of the mitochondrial genome in the oriental white stork, Ciconia boyciana.
Genes Genet. Syst. 75: 25–32. [Medline] [CrossRef]

NOTE

Biochemistry

Characterization of a Canine Tetranucleotide Microsatellite Marker Locatedin the First Intron of the Tumor Necrosis Factor Alpha Gene
Masashi WATANABE1)**, Kazuaki TANAKA2)**, Tatsuya TAKIZAWA2), Kazuhito SEGAWA1), Sakurako NEO1),
Ryo TSUCHIYA1), Michiko MURATA3), Masaru MURAKAMI3) and Masaharu HISASUE1)*
1)Laboratory of Veterinary Internal Medicine II, Faculty of Veterinary Medicine, Azabu University, Chuo-ku, Sagamihara, Kanagawa

252–5201, Japan
2)Laboratory of Animal Biotechnology, Faculty of Veterinary Medicine, Azabu University, Chuo-ku, Sagamihara, Kanagawa 252–5201,

Japan3)Laboratory of Molecular Biology, Faculty of Veterinary Medicine, Azabu University, Chuo-ku, Sagamihara, Kanagawa 252–5201,

Japan(Received 18 June 2013/Accepted 21 August 2013/Published online in J-STAGE 13 September 2013)
ABSTRACT. A polymorphic tetranucleotide (GAAT)nPLFURVDWHOOLWHLQWKH¿UVWLQWURQRIWKHFDQLQHWXPRUQHFURVLVIDFWRUDOSKD TNFA) gene wascharacterized in this study; 139 dogs were analyzed: 22 Beagles, 26 Chihuahuas, 20 Miniature Dachshunds, 24 Miniature Poodles, 22 PemEURNH:HOVK&RUJLVDQG6KLED,QXV:HGHWHFWHGWKHSUHVHQFHRIWKHDOOHOHV *$$7 5, (GAAT)6, (GAAT)7 and (GAAT)8, including 9
RIWKHH[SHFWHGJHQRW\SHV7KHH[SHFWHGKHWHUR]\JRVLW\ He DQGWKHSRO\PRUSKLFLQIRUPDWLRQFRQWHQW PIC YDOXHRIWKLVPLFURVDWHOOLWH
ORFXVYDULHGIURPWRDQGIURPWRUHVSHFWLYHO\DPRQJWKHEUHHGV7KHDOOHOLFIUHTXHQF\GLIIHUHGJUHDWO\DPRQJ
EUHHGVEXWWKLVPLFURVDWHOOLWHPDUNHUZDVKLJKO\SRO\PRUSKLFDQGFRXOGEHDXVHIXOPDUNHUIRUWKHFDQLQHTNFA gene.
KEY WORDS: canine TNFA gene, tetranucleotide microsatellite.

doi: 10.1292/jvms.13-0316; J. Vet. Med. Sci. 76(1): 119–122, 2014

7XPRUQHFURVLVIDFWRUDOSKD 71)$ LVDPXOWLIXQFWLRQDO
SURLQÀDPPDWRU\ F\WRNLQH ZKLFK LV VHFUHWHG PDLQO\ E\monocytes and macrophages (Online Mendelian Inheritancein Man (OMIM, 191160),. In humans, genetic variations in
the TNFA gene are associated with various disorders, e.g.,
VHSWLF VKRFN >@ UKHXPDWRLG DUWKULWLV >@ F\VWLF ¿EURVLV>@LQÀDPPDWRU\ERZHOGLVHDVHV>@DQGLQVXOLQUHVLVWDQFH
syndrome [8].
,QWKHYHWHULQDU\¿HOG71)$LVDOVRFRQVLGHUHGWREHRQH
RIWKHPRVWLPSRUWDQWF\WRNLQHVLQYROYHGZLWKPDQ\LQÀDPPDWRU\GLVHDVHVLQFOXGLQJEDFWHULDODQGSURWR]RDQLQIHFWLRQVDQGVHSVLV>@)RUH[DPSOHLQFUHDVHGH[SUHVVLRQRITNFA mRNA in colonic mucosa has been reported on dogs
with idiopathic lymphocytic-plasmacytic colitis [16].
The canine TNFA gene is located on dog chromosome 12&DQ)DPGRJJHQRPHDVVHPEO\NW_003726081), and it
FRPSULVHVH[RQVDQGLQWURQV+RZHYHUWKHJHQHWLFSRO\morphisms in the canine TNFAJHQHKDYHQRWEHHQFODUL¿HG
0LFURVDWHOOLWHV DUH WDQGHP UHSHDWHG VHTXHQFHV XVXDOO\GLWULDQGWHWUDQXFOHRWLGHPRWLIVZKLFKGLVSOD\KLJKOHYHOV*CORRESPONDENCE TO: HISASUE 0 /DERUDWRU\ RI 9HWHULQDU\ ,QWHUQDO 0HGLFLQH ,, )DFXOW\ RI9HWHULQDU\ 0HGLFLQH$]DEX 8QLYHUVLW\ ±± )XFKLQREH &KXRNX 6DJDPLKDUD .DJDJDZD
252–5201, Japan.
e-mail: hisasue@azabu-u.ac.jp
7KH¿UVWWZRDXWKRUVFRQWULEXWHGHTXDOO\WRWKLVZRUN
7KH-DSDQHVH6RFLHW\RI9HWHULQDU\6FLHQFH
7KLVLVDQRSHQDFFHVVDUWLFOHGLVWULEXWHGXQGHUWKHWHUPVRIWKH&UHative Commons Attribution Non-Commercial No Derivatives (by-ncnd) License <http://creativecommons.org/licenses/by-nc-nd/3.0/>.

409

RI SRO\PRUSKLVP PDNLQJ WKHP LGHDO JHQHWLF PDUNHUV >18]. Microsatellites were thought to be evolutionarily neutral
DQG WR KDYH QR JHQHUDOL]HG IXQFWLRQ >@ ,Q UHFHQW \HDUV
however, many reports have indicated that microsatellites in
WKH SURPRWHU UHJLRQV DQG LQWURQV RI IXQFWLRQDO JHQHV H[HUW
JHQHWLFHIIHFWVLQPDQ\FDVHV>@7KXVPLFURVDWHOOLWHVLQNQRZQIXQFWLRQDOJHQHVDUHJRRGWDUJHWVIRU
analyzing genetic polymorphisms.
,QWKLVVWXG\ZHIRFXVHGRQD *$$7 nUHSHDWPRWLIORFDWHGLQWKH¿UVWLQWURQRIWKHFDQLQHTNFA gene and demonstrated that this microsatellite locus was highly polymorphic,
VRLWFRXOGEHDXVHIXOPDUNHUIRUJHQHWLFDQDO\VLV:KROH EORRG VZDEV RI WKH RUDO PXFRVD RU QDLO VDPSOHV
ZHUH FROOHFWHG IURP GRJV DW YHWHULQDU\ KRVSLWDOV DQG GRJ
grooming shops in Japan. The 139 samples obtained were
IURP%HDJOHV&KLKXDKXDV0LQLDWXUH'DFKVKXQGV
24 Miniature Poodles, 22 Pembroke Welsh Corgis and 25
Shiba Inus. This study was approved by the Institutional
Animal Care and Use Committee (Permission number:
1306094) and carried out according to the Azabu University$QLPDO ([SHULPHQWDWLRQ 5HJXODWLRQV '1$ ZDV H[WUDFWHG
IURPWKHVHVDPSOHVXVLQJD4XLFN*HQH'1$WLVVXHNLW )XML)LOP7RN\R-DSDQ RUD1XFOHR6SLQ%ORRG4XLFN3XUHNLW
(Macherey-Nagel, Duren, Germany), according to the manuIDFWXUHU¶VSURWRFROV
7KH SULPHUV XVHG IRU SRO\PHUDVH FKDLQ UHDFWLRQ 3&5 
DQDO\VLV ZHUH GHVLJQHG XVLQJ &DQ)DP $ NE '1$
IUDJPHQW FRQWDLQLQJ DERXW  ES XSSHU VWUHDP IURP WKHSXWDWLYH WUDQVFULSWLRQ VWDUW VLWH D ƍ XQWUDQVODWHG UHJLRQƍ875 WKH¿UVWH[RQDQGDERXWESRIWKH¿UVWLQWURQ
RI WKH FDQLQH TNFA JHQH ZDV DPSOL¿HG E\ 3&5 XVLQJ WKHIRUZDUGSULPHUGRJTNFA) ƍ$$*&&&&$&&&&77*-

120

M. WATANABE ET AL.

)LJ 6FKHPH RI WKH FDQLQH TNFA JHQH VKRZLQJ WKH UHJLRQ VHTXHQFHG DQG WKH SRO\PRUSKLVPVGHWHFWHG $  6WUXFWXUH RI WKH FDQLQH TNFA JHQH DERYH  DQG LWV VHTXHQFHG UHJLRQ EHORZ  7KHSURWHLQFRGLQJVHTXHQFHVDUHLQGLFDWHGE\GDUNER[HV7KHQXPEHULQJRIEDVHSRVLWLRQVZDVEDVHGRQWKHUHIHUHQFHVHTXHQFH1:B &DQ)DPFKURPRVRPH ,QWKHUHIHUHQFHVHTXHQFH
JDQGJZHUHWKHSXWDWLYHWUDQVFULSWLRQVWDUWVLWH 766 DQGWKHKHDGRIWKHWUDQVODWLRQLQLWLDWLRQFRGRQRIWKHTNFA gene, respectively. (B) The variations detected in the partial TNFA
gene in 7 Beagles.

&$&&77ƍ DQGWKHUHYHUVHSULPHUGRJTNFA5 ƍ&&$&$&&&&$&$7&7&7&&$&$&$ƍ  )LJ $). PCR was
SHUIRUPHG XVLQJ  μl reaction volumes, according to the
PDQXIDFWXUHU¶V LQVWUXFWLRQV IRU *R7DT *UHHQ 0DVWHU 0L[
3URPHJD)LWFKEXUJ:,86$ 7KHSURJUDPFRPSULVHG
LQLWLDOGHQDWXUDWLRQDW&IRUPLQIROORZHGE\F\FOHVRIGHQDWXUDWLRQDW&IRUVHFDQQHDOLQJDW&IRU
VHFDQGH[WHQVLRQDW&IRUVHFZLWKD¿QDOH[WHQVLRQDW&IRUPLQ7KHDPSOL¿HGSURGXFWVZHUHSXUL¿HGXVLQJD
4,$TXLFN3&53XUL¿FDWLRQNLW 4LDJHQ+LOGHQ*HUPDQ\ 
7KHSXUL¿HG'1$ZDVVHTXHQFHGGLUHFWO\XVLQJD%LJ'\H
7HUPLQDWRUYHUVLRQ&\FOH6HTXHQFLQJNLW $SSOLHG%LRV\VWHPV )RVWHU &LW\ &$ 86$  ZLWK GRJTNFA) DQGdog-TNFA5SULPHUV)LUVWZHDQDO\]HGWKH'1$VHTXHQFHV RI  %HDJOHV +RZHYHU  RI WKH  LQGLYLGXDOV SURGXFHGSRRUUHVXOWVEHFDXVHRIZLGHVSUHDGGRXEOHSHDNIURPthe dog_TNFA_R1 primer side. As a result, we determined
WKHWDUJHWVHTXHQFHVVXFFHVVIXOO\LQLQGLYLGXDOVRQO\$IWHUPXOWLSOHDOLJQPHQWVRIWKHSDUWLDOTNFAVHTXHQFHVRI WKH  %HDJOHV ZLWK WKH FDQLQH UHIHUHQFH VHTXHQFH ZH
IRXQGSRO\PRUSKLFVLWHVDF\WRVLQHGLUHFWUHSHDWYDULDWLRQLQWKHƍJHQHÀDQNLQJUHJLRQ J& B DQDGHQLQHWRF\WRVLQHVXEVWLWXWLRQLQWKHƍ875 J$!& 
DQG *$$7 PLFURVDWHOOLWH YDULDWLRQV LQ WKH ¿UVW LQWURQ
J*$$7 B  DQG J*$$7 B  )LJ$ 1H[WZHLGHQWL¿HGTNFAYDULDWLRQV71)$%HDJOH
 GRJ  71)$%HDJOH  GRJV  DQG 71)$%HDJOH 
GRJV  LQ WKH  LQGLYLGXDOV )LJ % 7KHVH QXFOHRWLGH VHTXHQFHVZHUHGHSRVLWHGLQWKH''%-(0%/DQG*HQ%DQN
nucleotide databases under accession numbers AB819627–
AB819629.

410%DVHG RQ WKHLU HOHFWURSKHURJUDPV ZH DOVR IRXQG WKDW
WKH VXERSWLPDO VHTXHQFLQJ UHVXOWV IRU WKH  %HDJOHV UHVXOWHG IURP WKH KHWHUR]\JRVLW\ He  LQ GLIIHUHQW QXPEHUVRI *$$7  UHSHDWV LQ WKH > *$$7 5–8 (GAT) (GAAT) 2]
PLFURVDWHOOLWHPRWLILQWKH¿UVWLQWURQ )LJ 7RJHQRW\SHWKLVPLFURVDWHOOLWHORFXVZHGHVLJQHGWKHIROORZLQJSULPHU
VHW &)$TNFA675) ƍ**$$*$7*&7&$7**$77*&7ƍ  DQG &)$TNFA6755 ƍ7$&&&$&$&&&&$&$7&7&7ƍ  3&5 ZDV SHUIRUPHG XVLQJ
25 μl UHDFWLRQ YROXPHV DFFRUGLQJ WR WKH PDQXIDFWXUHU¶VLQVWUXFWLRQVIRU*R7DT0DVWHU0L[ 3URPHJD 7KHSURJUDPFRPSULVHGLQLWLDOGHQDWXUDWLRQDW&IRUPLQIROORZHGE\HLWKHURUF\FOHVRIGHQDWXUDWLRQDW&IRUVHF
DQQHDOLQJDW&IRUVHFDQGH[WHQVLRQDW&IRUVHF
ZLWKD¿QDOH[WHQVLRQDW&IRUPLQ7KH3&5SURGXFWV
were electrophoresed on a 3.0% agarose gel and visualized
E\ HWKLGLXP EURPLGH VWDLQLQJ )LJ   )RXU DOOHOHV LH
(GAAT)5, (GAAT)6, (GAAT)7 and (GAAT)8, were detected
LQ WKH &)$TNFA-STR1 tetranucleotide microsatellite
ORFXVDQGWKHOHQJWKVRIWKHLU3&5SURGXFWVZHUH
 DQG  ES UHVSHFWLYHO\ 6XEVHTXHQWO\ ZH VXUYH\HG
WKHDOOHOHGLVWULEXWLRQRIWKLVPLFURVDWHOOLWHPDUNHULQGRJ
EUHHGV :H XVHG WKH )$0ODEHOHG &)$TNFA-STR1) SULPHU DQG WKH IUDJPHQW OHQJWKV RI WKH 3&5 SURGXFWV
were determined automatically using an ABI 3130 genetic
DQDO\]HU ZLWK *HQH0DSSHU9HU  $SSOLHG %LRV\VWHPV 
Table 1 shows the genotypic and allele distribution in 6 dog
EUHHGV )RXU DOOHOHV ZHUH SUHVHQW LQ  %HDJOHV ZKHUHDVRQO\  DOOHOHV ZHUH GHWHFWHG LQ  GRJV IURP WKH RWKHU 
breeds. This is because the (GAAT)8DOOHOHZDVDEVHQWIURPChihuahuas, Miniature Dachshunds, Miniature Poodles,121

TETRANUCLEOTIDE MICROSATELLITE IN DOG TNFA

)LJ *HQRW\SHGSDWWHUQVRIWKHWHWUDQXFOHRWLGHPLFURVDWHOOLWHSRO\PRUSKLVPLQWKH
¿UVWLQWURQRIWKHFDQLQHTNFA gene on a 3.0% agarose gel. The genotypes relate to
WKHXSSHUODQHVRIWKHJHO0LVDES'1$ODGGHUPROHFXODUVL]HPDUNHU 7R\RER
Osaka, Japan).
7DEOH *HQRW\SLFDQGDOOHOLFIUHTXHQF\RI *$$7 n tetranucleotide microsatellite in canine TNFA gene among dog breeds%UHHGV QXPEHURIDQLPDOV$OOHOLFIUHTXHQF\

Genotype
5

6

7

8

5/6

5/7

5/8

6/7

6/8

7/8

5

6

7

8

Beagle (22)1
Miniature Dachshundd) (20) 8
Chihuahuad) (26)12
Miniature Poodle (24)6Pembroke Welsh Corgi (22) 8
Shiba Inu (25)
13

0
0
2
1
1
0

2
2
0
0
0
0

4
0
0
0
0
0

5
2
9
6
5
2

5
7
3
11
4
10

1
0
0
0
0
0

1
1
0
0
4
0

0
0
0
0
0
0

3
0
0
0
0
0

0.295
0.625
0.692
0.604
0.568
0.760

0.136
0.075
0.250
0.167
0.250
0.040

0.295
0.300
0.058
0.229
0.182
0.200

0.273
0.000
0.000
0.000
0.000
0.000

Hea)

PICb) Deviations
RI+:(c)

0.749
0.527
0.464
0.566
0.595
0.389

0.682
0.438
0.391
0.493
0.516
0.333

P<0.05NSe)
NS
P<0.05NSNS

D ([SHFWHGKHWHUR]\JRVLW\E 3RO\PRUSKLFLQIRUPDWLRQFRQWHQWF 'HYLDWLRQVRI+DUG\:HLQEHUJ(TXLOLEULXPG $OOWKHLQGLYGXDOVKDGORQJFRDWH
H 1RWVLJQL¿FDQWPembroke Welsh Corgis and Shiba Inus. (GAAT)5 was the
PRVW FRPPRQ DOOHOH LQ WKH  GRJ EUHHGV$Q H[DFW WHVW RI
WKH +DUG\±:HLQEHUJ HTXLOLEULXP XVLQJ D 0DUNRY FKDLQ
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ACKNOWLEDGMENTS. We thank Dr. Hidekatsu ShiPDNXUDIURPWKH)LUVW'HSDUWPHQWRI0LFURELRORJ\)DFXOW\RI9HWHULQDU\0HGLFLQH$]DEX8QLYHUVLW\IRUFROOHFWLQJFDnine DNA samples. This work was supported by the MEXT
3URJUDP IRU WKH 6WUDWHJLF 5HVHDUFK )RXQGDWLRQ DW 3ULYDWH
Universities, 2011–2015. The authors would like to thank411(QDJR ZZZHQDJRMS IRUWKH(QJOLVKODQJXDJHUHYLHZ
5()(5(1&(6  $RNL 7 .RFK . 6 DQG /HIIHUW + / $WWHQXDWLRQ RI
JHQH H[SUHVVLRQ E\ D WULQXFOHRWLGH UHSHDWULFK WUDFW IURP WKHWHUPLQDO H[RQ RI WKH UDW KHSDWLF SRO\PHULF LPPXQRJOREXOLQ
receptor gene. J. Mol. Biol. 267: 229–236. [Medline] >&URVV5HI@
2. Botstein, D., White, R. L., Skolnick, M. and Davis, R. W. 1980.
&RQVWUXFWLRQ RI D JHQHWLF OLQNDJH PDS LQ PDQ XVLQJ UHVWULFWLRQ IUDJPHQW OHQJWK SRO\PRUSKLVPV Am. J. Hum. Genet. 32:
314–331. [Medline]
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0RJD\]HO3--U:DQJ;DQG&XWWLQJ*59DULDQWV
LQ PDQQRVHELQGLQJ OHFWLQ DQG WXPRXU QHFURVLV IDFWRU DOSKDDIIHFW VXUYLYDO LQ F\VWLF ¿EURVLV J. Med. Genet. 44: 209–214.
[Medline] >&URVV5HI@
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analysis. Evol. Bioinform. Online 1: 47–50.
5. Gardezi, A. Z., Ziaei, Y. Z. and Marashi, S. M. 2008. MicrosatelOLWHSRO\PRUSKLVPRIWKHKXPDQOHSWLQJHQHDQGULVNRIREHVLW\
J. Crit. Care 23: 440–444. [Medline] >&URVV5HI@
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Matta, M. A., Souza, S. M., Lana, M., Chiari, E., Galvão, L. M.
DQG%DKLD07'HYHORSPHQWRIFKURQLFFDUGLRP\RSDWK\LQ
FDQLQH&KDJDVGLVHDVHFRUUHODWHVZLWKKLJK,)1JDPPD71)DOSKDDQGORZ,/SURGXFWLRQGXULQJWKHDFXWHLQIHFWLRQSKDVHVet. Immunol. Immunopathol. 130: 43–52. [Medline] >&URVV5HI@
7. Hearne, C. M., Ghosh, S. and Todd, J. A. 1992. Microsatellites
IRUOLQNDJHDQDO\VLVRIJHQHWLFWUDLWVTrends Genet. 8: 288–294.
[Medline]

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8. Ishii, T., Hirose, H., Saito, I., Nishikai, K., Maruyama, H. and
6DUXWD 7  7XPRU QHFURVLV IDFWRU DOSKD JHQH *$
SRO\PRUSKLVP LQVXOLQ UHVLVWDQFH DQG IDVWLQJ SODVPD JOXFRVH
in young, older, and diabetic Japanese men. Metabolism 49:
1616–1618. [Medline] >&URVV5HI@  .DVKL< .LQJ ' DQG 6ROOHU 0  6LPSOH VHTXHQFH UHSHDWVDVDVRXUFHRITXDQWLWDWLYHJHQHWLFYDULDWLRQTrends Genet.
13: 74–78. [Medline] >&URVV5HI@
10. Klesert, T. R., Otten, A. D., Bird, T. D. and Tapscott, S. J. 1997.
7ULQXFOHRWLGHUHSHDWH[SDQVLRQDWWKHP\RWRQLFG\VWURSK\ORFXVUHGXFHV H[SUHVVLRQ RI DMAHP. Nature Genet. 16: 402–406.
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 0HORQL5$OEDQqVH95DYDVVDUG37UHLOKRX)DQG0DOOHWJ. 1998. A tetranucleotide polymorphic microsatellite, located
LQ WKH ¿UVW LQWURQ RI WKH W\URVLQH K\GUR[\ODVH JHQH DFWV DV D
transcription regulatory element in vitro. Hum. Mol. Genet. 7:
423–428. [Medline] >&URVV5HI@
13. Menezes-Souza, D., Corrêa-Oliveira, R., Guerra-Sá, R., GiunchetWL5&7HL[HLUD&DUYDOKR$0DUWLQV)LOKR2$2OLYHLUD*&DQG5HLV$%&\WRNLQHDQGWUDQVFULSWLRQIDFWRUSUR¿OHVLQWKHVNLQRIGRJVQDWXUDOO\LQIHFWHGE\Leishmania (Leishmania)
chagasi presenting distinct cutaneous parasite density and clinicalstatus. Vet. Parasitol. 177: 39–49. [Medline] >&URVV5HI@

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with septic shock susceptibility and mortality: a multicenter
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Amos, C. I., Zhu, D. K., Ward, R. H., Clegg, D. O., Shanahan,) 0ROOR\ 0 * DQG 2¶*DUD )  *HQHWLF YDULDELOLW\ LQ
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0LOOHU+53(YDOXDWLRQRI7K7KDQGLPPXQRVXSSUHVVLYHF\WRNLQHP51$H[SUHVVLRQZLWKLQWKHFRORQLFPXFRVD
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,/DQG71)ĮFKDQJHVLQDFDQLQHPRGHORIVHSVLVLQGXFHGE\HQGRWR[LQVet. Immunol. Immunopathol. 146: 143–149. [Medline] >&URVV5HI@
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polymorphisms which can be typed using the polymerase chain
reaction. Am. J. Hum. Genet. 44: 388–396. [Medline]
19. Wei, J., Ramchand, C. N. and Hemmings, G. P. 1995. Association
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cell biochemistry and function
Cell Biochem Funct 2014; 32: 236–240.
Published online 20 September 2013 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/cbf.3006

Downregulation of Pgc-1α expression by tea leaves and theirby-products
Erika Shibuya1, Masaru Murakami2, Makoto Kondo3, Yasutomi Kamei4, Shozo Tomonaga1, Tohru Matsui1
and Masayuki Funaba1*1Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara, Japan
3
Graduate School of Bioresources, Mie University, Tsu, Japan
4
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan
2

Previous studies indicate that muscle Pgc-1α expression governs the proportion of muscle ﬁbre types. As a ﬁrst step in using diet to manipulate theproportion of muscle ﬁbre types by using Pgc-1α expression, the present study investigates the modulation of Pgc-1α expression by feedstuffs. A
luciferase-based Pgc-1α reporter construct (Pgc-1α(-2553)-luc) that contains the mouse Pgc-1α promoter (2553 to +78 bp) was prepared. A
screen of ethanol extracts from 33 feedstuffs indicated that oolong tea and roasted green tea extracts decreased Pgc-1α(-2553)-luc expression
in C2C12 myoblasts. The transcriptional repression of Pgc-1α by tea leaf extracts was reproduced in hepatic HepG2 cells. We further examinedthe effects of the alcohol extracts of tea waste and its silage on Pgc-1α transcription; the tea waste silage extract inhibited Pgc-1α transcription.
Treatment with the extracts of raw tea leaves, tea waste and tea waste silage effectively decreased Pgc-1α mRNA levels during myogenesis ofmyosatellite cells. The present results suggest that tea leaves and their by-products could be used to modulate proportions of muscle ﬁbre types.
Copyright © 2013 John Wiley & Sons, Ltd.
key words—Pgc-1α; skeletal muscle; tea leaf; muscle ﬁbre type; feedstuffs

INTRODUCTION

Metabolic changes related to muscle Pgc-1α expression
can be partly explained by the modulation of muscle ﬁbretypes. Muscle ﬁbres are divided into two types, slow twitch
and fast twitch. Reddish slow-twitch myoﬁbres contain ahigh number of mitochondria and use oxidative metabolism
as their primary energy source, whereas whitish fast-twitch
myoﬁbres contain more glycogens and predominantly use
glycolytic metabolism.10 Mouse muscles forced to express
Pgc-1α state contained more slow-twitch muscle ﬁbres,11
whereas gene targeting of skeletal muscle Pgc-1α increased
expression of fast-twitch muscle ﬁbre-speciﬁc myosin heavy
chain (Myhc) 2b and decreased the expression of Myhc 1,
which is slow-twitch muscle ﬁbre-speciﬁc.9
Pgc-1α activity is closely linked to its expression level,
because it is primarily regulated at the transcriptional level.
MEF2 activates Pgc-1α gene transcription, and MEF2
repression decreases Pgc-1α mRNA levels, leading to thedownregulation of genes involved in fatty acid oxidation in
the heart.12 In addition, selective expression of constitutively
active CAMK IV in skeletal muscle increases Pgc-1αtranscription, mRNA levels and mitochondrial DNA copy
number.13 Therefore, Pgc-1α activity can be potentiallymonitored by evaluation of its mRNA levels. The objectiveof this study is to identify feedstuffs that affect Pgc-1α
expression. We screened for Pgc-1α expression using luciferase under the control of the Pgc-1α promoter as a reporter.

Pparγ co-activator-1α (Pgc-1α) was originally identiﬁed as aco-activator of Pparγ; Pgc-1α enhances the expression of the
Ucp1 gene, which encodes an uncoupling protein found in
the mitochondria of brown adipocytes and is essential togenerating heat by nonshivering thermogenesis.1 Subsequent
analyses in cultured cells and transgenic mice haveestablished that the biological function of Pgc-1α is to regulate
mitochondrial oxidative metabolism as well as mitochondrial
biogenesis in diverse cell types.2–4 In skeletal muscle, Pgc-1α
expression is enhanced by exercise in humans and rodents5–7
and is involved in the maintenance of muscle function;transgenic mice with enhanced Pgc-1α expression in skeletal
muscles preserve muscle integrity and function during ageing.
This phenotype is related to the maintenance of the activity ofenzymes involved in oxidative phosphorylation, the reductionof apoptosis, autophagy and proteasomal degradation.8 In
addition, the transgenic mice exhibited resistance to agerelated obesity and increased insulin sensitivity.8 In contrast,
reduced muscle function and exercise capacity were detectedin skeletal muscle-speciﬁc Pgc-1α knock-out mice.9*Correspondence to: Masayuki Funaba, Division of Applied Biosciences,
Graduate School of Agriculture, Kyoto University, Kitashirakawa
Oiwakecho, Kyoto 606-8502, Japan. E-mail: mfunaba@kais.kyoto-u.ac.jp

Received 12 June 2013
Revised 21 August 2013
Accepted 28 August 2013

Copyright © 2013 John Wiley & Sons, Ltd.

413

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regulatory expression of pgc-1α by tea leaf
Here, we show that tea leaves and their by-products containfactors that downregulate Pgc-1α expression through transcriptional inhibition.

(RD-400, Yamato Scientiﬁc Co., Ltd, Tokyo, Japan). The
samples were stored under N2 gas at 20 °C until analysis.
RNA isolation and RT-qPCR
Total RNA isolation from myosatellite cells and cDNA
synthesis were performed using TRIZOL (Invitrogen)and the ReverTra Ace qPCR RT kit (Toyobo, Osaka,
Japan) respectively according to the manufacturers’
protocols. The cDNA was reverse transcribed from 5
ng of total RNA and was used as a template for reverse
transcription-quantitative polymerase chain reaction(RT-qPCR) as described previously.15 The oligonucleotide
primers were 5’-TGTGGAACTCTCTGGAACTGC-3’
and 5’-GCCTTGAAAGGGTTATCTTGG-3’ for Pgc-1α
and 5’-CTAAGGCCAACCGTGAAAAG-3’ and 5’ACCAGAGGCATACAGGGACA-3’ for β-actin. The
Ct values were determined, and the abundance of gene transcripts was analysed using the ▵▵Ct method by using β-actin
as the normalization gene.

MATERIALS AND METHODS
Cell culture
Animal experiments were approved by the Kyoto University
Animal Experiment Committee. Myosatellite cells wereisolated from rat plantaris muscles; male Wistar rats
weighing 200–300 g were sacriﬁced by exsanguination
under isoﬂurane anaesthesia, and the plantaris muscle wascollected. Muscles were washed with phosphate-bufferedsaline and minced with scissors in digestion buffer
containing 0.1% (w/v) collagenase I (Wako Pure Chemical
Industries, Ltd., Osaka, Japan) and 1,000 U/ml dispase(Invitrogen, Grand Island, NY, USA) in 140 NaCl, 5.4
KCl, 0.34 Na2HPO4, 0.44 KH2PO4, 0.81 MgSO4∙7H2O,
1.3 CaCl2, 4.2 NaHCO3 and 5.6 mM glucose supplemented
with 10 U/mL penicillin, 100 μg/mL streptomycin and2.5 μg/mL amphotericin B, followed by enzyme digestion
shaking at 100 rpm for 60 min at 37 °C. After ﬁltering
through 50 μm nylon mesh, cells were washed with
Dulbecco’s modiﬁed Eagle’s medium (DMEM) with 10%
fetal bovine serum and antibiotics twice and seeded on cell
culture dishes. On day one post conﬂuence, cells weredifferentiated into myotubes by using differentiation
medium, i.e. DMEM with 2% horse serum and antibiotics.
Ethanol extracts of feedstuffs (20 μg/mL) or forskolin(10 μM) were simultaneously added to the differentiation
medium. Cells were harvested 12 days post-differentiation
to analyse Pgc-1α expression.
C2C12 cells, COS7 cells and HepG2 cells were cultured
in DMEM with 10% fetal bovine serum and antibiotics.
The DMEM used for C2C12 cells contained 1 g/L of
glucose, whereas the medium used for COS7 and HepG2 cells
contained 4.5 g/L of glucose. For luciferase-based reporter
assays, plasmid vectors were transiently transfected using
Lipofectamine LTX reagent (Invitrogen) for C2C12 cells or
PolyFect transfection reagent (Qiagen, Valencia, CA, USA)
for COS7 and HepG2 cells according to the manufacturers’
protocol. At 24 h post-transfection, cells were treated with orwithout the extracts from feedstuffs (20 μg/mL) or forskolin(10 μM) for 24 h.

Plasmids and reporter assays
The DNA fragment encompassing 2554 and +79 bp of themouse Pgc-1α promoter was ampliﬁed and cloned into thepGL4 basic vector containing the ﬁreﬂy luciferase reporter
gene (Pgc-1α(-2553)-luc); nt +1 is the transcription
initiation site. The reporter construct contains the promoter
region used in study by Amat et al.,16 who showedtranscriptional regulation of Pgc-1α by MyoD. The coding
region of mouse MyoD was cloned into pcDNA3. The
product was veriﬁed by nucleotide sequencing. Cells weretransiently transfected with the indicated expression vectors:
Pgc-1α(-2553)-luc and a plasmid expressing Renilla luciferase under the control of thymidine kinase (pRenilla-luc).
Luciferase activity was normalized to Renilla luciferase
activity, and the ﬁreﬂy luciferase activity in the cell lysate
treated with vehicle was set at 1.
Statistical analyses
The data are expressed as the mean ± SEM. Data from thereporter assays and gene expression data were logtransformed to provide an approximation of a normal
distribution before analysis. Differences between control
cells and cells treated with the feedstuff extracts were
examined using an unpaired t-test. Differences of P < 0.05
were considered signiﬁcant.

Preparation of ethanol extract
A total of 41 feedstuffs were used. Wet tea wastes wereobtained from a local beverage company that produces
canned and bottled tea drinks. Tea waste was ensiled inlaboratory silos by using the method described by Nishino
et al.14 Two grammes of food was mixed with 40 mL ofethanol and vigorously shaken for 60 min at room temperature
followed by ﬁltration through ﬁlter paper. For tea waste and its
silage, the ethanol extraction was performed after freeze
drying the samples. The ethanol extracts were concentrated to~20 mg/mL ethanol extract by using a centrifugal evaporator

RESULTS AND DISCUSSION
To evaluate the transcriptional activity of Pgc-1α, a luciferasebased reporter assay using the Pgc-1α promoter were
performed in C2C12 myoblasts; ethanol extracts from 33
feedstuffs were evaluated. The extracts were evaluated at aconcentration of 20 μg/mL, except for the red bell pepper
extract. Because the red bell pepper extract had a cytotoxic
effect in C2C12 cells, the transcriptional activity was examined at a concentration of 2 μg/mL. The reporter assay screen
Cell Biochem Funct 2014; 32: 236–240.

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e. shibuya ET AL.

was replicated in at least three independent experiments.
Representative results are shown in Figure 1. Luciferase
expression was signiﬁcantly lower in cells treated with oolong
tea or roasted green tea extracts of than in control cells. Noneof the extracts reproducibly increased luciferase expression
more than twofold (data not shown).
Next, we examined whether the inhibitory effects of tea
leaf extract can be detected in the other cells. Pgc-1αtranscription is stimulated by forskolin, an activator of adenylate cyclase17 and by MyoD, a muscle-speciﬁc transcription
factor.16 However, Pgc-1α transcription was unaffected byforskolin in COS7 cells, even when MyoD was co-expressed(Figure 2A). In contrast, HepG2 hepatoma cells were responsive to forskolin irrespective of MyoD expression (Figure 2A),
indicating that Pgc-1α transcription can be evaluated in
HepG2 cells. The inhibitory effects of tea leaf extracts on

Pgc-1α transcription were weaker in HepG2 cells (Figure 2B)
relative to C2C12 cells (Figure 1) but were detected in green
tea, black tea, roasted green tea and oolong tea extracts. These
results suggest that tea leaves contain factor(s) that inhibit
Pgc-1α transcription; the activity of the tea leaves is not
limited to C2C12 myogenic cells but is also detected in liver
cells. Pgc-1α orchestrates a complex program of metabolicchanges that occur during the transition from a fed liver to afasted liver, including gluconeogenesis; these effects on
fasting adaptation are achieved through co-activation ofhepatic transcription factors such as hepatic nuclear factor
4α, Pparα, glucocorticoid receptor, Foxo1 and the liver X
receptor.18 Therefore, the tea leaves may also be able tomodulate hepatic glucose metabolism.
Effects of tea waste and its silage on Pgc-1α transcription
were further evaluated in C2C12 cells (Figure 3). The tea

Figure 1. Inhibition of Pgc-1α transcription by tea leaf extracts in C2C12 cells. C2C12 cells were transiently transfected with Pgc-1α(-2553)-luc and pRenilla-luc,
and treated with vehicle or feedstuff extracts for 24 h. The ﬁreﬂy luciferase activity was normalized to the Renilla luciferase activity, and the ﬁreﬂy luciferase activity
in cell lysates treated with vehicle was set to 1. Data are expressed as the mean ± SE (n = 3). **: P < 0.01 vs. vehicle. (1) ﬁsh meal, (2) glycated rice meal, (3) chicken
meal, (4) wine residues, (5) yellow grease, (6:) red bell pepper extract, (7) dried distiller’s grains with solubles, (8) black soybean branch, (9) cassava starch pulp, (10)skim milk, (11) rapeseed meal, (12) corn gluten meal, (13) black soybean leaf, (14) corn germ meal, (15) wheat ﬂour, (16) shochu distillery by-product, (17) tall fescue,(18) soybean meal, (19) corn gluten feed, (20) yogurt, (21) rice alcohol cake, (22) meat and bone meal, (23) dehulled soybean meal, (24) sake kasu, (25) butter oil, (26)soybean ﬂour, (27) feather meal, (28) wine lees, (29) defatted rice bran, (30) soy protein concentrate, (31) wheat bran, (32) oolong tea and (33) roasted green tea

oolong tea

Tea leafroostedgreen tea

MyoD - + - +Cell COS7 HepG2

black teaBgreen teaA

Figure 2. Transcriptional repression of Pgc-1α(-2553)-luc by tea leaf extracts in HepG2 cells. (A) COS7 cells or HepG2 cells were transiently transfected with
Pgc-1α(-2553)-luc and pRenilla-luc with or without the MyoD expression plasmid and treated with vehicle or forskolin (10 μM) for 24 h. (B) C2C12 cells weretransiently transfected with Pgc-1α(-2553)-luc and pRenilla-luc and treated with vehicle or the indicated extract for 24 h. Fireﬂy luciferase activity was
normalized to Renilla luciferase activity, and the ﬁreﬂy luciferase activity in the cell lysates treated with vehicle was set to 1. Data are expressed as themean ± SE (n = 3). **: P < 0.01 vs. vehicle
Cell Biochem Funct 2014; 32: 236–240.

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regulatory expression of pgc-1α by tea leaf

vehiclewaste extracts from green tea, black tea and oolong tea did not
signiﬁcantly decrease expression of the Pgc-1α(-2553)-luc
construct, but the silage extract effectively repressed Pgc-1αtranscription; the effects of oolong tea waste silage were notstatistically signiﬁcant but had a tendency to repress expression (P = 0.08).
Screening feedstuffs using Pgc-1α(-2553)-luc indicatesthat the extracts from tea leaves and their by-products cause
inhibitory effects on Pgc-1α transcription. We further
veriﬁed whether these feedstuffs actually downregulate
Pgc-1α expression in myogenic cells; cells were treated during myogenesis with or without the extracts of tea leaves ortheir by-products, and Pgc-1α mRNA levels were examinedby RT-qPCR. Because C2C12 myoblasts and myotubes did

not express Pgc-1α mRNA signiﬁcantly (data not shown),
we used primary myosatellite cells from rat plantaris muscle.
Consistent with the reporter assay results (Figure 1), theoolong tea leaf extract decreased Pgc-1α mRNA expression,
whereas cassava starch pulp or dehulled soybean meal
extracts did not affect Pgc-1α expression (Figure 4A).
Forskolin treatment increased Pgc-1α expression as expected(Figure 4A). The extracts from tea waste silage also had theability to down-regulate Pgc-1α expression (Figure 4B).
Unlike Pgc-1α transcription, Pgc-1α mRNA level was
decreased also by the tea waste extract (Figure 4B); theinconsistent result between the reporter assay and expression
at the mRNA level may be due to (1) transcriptional repression via a region not contained in the Pgc-1α reporter geneor (2) post-transcriptional regulation such as mRNA stability.
The present study identiﬁes tea leaves and their by-products
as negative regulators of Pgc-1α expression. As described
previously, muscle ﬁbre type is modulated by the expression
level of Pgc-1α; higher expression of Pgc-1α increases the
proportion of slow-twitch oxidative muscle ﬁbre type,11
whereas the suppression of Pgc-1α expression increases fasttwitch glycolytic muscle ﬁbre type.9 Therefore, tea leaves
and their by-products may act as a switch of muscle ﬁbretypes, although future studies should be carried out to clarify
whether they affect muscle ﬁbre types through modulationof Pgc-1α expression in vivo.
Pgc-1α transcription is accelerated through calcium signaling pathway in skeletal muscle; activation of calcineurin
stimulates Pgc-1α transcription mediated by activated
CREB and MEF2.18 In addition, Pgc-1α expression is enhanced by glucagon and glucocorticoid signaling in liver.18
Although mechanisms underlying why tea leaves and theirby-products inhibit Pgc-1α transcription are unclear, the inhibition may be elicited by negative regulation of the transcriptional activation.

raw silage raw silage raw silage
blue teagreen tea
black tea
waste
waste
waste

Figure 3. Transcriptional repression of Pgc-1α(-2553)-luc by extracts of tea
waste and its silage in C2C12 cells. C2C12 cells were transiently transfectedwith Pgc-1α(-2553)-luc and pRenilla-luc and were treated with vehicle orthe indicated extract for 24 h. Fireﬂy luciferase activity was normalized toRenilla luciferase activity, and the ﬁreﬂy luciferase activity in the cell lysates
treated with vehicle was set to 1. Data are expressed as the mean ± SE
(n = 3). † and **: P < 0.10 and P < 0.01, respectively, vs. vehicle

vehicleforskolin

dehulled soybean mealcassava starch pulp

oolong tea

BvehicleA

raw silage raw silage raw silage
green tea
black tea oolong tea

Figure 4. Downregulation of Pgc-1α expression by extracts of tea waste and its silage in primary myosatellite cells. Myosatellite cells from rat plantaris muscle
were differentiated into myotubes by reducing the serum concentration of the culture medium. After simulating differentiation, the cells were treated with theindicated extract or forskolin 12 days. Pgc-1α mRNA levels were examined by reverse transcription-quantitative polymerase chain reaction and normalized to β-actinmRNA levels. The expression level in cells treated with vehicle was set to 1. Data are expressed as the mean ± SE (n = 6). **: P < 0.01 vs. vehicle
Cell Biochem Funct 2014; 32: 236–240.

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e. shibuya ET AL.

Considering that Pgc-1α expression levels in skeletal
muscle are positively correlated with the health beneﬁts described previously,8,9 tea leaves and their by-products may
have a negative effect on skeletal muscle Pgc-1α-mediatedimprovement of whole-body health in humans. However,the downregulation of Pgc-1α expression may be beneﬁcial
to beef production. Beef colour is one important factor in
determining the wholesale price of beef in Japan. The dark
red colour of beef decreases the commodity value of beefin Japan; Japanese consumers prefer lightly coloured beef.
Muscle ﬁbre type affects beef colour; longissimus muscle
and psoas major muscle are a mixture of fast-twitch andslow-twitch muscle ﬁbres; slow-twitch muscle ﬁbres are
reddish, and fast-twitch muscle ﬁbres are whitish. Thus,
the tea leaves and their by-products could potentially enable
the production of the preferred colour of beef throughincreased proportion of fast-twitch muscle ﬁbres mediatedby the downregulation of Pgc-1α expression. Furthermore,considering the correlation between slow-twitch muscle
ﬁbre content and cooking loss, a beef quality trait,19 tea
leaves and their by-products may be effective at improving
beef quality by decreasing cooking loss. Effects of muscle
ﬁbre type in beef on human nutrition are unknown. Comparison of amino acid composition between fast-twitch myosin
and slow-twitch myosin indicates no substantial difference(data not shown).
Canned and bottled tea drinks have become a favourite beverage in Japan. Over 100 000 t of tea waste per year are emitted
from beverage companies; however, much of these wastes areburned or composted.14 Tea waste can be preserved by ensilingbecause of its high moisture content, thus tea waste silage
would be a practical feedstuff to improve beef production.
Downregulation of Pgc-1α expression by tea leaf byproducts may also lead to an improvement of fattening
efﬁcacy, i.e. weight gain per feed intake. Transgenic micewith elevated muscle Pgc-1α expression are resistant toage-related obesity;8 one of the underlying the events in thisprocess is increased energy expenditure resulting from Irisin
production and secretion from the skeletal muscle inresponse to the upregulation of skeletal muscle Pgc-1α
expression.20 Future studies should identify the speciﬁc
molecule(s) in the extract of tea leaves and their by-productsthat represses Pgc-1α expression, and how these molecule(s)induce transcriptional repression. Considering the abundanceof various phenols, polyphenols and tannins in tea leaves,21,22
these compounds could be candidates.

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17. Karamitri A, Shore AM, Docherty K, Speakman JR, Lomax MA.
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CONFLICT OF INTEREST
The authors have declared that there is no conﬂict of interest.
ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid for ScientiﬁcResearch (23380158) from the Japan Society for the Promotion of Science and a research project grant awarded by the
Azabu University.

Cell Biochem Funct 2014; 32: 236–240.

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Cytokine 64 (2013) 138–145

Contents lists available at SciVerse ScienceDirect

Cytokine
journal homepage: www.journals.elsevier.com/cytokine

Bmp4 expressed in preadipocytes is required for the onset of adipocyte
differentiation
Masashi Suenaga a, Norio Kurosawa a, Hiroki Asano a, Yohei Kanamori a, Takenao Umemoto a,
Hirofumi Yoshida a, Masaru Murakami b, Hiroyuki Kawachi a,1, Tohru Matsui a, Masayuki Funaba a,⇑ab

Division of Applied Biosciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan
Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara 229-5201, Japana r t i c l e

i n f oArticle history:
Received 23 February 2013
Received in revised form 3 July 2013
Accepted 8 July 2013
Available online 2 August 2013
Keywords:
Bmp4
Adipocyte differentiation
Preadipocytes
Interleukin-11

a b s t r a c t
We previously revealed that endogenous bone morphogenetic protein (Bmp) activity is required for lipid
accumulation in 3T3-L1 adipocytes. The present study characterized the role of endogenous Bmp activity
in preadipocytes. Endogenous Bmp activity was monitored by analyzing the level of phosphorylation of
Smad1/5/8, downstream molecules in the Bmp pathway. Higher levels of phosphorylated Smad1/5/8
were detected in adipogenic cells but not in non-adipogenic cells prior to differentiation induction.
The inhibition of the Bmp pathway during this period decreased the expression of Pparc2 and C/ebpa,
which are transcription factors responsible for adipocyte differentiation. The expression of these transcription factors were also down-regulated by Bmp4 knockdown. In addition, endogenous Bmp4 was
required for the repression of Intrleukin-11 expression. Endogenous Bmp4 in preadipocytes is indispensable for the onset of the adipogenic program, and may help to maintain the preadipocytic state during
adipocyte differentiation.
 2013 Elsevier Ltd. All rights reserved.

1. Introduction
In addition to its role as the major energy-storage tissue, adipose tissue integrates a wide array of homeostatic processes bysecreting various cytokines. Because excess and deﬁcient levels
body fat are related to various (patho-)physiological conditions,
optimal body fat mass is essential for the maintenance of health[1]. The amount of adipose tissue is determined by the size andnumber of adipocytes. Adipocyte size largely reﬂects the amountof stored triglycerides, whereas adipocyte number is regulated by
the commitment of mesenchymal stem cells in the adipocyte lineage and by the proliferation and differentiation of preadipocytes.
Peroxisome proliferator-activated receptor c (Pparc, a nuclear hormone receptor, is the dominant regulator for adipocyte differentiation, and various molecules including growth factors andhormones affect adipogenesis through the modulation of Pparc
expression and activity [2].
Bone morphogenetic proteins (Bmps), members of the transforming growth factor-b (Tgf-b) family, regulate diverse physiological processes including adipose tissue formation [3]. The role ofBmps in the commitment of pluripotent stem cells to the adipocyte⇑ Corresponding author. Tel.: +81 75 753 6055; fax: +81 75 753 6344.
E-mail address: mfunaba@kais.kyoto-u.ac.jp (M. Funaba).
Present address: Department of Animal Bioscience, Faculty of Bioscience,
Nagahama Institute of Bio-Science and Technology, Nagahama 526-0829, Japan.
1lineage (adipocyte progenitors) has been well established [4]. The
addition of Bmp4 to the culture medium stimulated the adipocyte
differentiation of multipotential C3H10T1/2 cells [5,6] and mouseembryonic ﬁbroblasts [7]. A33 cells, a subline of C3H10T1/2 cells
treated with the DNA methylation inhibitor 5-azacytidine, werecommitted to the adipocyte lineage and highly expressed and secreted Bmp4 [8]. In contrast, the roles of Bmps in the differentiation of preadipocytes into adipocytes are controversial; treatment
with Bmp2 decreased insulin-induced lipid accumulation in 3T3F442A preadipocytes [9], whereas Bmp7 stimulated the adipocyte
differentiation of 3T3-L1 preadipocytes [10].
3T3-L1 preadipocytes are frequently used as a cell model for
adipocyte differentiation. In a standard differentiation program,
growth-arrested conﬂuent 3T3-L1 preadipocytes are treated withthe appropriate hormonal agents, which induce synchronous reentrance into the cell cycle and undergo at least 2 rounds of mitosis,
referred to as mitotic clonal expansion. Subsequently, the preadipocytes exit the cell cycle and begin to differentiate into adipocytes[11]. We previously analyzed endogenous Bmp activity during theadipocyte differentiation of 3T3-L1 activity was higher before differentiation induction, and the inhibition of this activity decreased
lipid accumulation [12]. These results suggest a novel activity of
Bmp in preadipocytes, but the following points remained to beclariﬁed: (1) the levels of endogenous Bmp activity in the otheradipogenic and non-adipogenic cells, (2) the effects of exogenous
Bmp after differentiation induction in 3T3-L1 cells, (3) the

1043-4666/$ - see front matter  2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.cyto.2013.07.011

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M. Suenaga et al. / Cytokine 64 (2013) 138–145
Table 1
Oligonucleotide PCR primers for RT-qPCR.
Oligonucleotide

GenBank accession number0

Actr2aActr2b
Alk2
Alk3
Bmp4
Bmpr2
C/ebpaFabp4
Hprt1
Interleukin-11
Lox
Pparc2Pref-1
Zfp4230

5 -primer

3 -primer

50 -CCCTCCTGTACTTGTTCCTACTCA-30
50 -GCTCAGCTCATGAACGACT-30
50 -AGGGCTCATCACCACCAAT-30
50 -TGACCTGGGCCTAGCTGTTA-30
50 -GAGGAGTTTCCATCACGAAGA-30
50 -TGGGAGGTGTTTATGAGGTGT-30
50 -CAAGAACAGCAACGAGTACCG-30
50 -AAGGTGAAGAGCATCATAACCCT-30
50 -TCCTCCTCAGACCGCTTTT-30
50 -CGCCGTTTACAGCTCTTGA-30
50 -CAGGCTGCACAATTTCACC-30
50 -TGCTGTTATGGGTGAAACTCTG-30
50 -CGGGAAATTCTGCGAAATAG-30
50 -CGTGAAGTTCGAGAGTGCTG-30

50 -GCAATGGCTTCAACCCTAGT-30
50 -CTCTGCCACGACTGCTTGT-30
50 -GCCACTTCCTGATGTACACG-30
50 -TTCAGGCTTTCATCCAGCA-30
50 -GCTCTGCCGAGGAGATCA-30
50 -GAAAAGCCATCTGGTAATCTGG-30
50 -GTCACTGGTCAACTCCAGCAC-30
50 -TCACGCCTTTCATAACACATTCC-30
50 -CCTGGTTCATCATCGCTAATC-30
50 -CAGGGGGATCACAGGTTG-30
50 -CAAACACCAGGTACGGCTTT-30
50 -CTGTGTCAACCATGGTAATTTCTT-30
50 -TGTGCAGGAGCATTCGTACT-30
50 -GGCACTTGATACACTGGTACGTC-30

identities of the molecule(s) responsible for the higher Bmp activity prior to differentiation induction in 3T3-L1 cells, and (4) the
possible target process(es) involved in the progression of the adipogenic program. In this study, we explored these unknowns tounderstand the precise role of Bmp during adipogenesis.
2. Materials and methods
2.1. Materials
The following reagents were purchased: dorsomorphin (compound C: 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine) was from Calbiochem (La Jolla, CA,USA);LDN-193189 was from Stemgent (San Diego, CA, USA);puriﬁed Tgf-b1 was from Becton Dickinson (Franklin Lakes, NJ, USA);recombinant Activin A, Bmp2 and Bmp4 were from R & D Systems(Minneapolis, MN, USA);rabbit polyclonal antibody against phospho-Smad1(Ser463/Ser465)/Smad5(Ser463/Ser465)/Smad8(Ser426/Ser428) (#9511) and rabbit polyclonal antibody against
phospho-Smad2 (Ser465/Ser467) were from Cell Signaling
Technology (Danvers, MA, USA);and rabbit monoclonal antibody
against Smad1 (ab33902), rabbit polyclonal antibody against
Smad2 (ab63576), and mouse monoclonal antibody against andb-actin (AC-15) were from Abcam (Cambridge, MA, USA).
2.2. Cell culture
3T3-L1 preadipocytes were cultured and differentiated as described previously [12]. Bovine stromal–vascular (SV) cells wereisolated from perirenal adipose tissues as follows. The tissues from
28- to 32-month-old Japanese Black steers were digested by with
type I collagenase (1 mg/ml) in Hank’s balanced salt solution containing penicillin (100 U/ml), streptomycin (100 lg/ml) andamphotericin B (250 ng/ml) and subsequently ﬁltered through a
250 lm nylon mesh ﬁlter to remove undigested tissue fragments
and debris. After centrifugation, the cell pellet consisting of SV cells
containing preadipocytes was washed with growth medium, i.e.,
Dulbecco’s modiﬁed Eagle’s medium (DMEM) containing 10%
FBS, ascorbic acid phosphate magnesium salt (100 lM) and antibiotics, followed by resuspension in the growth medium containing
10% DMSO and storage in liquid nitrogen. The adipocyte differentiation of the SV cells was induced as described previously [13]. Twodays after reaching conﬂuence (day 0), cells were cultured in thegrowth medium in the presence of differentiation inducers (3-isobutyl-1-methylxanthine (0.5 mM), dexamethasone (0.25 lM) andinsulin (10 lg/ml)) for 2 days, followed by culture with insulin(5 lg/ml) in the growth medium. To investigate the effects of419

M65287
M84120
L15436
Z23154
NM_007554
U78048NM_007678
NM_024406
NM_013556
NM_008350
NM_010728
NM_011146
NM_010052
NM_033327

dorsomorphin and LDN-193189 on adipocyte differentiation, dorsomorphin (10 lM) or LDN-193189 (100 nM) dissolved in DMSO
was added to the culture medium for the indicated period; treatments with the concentration of the inhibitors effectively blocked
Bmp-induced phosphorylation of Smad1 and transcriptional activation of a Bmp-responsive gene Id1 [14,15]. An equal concentration of DMSO (0.1%) was used as a control. Lipid accumulation
was examined by Oil Red O staining on day 8. The images were obtained by scanning stained wells (GT-9400UF;EPSON, Tokyo, Japan). Subsequently, dye was extracted with 2-propanol. The
absorbance of the solution was measured at 510 nm for quantiﬁcation [12].
2.3. Western blotting
To examine changes in phosphorylated Smad1/5/8 expression
over time, cells were recovered in 200 mM phosphate buffer, pH
7.4, 2 M NaCl, 2 mM Na3VO4, 1 mM PMSF and 1% aprotinin andthen lysed by ultrasonication. DNA content was measured by themethod of Labarca and Paigen [16]. Samples containing equalamounts of DNA were subjected to SDS–PAGE, followed by immunoblotting as described previously [17]. The reacted proteins werevisualized using the ECL Plus Western Blotting Detection System(GE Healthcare) according to the manufacturer’s protocol. After
stripping the antibodies and the detection reagents, the membranes were reprobed with anti-Smad1 antibody or anti-b-actin
antibody.
To examine effects of protein kinase inhibitors on ligand-induced Smad phosphorylation, cells were cultured in medium with
0.2% FBS for 4 h, treated with vehicle (DMSO), dorsomorphin(5 lM) or LDN-193189 (100 nM) with 0.2% FBS for 20 min, andsubsequently stimulated with Tgf-b1 (200 pM), Activin A (4 nM)
or Bmp2 (4 nM) in the presence of the kinase inhibitor for 1 h.
2.4. RT-qPCR
RNA isolation and RT-qPCR were performed as described previously [12]. The oligonucleotide primers for RT-qPCR are presented
in Table 1. The Ct value was determined, and the abundances ofgene transcripts were calculated from the Ct value using Hprt1 as
the normalization gene.
2.5. Double-stranded RNA transfection
Double-stranded RNAs (dsRNAs) targeting the expression of
Bmp4 and green ﬂuorescent protein (GFP) were synthesized by BONAC corporation (Kurume, Japan). The coding sequence of the140

M. Suenaga et al. / Cytokine 64 (2013) 138–145

A

Fig. 1. Endogenous Bmp activity is higher prior to differentiation induction in 3T3L1 cells. Phosphorylation status of Smad1/5/8 during adipocyte differentiation in
3T3-L1 and NIH-3T3 cells. 3T3-L1 and NIH-3T3 cells were stimulated withdifferentiation inducers for 2 days starting on day 0. Samples containing 1 lg of
DNA were subjected to Western blotting to detect phosphorylated Smad1/5/8 at Cterminal serines (upper panel). The membranes were re-blotted with an antibody
against Smad1 (middle panel) or b-actin (lower panel).

dsRNA for GFP is as described previously [16]. Four oligonucleotides were designed to target Bmp4, 5’-GUUGAAAAAUUAUCAGGAGAU-3’ (set 1), 5’-CUCCUGAUAAUUUUUCAACAC-3’ (set 1), and 5’CAGACUAGUCCAUCACAAUGU-3’ (set 2), and 5’-AUUGUGAUGGACUAGUCUGGU-3’ (set 2). To make dsRNA, the two oligonucleotidesof each set were mixed, and 1:1 mixtures of the dsRNA were prepared. dsRNA transfection was performed using the Lipofectamine
RNAiMAX regent (Invitrogen: Carlsbad, CA, USA) according to the
manufacturer’s protocol.
2.6. Plasmids and reporter assay
BRE-luc was kindly provided by Dr. P. ten Dijke. The other reporter genes were constructed by a PCR-based method, and all
constructs were veriﬁed by DNA sequencing. Luciferase-based reporter assays were conducted as described previously [18]. 3T3L1 cells were transiently transfected with the indicated ﬁreﬂyluciferase-based reporter construct using Lipofectamine LTX reagent (Invitrogen). A Renilla reniformis luciferase vector driven by
the thymidine kinase promoter (Renilla-luc; Promega, Madison,
WI, USA) was co-transfected to serve as an internal control fortransfection efﬁciency. At 24 h after transfection, the cells weretreated with Bmp4 (4 nM) for 16 h. Fireﬂy luciferase activity was
normalized to Renilla luciferase activity, and the luciferase activity
in the cell lysate in the absence of ligand was set to 1.

A

B

C

D

B

C

Fig. 3. Endogenous Bmp activity prior to differentiation induction is required for
efﬁcient lipid accumulation in 3T3-L1 cells. (A) Smad phosphorylation in responseto the Tgf-b family in 3T3-L1 cells treated with dorsomorphin or LDN-193189. 3T3L1 cells at 70% conﬂuence were pre-treated with dorsomorphin (DM: 5 lM), LDN193189 (LDN: 100 nM) or DMSO as a vehicle control (V) for 20 min, followed bytreatment with Tgf-b1 (200 pM), Activin A (4 nM) or Bmp2 (4 nM) for 1 h.
Phosphorylated Smad1/5/8 (upper panel) and Smad2 (third panel) were examined byWestern blotting, and the membranes were then re-blotted for total Smad1 (second
panel) and Smad2 (fourth panel). (B) Effects of dorsomorphin and LDN-193189 on
Bmp-mediated transcription. 3T3-L1 cells were transiently transfected with BREluc and Renilla-luc and treated with dorsomorphin (DM: 5 lM), LDN-193189 (LDN:
100 nM) or DMSO as a vehicle control (V) for 20 min, followed by treatment with orwithout Bmp4 (4 nM) for 16 h. Fireﬂy luciferase activity was normalized to Renillaluciferase activity, and the luciferase activity in cell lysates in the absence of Bmp4
and inhibitors was set to 1. Data were expressed as the mean ± SE of triplicates froma representative experiment. (C) Suppression of lipid accumulation by the inhibition of Bmp activity in 3T3-L1 cells. Dorsomorphin (DM: 5 lM), LDN-193189 (LDN:
100 nM) or DMSO as a vehicle control (V) was added to the culture medium fromday 2 to day 0, and lipid accumulation on day 8 was examined by Oil Red O
staining (upper panel). After staining, the dye was extracted with 2-propanol, andthe amount was quantiﬁed by measuring the absorbance at 510 nm. The
absorbance of the extracted dye from cells treated with vehicle was set to 100.
Data are expressed as the mean ± SE of quadruplicates. P < 0.01 vs. vehicle. (Forinterpretation of the references to colour in this ﬁgure legend, the reader is referredto the web version of this article.)

E

Fig. 2. Expression of Bmp receptors dynamically changes during adipogenesis in 3T3-L1 cells. Time-course changes in the gene expression of Alk2 (A), Alk3 (B), Actr2a (C),Actr2b (D) and Bmpr2 (E) during adipogenesis were examined by RT-qPCR analyses in 3T3-L1 cells. The gene transcript levels were examined on day 4, 2, 1, 0, 1, 2, 4, 6
and 8, and are expressed as ratios relative to Hprt1 expression, with the level in 3T3-L1 cells on day 4 set to 1. Data are expressed as the mean ± SE of quadruplicates.

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M. Suenaga et al. / Cytokine 64 (2013) 138–145

A

B

Fig. 4. Exogenous Bmp does not affect lipid accumulation in 3T3-L1 cells (A and B)
Limited effect of exogenous Bmp on lipid accumulation in 3T3-L1 cells. Bmp2
(4 nM) was added to the culture medium for the indicated period (A), and lipid
accumulation on day 8 was examined by Oil Red O staining (B, upper panel). After
staining, the dye was extracted with 2-propanol, and the amount was quantiﬁed bymeasuring the absorbance at 510 nm. The absorbance of the extracted dye from
cells treated with vehicle was set to 100 (B, lower panel). Data are expressed as themean ± SE of quadruplicates.

141

to Bmp just before the differentiation stimulation and lower after
the stimulation.
Dorsomorphin was identiﬁed as an inhibitor of the Bmp pathway by screening for compounds that disrupt dorsoventral axis
formation, which is governed by Bmp, in zebraﬁsh [14]. Subsequently, LDN-193189 was synthesized as a structurally related
compound capable of efﬁciently inhibiting Bmp4-induced
Smad1/5/8 phosphorylation in murine pulmonary artery smoothmuscle cells [21]. To examine the ability of the compounds to inhibit Bmp signaling in 3T3-L1 cells, ligand-induced Smad phosphorylation (Fig. 3A) and Bmp-mediated transcriptional activation(Fig. 3B) were evaluated. Pretreatment with dorsomorphin or
LDN-193189 effectively inhibited the Bmp2-induced phosphorylation of Smad1/5/8 (Fig. 3A, upper panel, lanes 4, 8 and 12). As in theother cell types [18,22–25], treatment of 3T3-L1 cells with Tgf-b1
induced the phosphorylation of Smad1/5/8 (Fig. 3A, upper panel,lanes 1 and 2), which was also partially inhibited by pretreatment
with dorsomorphin but not with LDN-193189 (Fig. 3A, upper panel, lanes 6 and 10). The phosphorylation and activation of Smad2
is a canonical Tgf-b1 and Activin A pathway [19]. Dorsomorphin
and LDN-193189 did not affect the Tgf-b1- or Activin A-induced
phosphorylation of Smad2 (Fig. 3A, third panel, lanes 2, 3, 6, 7,
10 and 11). Luciferase-based reporter assays also showed that both
dorsomorphin and LDN-193189 effectively blocked Bmp4-induced
expression of BRE-luc, a sensitive Bmp-responsive reporter gene[26] (Fig. 3B). These results indicate that dorsomorphin andA

2.7. Statistical analyses
Data are expressed as the mean ± SEM. Differences between
treatments were examined by Student’s t-test. Differences of
P < 0.05 were considered signiﬁcant.

B

3. Results and discussion
Bmp transmits signals through the phosphorylation of Smad1/
5/8 at the C-terminus after complex formation with ligand, type I
receptors and type II receptors [19,20]. Phosphorylated Smad1/5/
8 levels were monitored to evaluate endogenous Bmp activity during adipocyte differentiation (Fig. 1). Consistent with a previous
study [12], the intensity of the bands was higher for 3T3-L1 preadipocytes on day 2 (lane 1) and gradually decreased with the progression of differentiation (lanes 2–8). NIH-3T3 cells did not
signiﬁcantly differentiate into cells with lipid droplets, even if cells
were treated using the differentiation protocol for 3T3-L1 cells(data not shown). Unlike 3T3-L1 cells, NIH-3T3 cells did not exhibit
higher levels of phosphorylated Smad1/5/8 expression prior totreatment with differentiation inducers (Fig. 1, lane 9), although
the expression levels on days 1, 2 and 10 were relatively higher(lanes 11, 12 and 16).
We next evaluated time-course changes in expression of Bmp
receptors during adipogenesis in 3T3-L1 cells (Fig. 2). Alk2, Alk3
and Alk6 are type I receptors for Bmp, whereas Actr2a, Actr2band Bmpr2 function as type II receptors for Bmp [19,20]. No significant expression of Alk6 was detected throughout the adipogenesisof 3T3-L1 cells (data not shown). Expression level of all the Bmp
receptors, except for Actr2b, tended to increase from day 4 to
day 0, and decrease in response to the differentiation stimulation.
Thereafter, the decreased level continued at least to day 8. Actr2b
expression tended to increase day 4 to day 1, and increase again
from day 6 to day 8. Changes in Bmp receptor expression during
adipogenesis suggest that responsiveness to Bmp changes before
and after the differentiation stimulation, i.e., higher responsiveness

421

CFig. 5. Endogenous Bmp activity prior to differentiation induction is also required
for lipid accumulation in bovine SV cells (A) Phosphorylation status of Smad1/5/8
during adipocyte differentiation in bovine SV cells. Bovine SV cells were stimulatedwith differentiation inducers for 2 days starting on day 0. Samples containing 1 lg
of DNA were subjected to Western blotting to detect Smad1/5/8 phosphorylated at
C-terminal serines (upper panel). The membranes were re-blotted with an antibody
against Smad1 (middle panel) or b-actin (lower panel). (B and C) Suppression of lipid
accumulation by the inhibition of Bmp activity in bovine SV cells. Dorsomorphin(5 lM) or DMSO as a vehicle control was added to the culture medium for theindicated period (B), and lipid accumulation on day 8 was examined by Oil Red O
staining (C, upper panel). After staining, the dye was extracted with 2-propanol, andthe amount was quantiﬁed by measuring the absorbance at 510 nm. The
absorbance of the extracted dye from cells treated with vehicle was set to 100 (C,lower panel). Data are expressed as the mean ± SE of quadruplicates. and P < 0.05
and 0.01, respectively, vs. vehicle. (For interpretation of the references to colour inthis ﬁgure legend, the reader is referred to the web version of this article.)142

M. Suenaga et al. / Cytokine 64 (2013) 138–145

A

B

C

Fig. 6. Inhibition of Bmp activity prior to differentiation induction inhibits
adipocyte differentiation in 3T3-L1 cells Effects of inhibition of Bmp activity prior
to differentiation induction on adipocyte differentiation. Dorsomorphin (5 lM) or
DMSO as a vehicle control was added to the culture medium from day -2 to day 0.
The gene transcript levels of Pparc2 (A), C/ebpa (B) and Fabp4 (C) on day 8 were
measured by RT-qPCR and are expressed as ratios relative to Hprt1 expression, withthe level in 3T3-L1 cells treated with vehicle set to 100. Data shown are themean ± SE (n = 4). P < 0.01 vs. control.

LDN-193189 selectively inhibit Bmp-induced phosphorylation of
Smad1/5/8 in 3T3-L1 cells.
To examine the role of the high Bmp activity in preadipocytes,
3T3-L1 cells were treated with dorsomorphin or LDN-193189 before differentiation induction, i.e., from day 2 to day 0, and lipid
accumulation was evaluated on day 8. Although LDN-193189
was less effective, both inhibitors inhibited lipid accumulation, as
demonstrated by lower lipid levels on day 8 (Fig. 3C). The
reason of the less potency of LDN-193189 on lipid accumulation,AD

irrespective of compatible inhibition of Bmp4-mediated transcriptional activation between dorsomorphin and LDN-193189, is currently unknown. Dorsomorphin inhibits not only the Bmp
pathway but also the AMPK pathway; dorsomorphin, also known
as compound C, is originally identiﬁed as the AMPK inhibitor[27]. It is possible that the AMPK activity is involved in lipid accumulation of 3T3-L1 cells; endogenous AMPK activity was detected
in 3T3-L1 preadipocytes [12]. Whatever the reason may be, theseresults deﬁnitely suggest that the high Bmp activity in preadipocytes is necessary for lipid accumulation. Alternatively, decreasing
Bmp activity in response to differentiation induction may be required for efﬁcient lipid accumulation. Thus, the effects of additional Bmp on lipid accumulation were examined in 3T3-L1 cells(Fig. 4). Bmp2 was added to the culture medium for various periodsof time (Fig. 4A), and lipid accumulation was evaluated by Oil Red
O staining on day 8. The presence of additional Bmp2 after differentiation induction did not affect lipid accumulation in 3T3-L1
cells (Fig. 4B). As described above, the inconsistent results havebeen reported on the role of exogenous Bmps in adipogenesis[9,10]. The precise reason of the results is unknown; the discrepancy may relate to subtle differences in cell systems or culture
conditions.
The role of endogenous Bmp activity in adipogenic cells otherthan 3T3-L1 cells was next evaluated. Bovine SV cells can be differentiated into adipocytes in response to treatment with the differentiation inducers used on 3T3-L1 cells [13]. Endogenous Bmp
activity was relatively constant during the adipocyte differentiation of SV cells (Fig. 5A). As in 3T3-L1 cells, treatment with dorso-

B

C

E

Fig. 7. Down-regulation of Bmp4 prior to differentiation is sufﬁcient to inhibit adipocyte differentiation in 3T3-L1 cells (A) The down-regulation of Bmp4 expression in 3T3L1 cells. 3T3-L1 cells were transfected with dsRNA for Bmp4 or GFP on day 3. The gene transcript level of Bmp4 on day 0 was measured by RT-qPCR and expressed as a ratio
relative to Hprt1 expression, with the level in 3T3-L1 cells treated with dsRNA for GFP set to 100. Data shown are the mean ± SE (n = 4). P < 0.01 vs. GFP. (B) Suppression oflipid accumulation in 3T3-L1 cells transfected with dsRNA for Bmp4. 3T3-L1 cells were transfected with dsRNA for Bmp4 or GFP as a negative control on day -3, and lipid
accumulation on day 8 was examined by Oil Red O staining (upper panel). After staining, the dye was extracted with 2-propanol, and the amount was quantiﬁed by themeasuring absorbance at 510 nm. The absorbance of the extracted dye from cells treated with dsRNA for GFP was set to 100. Data are expressed as the mean ± SE ofquadruplicates. P < 0.01 vs. GFP. (C-E) Inhibition of adipocyte differentiation by knockdown of Bmp4. 3T3-L1 cells were transfected with dsRNA for Bmp4 or GFP on day 3.
The gene transcript levels of Pparc2 (C), C/ebpa (D) and Fabp4 (E) on day 8 were measured by RT-qPCR and expressed as ratios relative to Hprt1 expression, with the level in
3T3-L1 cells treated with dsRNA for GFP set to 100. Data shown are the mean ± SE (n = 4). P < 0.01 vs. GFP.

422

M. Suenaga et al. / Cytokine 64 (2013) 138–145

morphin prior to differentiation induction but not after differentiation induction decreased lipid accumulation in SV cells (Fig. 5B
and C). All of these results suggest that endogenous Bmp activity
before differentiation induction is necessary for lipid accumulation
during the adipogenic program.
To identify the process that endogenous Bmp activity regulates,
we next examined the expression of genes regulating adipocyte
differentiation. Pparc, a dominant and essential regulator of adipocyte differentiation [28–30], controls the transcriptional pathwayof adipogenesis in concert with CCAAT/enhancer-binding proteina(C/ebpa [31]. The inhibition of endogenous Bmp activity downregulated the expression of Pparc2 and C/ebpa (Fig. 6A and B). Consistent with these results, the expression of Fabp4, a late differentiation marker of adipogenesis and a representative target gene of
C/ebpa and Pparc [32], was decreased in 3T3-L1 cells pretreatedwith dorsomorphin (Fig. 6C). These results suggest that endogenous Bmp activity prior to differentiation induction is responsible
for adipocyte differentiation.
Our previous study indicated that the expression of Bmp4 washigher in preadipocytes and decreased after day 1 [12]. In viewof the parallel changes in Bmp4 expression and the phosphorylation of Smad1/5/8, Bmp4 may be responsible for the higher Bmp
activity before differentiation induction. The down-regulation of
Bmp4 expression by transfection of cells with dsRNA for Bmp4 on
day 3 to day 0 decreased lipid accumulation on day 8 (Fig. 7A
and B). The treatment also decreased the expression of Pparc2, C/
ebpa and Fabp4 (Fig. 7C–E), suggesting that higher Bmp4 activity
in preadipocytes is indispensable for adipocyte differentiation.
We further explored the molecular process regulated by Bmp4
in 3T3-L1 cells. The gene expression of molecules involved in adipocyte differentiation, including Zfp423, Interleukin-11, Pref-1 andlysyl oxidase (Lox), was examined. Zfp423 was identiﬁed as a gene143

highly expressed in ﬁbroblasts committed to the adipocyte lineage
and was found to be responsible for adipocyte differentiation[32,33]. Analysis of the nucleotide sequence from 3 kb to thetranscriptional initiation site of the Zfp423 gene revealed 6 possible
Bmp-responsive elements (BRE: GCCGnCGC) in Drosophila cells[34] and mammalian cells [35] in the region from nt –883 to nt –800;5 of these BREs partially overlapped (Fig. 8A). Zfp423 transcription was not decreased by treatment with Bmp inhibitors, and
Bmp4 addition did not increase the expression of Zfp423-luc, evenwhen the possible BREs were isolated (Fig. 8A and B). Furthermore,treatment with dorsomorphin or dsRNA for Bmp4 did not signiﬁcantly decrease Zfp423 expression (Fig. 8C). Thus, we concluded
that Zfp423 is not a target gene for endogenous Bmp activity inpreadipocytes.
Interleukin-11 was cloned based on its inhibitory activity on the
differentiation of 3T3-L1 preadipocytes [36]. The knockdown of
Bmp4 signiﬁcantly increased the expression of Interleukin-11 on
day 0 and day 2 in 3T3-L1 cells (Fig. 9A). Pref-1 is highly expressedin preadipocytes, and down-regulation of the expression of Pref-1
triggers the progression of the adipogenic program [37–41]. Transfection with dsRNA for Bmp4 signiﬁcantly decreased the expression of Pref-1 on day 0 and day 2 (Fig. 9B). Lox was identiﬁed asa gene that mediates Bmp-induced commitment to the adipocyte
lineage [6]. Transfection with dsRNA for Bmp4 did not decrease
Lox expression (data not shown).
Our previous study revealed that endogenous Bmp activity inpreadipocytes is required for lipid accumulation in adipocytes[12]. Here, we extend this information: higher expression of
Bmp4 in preadipocytes is indispensable for adipocyte differentiation. Previous studies revealed that a number of molecules are involved in the regulation of adipocyte differentiation, and most ofthese studies evaluated effects of the factors by treating with dif-

A

B

C

Fig. 8. Zfp423 transcriptional regulation is independent of Bmp activity (A) 3T3-L1 cells were transiently transfected with the indicated reporter gene and Renilla-luc andtreated with or without Bmp4 (4 nM) for 16 h. Fireﬂy luciferase activity was normalized to Renilla luciferase activity, and the luciferase activity in cell lysates in the absence of
inhibitors and Bmp4 was set to 1. Data were expressed as the mean ± SE of triplicates from a representative experiment. (B) 3T3-L1 cells were transiently transfected with
Zfp423(-913)-luc and Renilla-luc and treated with dorsomorphin (5 lM), LDN-193189 (100 nM) or DMSO as vehicle for 16 h. (C) 3T3-L1 cells were transfected with dsRNA for
Bmp4 or GFP on day 3. The gene transcript level of Zfp423 on day 8 was measured by RT-qPCR and expressed as a ratio relative to Hprt1 expression, with the level in 3T3-L1
cells treated with dsRNA for GFP set to 100. Data shown are the mean ± SE (n = 4).

423

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M. Suenaga et al. / Cytokine 64 (2013) 138–145

A

B

Fig. 9. Down-regulation of Bmp4 increases and decreases the expression ofInterleukin-11 and Pref-1, respectively, in 3T3-L1 cells Increased expression ofInterleukin-11 (A) and decreased expression of Pref-1 (B) in response to the downregulation of Bmp4 in 3T3-L1 cells. (A and B) 3T3-L1 cells were transfected withdsRNA for Bmp4 or GFP on day 3. The gene transcript levels of Interleukin-11 (A)and Pref-1 (B) on day 0 and day 2 were measured by RT-qPCR and expressed asratios relative to Hprt1 expression, with the level in 3T3-L1 cells treated with dsRNAfor GFP set to 100. Data shown are the mean ± SE (n = 4). and P < 0.05 and 0.01,
respectively, vs. GFP.

ferentiation inducers [2]. In contrast, little information is available
on factors affecting preadipocytic properties. The present study
indicates the importance of states prior to differentiation induction
for adipocyte differentiation. The provided basic information
potentially contributes to understanding the control of the numberof adipocytes.
Treatment with Interleukin-11 inhibited the adipocyte differentiation of 3T3-L1 preadipocytes, irrespective of the down-regulation of Pref-1 expression [42]. In view of the well-deﬁned role ofPref-1 as an inhibitor of adipocyte differentiation [35,37], these results suggest that Interleukin-11 negatively regulates adipogenesis
in a Pref-1-independent manner. Bmp4 produced in preadipocytes
may stimulate adipocyte differentiation through the repression ofInterleukin-11 expression; the expression levels of Interleukin-11
and Pref-1 were increased and decreased, respectively, in 3T3-L1
cells with down-regulated Bmp4 expression.
The present study revealed that higher expression of Bmp4 is
required for the progression of the adipogenic program in preadipocytes that are already committed to the adipogenic lineage. Previous studies indicated that Bmp4 stimulates the commitment ofmesenchymal stem cells to the adipocyte lineage [5–7]. In conclusion, there are at least two roles of Bmp in adipocyte differentiation: a role in inducing the commitment of preadipocytes and arole in maintaining preadipocytic properties, as revealed in thepresent study.
Acknowledgements
We thank Dr. Peter ten Dijke for providing plasmid. This work
was supported by Kakenhi from The Japan Society for the Promotion of Science.
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Fish Sci (2013) 79:935–941
DOI 10.1007/s12562-013-0674-x

ORIGINAL ARTICLE

Biology

Genetic characterization of the progeny of a pair of the tetraploidsilver crucian carp Carassius auratus langsdorﬁi
Jie Dong • Masaru Murakami • Takafumi Fujimoto •Etsuro Yamaha • Katsutoshi Arai

Received: 30 May 2013 / Accepted: 30 September 2013 / Published online: 19 October 2013
 The Japanese Society of Fisheries Science 2013

Contrary, tetraploid silver crucian carp gave eight rDNA
signals. These results suggest that gynogenetic development in eggs spawned by tetraploid females should betriggered by tetraploid males’ homospeciﬁc sperm.

Abstract Silver crucian carp Carassius auratus langsdorﬁi comprises a diploid-polyploid complex in wild
Japanese populations. Bisexually reproducing diploids are
sympatrically distributed with gynogenetically developing
triploids and tetraploids. Triploid and tetraploid males arevery rare among Japanese silver crucian carp due to theirgynogenetic reproduction. We examined the genetic characteristics of progeny that arose in a tank by natural
spawning of a tetraploid silver crucian carp pair. The
ploidy status of 120 samples randomly collected from theseprogeny was determined to be tetraploid by DNA content
ﬂow cytometry. DNA ﬁngerprints from a random ampliﬁed
polymorphic DNA assay indicated that almost all theprogeny examined had genotypes identical to the maternal
tetraploid female with no paternally derived fragments.
Selected specimens’ cytogenetic analyses revealed that theprogeny examined had tetraploid chromosome numbers,
categorized into 40 metacentric, 80 submetacentric, and 80
subtelocentric or telocentric chromosomes, which werearranged into quartets and six supernumerary microchromosomes. Fluorescence in situ hybridization signals weredetected in four homologous chromosomes in all analyzed
metaphases prepared from diploid goldﬁsh specimens.

Keywords Silver crucian carp Tetraploid
Gynogenesis Flow cytometry RAPD markers
FISH

Introduction
Silver crucian carp C. auratus langsdorﬁi comprises a
diploid-polyploid complex in the wild populations ofJapan. Bisexually reproducing diploids (2n = 100) aresympatrically distributed with gynogenetically developing
triploids (3n = 156) and tetraploids (4n = 206) [1–3].
Frequencies of tetraploids are much lower than those of
sympatric diploids and triploids [4, 5], and these tetraploids
are considered to arise from triploids by accidental incorporation of a haploid sperm nucleus into a triploid egg[6–8]. The occurrence of relatively rare tetraploids from
triploids, and the reproductive characteristics of such tetraploid progeny will provide good insights into mechanisms responsible for the origin, diversiﬁcation andmaintenance of such a diploid-polyploid complex in nature.
Thus, the wild tetraploid Japanese silver crucian carp C.
auratus langsdorﬁi has been considered excellent material
to disclose the biological signiﬁcance of polyploidy in ﬁshreproduction and evolution.
Because unreduced eggs of both triploids and tetraploids
develop by gynogenesis (sperm-dependent parthenogenesis) without any genetic contributions from males, theoffspring are theoretically an all-female population due tono contribution by male-determinant Y chromosomes.

J. Dong T. Fujimoto K. Arai (&)
Faculty and Graduate School of Fisheries Sciences,
Hokkaido University, Hakodate, Hokkaido, Japane-mail: araikt@ﬁsh.hokudai.ac.jp
M. Murakami
School of Veterinary Medicine, Azabu University, Sagamihara,
Kanagawa, JapanE. Yamaha
Nanae Fresh-Water Laboratory, Field Science Center for
Northern Biosphere, Hokkaido University, Nanae,
Hokkaido, Japan

123
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Fish Sci (2013) 79:935–941

2003, and then transferred and kept at Azabu University.
The rearing population of C. a. langsdorﬁi in the Gunma
Prefectural Fisheries Station was originated from a wild
population of the Jounuma Lake, Gunma Prefecture. The
tetraploid male was transferred to the Nanae Freshwater
Laboratory, Hokkaido University, Nanae, Hokkaido in
2004. No sampling and rearing record of the female C. a.
langsdorﬁi tetraploid individual was available, but it has
been reared together with the tetraploid male in the sametank. The tetraploid pair ﬁrst reproduced in the tank in
May, 2009 and a large number of progeny have appeared in
the tank since then.
A total of 120 individuals were randomly sampled fromthese progeny. Due to their small body sizes (body length,36–54 mm), sex could not be determined for each individual by naked-eye observation. Fin clips were collected
for somatic cellular DNA content determinations andstored in 100 % ethanol at -30 C for genomic DNA
extraction. Samples of goldﬁsh Carassius auratus auratus
which were kept in the aquarium of the Environment
Control Experiment Building, Faculty and Graduate Schoolof Fisheries Sciences, Hokkaido University, Hakodate
City, Hokkaido, were also used as a source of diploid
control samples for these analyses.

Although a high male incidence has been observed among
Chinese silver crucian carp C. auratus gibelio, because
these triploid forms undergo a mixture of allogynogenesis(gynogenesis triggered by heterospeciﬁc sperm from otherspecies) and bisexual gonochoristic reproduction [9], both
triploid and tetraploid males are very rare in the Japanesesilver crucian carp C. auratus langsdorﬁi population[10, 11].
In 2003, we happened to discover a tetraploid maleamong samples from a rearing population in the Gunma
Prefecture Fisheries Experiment Station. This male has
been reared in the Nanae Fresh-Water Laboratory, Hokkaido University, Nanae, Hokkaido, since 2004. This maleis kept in a tank along with a conspeciﬁc tetraploid female
whose sampling site and year were not recorded. They
reproduced in this tank by natural spawning, after which alarge number of progeny appeared.
There are three possible genetic results for the progenyof a pair of tetraploids. (1) When some reduced diploideggs are laid by a tetraploid female and then fertilized with
diploid sperm from a tetraploid male, the resulting progeny
are likely to be tetraploids with different genotypes. (2)Hexaploid progeny with different genotypes may result
when unreduced tetraploid eggs accidentally incorporate a
diploid sperm nucleus from a tetraploid male. (3) If unreduced tetraploid eggs of a tetraploid female initiate gynogenetic development due to the intrusion of diploid spermfrom a tetraploid male, all of these progeny will grow to asecond generation of gynogenetic tetraploids.
In the present study, 120 individuals were randomly
selected from the large population of progeny arising froma pair of tetraploids and were assessed for their ploidy
status by ﬂow cytometry analysis. Then, using selected
samples, the isogenic characteristics of these progeny wereveriﬁed by random ampliﬁed polymorphic (RAPD) DNAPCR ﬁngerprints. Chromosomes of these progeny werealso prepared and assessed by conventional karyotyping
and rDNA detection using ﬂuorescence in situ hybridization (FISH).

Flow cytometry
Ploidy status of 120 individuals was determined by ﬂow
cytometry analysis using a previously described procedure[12]. Brieﬂy, ﬁn tissues were minced in DNA-extraction
buffer (Cystain DNA 2 step, Partec GmbH, Münster,Germany) and then incubated at room temperature for
20 min. Samples were ﬁltered through a 50-lm mesh ﬁlter(Cell TRICS disposable ﬁlter units, Partec GmbH), after
which 5 volumes of staining solution (Cystain DNA 2 step,
Partec GmbH) containing DAPI (40 ,6-diamidino-2-phenylindole dihydrochloride) were added. DNA contents werethen analyzed using a CyFlow ploidy analyzer (PartecGmbH). Ploidy status was determined by comparing therelative DNA content against standard goldﬁsh diploid
DNA content (2C).

Materials and methods
DNA Preparation and RAPD-PCR Reactions
EthicsTotal genomic DNA was isolated from ﬁn-clip samples of
100 individuals by the phenol–chloroform method. RAPD
ampliﬁcation was performed as described previously [13].
From a total of 40 commercially available decamer random
primers (Kits A and B, Operon Technologies, Alameda,
CA, USA), three primers, OPA-07 (Kit A-7: 50 -GAAACG
GGTG-30 ), OPB-05 (Kit B-5: 50 -TGCGCCCTTC-30 ), and
OPB-07 (Kit B-7: 50 -GGTGACGCAG-30 ), were selectedon the basis of a preliminary screening for stable

Research and handling of the animals were performed inline with the Guide for the Care and Use of Laboratory
Animals at Hokkaido University.
Source of samples
A tetraploid male C. a. langsdorﬁi was found in a rearing
population of the Gunma Prefectural Fisheries Station in

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937

isothiocyanate, Roche) conjugate. Finally, the slides were
mounted in 100 ll of DABCO (1,4-Diazabicyclo-octane)
antifade solution containing DAPI for counterstaining. The
slides were observed under a Nikon ECLIPSE E800
microscope and images were captured with a Pixera Penguin 150CL-CU CCD camera (Pixera, San Jose, CA,USA).

ampliﬁcation, reproducible fragment patterns, and thepresence of polymorphisms between parents. The RAPDPCR mixture contained 1.0 ll of a DNA template (100 ng/ll), 2.0 ll of 10 9 PCR buffer (TaKaRa, Japan), 1.6 ll ofa dNTP mixture (TaKaRa), 0.2 ll of rTaq polymerase(TaKaRa), 15.2 ll of DDW, and 1.0 ll of a random primer(20 lM). Ampliﬁcation conditions were 3 min at 95 C as
an initial step, followed by 35 cycles of 95 C for 30 s,
36 C for 1 min for primer annealing, and 72 C for 1 minfor elongation. This was followed by a single ﬁnal step ofprimer extension at 72 C for 7 min. Approximately 9 llof ampliﬁcation products were separated by 1.5 % agarose
gel electrophoresis in TBE buffer (89 mM Tris, 89 mMboric acid, 2.2 mM EDTA). Gels were stained with ethidium bromide and photographed on a UV trans-illuminatorusing a gel documentation system (Print-graph AE-6915H,
Atto, Tokyo).

Results
After testing all progenies (n = 120), a prominent tetraploid-range DNA content peak (Fig. 1b) was detected
when compared with diploid goldﬁsh as a control (Fig. 1a).
RAPD-PCR was ﬁrst performed for 100 (nos. 1–100) out of
120 tetraploid samples using OPA-07. No paternal contribution was detected by this primer, and the ﬁngerprint
pattern of progeny was identical to that of the maternal
tetraploid female. Furthermore, no variations were detectedamong these progeny. Similar isogenic genotypes and all-

Chromosome preparation and karyotype analysis
Immature diploid goldﬁsh and tetraploid silver crucian carp
(total length: 40–65 mm; body weight: 1.0–3.0 g) wereintraperitoneally injected with 50–150 ll of 0.001 % colchicine 3 h prior to sacriﬁce. Gill tissues were removed andminced ﬁnely with forceps and suspended in goldﬁsh
Ringer’s solution (150 mM NaCl, 3.0 mM KCl, 3.5 mM
MgCl2, 5.0 mM CaCl2, 10 mM HEPES, pH 7.5). Aftercentrifugation at 1009g for 12 min, the cells were resuspended in 0.075 M KCl hypotonic solution for 40–60 min
at room temperature. Then, the cells were ﬁxed with chilled
Carnoy’s solution (methanol:acetic acid = 3:1) and stored
at -20 C. One droplet of a cell suspension was air-driedon a slide, which had been cleaned in advance with chilled
50 % ethanol. After Giemsa staining, karyotypes wereanalyzed on the basis of metaphase chromosomes,
according to Levan et al. [14].
Fluorescence in situ hybridization
FISH was performed using the 5.8S ? 28S rDNA probe
according to the protocol described by Fujiwara et al. [15]with minor modiﬁcations. Brieﬂy, human 5.8S ? 28S
rDNA was labeled with biotin-16-dUTP using a nicktranslation kit (Roche, Germany). Chromosome metaphasespread slides (pretreated with RNase) were denatured in
70 % formamide/2 9 SSC (pH 7.0) at 70 C for 2 min,dehydrated in cold 70 and 100 % ethanol for 10 min each,and then air dried. Then, 150–200 ng of labeled rDNA
probe was denatured at 75 C for 10 min and quickly
placed on ice before hybridization with each chromosome
slide. After washing in 50 % formamide/2 9 SSC,
2 9 SSC, 1 9 SSC, and 4 9 SSC, ﬂuorescent signals
were generated using an avidin–FITC (N-ﬂuorescein

Fig. 1 Flow cytometry results for relative DNA contents in ﬁn cells
from control diploid goldﬁsh Carassius auratus auratus 2C (a) and atetraploid silver crucian carp Carassius auratus langsdorﬁi progeny
4C (b)

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Fish Sci (2013) 79:935–941

female inheritance by progeny were also veriﬁed in thesame sample set (nos. 1–16) by RAPD-PCR using OPB-05(Fig. 2).
Fingerprint pattern using OPB-07 primer exhibited nocontribution of fragments speciﬁc to the father in all thesamples examined (n = 16). Samples, except for sample#3, demonstrated DNA ﬁngerprints identical to theirmother, but a slight variation was found in electropherogram in one out of 16 progeny examined: one maternallyderived fragment was faint or lacking when compared withother progeny (Fig. 2). However, this variant individual did
not show any polymorphism in ﬁngerprints ampliﬁed byother primers. These results indicated that almost all thesecond generation progeny of the tetraploid female were
genetically isogenic and thus likely a gynogeneticallygenerated clone without any contribution of paternal genome, and thus almost all the samples examined were likely
genetically identical.

Table 1 Chromosome distributions in somatic cells of diploid
goldﬁsh Carassius auratus auratus and tetraploid silver crucian carp
Carassius auratus langsdorﬁi
FishGoldﬁshSilvercruciancarp

Sampleno.

No. of cells with chromosomes
97

98

99

100

204

205

206

Total

1

0

0

0

12

–

–

–

12

2

0

0

0

13

–

–

–

13

3

1

0

0

11

–

–

–

12

1

–

–

–

–

0

1

9

10

2

–

–

–

–

1

0

10

11

3

–

–

–

–

1

0

11

12

Table 1 shows the chromosome distributions determinedfor diploid goldﬁsh and tetraploid silver crucian carp
samples. Tetraploid progeny (n = 3) had a modal numberof chromosomes of 4n = 206 (Fig. 3c), whereas control
goldﬁsh (n = 3) had a modal number of 2n = 100(Fig. 3a). In goldﬁsh, chromosomes were categorized into
20 metacentric (M), 40 submetacentric (SM), and 40
subtelocentric or telocentric (ST/T) chromosomes thatwere arranged into 10 M, 20 SM, and 20 ST/T pairs in theirkaryotypes (Fig. 3b). In tetraploid progeny, the mostinformative spreads gave karyotypes that were categorized
into 40 M, 80 SM, 80 ST/T, and six supernumerary microchromosomes (m). Regular-sized chromosomes werearranged into 10 M, 20 SM, and 20 ST/T quartets in tetraploid karyotypes (Fig. 3d).
FISH signals using the 5.8S ? 28S rDNA probe weredetected in four chromosomes in all analyzed metaphases
prepared from three goldﬁsh samples (Fig. 4a). Tetraploidprogeny had rDNA signals in eight chromosomes (Fig. 4b).
However, the exact morphologies of rDNA bearing chromosomes and the exact locations of rDNA signals were
difﬁcult to identify.

Discussion
Tetraploid-range DNA contents and isogenic RAPD
genotypes shown in these progeny clearly indicated theoccurrence of gynogenetic development in tetraploid eggs
that were spawned by a tetraploid female after fertilization by a tetraploid male sperm. These results indicatedthat sperm from the tetraploid male had triggered gynogenesis in unreduced tetraploid eggs spawned by thetetraploid female. In the Chinese silver crucian carp C.
auratus gibelio both allogynogenetic and gonochoristic
reproduction are involved [16]. In this species, gynogenesis is initiated only by fertilization with heterospeciﬁc
sperm from other species (allogynogenesis), whereas

Fig. 2 Random ampliﬁed polymorphic DNA (RAPD)-PCR proﬁles
using OPA-07 (top), OPB-05 (middle), and OPB-07 (bottom) primers
for silver crucian carp Carassius auratus langsdorﬁi, tetraploidparents ($ and #), and their progeny (nos. 1–10). M indicatesmolecular size markers

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Fig. 3 Mitotic metaphasespread (a) and corresponding
karyotype of goldﬁsh Carassius
auratus auratus with 100
chromosomes (b). Mitotic
metaphase spread (c) andcorresponding karyotype ofsilver crucian carp Carassius
auratus langsdorﬁi (d) with 206
chromosomes. Arrows indicate
microchromosomes. Scale bars
10 lm

Fig. 4 Mitotic metaphase chromosomes identiﬁed after ﬂuorescencein situ hybridization (FISH) probing with 5.8S ? 28S rDNA
sequences. Arrows indicate four ﬂuorescent signals (green) in goldﬁshCarassius auratus auratus (a) and eight ﬂuorescent signals (green) in

tetraploid silver crucian carp Carassius auratus langsdorﬁi (b). All
metaphase chromosomes were counterstained with 40 ,6-diamidino-2phenylindole dihydrochloride (DAPI). Scale bars 10 lm

normal bisexual fertilization occurs when eggs are fertilized with homospeciﬁc sperm (gonochorism) [16]. As
shown in the present study, eggs from a tetraploid female
initiated gynogenetic development even after fertilization
with homospeciﬁc sperm from a tetralploid male. Among
120 individuals examined, no hexaploid individuals arose
due to the incorporation of a sperm nucleus from thetetraploid male.

However, an exceptional progeny with a slight difference
was detected by RAPD-PCR using OPB-07 in 16 samples
examined. This result suggests that the tetraploid female
presumably produced large numbers of unreduced clonaltetraploid eggs as well as a very small number of exceptional
tetraploid eggs with genetic variation. Such variant eggs canbe explained by unusual meiotic recombination during
oogenesis of the tetraploid female. Genetic and cytogenetic

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results showing the involvement of meiotic recombinationby the formation of synapsed bivalents and partially paired
trivalents have been reported in gynogenetic triploid silver
crucian carp [17, 18]. A few tetraploid eggs may undergoparing and recombination between two homologous and/or
homoeologous chromosomes, because they essentially havefour sets of chromosomes with relatively higher similarities
or afﬁnities. The other possibility may be attributed to
unstable reproducibility of RAPD-PCR itself, in which thepurity of template DNA often affects ampliﬁcation of fragments [19]. Therefore, further study is required to conﬁrm
the presence or absence of genetic variation among tetraploid eggs using more sensitive and reliable genetic markers
such as a microsatellite DNA.
The karyotypes of the goldﬁsh examined here were very
similar to those previously reported for diploid C. auratus
subsp. (2n = 100) and diploid Miyazaki race silver crucian
carp C. auratus langsdorﬁi (2n = 100) [1], which had
20 M ? 40 SM ? 40 ST/T chromosomes. The karyotype ofa tetraploid silver crucian carp C. auratus langsdorﬁi(4n = 206), obtained from the Kanto district was reported to
be 44 M ? 82 SM ? 80 T [1], similar to our present results.
They had 206 chromosomes, but six supernumerary chromosomes were not distinguished from 200 regular chromosomes. The presence of six microchromosomes found in
the present study suggested that a gynogenetic tetraploid
may have arisen from a triploid with 3n = 156 chromosomes [1] by incorporation of haploid chromosomes.
An rDNA locus with 5.8S ? 18S ? 28S genes and thatwith 5S genes are located on different chromosomes inmost taxa [20]. In triploid silver crucian carp C. auratus
gibelio, 5S rDNA signals were detected in three STs [21].
In the present study, rDNA signals were detected in fourchromosomes of diploid goldﬁsh with 2n = 100, whereas
eight signals were detected in chromosomes of tetraploidsilver crucian carp with 4n = 206. These FISH results
indicated that the progeny of silver crucian carp must havea tetraploid karyotype, including four sets of chromosomes.
However, the exact morphologies of rDNA-bearing chromosomes have not been determined and additional
molecular cytogenetic studies will be required in the near
future.
Gynogenesis usually occurs by preventing the decondensation of a sperm nucleus due to a failure in the
breakdown of the sperm nuclear envelope [22, 23] in ﬁsh
species that spawn unreduced diploid or triploid eggs [13,24–26]. Unreduced diploid eggs are generated by themechanism of premeiotic endomitosis (chromosome
doubling without cytokinesis) in the clonally reproducing
diploid loach Misgurnus anguillicaudatus [13], whereas
clonal triploid eggs are formed by apomixis (i.e., ameiotic
division of oocytes by skipping the ﬁrst meiotic division)in the silver crucian carp C. auratus gibelio [24, 25] andC. auratus langsdorﬁi [26], because the formation of a
tripolar spindle may physically inhibit the ﬁrst meiotic
division.
The occurrence of a tripolar spindle is presumably
related to the presence of three sets of chromosomes ingynogenetic triploid silver crucian carp. If so, then what
would occur in unreduced tetraploid eggs that include four
sets of chromosomes? This is an enigma that needs to beresolved. In tetraploid eggs with an even number of chromosome sets, normal synapsis and subsequent meiosis canbe predicted, although tetraploid silver crucian carp produce unreduced tetraploid eggs that develop by
gynogenesis.
In tetraploid silver crucian carp, the cytological mechanisms responsible for the formation of unreduced tetraploideggs and the initiation of gynogenesis remain to be determined. Our tetraploid population is a reliable set of experimental animals for examining these problems. To identify
these mechanisms, the presence or absence of multipolarand other unusual spindles must be investigated in theoocytes of tetraploid silver crucian carp, which can be
induced to undergo ﬁnal maturation by administering 17a20b-dihydroxy-4-pregnene-3-one during in vitro culture[13, 26]. The conﬁguration of meiotic chromosomes is also
informative for determining the reproductive mode [13].
Furthermore, the second generation progeny of tetraploid silver crucian carp are reliable for generating sexreversed tetraploid males, which are a genetic resource ofdiploid spermatozoa for further ploidy manipulations. A
dimorphism of reproductive mode in the tetraploid silver
crucian carp, showing gynogenetic unreduced oogenesis
and reduced spermatogenesis, may provide an excellent
model for reproductive biology in ﬁshes.

References
1. Kobayasi H, Kawashima Y, Takeuchi N (1970) Comparative
chromosome studies in the genus Carassius, especially with aﬁnding of polyploidy in the ginbuna (C. auratus langsdorﬁi) (inJapanese with English abstract). Japanese J Ichthyol 17:153–160
2. Kobayasi H, Ochi H (1972) Chromosome studies of the hybrids,
ginbuna C. auratus langsdorﬁi 9 kinbuna C. auratus subsp andginbuna 9 loach Misgurnus anguillicaudatus (in Japanese withEnglish abstract). Zool Mag 81:67–71
3. Kobayasi H, Nakano K, Nakamura M (1977) On the hybrids, 4nginbuna (Carassius auratus langsdorﬁi) 9 kinbuna (C. auratus
subsp.), and their chromosomes (in Japanese with English
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4. Onozato H, Torisawa M, Kusama M (1983) Distribution of thegynogenetic polyploid crucian carp Carassius langsdorﬁi in
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5. Maeda K, Nishimura D, Maemura H, Morishima K, Zhang Q,
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auratus gibelio) as revealed by RAPD assays. J Mol Evol
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18. Zhang F, Oshiro T, Takashima F (1992) Chromosome synapsis
and recombination during meiotic division in gynogenetic triploid ginbuna, Carassius auratus langsdorﬁi. Japan J Ichthyol
39:151–155
19. McClelland M, Welsh J (1994) DNA ﬁngerprinting by arbitrarily
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sequences and chromosomal assignment of the genes to theextreme terminal region of chromosome 19. Cytogenet Cell
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21. Zhu HP, Ma DM, Gui JF (2006) Triploid origin of the gibel carp
as revealed by 5S rDNA localization and chromosome painting.
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H, Yamaha E, Arai K (2007) Cytological mechanisms of gynogenesis and sperm incorporation in unreduced diploid eggs of theclonal loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae).
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23. Yamashita M, Onozato H, Nakanishi T, Nagahama Y (1990)
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24. Cherfas NB (1966) Natural triploidy in females of the unisexualform of silver crucian carp (Carassius auratus gibelio Bloch).
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26. Yamashita M, Jiang JQ, Onozato H, Nakanishi T, Nagahama Y(1993) A tripolar spindle formed at meiosis I assures the retentionof the original ploidy in the gynogenetic triploid crucian carp,
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markers (in Japanese with English abstract). Nippon Suisan
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8. Murakami M, Matsuda C, Fujitani H (2001) The maternal originsof the triploid ginbuna (Carassius auratus langsdorﬁi): phylogenetic relationship within the C. auratus taxa by partial mitochondrial D-loop sequencing. Genes Genet Syst 76:25–32
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Brykov VA, Gui JF (2013) High male incidence and evolutionary
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13. Itono M, Morishima K, Fujimoto T, Bando E, Yamaha E, Arai K(2006) Premeiotic endomitosis produces diploid eggs in thenatural clone loach, Misgurnus anguillicaudatus (Teleostei:Cobitidae). J Exp Zool 305A:513–523
14. Levan A, Fredga K, Sandberg AA (1964) Nomenclature forcentromeric position on chromosomes. Hereditas 52:201–220
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日本食品微生物学会雑誌
160
日食微誌 Vol. 30 Jpn.
No. J.
3 Food2013 Microbiol., 30(3), 160‒164, 2013

＝ Short Paper ＝

Development of Duplex SYBR Green Real-Time PCR for
Rapid and Simultaneous Detection of 16 Speciﬁc
Genes of 16 Major Foodborne Bacteria
Natsuko IIDA1, 3, Hiroshi FUKUSHIMA2, Midori HIROI1, Miya YAGI1, Takashi KANDA1,
Masaru MURAKAMI3, † and Kanji SUGIYAMA1(1 Shizuoka Institute of Environment and Hygiene, Kita-ando, Aoi-ku, Shizuoka 420‒8637)(2 Shimane Prefectural Livestock Technology Center, Kosi-cho, Izumo, Shimane 693‒0031)(3 Azabu University School of Veterinary Medicine, Fuchinobe, Chuo-ku,
Sagamihara 252‒5201; † Corresponding author)(Received: April 24, 2013)(Accepted: June 17, 2013)
Key words: foodborne bacteria, duplex SYBR Green real-time PCR, simultaneous screening

time PCR assay system for simultaneously detecting 16 speciﬁc genes of 16 common foodborne
bacteria occurring mainly in Japan. The target
foodborne pathogens were selected on the basis ofdata on foodborne outbreaks5, 6) in Shizuoka Prefecture collected in the past 10 years. Of these, thefollowing are considered the main causative pathogens of foodborne outbreaks:,, enterohaemor(EHEC), enteropathogenic
rhagic(EPEC), enteroaggregative(EAEC),
emetic toxin-producing, TDH-positive, andspp. The aim of this study
was to develop a simple, practical, labor-saving,and eﬀective method for food poisoning examination in the ﬁeld.

Introduction
In foodborne outbreaks, public health administrators must respond promptly and eﬀectively
to prevent the spread of pathogens and the recurrence of food poisoning. To do this, it is necessary
to identify the causative agents of outbreaks assoon as possible. Traditional culture methods thathave been routinely used require several days toidentify foodborne bacteria. Traditional culture
methods are time-consuming and laborious, since
the necessary culture conditions and nutrient media diﬀer from bacteria to bacteria. Thus, simple
and reasonable methods for simultaneously detecting various food-poisoning bacterial species
are preferable.
Recently, speciﬁc gene sequences have been
identiﬁed in various pathogenic bacteria, and reports on PCR methods targeting the gene sequences of foodborne or waterborne pathogens
have been extensively studied4, 7, 9). In food poisoning tests, these PCR methods are useful for
screening prior to culture. For instance, Fukushima
developed a method for simultaneously
detecting 24 speciﬁc genes of foodborne bacteriausing a relatively low-cost multiplex real-time
PCR system3). However, the system is considered
a little laborious and somewhat impractical to use
in the ﬁeld.
Here, we designed a modiﬁed duplex real-

Materials and Methods
The 11 species of foodborne bacteria (
,
spp.,
spp.,
,
,
,
,
,
, and
), including ﬁve groups of[EHEC, EPEC, EAEC, enteroinvasive(EIEC), and enterotoxigenic(ETEC)](emetic toxin-producand two groups of) wereing
and enterotoxigenic
used as control for PCR analysis.

e-mail: murakami@azabu-u.ac.jp

DNA was extracted from bacterial cultures

433

161

and stool samples with a QIAamp DNA Stool Minikit (Qiagen) according to the manufacturerʼs instructions.

in feces and then assessed its usefulness. The present PCR system allows us to simultaneously analyze 16 speciﬁc genes of foodborne pathogens in
up to nine fecal samples. There are many kinds offoodborne bacteria. In this study, we selected only
16 major foodborne bacteria and set a larger mdistance in duplex PCR between the target geneʼs
products in this study than in a previous study3).
The Tm diﬀerences were suﬃcient to unequivocally distinguish between two given foodborne
bacteria. The speciﬁcity of the present PCR assay
was veriﬁed using three strains for each of the 16
target genes (a total of 48 strains). The detection
limit, ranging from 103 to 106 CFU per gram ofstool sample (CFU/g), was determined to be as
andfollows: 103 CFU/g forand 104 to 105
ETEC, 106 CFU/g for
CFU/g for the other foodborne bacteria. This sensitivity was suﬃcient for practical screening.
The set of eight PCR duplex assays used inthis study was evaluated to detect the causative
bacteria in feces in 12 cases of foodborne-related
outbreaks in Shizuoka Prefecture (Table 2). In all
the 12 cases examined, the duplex PCR assay detected the causative bacteria from at least one ofthree fecal samples. Figure 1 shows the detectionof a-speciﬁc gene using primer set B(Table 1) in case 3. In the duplex PCR assay withthis primer set B, each PCR product showed a different Tm curve.
was detected in six ofseven stool specimens (S1‒S7) from the symptomatic patients. Consistent with these moleculardata,
was isolated from six corresponding samples by the culture method (Table 2). The
presence of causative agents presumed from theduplex PCR assay was thus conﬁrmed from theresults of bacterial culture. In case 3,-posi(EAEC)
tive
and-positive
were also simultaneously detected using primer
set F from one of seven fecal specimens andprimer set G from three of seven fecal specimens.
Such occurrence of multiple foodborne bacterial
pathogens was observed in 8 of 12 cases, whichwere demonstrated using the present duplex PCR
assay system. In case 10,
was rapidly and successfully detected in two patients by the PCR method, but not by the routine
culture method. In cases 9 and 12, the number of
positive results obtained by the PCR assay wasless than that obtained by the conventional culture method. This might be due to the insuﬃcient

The 16 primer pairs were used to detect theabove 11 species of foodborne bacteria. These
primer pairs for 16 target genes were selectedfrom previous reports (see Table 1 references).
The primer sequence is listed in Table 1.

Duplex SYBR Green real-time PCR was performed in 8 lines (12 wells per line) on a 96-wellreaction plate using ABI7500 Real-Time PCR systems (Applied Biosystems). One line included anegative control (water), two lines included positive controls, and nine lines included template
DNA samples from nine fecal samples. Real-time
PCR was performed for a total volume of 20 μ .
Each reaction well contained 10 μ of SYBR Premix DimerEraser (TaKaRa, Japan), 0.4 μ of ROX
Reference Dye II (50×), 4.4 μ of PCR-grade H2O,
0.8 μ of a 10 μM primer set for each of two target
genes, and 2 μ of sample DNA in a 20 μ PCR
mixture. The assay cycling proﬁle was one cycleof 95℃ for 30 sec followed by 30 cycles of denaturation at 95℃ for 5 sec and annealing at 55℃ for
34 sec and then at 72℃ for 40 sec, and ﬁnally adissociation stage of one cycle each at 95℃ for 15
sec, at 60℃ for 60 sec, and at 95℃ for 15 sec. The
sizes and melting point temperatures ( mʼs) of the
PCR products are listed in Table 1.
Reaction speciﬁcity was determined on thebasis of the m of the ampliﬁcation products. The
diﬀerence in Tm between two of the expected
PCR products in a duplex reaction was set at
more than 1.9℃.

Twelve foodborne-related outbreak casesthat occurred in Shizuoka Prefecture from 2009 to2012 were examined using duplex real-time PCR
analysis. Fecal samples (侑200 mg) from diarrhea
or symptomatic patients were treated with a
QIAamp DNA Stool Mini kit.
Results and Discussion
We developed a modiﬁed real-time PCR
screening system for eight duplex assays using a
96-well reaction plate to detect foodborne bacteria

434

435

b

a

EHEC

16

14

EHEC
invA139
invA141
SG-F3
SG-R3

spp.

FemB-fw
FemB-rv
yadA-X-F
yadA-X-R

Tdh199-F
Tdh199-R
ST-f
ST-r

ces-TM-F
ces-TM-R
LT-1
LT-2

15a

and

spp.

EAST-1-S
EAST-1-AS
JMS1F
JMS1R

ETEC

ETEC

EIEC andeae-F2eae-R
virA-FvirA-R

13a EAEC

12

11a

10

9a

8

7a

6

5a EHEC and EPEC

Nine main foodborne bacteria
Melting point temperatures value of PCR products

H

G

F

E

D

C

4

Speciﬁc

AB-F
AB-R
JMS2-F
JMS2-R

2

3aPrimername
GAP-11GAP-12LM-hly-1
LM-hly-2

Targetgene

1aSpecies

GTGAAATTATCGCCACGTTCGGGCAA
TCATCGCACCGTCAAAGGAACC
GCACTTATGGCAGTATTTGCAGC
GCATCTTTTAAGCCTTCTGGTCGCCATCAACACAGTATATCCGAGTGACGGCTTTGTAGTCC
GTCACAGTAACAAACCGTAACA
TCGTTGACTACTTCTTATCTGGAAATTAACGAAATGGGCAGAAACATGCGCAACACCCTGAACTTCCAGAACCAATTGCAATGCCTCTTTAAACAGCTTGTTCCAGCCA

GGTACTAAATGGCTGACATC
CCACTACCACTCTCATATGCGCTAAACCAGTARGGTCTTCAAAA
CCCGGTACARGCAGGATTACAACAGATGTTTGCGACGATGCAACTTTCGGCGTGATACCCATT
AGCAGGTTTCCCACCGGATCACCAGTGCTCAGATTCTGGGTCTCCATTGATCAGGATTTTTCTGGTGATACTCATGCGGAAATAGCCGTTA
CTGCATTCTGGCAATCTCTTCACATGATGAGCTAACTTCGTAAGCCCTCC

CTGAATTTGATACCTTAAGTGCAGC
AGGCACGCCTAAACCTATAGCTCGACCCCTCTTGAACATAGATAGACATCAAGCCCTCGT

GGTTCATTAATTGAAACTGGTG
AACGCCAATCATATAAATTACAGCCGGAGGTTCCGCAAAAGATG
CCTCCAGAGTGATCGATGTT

Sequence(5' → 3')

152

284

95

106

100

93

147

251

275

65

215

102

108

86

234

154

Product size
(bp)

79.6

85.0

79.6

83.5

78.7

81.2

76.6

79.3

80.4

78.5

80.4

78.1

80.4

77.0

79.1

75.9

b
m

2)

4)

3)

3)

2)

3)

8)

2)

3)

3)

2)

3)

3)

3)

1)

3)

References

Eight primer sets of duplex SYBR Green real-time PCR with two primer pairs for two target genes used to simultaneously and rapidly screen various foodborne bacteria

日食微誌

B

A

Primerset

Table 1.

162
Vol. 30 No. 3 2013

163
Table 2.

Examination by duplex real-time PCR assay and bacterial culture in 12 foodborne-related outbreaks that
occurred from 2009 to 2012 in Shizuoka Prefecture.
Positive detection

No. ofexamined
samplesConventional
culture method

7
3

5
1

7

6

4
7

3
7

Supper4

3

Restaurant
Restaurant

Catering
Supper

6
4

3
2

9

School

Unknown(TW78)

9

8

10
11

SchoolCaterer

Unknown
Catering

5a

5
5

2
5

12

Caterer

Catering

8

8

Case
No.

Facility

Source ofinfection

1
2

Hospital
Restaurant

Lunch
Supper3

Restaurant

Supper4
5

Restaurant
Hospital

Lunch
Lunch

6

Hotel

7
8

Fig. 1.

Isolatedbacteria(TW9)(TW12)S. Nagoya

S. Enteritidis

This study samples(Detected gene)
5(
1(
2(
6(
1(
3(
4(
7(
2(
3(
1(
3(
3(
1(
6(
1(
2(
3(
2(
4(
2(

)

speciﬁc)

)

)
)

)

)

)

speciﬁc)))))

speciﬁc)
speciﬁc)

)

)
)
)
)
)

Melting point temperature ( m) curves for the products of duplex PCR using primer set B from seven samples incase 3 of the foodborne outbreak (see Table 2).
When a few target bacterial cells were present in the reaction well, m became lower than the expected value by
approximately 1℃ . The mʼs of S2 and S3 were within allowable tolerance.

essentially consistent with those of the culture
method (Table 2). These data show that the proposed PCR method is useful for the rapid identiﬁcation of the causative pathogens of foodborne
outbreaks. Moreover, the system can rapidly, accurately, and safely detect various bacterial species, including, which require long culture times, as well as
and ETEC,

DNA extraction sinceandare Gram-positive bacteria with a thick cell wall.
Analysis using the screening system (DNAextraction from feces and duplex real-time PCR
reactions) was performed approximately 3 to 4
hours, faster than the conventional culture assay,
where causative pathogens were isolated after 2
to 4 days. The results of the PCR assay were also

436

164

日食微誌

Vol. 30 No. 3 20132)

which produce toxins.
The rapid and comprehensive presumption offood-poisoning pathogens, which can be obtainedusing the present simultaneous screening system
by duplex real-time PCR assay, will enable public
administrators to promptly and eﬃciently provide
hygiene instructions in foodborne outbreaks.
In conclusion, the screening system describedhere enables the simultaneous analysis of 16 speciﬁc target genes of foodborne pathogens in up tonine stool specimens by using a set of eight duplex real-time PCR assays with a 96-well reaction
plate. This PCR assay system is useful for detecting causative bacteria in fecal specimens obtained
from symptomatic patients and is suitable for the
rapid and presumptive diagnosis of food-poisoning
pathogens in fecal specimens of foodborne outbreaks in Japan.

Fukushima, H., Katsube, K., Tsunomori, Y., Kishi, R.,
Atsuta, J. and Akiba, Y.: Comprehensive and rapid
real-time PCR analysis of 21 foodborne outbreaks. Int. J.
Microbiol., Article ID 917623, 13 pages (2009).

3)

Fukushima, H., Kawase, J., Etoh, Y., Sugama, K., Yashiro,S., Iida, N. and Yamaguchi, K.: Simultaneous screening of
24 target genes of foodborne pathogens in 35 foodborne
outbreaks using multiplex real-time SYBR Green PCR
analysis. Int. J. Microbiol., Article ID 864817, 17 pages(2010).

4)

Fukushima, H., Tsunomori, Y. and Seki, R.: Duplex realtime SYBR Green PCR assays for detection of 17 spe-

5)cies of food- or waterborne pathogens in stools. J. Clin.
Microbiol., 41, 5134‒5146 (2003).
Health and Welfare Department, Shizuoka Prefecture:
Food poisoning in Shizuoka Prefecture 2004 (in Japa-

nese).
)
6 Health Department, Shizuoka Prefecture: Food poisoning in Shizuoka Prefecture 2009 (in Japanese).
)
7 Matsuki, T., Watanabe, K., Fujimoto, J., Takada, T. andTanaka, R.: Use of 16S rRNA gene-targeted group-speciﬁc primers for real-time PCR analysis of predominantbacteria in human feces. Appl. Environ. Microbiol., 70,7220‒7228 (2004).

Acknowledgment
We thank the staﬀ of health centers in Shizuoka Prefecture for performing the sample collection.
References
1) Bubert, A., Riebe, J., Schnitzler, N., Schönberg, A., Goebel, W. and Schubert, P.: Isolation of catalase-negative
strains from listeriosis patientsand their rapid identiﬁcation by anti-p60 antibodies
and/or PCR. J. Clin. Microbiol., 35, 179‒183 (1997).

8)

Nguyen, T. V., Van, P. L., Huy, C. L., Gia, K. N. and Weintraub, A.: Detection and characterization of diarrheagenic
from young children in Hanoi,
Vietnam. J. Clin. Microbiol., 43, 755‒760 (2005).

9)

Ott, S. J., Musfeldt, M., Ullmann, U., Hampe, J. and Schreiber, S.: Quantiﬁcation of intestinal bacterial populations by real-time PCR with a universal primer set andminor groove binder probes: a global approach to theenteric ﬂora. J. Clin. Microbiol., 42, 2566‒2572 (2004).

Duplex SYBR Green real-time PCR 法による食中毒起因菌 16 遺伝子の一斉迅速検査法の開発
飯田奈都子 1, 3・福 島 博 2・廣井みどり 1・八 木 美 弥 1
神 田 隆 1・村 上 賢 3, †・杉 山 寛 治 1
1 静岡県環境衛生科学研究所，2 島根県畜産技術センター，3 麻布大学獣医学部，† 責任著者
細菌性食中毒検査の迅速化・省力化のため，duplex real-time PCR 法を用いた食中毒起因菌のより実践的な一斉スクリーニング法を考案した．本法は，食中毒患者の糞便検体から抽出されたDNA を対象とし，Duplex SYBR Green real-time PCR の 8 反応系を同時に行い，発生頻度の高い
食中毒起因菌の 16 遺伝子を一斉に検索する．菌種の同定は，融解曲線分析の m 値との比較より，判定することが可能である．実際の食中毒事例の患者糞便検体に本法を導入したところ，培養法と同等の成績を約 3 〜 4 時間で得ることができた．本法は，検査の効率化のみならず，食中毒事件へのより迅速な行政対応を可能にし , 健康危機管理の一端を担う方法として活用が期待される．

437

Biochimica et Biophysica Acta 1830 (2013) 5027–5035

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen

Regulation of brown adipogenesis by the Tgf-β family: Involvement of
Srebp1c in Tgf-β- and Activin-induced inhibition of adipogenesis
Hirofumi Yoshida a, Yohei Kanamori a, Hiroki Asano a, Osamu Hashimoto b, Masaru Murakami c,
Teruo Kawada d, Tohru Matsui a, Masayuki Funaba a,⁎aDivision of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Laboratory of Experimental Animal Science, Kitasato University School of Veterinary Medicine, Towada 034-8628, Japanc
Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Chuo-ku, Sagamihara 252-5201, Japand
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 611-0011, Japanba r t i c l e

i n f oArticle history:
Received 13 February 2013
Received in revised form 12 June 2013
Accepted 30 June 2013
Available online 10 July 2013
Keywords:
Brown adipocyte
Tgf-β family
Tgf-β
ActivinBmp

a b s t r a c t
Background: Brown adipocytes generate heat through the expression of mitochondrial Ucp1. Compared with theinformation on the regulatory differentiation of white preadipocytes, the factors affecting brown adipogenesis
are not as well understood. The present study examined the roles of the Tgf-β family members Bmp, Tgf-β
and Activin during differentiation of HB2 brown preadipocytes.
Methods: Endogenous Bmp activity and effects of exogenous Tgf-β family members were examined. Role of
Srebp1c in brown adipogenesis was further explored.
Results: Although Bmp7 has been suggested to be a potent stimulator of brown adipogenesis, it affected neither
the expression of brown adipocyte-selective genes nor Ucp1 induction in response to a β adrenergic receptor agonist. Unlike in 3T3-L1 white preadipocytes, endogenous Bmp activity was not required for brown adipogenesis;
treatment with inhibitors of the Bmp pathway did not affect differentiation of preadipocytes. Administration of
Tgf-β1 or Activin A efﬁciently decreased the insulin-induced expression of brown adipocyte-selective genes.
Tgf-β1 and Activin A decreased the expression of Pparγ2 and C/ebpα, suggesting the inhibition of adipogenesis.
The Tgf-β- and Activin-induced inhibition of brown adipogenesis was mediated by the repression of Srebp1c
expression; Tgf-β1 and Activin A blocked Srebp1c gene induction in response to the differentiation induction,and knock-down of Srebp1 expression inhibited brown adipogenesis.
Conclusion: Endogenous Bmp is dispensable for brown adipogenesis, and Srebp1c is indispensable, which is
negatively regulated by Tgf-β and Activin.
General signiﬁcance: Control of activity of the Tgf-β family is potentially useful for maintenance of energy
homeostasis through manipulation of brown adipogenesis.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
There are two major types of adipocytes in mammals: white andbrown. White adipocytes are specialized for the storage of excess energy, containing all of the enzymatic machinery necessary to build triacylglycerols from fatty acids synthesized de novo or from fatty acids
imported from circulating lipoproteins [1]. In contrast, brown adipocytes dissipate chemical energy in the form of heat against cold exposure [2]. This thermogenic function of brown fat results from theexpression of a series of genes related to a high mitochondrial content
and the elevated cellular respiration that is largely uncoupled from⁎ Corresponding author at: Division of Applied Biosciences, Graduate School of
Agriculture, Kyoto University, Kitashirakawa Oiwakecho, Kyoto 606-8502, Japan.
Tel.: + 81 75 753 6055; fax: + 81 75 753 6344.
E-mail address: mfunaba@kais.kyoto-u.ac.jp (M. Funaba).
0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbagen.2013.06.036

438

ATP synthesis [3]. This uncoupling occurs through the mitochondrial
uncoupling protein 1 (Ucp1), a mammalian brown adipocyte-speciﬁc
protein that promotes proton leak across the inner mitochondrial membrane [2,4]. Recent ﬁndings that adult humans have functional brown
adipocytes have initiated studies of the origin and regulatory differentiation of brown preadipocytes [5,6]. However, compared with the information known about white adipogenesis, knowledge of the regulationof brown adipogenesis is still limited.
Despite having a different physiological role from white adipocytes, brown adipocytes share many molecular components withwhite adipocytes [7–9]. Peroxisome proliferator-activated receptor(Ppar) γ and CCAAT-enhancer binding protein (C/ebp) α, transcription factors, potently regulate differentiation of preadipocytes into
adipocytes; Pparγ-null mice exhibited extremely lower weight ofwhite fat as well as brown fat [10], and C/ebpα was required fordevelopment of functional white and brown fat [11,12]. Pparγ and5028

H. Yoshida et al. / Biochimica et Biophysica Acta 1830 (2013) 5027–5035

C/ebpα stimulate the transcriptional activation of the large group ofgenes that produce the adipocyte phenotype [13]. In contrast, expression of genes related to mitochondrial biogenesis and oxidative metabolism highly expressed in brown adipocytes is mainly regulated
by a transcriptional co-activator Pgc1α, a Pparγ-binding partner predominantly expressed in the brown fat [3].
Members of the transforming growth factor-β (Tgf-β) family
potently regulate commitment, differentiation and maturation ofmesenchymal cells [14]. This family also participates in the development of mature white adipocytes; treatment with Tgf-β or Activin
A, representative members of the Tgf-β family, inhibited the transcriptional activity of C/ebpβ, the expression of Pparγ and lipid accumulation in 3T3-L1 white adipocytes, indicating inhibitory effects on
adipogenesis [15,16]. Endogenous activity of bone morphogenetic
protein (Bmp), another representative member of the Tgf-β family,
in white preadipocytes was higher before differentiation induction,and this activity was indispensable for adipogenesis [17]. In contrast,
exogenous Bmp regulates white adipogenesis in a cell contextdependent manner; treatment with Bmp2 decreased insulin-induced
lipid accumulation in 3T3-F442A adipocytes [18], whereas Bmp7 stimulated differentiation of 3T3-L1 preadipocytes [19]. Furthermore, Bmp4
potentiated the commitment of pluripotent mesenchymal C3H10T1/2
cells to adipocyte-lineage cells [20,21].
Here, we explored the roles of the Tgf-β family in brown adipogenesis using HB2 preadipocytes [22]. Our results indicate that1) both endogenous and exogenous Bmp do not affect brown adipogenesis, 2) Tgf-β and Activin inhibit differentiation of brown
preadipocytes, and 3) Tgf-β and Activin block insulin-induced expression of sterol regulatory element-binding protein (Srebp) 1c, which
is necessary for brown adipogenesis. The present results indicate theroles of Tgf-β and Activin as negative regulators of brown adipogenesis
through the inhibition of differentiation into fat cells.
2. Materials and methods

immortalization are clear in HB2 brown preadipocytes, as comparedwith brown preadipocyte lines transformed with SV40 T-antigen[23–25]. Thus, we used HB2 brown preadipocytes in this study.
HB2 cells were grown in growth medium (Dulbecco's modiﬁed
Eagle's medium (DMEM) with heat-inactivated 10% FBS, 100 U/ml
penicillin and 100 μg/ml streptomycin) at 37 °C in a humidiﬁed 5%
CO2 atmosphere. To examine the roles of endogenous Bmp activity,
HB2 cells were differentiated with the differentiation protocol of
3T3-L1 cells [26]: two days after reaching conﬂuence (day 0), thecells were cultured in the growth medium in the presence of differentiation inducers (Dex [0.25 μM], IBMX [0.5 mM] and insulin [10 μg/ml])
for 2 days, followed by culture with insulin (5 μg/ml) in the growth
medium. In addition, T3 (50 nM) was added to the medium; T3 is frequently used during brown adipocyte differentiation in vitro [27–31].
To determine the minimal factors for brown adipogenesis, cells were
cultured in the growth medium in the presence or absence of T3(50 nM) and insulin (20 nM). The Bmp inhibitors and Tgf-β family
members were added at the following concentrations: dorsomorphin(5 μM), LDN-193189 (100 nM), Bmp (3.3 nM), Tgf-β1 (200 pM) andActivin A (4 nM). To examine the effects of cAMP stimulation, thecells were treated on day 8 with or without isoproterenol (10 μM)
for 4 h. Oil Red O staining on day 6 or day 8 after the differentiationinduction and the subsequent quantiﬁcation of dye were performed
as described previously [17].

2.3. Western blotting
Western blot analyses were performed as described previously [32].
The immunoreactive proteins were visualized using the ECL AdvanceWestern blotting detection system (GE Healthcare, Buckinghamshire,UK) according to the manufacturer's protocol. After stripping the antibodies and the detection reagents, the membranes were reprobedwith antibodies against Smad1, Smad2 or β-actin.

2.1. Materials
2.4. RT-quantitative PCR
The following reagents were purchased or provided: dorsomorphin(compound C: 6-[4-(2-piperidin-1-yl-ethoxy) phenyl]-3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine) was from Calbiochem (La Jolla, CA, USA);LDN-193189 was from Stemgent (San Diego, CA, USA); dexamethasone(Dex), 3-isobutyl-1-methylxanthine (IBMX), insulin, triiodothyronine(T3) and isoproterenol were from Sigma (St. Louis, MO, USA); puriﬁed
Tgf-β1 was from Becton Dickinson (Franklin Lakes, NJ, USA); recombinant Activin A, Bmp4 and Bmp7 were from R & D Systems (Minneapolis,
MN, USA); troglitazone was from Daiichi-Sankyo (Tokyo, Japan); a
rabbit polyclonal antibody against phospho-Smad1 (Ser463/Ser465)/
Smad5 (Ser463/Ser465)/Smad8 (Ser426/Ser428) (#9511) and a rabbit
polyclonal antibody against phospho-Smad2 (Ser465/Ser467) werefrom Cell Signaling Technology (Danvers, MA, USA); and a rabbit monoclonal antibody against Smad1 (ab33902), rabbit polyclonal antibodies
against Pparγ2 (ab45278), Smad2 (ab63576) and Ucp1 (ab10983),
and a mouse monoclonal antibody against β-actin (AC-15) were from
Abcam (Cambridge, MA, USA).

RNA isolation and RT-quantitative PCR (qPCR) were performed as
described previously [33,34]. The oligonucleotide primers for RT-qPCR
are presented in Supplementary Table 1. The Ct value was determined,
and the abundance of gene transcripts was analyzed using the ΔΔCt
method using 18S rRNA as the normalization gene.

2.5. siRNA transfection
To target the expression of Srebp1 and green ﬂuorescent protein(GFP) as a control, the siRNAs of the respective genes were synthesized by Bonac Corp. (Kurume, Japan). Two oligonucleotides were usedin combination to inhibit the expression of Srebp1, 5′-CCUACGAAGUGCACACAAAdTdT-3′ and 5′-UUUGUGUGCACUUCGUAGGdTdT-3′.
The siRNA for GFP was described previously [34]. Overall, siRNA(60 pmol) was transfected in a 12-well plate using Lipofectamine
RNAiMAX (Invitrogen) according to the manufacturer's protocol.

2.2. Cell culture
HB2 brown preadipocytes, which were kindly provided by
Dr. M. Saito (Tenshi University), were established from the stromal
vascular fraction of the interscapular brown fat of mice deﬁcientof a tumor-suppressor gene p53 [22]. Three cell lines immortalized
brown preadipocytes have been reported; two were established frombrown adipose tissue tumors induced by expression of SV40 T-antigen
in transgenic mice [23,24], and one was immortalized the stromal
vascular cells of the interscapular brown fat by infection with virus expressing SV40 T-antigen [25]. Genetic background and the cause of

2.6. Statistical analyses
Data are expressed as the mean ± SEM. Data on gene expression
were log-transformed to provide an approximation of a normal distribution before analysis. Data in Fig. 3 were analyzed by two-way
ANOVA; the statistical model included effects of T3, insulin and theirinteraction. As for the other data, differences between the control
group and the tested group were examined using unpaired t-test. Differences of P b 0.05 were considered signiﬁcant.

439

H. Yoshida et al. / Biochimica et Biophysica Acta 1830 (2013) 5027–5035

3. Results

5029

program, indicating the differential role of endogenous Bmp in brown
adipogenesis from that in white adipogenesis.

3.1. Endogenous Bmp activity is not required for the onset of theadipogenic program in HB2 brown preadipocytes

3.2. Neither Bmp4 nor Bmp7 affects brown adipogenesis-related gene
expression in HB2 cells

We previously revealed that endogenous Bmp activity is higher
prior to the induction of differentiation in 3T3-L1 white preadipocytes and that this activity is required for proper adipogenesis[17]. To examine the activity and role of endogenous Bmp during
brown adipogenesis, HB2 brown preadipocytes were differentiatedusing the protocol for 3T3-L1 cells [26] supplemented with T3, a
procedure that has been reported to differentiate HB2 brown preadipocytes [35]. Endogenous Bmp activity was evaluated by phosphorylated Smad1/5/8 levels, as Bmp induces phosphorylation of
Smad1/5/8 at the carboxyl-terminal serines after complex formation
with the type I and type II receptors [36]. Time-course changes in
the phosphorylation levels of Smad1/5/8 in HB2 cells were relatively
constant throughout the differentiation process (Fig. 1A) and werelower than the phosphorylation levels in 3T3-L1 preadipocytes (datanot shown). In addition, treatment with dorsomorphin or LDN193189, kinase inhibitors for the Bmp type I receptors [37,38], did
not affect lipid accumulation (Fig. 1B) or the expression of Pparγ2
and C/ebpα, regulators of brown and white adipogenesis [7–9,13],on day 8 (Fig. 1C). Brown adipocyte-speciﬁc Ucp1 is transcriptionallyactivated in response to β3 adrenergic receptor activation [2]. Ucp1
expression was not affected by the inhibitors, irrespective of treatment with the β adrenergic receptor agonist isoproterenol (Fig. 1D).
These results suggest that endogenous Bmp activity in HB2 brown
preadipocytes is low and does not require the onset of the adipogenic

Previously, exogenous Bmp7, but not Bmp2, 4, 5 and 6, stimulateddifferentiation of a brown preadipocyte cell line in the presence of T3
and insulin [29]. We next explored whether the ability of exogenous
Bmp7 could have a similar effect on the HB2 brown preadipocytes.
Responsiveness to exogenous Bmp was ﬁrst examined by evaluationof Smad phosphorylation levels. Smad1/5/8 was shown to be phosphorylated by treatment with Bmp4 or Bmp7 (Fig. 2A). Treatment
with Bmp4 or Bmp7 signiﬁcantly increased lipid accumulation on day
8 (Fig. 2B), but neither Bmp7 nor Bmp4 affected the expression of
Pparγ2 (Fig. 2C). The expression of fatty acid-binding protein (Fabp) 4
and resistin, molecules expressed in mature adipocytes [39,40], wasalso unaffected by treatment with Bmp4 or Bmp7 (Fig. 2D–E). Furthermore, the Bmp treatments did not affect the basal or the induced levelsof Ucp1 (Fig. 2F).
3.3. Insulin is essential for brown adipogenesis in HB2 cells
The lack of effects of endogenous and exogenous Bmp on brown
adipogenesis may result from the presence of additional reagentsother than the essential factors required for brown adipogenesis in
the culture conditions; as the result, it is possible that the effects of
Bmp are masked. Thus, we explored the essential factors that are necessary for brown adipogenesis.

B

A
day:

-4 -2 0 1 2 4 68

1 2 3 4 5 6

8pSmad1/5/8
Smad1

arbitrary unit

100
7

50

0

C

Ppar γ 2

relative mRNA level

LDN

V

DM

LDN

V

0.5

LDN

-Iso
+Iso

20
2
1
0

C/ebp α

DM

Ucp1

40

1.0

0.0

V

D

1.5

DM

relative mRNA level

2.0

V

DM

LDN

Fig. 1. Endogenous Bmp activity is lower and is dispensable for brown adipogenesis. (A) Phosphorylation status of Smad1/5/8 during differentiation of HB2 brown preadipocytes.
HB2 cells were stimulated by differentiation inducers containing Dex (0.25 μM), IBMX (0.5 mM), insulin (10 μg/ml) and T3 (50 nM) on day 0 for 2 days, followed by culture withinsulin (5 μg/ml) and T3 (50 nM). Samples containing 1 μg DNA were subjected to Western blotting to detect Smad1/5/8 phosphorylated at C-terminal serines (upper panel).
The membranes were then re-blotted with an antibody against Smad1 (lower panel). (B−D) Effects of inhibition of Bmp activity on lipid accumulation (B) and expression of
Pparγ2, C/ebpα (C) and Ucp1 (D). Dorsomorphin (DM: 5 μM), LDN-193189 (LDN: 100 nM) or DMSO as vehicle (V) was added to the culture medium from day −2 to day 0.
(B) Lipid accumulation was examined on day 8 by Oil Red O staining (upper panel). After staining, the dye was extracted in 2-propanol, and the amount was quantiﬁed by measuring absorbance at 510 nm. The absorbance of the extracted dye from cells treated with vehicle was set at 100. Data are expressed as the mean ± SE (n = 4). (C) The gene transcript levels of Pparγ2 and C/ebpα on day 8 were measured by RT-qPCR and expressed as ratios to 18S rRNA, with the level in HB2 cells treated with vehicle set to 1. Data shown are
the mean ± SE (n = 4). (D) HB2 cells on day 8 were treated with or without isoproterenol (Iso: 10 μM) for 4 h. The gene transcript level of Ucp1 was measured by RT-qPCR and
expressed as a ratio to 18S rRNA, with the level in HB2 cells treated with or without isoproterenol set to 1. Data shown are the mean ± SE (n = 4).

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A

pSmad1/5/8
Smad1
1 2 3

200

arbitrary unit

Ligand: -

Bmp4
Bmp7

B
** **

150
100
50
0

Pparγ2

‡

4

Fabp4

7

E

‡

10

‡
5

0

Bmp: - - 4 7
T3, Ins: +†10

Resistin

F

30

15

‡

‡

5

relative mRNA level

relative mRNA level

15

D
relative mRNA level

C

-

‡
20

‡

‡

10

0

0

Bmp: - - 4 7
T3, Ins: +

Ucp1
500

Bmp: - - 4 7
T3, Ins: +relative mRNA level

Bmp:

-Iso
+Iso

‡

‡ †

300
100

10

† †

‡

0

Bmp: T3, Ins: -

-

4
+

7

Fig. 2. Neither Bmp7 nor Bmp4 enhances expression of brown adipogenesis-related genes. (A) Smad phosphorylation in response to exogenous Bmp. HB2 cells were treated with
Bmp4 (3.3 nM) or Bmp7 (3.3 nM) for 1 h. Phosphorylated Smad1/5/8 was examined by Western blotting, followed by re-blotting with an antibody against Smad1. (B–F) Effects ofexogenous Bmp on lipid accumulation (B) and expression of genes related to brown adipogenesis (C–F) in HB2 cells. Bmp4 (3.3 nM) or Bmp7 (3.3 nM) was added to the culture
medium supplemented with T3 (50 nM) and insulin (Ins: 20 nM). (B) Lipid accumulation was examined on day 8 by Oil Red O staining (upper panel). After staining, the dye was
extracted in 2-propanol, and the amount was quantiﬁed by measuring absorbance at 510 nm. The absorbance of the extracted dye from cells treated without Bmp was set at 100.
Data are expressed as the mean ± SE (n = 4). **: P b 0.01 vs. cells treated without Bmp. (C–E) The gene transcript levels of Pparγ2 (C), Fabp4 (D) and Resistin (E) on day 8 were
measured by RT-qPCR and expressed as ratios to 18S rRNA, with the level in HB2 cells treated with the growth medium alone set to 1. Data shown are the mean ± SE (n = 4).
(F) HB2 cells on day 8 were treated with or without isoproterenol (Iso: 10 μM) for 4 h. The gene transcript level of Ucp1 was measured by RT-qPCR, and expressed as a ratio
to 18S rRNA, with the level in HB2 cells treated with the growth medium alone set to 1. Data shown are the mean ± SE (n = 4). † and ‡: P b 0.05 and P b 0.01, respectively, vs.
cells treated without Bmp, T3 and insulin.

Irie et al. [22] showed that HB2 cells differentiated into brown adipocytes after 5 days in the presence of T3 and insulin in the growth
medium. We examined effects of T3 or insulin or both on lipid accumulation (Fig. 3A), basal Ucp1 expression (Fig. 3B), expression of
Pgc1α (Fig. 3C) and Cidea (Fig. 3D), genes expressed predominantly
in brown fat and involved in the regulatory expression and activityof Ucp1 [3,41,42], and Pparγ2 expression (Fig. 3E) on day 8. Insulin
increased lipid accumulation, whereas T3 slightly but signiﬁcantly decreased it (Fig. 3A and F). Expression of Ucp1, Pgc1α, Cidea andPparγ2 was signiﬁcantly increased by treatment with insulin alone(Fig. 3B–F). Insulin-induced expression of Ucp1 was also veriﬁed at
the protein level; signiﬁcant Ucp1 expression was detected in thepresence of insulin (Fig. 3G, lane 2). The expression level of Ucp1 wasfurther increased in the presence of troglitazone, a thiazolidinedionethat activates the transcriptional activity of Pparγ [43] (Fig. 3G, lanes
4–6). Consistent with the expression at the mRNA level, isoproterenol
treatment also enhanced the expression of Ucp1 induced by insulin
and troglitazone (Fig. 3G, lanes 7–12). Unexpectedly, T3 inhibited
insulin-induced Ucp1 expression (Fig. 3G, lane 3), which contrasted
with the evaluation at the mRNA level. All of these results indicatethat insulin but not T3 is a minimal factor for inducing differentiationin HB2 cells and that T3 instead inhibits insulin-induced differentiationof brown preadipocytes. Previous studies showed that T3 was requiredfor optimal Ucp1 expression in primary cultures of brown adipocyte
precursors [27,28,44]; thyroid hormones in FBS may be sufﬁcient during brown adipogenesis of HB2 cells.
We further evaluated the effects of exogenous Bmp in insulinmediated brown adipogenesis. Similar to the effects on the culturesin the presence of T3 and insulin (Fig. 2B and F), both Bmp4 and Bmp7
potentiated an increase in lipid accumulation but did not increase

basal and inducible Ucp1 expression in response to isoproterenol treatment on day 8 (Supplementary Fig. 1).

3.4. Tgf-β1 and Activin A inhibit brown adipogenesis in HB2 cells
Tgf-β and Activin A inhibit differentiation of 3T3-L1 white preadipocytes [15,16], suggesting that these growth factors may also inhibit brown adipogenesis. We showed that Smad2, a canonical signal
mediator of Tgf-β and Activin [45], was phosphorylated by treatment
with Tgf-β1 or Activin A (Fig. 4A), suggesting that HB2 cells arepotentially target cells to Tgf-β and Activin. Furthermore, treatment
with Tgf-β1 or Activin A for 8 days inhibited lipid accumulation(Fig. 4B), basal Ucp1 expression (Fig. 4C), and expression of the otherbrown adipocyte-selective genes such as Pgc1α, Cidea and Cox7a(Fig. 4C). In addition, the expression of Pparγ2, C/ebpα and Fabp4 wassigniﬁcantly decreased by treatment with Tgf-β1 or Activin A (Fig. 4D).
To characterize the inhibition of brown adipogenesis by Tgf-β andActivin, time-course changes in the expression of genes related tobrown adipogenesis and effects of Tgf-β and Activin were evaluated.
Gene transcript levels were increased after the insulin treatment within
2 days for Pparγ2 and within 4 days for Ucp1, Pgc1α and C/ebpα; Tgf-β1
and Activin A completely inhibited the gene induction (Fig. 5A–D).
The blockage of insulin-induced expression of Ucp1 and Pparγ2 by
Tgf-β1 and Activin A was also veriﬁed at the protein level (Fig. 6A).
Furthermore, the isoproterenol-induced Ucp1 expression was blunted
by Tgf-β1 or Activin A, which was veriﬁed at the mRNA level (Fig. 6B)
and at the protein level (Fig. 6C). These results clearly indicate that
Tgf-β and Activin function as inhibitors for the progression of brown
adipogenesis.

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5031

A

200
100

-

B

+
-

Ucp1

+

C
relative mRNA level

relative mRNA level

20

10

0

D

Pgc1α
4

30

T3:
Ins:

+
+

- + - +
- - ++

2
1
0Pparγ2

20

3

T3:
Ins:

E

Cidea

15
10
5
0

- + - +
- - ++

relative mRNA level

0

T3:
Ins:

relative mRNA level

arbitrary unit

300

T3:
Ins:

5.0

2.5

0.0

- + - +
- - ++

T3:
Ins:- + - +
- - ++GF
T3
Oil red O

Ucp1
Pgc1α
Cidea
Pparγ2

0.006
NS
NS
NS
NS

Iso:
+
Tro:
+
+
Ins: - + + - + + - + + - + +T3: - - + - - + - - + - - +Ucp1

Effects of
Ins T3×Ins
0.001
0.005
0.002
0.02
0.001

NS
NS
NS
NS
NS

β -actin
1 2 3

4

5

6

7 8

9 10 11 12

NS: P > 0.05.
Fig. 3. Insulin is essential for brown adipogenesis. Effects of T3 and insulin on lipid accumulation (A) and expression of genes related to brown adipogenesis (B–E and G). (A–E) T3(50 nM) or insulin (Ins: 20 nM) was added to the culture medium. (B) Lipid accumulation was examined on day 8 by Oil Red O staining (upper panel). After staining, the dye was
extracted in 2-propanol, and the amount was quantiﬁed by measuring absorbance at 510 nm. The absorbance of the extracted dye from cells treated without T3 and insulin was set
at 100. Data are expressed as the mean ± SE (n = 4). (B–E) The gene transcript levels of Ucp1 (B), Pgc1α (C), Cidea (D) and Pparγ2 (E) on day 8 were measured by RT-qPCR and
expressed as ratios to 18S rRNA, with the level in HB2 cells treated without T3 and insulin set to 1. Data shown are the mean ± SE (n = 4). (F) Results of two-way ANOVA on data oflipid accumulation and gene expression are shown (G) T3 (50 nM), insulin (20 nM) or troglitazone (Tro: 10 μM) was added to the culture medium for 8 days prior to treatment
with or without isoproterenol (Iso: 10 μM) for 4 h. Ucp1 expression (upper panel) was examined by Western blotting, followed by re-blotting by an anti-β-actin antibody(lower panel).

However, when HB2 cells were differentiated under the conditions
according to the modiﬁed protocol for 3T3-L1 cell differentiation as
described above, the inhibitory activity of Tgf-β was less effective
than that observed in HB2 cells differentiated with insulin alone; thedecrease in lipid accumulation and expression of Ucp1, Cidea, Cox7a,Pparγ2 and C/ebpα were smaller compared with cells differentiatedby insulin alone (Supplementary Fig. 2). In addition, no inhibitory
effects of Activin A could be detected (Supplementary Fig. 2).
3.5. Srebp1c expression is responsible for brown adipogenesis, which is
down-regulated by Tgf-β1 and Activin A
Given that Tgf-β and Activin blocked insulin-mediated inductionof adipogenic genes such as Pparγ2 and C/ebpα, we concluded thatthese growth factors inhibit differentiation into fat cells. To explore
how Tgf-β and Activin inhibit adipogenesis, we examined timecourse changes in expression of several genes in more depth. We
focused on genes that demonstrated modulation of expression levels
within 2 days after insulin treatment, speciﬁcally those moleculeswith possible pro-adipogenic activity showing increases in expression

442

and those molecules with putative inhibitory effects on adipogenesis
showing decreases in expression. As the candidates with proadipogenic activity, we chose C/ebpβ, Krüeppel-like factor (Klf) 5,
lysyl oxidase (Lox) and zinc ﬁnger protein 423 (Zfp423). Increased
expression of C/ebpβ and Klf5 stimulated adipogenesis [46,47], andmesenchymal stem cells and ﬁbroblasts were committed to theadipocyte-lineage cells by expression of Lox and Zfp423, respectively[21,48]. The transcript levels of these genes were not increased within
2 days after the insulin stimulation (Supplementary Fig. 3). We also
examined expression of Foxo1, Klf2 and necdin as the putative genes
involved in the inhibition of adipogenesis. Expression of Foxo1 andKlf2 inhibited adipocyte differentiation in 3T3-L1 cells [49,50]. Necdin
expression was higher in brown preadipocytes that cannot differentiate, and knock-down of Necdin restored adipogenesis [51]. The expression of these genes was not downregulated within 2 days in
response to insulin stimulation, although expression of Klf2 graduallytended to be decreased during the culture period (Supplementary
Fig. 4). Thus, we conclude that these genes are not candidates thattransmit the insulin signal through the regulation of their expression
level in order to induce differentiation into brown adipocytes.

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H. Yoshida et al. / Biochimica et Biophysica Acta 1830 (2013) 5027–5035

B
β

A

C

D
β

β

γ

α

α

Fig. 4. Tgf-β1 and Activin A inhibit brown adipogenesis. (A) Smad phosphorylation in response to exogenous Tgf-β and Activin. HB2 cells were treated with Tgf-β1 (200 pM) or
Activin A (4 nM) for 1 h. Phosphorylated Smad2 was examined by Western blotting, followed by re-blotting with an antibody against Smad2 (fourth panel). (B–D) Effects of
Tgf-β1 and Activin A on insulin-mediated lipid accumulation (B) and expression of genes related to brown adipogenesis (C and D). Tgf-β1 (200 pM) or Activin A (4 nM) was
added to the culture medium supplemented with insulin from day 0 to 8. (B) Lipid accumulation was examined on day 8 by Oil Red O staining (upper panel). After staining, thedye was extracted in 2-propanol, and the amount was quantiﬁed by measuring absorbance at 510 nm. The absorbance of the extracted dye from cells treated without ligand
was set at 100. Data are expressed as the mean ± SE (n = 4). * and **: P b 0.05 and P b 0.01, respectively, vs. cells treated without Tgf-β1 and Activin A. (C and D) The gene transcript levels of Ucp1, Pgc1α, Cidea and Cox7a (C), and Pparγ2, C/ebpα and Fabp4 (D) on day 8 were measured by RT-qPCR and expressed as ratios to 18S rRNA, with the level in HB2cells treated without Tgf-β1 and Activin A set to 1. Data shown are the mean ± SE (n = 4). **: P b 0.01 vs. cells treated without Tgf-β1 and Activin A.

A

B

Ucp1
12

Pgc1α
20

relative mRNA level

relative mRNA level

None
Tgf-β1
Activin A

8

4

0

None
Tgf-β1
Activin A

15
10
5
0

-4

0

4

8

-4

0

day

D

Pparγ2
1500

None

1000

Tgf-β1
Activin A

8

C/ebpα75relative mRNA level

relative mRNA level

C

4

day

500
80
40
0

None
Tgf-β1
Activin A

50

25

0
-4

0

4

8

-4

0

4

8

day

day

Fig. 5. Tgf-β1 and Activin A inhibit the onset of brown adipogenesis. Time-course changes in the expression of Ucp1 (A), Pgc1α (B), Pparγ2 (C) and C/ebpα (D) related to treatment with Tgf-β1
and Activin A. Tgf-β1 (200 pM) or Activin A (4 nM) was added to the culture medium supplemented with insulin (20 nM) from day 0 to 8. The gene transcript levels of Ucp1 (A), Pgc1α (B),Pparγ2 (C) and C/ebpa (D) were measured by RT-qPCR and expressed as ratios to 18S rRNA, with the level in HB2 cells on day −4 set to 1. Data shown are the mean ± SE (n = 4).

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A
4

6

- T A

8
T

-

A

Ppar γ 2β-actin1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

B
relative mRNA level

C

Ucp1

50
40

-

Iso:
Ligand: Ucp1

-Iso
+Iso

+
- T A

T A

β-actin1 2 3 4 5 6

30

**

10
5
0

Ligand:-** ** *T

A

Fig. 6. Tgf-β1 and Activin A blunt responsiveness to β3 adrenergic receptor activation.
Effects of Tgf-β1 and Activin A on expression of Ucp1 and Pparγ2. Tgf-β1 (200 pM) or
Activin A (4 nM) was added to the culture medium supplemented with insulin fromday 0 to 8. (A) Time-course changes in expression of Ucp1 (upper panel) and Pparγ2(middle panel) were examined by Western blotting, followed by re-blotting with ananti-β-actin antibody (lower panel). (E and F) HB2 cells on day 8 were treated withor without isoproterenol (Iso: 10 μM) for 4 h. (B) The gene transcript level of Ucp1
was measured by RT-qPCR and expressed as a ratio to 18S rRNA, with the level in HB2cells treated without Tgf-β1 and Activin A and isoproterenol set to 1. Data shown are
the mean ± SE (n = 4). * and **: P b 0.05 and P b 0.01, respectively, vs. respective
cells treated with or without isoproterenol and treated without Tgf-β1 and Activin A.
(C) Ucp1 expression (upper panel) was examined by Western blotting, followed byre-blotting with an anti-β-actin antibody (lower panel).

B

60

None
Tgf-β1
Activin A

40
20
3
2
1
0
-4

0

4

8

None
Tgf-β1
Activin A

40
20

100

3
2
1
0
-4

0

4

0
siRNA:

8

F

E
1.5
GFP
si-Srebp1
1.0

0.5

0.0

**

**

Ucp1 Pgc1α Cidea Cox7a

relative mRNA level

D

**

50

day

day

relative mRNA level

C

Srebp1a60

arbitrary unit

Srebp1c

relative mRNA level

relative mRNA level

A

1.5

GFP
si-Srebp1

1.0

siRNA:
Pparγ2Ucp1
β-actin

GFP
Srebp1

2- T A - T A

Srebp1

1- T A

We focused on the expression of Srebp1c because its expression is
rapidly increased (b6 h) in response to insulin stimulation in 3T3-L1
cells [52] and hepatocytes [53] and because it stimulates adipogenesis[54]. Time-course changes in Srebp1 indicated that Srebp1c (Fig. 7A)
but not Srebp1a (Fig. 7B) is increased within 2 days in response to theinsulin stimulation. Treatment with Tgf-β1 or Activin A completely
inhibited the insulin-induced Srebp1c expression but did not decrease the expression of Srebp1a (Fig. 7A and B).
We also examined insulin-induced genes (Insigs). In hepatocytes, insulin regulates Srebp activity at the mRNA and the posttranslational levels; insulin-induced downregulation of Insig-2a
expression enables the Srebp cleavage-activating protein/Srebp
complex to travel to the Golgi where the transcription factor domainof Srebp is released by two membrane-bound proteases, site-1 protease and site-2 protease [55]. A signiﬁcant expression of Insig-2a
could not be detected in HB2 cells or brown fat (Supplementary
Fig. 5A). In addition, the expression of Insig-1 and Insig-2b was not
rapidly decreased in response to the insulin stimulation, and treatment with Tgf-β1 or Activin A did not increase the expression of
any Insigs (Supplementary Fig. 5B and C). Thus, our results suggestthat unlike hepatocytes, insulin may stimulate Srebp1c expression
but not post-translational processing in brown preadipocytes, andTgf-β and Activin negatively regulate Srebp1c expression at themRNA level.
To understand the role of the induced Srebp1 in response to theinsulin stimulation, HB2 preadipocytes were transfected with siRNAfor Srebp1. The expression of Srebp1c was signiﬁcantly downregulated 4 days after the transfection as expected (Supplementary
Fig. 6), but the decreased expression was also detected at 10 days
after the transfection. The down-regulation of Srebp1c expression
resulted in decreases in lipid accumulation and expression of genes
related to brown adipogenesis on day 8 (Fig. 7C–F).

GFP

Day: -2 0
Ligand: - Ucp1

5033

1 2
0.5

*
0.0

*
**

Pparγ2 C/ebpα Fabp4

Fig. 7. Srebp1c induction is inhibited by Tgf-β1 and Activin A and is required for brown adipogenesis. (A and B) The gene transcript levels of Srebp1c (A) and Srebp1a (B) were
measured by RT-qPCR and expressed as ratios to 18S rRNA, with the level in HB2 cells on day −4 set to 1. Data shown are the mean ± SE (n = 4). (C–F) Effects of knock-downof Srebp1 on insulin-mediated lipid accumulation (C) and expression of genes related to brown adipogenesis (D–F). HB2 cells were transfected with double-stranded RNAi for
Srebp1 or GFP. (C) Lipid accumulation was examined on day 8 by Oil Red O staining (upper panel). After staining, the dye was extracted in 2-propanol, and the amount was quantiﬁed by measuring absorbance at 510 nm. The absorbance of the extracted dye from cells transfected with double-stranded RNAi for GFP was set at 100. Data are expressed as themean ± SE (n = 4). **: P b 0.01 vs. cells treated with siRNA for GFP. (D and E) The gene transcript levels of Ucp1, Pgc1α, Cidea and Cox7a (D), and Pparγ2, C/ebpα and Fabp4(E) on day 8 were measured by RT-qPCR and expressed as ratios to 18S rRNA, with the level in HB2 cells transfected with double-stranded RNAi for GFP set to 1. Data shown
are the mean ± SE (n = 4). * and **: P b 0.05 and P b 0.01, respectively, vs. cells treated with siRNA for GFP. (F) Expression of Ucp1 (upper panel) and Pparγ2 (middle panel)on day 8 was examined by Western blotting, followed by re-blotting with an anti-β-actin antibody (lower panel).

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4. Discussion
Although research on brown adipose tissue is advancing rapidly,
the current information on factors affecting brown adipogenesis is not
sufﬁcient. Because members of the Tgf-β family potently regulate differentiation of mesenchymal cells to adipocytes, myocytes, osteoblasts andchondrocytes [14], we expected that these members would also playroles in the differentiation of brown preadipocytes. The present study
illustrates that 1) endogenous Bmp activity is relatively constant during
brown adipogenesis, 2) endogenous Bmp activity prior to differentiation stimulation is not required for brown adipogenesis, 3) unlike previous results [29], Bmp7 does not act as an inducer of brown adipogenesis,
and 4) Tgf-β1 and Activin A act as inhibitors of adipogenesis, most likely
through blocking the induction of Srebp1c. These results provide basic
information on the regulation of brown adipogenesis by the Tgf-β
family. Our results indicate that control of activity of the Tgf-β family
is potentially useful for maintenance of energy homeostasis throughmanipulation of brown adipogenesis.
The role of Bmp in brown adipogenesis shown in this study is distinct from that shown in white adipogenesis [17]. Endogenous Bmp
activity prior to onset of the adipogenic program was required for
efﬁcient differentiation of white preadipocytes [17], whereas it was
not essential in brown preadipocytes. In vivo fate mapping revealed
that brown adipocytes but not white adipocytes arise from precursors
expressing Myf5, a myogenic molecule [56], indicating that cell lineage of brown adipocytes is distinct from that of white preadipocytes[56]. It is possible that the differences in the role of endogenous Bmp
between brown preadipocytes and white preadipocytes are related
to the differences of cell lineage.
The activity of Bmp7 during brown adipogenesis is also distinct
from that in a different cell line of brown preadipocytes establishedby use of SV40 T-antigen [29]. Treatment with various Bmp isoforms
during the differentiation of brown preadipocytes stimulated lipid
accumulation, and Bmp7 selectively upregulated the expression ofUcp1, suggesting a role for Bmp7 as an inducer of brown adipogenesis[29]. In the present study, both Bmp4 and Bmp7 increased lipid accumulation of HB2 adipocytes, which is consistent with the study by
Tseng et al. [29]. However, Bmp7 did not affect the basal expressionof Ucp1 or the Ucp1 induction in response to β3 adrenergic receptor
activation. Although the precise reason for the discrepant results is
unclear, there may be differences in the stage of brown preadipocytes(i.e., the extent of commitment as preadipocytes) between HB2 cells
and brown preadipocytes used by Tseng et al. [29]. In view of therole of Bmp as a molecule responsible for the commitment of mesenchymal stem cells to adipocyte-lineage cells [21] and osteoblastlineage cells [57], the brown preadipocytes used by Tseng et al. [29]may be relatively closer to mesenchymal stem cells, as comparedwith HB2 brown preadipocytes. In fact, Schulz et al. [58] showedthat Bmp7 treatment in Sca1+/CD45−/Mac1− stem cells promoted
commitment to brown preadipocytes and subsequent brown adipogenesis. Although the reason should be clariﬁed in future, the present
results explicitly indicate that Bmp7 during brown adipogenesis is
not required for the basal and the isoproterenol-induced expressionof Ucp1 in brown adipocytes.
Previous studies revealed the role of Bmp in brown adipocyte
development in vivo [29,59,60]; injection of Bmp7-treated mesenchymal stem cells developed a fat pad consisting of brown and white
adipocytes [29]. In addition, injection of Bmp2 expressing cells into
the skeletal muscle induced emergence of Ucp1-positive cells [59,60].
However, these results do not necessarily contradict from our data indicating dispensable role of Bmp7 in the basal and the induced expressionof Ucp1; the in vivo results [29,59,60] could be explained by the roleof Bmp as a stimulator of commitment of mesenchymal stem cells tobrown preadipocytes.
There are two distinct types of brown adipocytes. There are brown
adipocytes in classical brown fat and those in white fat called beige

adipocytes or brite adipocytes; beige adipocytes resemble white adipocytes in that they have extremely low basal expression of Ucp1,
but they express high Ucp1 similar to classical brown adipocytes in
response to cyclic AMP stimulation [61]. Recently, the origin of classical brown adipocytes has been revealed to be different from that ofbeige adipocytes [61]. Mice lacking functional Smad3, a signal mediator of Tgf-β and Activin signals [45], produce beige adipocytes [62],suggesting that endogenous Tgf-β and Activin inhibit commitment toor differentiation of beige preadipocytes. These results, taken together
with the results of the present study using brown preadipocytes originated from classical brown fat, indicate that the Tgf-β and Activin pathway negatively regulates brown adipogenesis, irrespective of brown
adipocyte types.
Srebp1c is a membrane-bound and tissue-restricted transcription
factor [63] that promotes white adipogenesis. Ectopic expression of
Srebp1 has been shown to stimulate differentiation of non-adipogenic
NIH-3T3 cells to adipocytes, whereas the expression of dominantnegative Srebp1 inhibited adipogenesis in 3T3-L1 cells [54]. Srebp1c
acts as a positive regulator for white adipogenesis through upregulationof Pparγ expression and an increase in the synthesis of putative
ligand(s) for Pparγ [64,65]. Despite the differences in the developmental origins of brown and white adipocytes [66], Pparγ expression isessential [10] and is part of a common transcriptional cascade thatis shared between the development of brown adipocytes and white
adipocytes [7–9]. Therefore, Srebp1c also regulates the process of conversion of preadipocytes to fat cells. In view of Srebp1c as a target molecule of Tgf-β and Activin, Tgf-β and Activin inhibit development of fat
cells, resulting in inhibition of the gene induction related to the functionof brown adipocytes.
Acknowledgements
We thank Dr. Masayuki Saito for providing cell line. This work was
supported by a Grant-in-Aid for Scientiﬁc Research (23580368) fromThe Japan Society for the Promotion of Science.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbagen.2013.06.036.
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Genetic Characteristics of CTX-M-Type Extended-Spectrum-␤Lactamase (ESBL)-Producing Enterobacteriaceae Involved in Mastitis
Cases on Japanese Dairy Farms, 2007 to 2011
Mamoru Ohnishi,a Alexandre T. Okatani,b Kazuki Harada,c Takuo Sawada,d Kenji Marumo,e Masaru Murakami,f Reiichiro Sato,gHidetake Esaki,h Keiko Shimura,h Hajime Kato,a Naoki Uchida,a Toshio Takahashid
Veterinary Clinical Laboratory, Nemuro District Agricultural Mutual Aid Association, Nakashibetsu, Hokkaido, Japana; Laboratory of Veterinary Public Health II, School of
Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japanb; Department of Veterinary Internal Medicine, Tottori University, Tottori-shi, Tottori, Japanc;Laboratory of Veterinary Microbiology, School of Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino, Tokyo, Japand; Department ofMicrobiology and Immunology, Showa University School of Medicine, Shinagawa, Tokyo, Japane; Laboratory of Molecular Biology, School of Veterinary Medicine, Azabu
University, Sagamihara, Kanagawa, Japanf; Laboratory of Veterinary Obstetrics and Gynecology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa,Japang; Research Institute for Animal Science in Biochemistry and Toxicology, Sagamihara, Kanagawa, Japanh

Sixty-ﬁve CTX-M-2/15/14 extended-spectrum-␤-lactamase-producing Enterobacteriaceae were isolated from 258,888 mastitic
milk samples from Japanese dairy farms between 2007 and 2011. CTX-M-2-producing Klebsiella pneumoniae and CTX-M-15producing Escherichia coli were the predominant strains isolated. There was no predominant clonal type, and clonal diversity
was found even in strains isolated from a single farm.

S

ince 2000, Escherichia coli and other Enterobacteriaceae species
producing CTX-M-type extended-spectrum ␤-lactamases(ESBLs) (CTX-M) have been commonly isolated from community-acquired extraintestinal infections in humans and their companion animals (1, 2, 3, 4), from food-producing animals (3, 5, 6,
7, 8), and from retail meats, including chicken, beef, and pork (3),worldwide. The CTX-M-type genes are assumed to have beentransferred separately to plasmids, including complex class 1 integrons and transposons (9), from chromosomes of different
Kluyvera species (i.e., Kluyvera ascorbata, K. georgiana, and K.
cryocrescens) that live in water, soil, and human and animal intestinal tracts; therefore, CTX-M has been divided into ﬁve clusters(CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25)
from base sequence homology (1, 9). CTX-M confers resistanceagainst penicillins, oxyimino-cephalosporins, and monobactams(1, 4). Recently, the CTX-M-15-producing E. coli ST131 (O25:
H4) clone has emerged as a multidrug-resistant pandemic strain
affecting humans worldwide (4).
Bovine mastitis is the most common disease affecting dairy
cattle (10). Both E. coli and Klebsiella pneumoniae often cause
life-threatening clinical mastitis (5, 11). The incidence of bovine
mastitis has been reported to be higher in Japan (30 to 35 cases per
100 cow-years at risk) (12) than in North America, Europe, and
New Zealand (10 to 30 cases per 100 cow-years) (10). Only a few
classes of antimicrobials are approved for the treatment of mastitis
in Japan; however, large amounts of antimicrobials are used formastitis treatment, creating selective pressure for drug-resistantorganisms (13). In our previous report, we showed that Japanese
dairy cattle might be a source of CTX-M-15/2/14- and CMY-2producing Enterobacteriaceae (7). However, few studies have reported the prevalence of Enterobacteriaceae producing CTX-M inbovine mastitis (5). The aims of this study were to determine thegenetic characteristics, antimicrobial susceptibility, and genetic
relatedness of ESBL- and plasmid-mediated AmpC ␤-lactamaseproducing Enterobacteriaceae isolated from bovine mastitis cases.
Screening of ESBLs. Bacterial cultures were carried out using
standard procedures on a total of milk samples from 258,888

September 2013 Volume 51 Number 9quarters obtained from 176,808 cows affected by (mainly clinical)
mastitis on 1,000 dairy farms in the Nemuro Subprefecture of
Hokkaido Prefecture, Japan, between February 2007 and April
2011 (14). Streptococcus spp. (mainly Streptococcus uberis) and Enterococcus spp., coagulase-negative staphylococci, Staphylococcus
aureus, E. coli, and Klebsiella spp. were the organisms most commonly isolated from culture-positive samples.
Of the isolates, 28,900 were identiﬁed as Gram-negative bacilli
and were submitted for susceptibility testing by disc diffusion according to the Clinical and Laboratory Standards Institute (CLSI)
guidelines (15); 419 isolates were identiﬁed as being cefazolin resistant and oxidase negative. These strains were then submitted for
CLSI combination disc ESBL conﬁrmatory tests (15) and a chromogenic oxyimino-cephalosporins hydrolysis test (Cica-␤-test I;
Kanto Chemical, Tokyo, Japan) to detect ESBLs, plasmidic AmpC
␤-lactamases, and metallo-␤-lactamases. Isolates with a positive
ESBL conﬁrmatory test and/or Cica-␤-test were screened for metallo-␤-lactamases using the sodium mercaptoacetic acid (SMA)
double-disc synergy test (SMA test) using two Kirby-Bauer discs
containing ceftazidime and one disc containing SMA (Eiken
Chemical, Tokyo, Japan). The ESBL test-positive Enterobacteriaceae isolates were identiﬁed using the ID 32 E API system (Sysmex
bioMérieux, Tokyo, Japan).
CTX-M genes and antimicrobial susceptibility. The ESBLand/or Cica-␤-test-positive and the SMA-test-negative isolates(n ⫽ 65) were analyzed by multiplex PCR for the presence of

Received 6 April 2013 Returned for modiﬁcation 6 May 2013
Accepted 4 July 2013
Published ahead of print 10 July 2013
Address correspondence to Mamoru Ohnishi, monishi@kind.ocn.ne.jp.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00920-13.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.00920-13

Journal of Clinical Microbiology

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p. 3117–3122

jcm.asm.org

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Ohnishi et al.

TABLE 1 Origins of CTX-M-2- and CTX-M-14 ESBL-producing Enterobacteriaceae isolates other than CTX-M-2-producing K. pneumoniae and E. coli
IsolateIsolation date, farm, and cow

Bacterial species

CTX-M genotype

TEM or SHV genotype

MCKo1
MCKo2
MCKo3
MCEa1
MCKo4
MCCk1
MCCk2
MCKo5
MCKo6
MCK45
MCK46

July 2008, F, 20
Sept. 2008, H, 22
May 2009, O, 30
Sep. 2009, H, 36
Jan. 2010, M, 42
Apr. 2010, U, 47
Apr. 2010, V, 48
Aug. 2010, F, 50
Sept. 2010, F, 53
Nov. 2010, Y, 57
Nov. 2010, Y, 57

K. oxytoca
K. oxytoca
K. oxytoca
E. aerogenes
K. oxytoca
C. koseri
C. koseri
K. oxytoca
K. oxytoca
K. pneumoniae
K. pneumoniae

CTX-M-2
CTX-M-2
CTX-M-2
CTX-M-2
CTX-M-2
CTX-M-2
CTX-M-2
CTX-M-2
CTX-M-2
CTX-M-14
CTX-M-14

OKP-A
TEM-1
TEM-1
Negative
Negative
Negative
Negative
SHV-1
Negative
Negative
Negative

blaCTX-M genes (16) and plasmid-mediated AmpC ␤-lactamase
genes (i.e., CMY, ACC, FOX, MOX, DHA, CIT, and EBC groups)(17). The CTX-M types of the CTX-M-positive isolates were identiﬁed by bidirectional sequencing using group-speciﬁc PCR prim-ers for blaCTX-M-1 group (18), blaCTX-M-2 group and blaCTX-M-9 group(19). AmpC-positive isolates were analyzed using type-speciﬁc
PCR primers (e.g., blaCMY-1 and blaCMY-2 genes), and blaTEM andblaSHV genes were analyzed and bidirectionally sequenced using

IsolateIsolation
date

Farm:
Cow

RAPD
Type

SHV, TEM

MCK10

Oct 07

H:11

R8

SHV-98, TEM-1

MCK12

Nov 07

J:14

R8

SHV-108

MCK15

Mar 08

F:16

R30

negative

MCK3A

Feb 07

B:2

R9

SHV-11

MCK3B

Feb 07

B:2

R9

SHV-11

MCK41

Apr 11

F:59

R24

OKP-A

MCK4B

May 07

C:3

R6

SHV-11

MCK4C

May 07

C:3

R6

SHV-11

MCK9

Oct 07

G:10

R7

SHV-11

MCK13

Jan 08

F:15

R15

SHV-11

MCK19

Jun 08

K:19

R2

SHV-98

MCK21

Aug 08

L:21

R1

SHV-11

MCK5

May 07

C:5

R1

SHV-11

MCK17

May 08

F:18

R25

OKP-A

MCK18

Jun 08

F:18

R25

OKP-A

MCK8

Sep 07

F:9

R25

SHV-1

MCK33

Feb 10

M:44

R27

negative

MCK27

July 09

Q:35

R3

SHV 28
SHV-28

MCK40

Oct 10

X:55

R29

negative

MCK24

Mar 09

F:28

R13

SHV-1

MCK7

Aug 07

F:8

R12

SHV-98MCK36

July 10

L:49

R10

SHV-148

MCK39

Oct 10

W:54

R10

negative

MCK14

Jan 08

F:16

R14

SHV-92

MCK23

Jan 09

F:26

R11

SHV-11

MCK25

Jun 09

M:31

R4

SHV-148

MCK26

July 09

M:33

R4

SHV-148

MCK35

Mar 10

F:46

R5

SHV-148

MCK28

Sep 09

M:31

R32

SHV-148

MCK11

Oct 07

I:13

R17

OKP-A

MCK29

Sep 09

R:38

R18

OKP-A

MCK20

July 08

F:17

R21

OKP-A

MCK30

N 09
Nov

S 39
S:39

R20

OKP A
OKP-A

MCK6

Aug 07

F:7

R19

SHV-52

MCK34

Mar 10

F:45

R23

SHV-83

MCK38

Sep 10

F:52

R16

SHV-1

MCK31

Dec 09

M:41

R22

OKP-A

MCK32

Feb 10

M:43

R22

OKP-A

MCK22

Oct 08

F:23

R26

SHV-11

MCK37

Augg 10

F:51

R28

SHV-28

MCK16

Mar 08

F:17

R31

OKP-A

FIG 1 RAPD-PCR of 41 K. pneumoniae isolates producing CTX-M-2. Cluster analysis was performed by the unweighted pair group method using arithmetic
averages with a 1.0% band position tolerance window and 1.0% optimization. DNA relatedness was calculated based on the Dice coefﬁcient. Thirty-two band
patterns were typed using similarity cutoff values of approximately ⱖ85%.

3118

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Journal of Clinical Microbiology

448

CTX-M Producers in Bovine Mastitis

Isolate; Year; Farm: Cow

ST (STcomplex), Serotype

MCE4; Dec 08; M: 24

ST58 (155), OUT: HUT

MCE6; Mar 09; M: 27

ST58 (155), OUT: HUT

PFGE

CTX-M

TEM,
SHV

V1

M-15

negativeV2

M-15

negative

type

MCE3; Oct 07; D: 12

ST23 (23), OUT: HUT

W

M-14

TEM-1

MCE1; May 07; D: 4

ST23 (23), OUT: HUT

W

M-14

negative

MCE8; Jul 09; M: 32

ST101 (101), O159: H45

X

M-15

negative

FCE3; 07; M: Cow 2

ST1167 (−), OUT: H19

R

M-15

negative

FCE5; 07; M: Cow 4

ST1167(−), O28ac: HUT

R

M-15

negative

MCE13; Dec 10; Z: 58

ST1126 (−), OUT: HUT

AD

M-15

TEM-1

MCE5; Dec 08; M: 25

ST648 (−), O74: HUT

AC

M-15

negative

FCE16; 09; M: Calf 11

ST2325 (−), OUT: HUT

U

M-15

negative

MCE11; Nov 09; T: 40

ST1415(−), OUT: HUT

Y

M-15

TEM-1

MCE2; Aug 07; E: 6

ST1284 (−), OUT: HUT

O

M-15

negative

MCE9; Aug 09; P: 34

ST10 (10), OUT: H5

Z1

M-15

TEM-1

MCE10; Sep 09; P: 37

ST10 (10), OUT: H5

Z2

M-15

TEM-1

FCE4; 07; M: Cow 3

ST540 (−), OUT: HUT

D

M-15

negative

MCE12; Oct 10; M: 56

ST88(23), OUT: H6

AA

M-2

TEM-1

MCE7 Apr
MCE7;
A 09;
09 N:
N 29

ST3499( ) OUT:
ST3499(−),
OUT HUT

AB

M 15
M-15

negativeti

FIG 2 PFGE patterns and cluster analysis of 13 CTX-M-producing E. coli isolates (MCE1 to 13) from mastitis cases, obtained using XbaI. Cluster analysis was
performed by the unweighted pair group method using arithmetic averages with a 1.0% band position tolerance window and 1.0% optimization. DNA
relatedness was calculated based on the Dice coefﬁcient. Twelve band patterns were typed using similarity cutoff values of ⱖ90%. The Salmonella strain
Braenderup CCUG50923 was used as a marker for assessing PFGE banding patterns. FCE3, -4, -5, and -16 were isolated from feces from cattle on farm M in ourprevious study (7).

previously described primers (19) (see Table S1 in the supplemental material). Comparison of nucleotide sequences and identiﬁcation of each CTX-M, TEM, and SHV type were carried out using
BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
For these 65 isolates, the MICs of 23 antimicrobials were determined by the CLSI broth microdilution method (15, 20) using
a custom-designed microtiter panel (Opt Panel MP; Kyokuto
Pharmaceutical, Tokyo, Japan). The 23 drugs were ampicillin,cefazolin, cefuroxime, ceftazidime, ceftazidime/clavulanic acid,cefotaxime, cefotaxime/clavulanic acid, ceftriaxone, cefpodoxime,
ceftiofur, cefquinome, cefepime, cefmetazole, moxalactam, imipenem, meropenem, aztreonam, gentamicin, amikacin, oxytetracycline, trimethoprim-sulfamethoxazole (SXT), enroﬂoxacin, and ciproﬂoxacin. Additional susceptibility tests for cefoxitin, kanamycin,
chloramphenicol, and levoﬂoxacin were performed by the CLSI discdiffusion method (15, 20). The breakpoints for veterinary pathogens
were used for 12 antimicrobial agents (15), and the breakpoints forhuman Enterobacteriaceae isolates were used for the other 12 antimicrobial agents (20). E. coli ATCC 25922 and Pseudomonas aeruginosa
ATCC 27853 were used as quality control strains.
Sixty-ﬁve of the 419 cefazolin-resistant isolates were identiﬁed as
CTX-M-producing strains. Fifty-one isolates (78.5%), which included 41 K. pneumoniae, 6 Klebsiella oxytoca, 2 Citrobacter koseri, 1 E.
coli, and 1 Enterobacter aerogenes isolate, harbored blaCTX-M-2; 10 E.
coli isolates (15.4%) harbored blaCTX-M-15; and 4 isolates (6.2%; 2 K.
pneumoniae and 2 E. coli isolates) harbored blaCTX-M-14. No isolates
contained the plasmidic AmpC gene (Table 1; Fig. 1 and 2).
Thirty-seven (90.2%) of 41 CTX-M-2-producing K. pneu-

moniae isolates also harbored genes encoding SHV-1/11/28/52/
83/92/98/108/148, OKP-A, or TEM-1. Four (66.6%) of 6 CTX-M2-producing K. oxytoca isolates also harbored blaTEM-1, blaSHV-1,
or blaOKP-A. Four (40.0%) of 10 CTX-M-15-producing E. coli isolates also harbored blaTEM-1, but no E. coli isolates harbored blaSHV(Table 1; Fig. 1 and 2). The gene sequences of the CTX-M-2/1/9,
TEM, and SHV groups were 99 to 100% homologous with those of
the CTX-M-2/15/14, TEM-1, and SHV subtypes which are available on GenBank.
The 65 CTX-M-producing Enterobacteriaceae were isolated
from 61 quarters of 58 mastitis cases on 25 dairy farms in the
Nemuro Subprefecture. Each of the 25 farms fed between 180 and
500 Holstein cattle with total mixed ration in free-stall barns orwith grass-silage and concentrates fed separately in tie-stall barns;
almost all used sawdust bedding. Their rolling yearly herd averages for milk production were 7,800 to 9,500 kg. The 58 affected
cows had either subclinical or local to systemic clinical mastitis.
Despite antimicrobial treatment, six cows were culled; and theremaining cows’ clinical signs resolved 3 to 10 weeks after onset.
The isolation rate of strains producing CTX-M-2/15/14 in bovine mastitis was 0.22% of the 28,900 Gram-negative bacillus isolates from the milk samples from 258,888 quarters of 176,808
cows. The CTX-M-2/15-producing K. pneumoniae and E. coli
strains were the most common ESBL producers causing bovine
mastitis in this study. In France, CTX-M-1/14-producing E. coli
and K. pneumoniae strains had an isolation rate of 0.4% (6 of 1,427
E. coli and K. pneumoniae isolates) from bovine mastitis cases (5).
There were no signiﬁcant (P ⬎ 0.05) differences between the isojcm.asm.org 3119

September 2013 Volume 51 Number 9

449

3120

jcm.asm.org

450

ⱕ2
ⱕ4
ⱕ4
ⱕ0.5/9.5
ⱕ0.25
ⱕ0.5

ⱕ2 to ⬎16
ⱕ4 to ⬎16
ⱕ4 to ⬎16
ⱕ0.5/9.5 to ⬎4/76
ⱕ0.25 to 2
ⱕ0.5 to 2

Gentamicine
Amikacin
OTETe
SXTe
Enroﬂoxacine
Ciproﬂoxacin
Cefoxitinf
Kanamycine,f
CHLf
Levoﬂoxacinf

ⱕ2
ⱕ4
⬎16
4/76
2
ⱕ0.5

⬎512
⬎256
⬎256
ⱕ2
0.5
128
ⱕ1
⬎64
⬎512
⬎512
⬎128
ⱕ8
ⱕ4
ⱕ8
ⱕ1
ⱕ2
16

MIC90
(␮g/ml)46 (93.9)
48 (98.0)
24 (49.0)
42 (85.7)
45 (91.8)
46 (93.9)
48 (98.0)
38 (77.6)
41 (83.7)
49 (100)

3 (6.1)000043 (87.8)g

43 (87.8)
47 (95.9)
48 (98.0)
49 (100)
48 (98.0)
—g

ⱖ16
ⱖ64
ⱖ16
ⱖ4/76
ⱖ2
ⱖ4c
ⱕ14 mmc
ⱕ13 mm
ⱕ13 mm
ⱕ13 mmc000

1 (2.0)
0
0

ⱖ16
ⱖ4c
ⱖ8
—d
ⱖ32c
ⱖ64c
ⱖ64c
ⱖ16
ⱖ4c
ⱖ16c
—d
1 (2.0)g
11 (22.4)—d2 (4.1)3 (6.1)1 (2.0)1 (2.0)004 (8.2)g

-g

ⱖ4c

0
0
0
3 (6.1)

Intermediate00045 (91.8)

Susceptible

No. of isolates (%)

ⱖ32
ⱖ32
ⱖ32c
ⱖ16c

Breakpoint(␮g/ml; mm)b14 (28.6)*B
7 (14.3)**B,*C
2 (4.1)0*B
0*B,**C
10 (20.4)*B, C
8 (16.3)
0*B

3 (6.1)**B,C
2 (4.1)1 (2.0)0
1 (2)6 (12.2)**B,*C
3 (6.1)48 (98.0)
49 (100)49 (100)45 (91.8)

49 (100)49 (100)49 (100)
1 (2.0)**B

Resistant (A)h

ⱕ2 to ⬎16
ⱕ4 to 8
ⱕ4 to ⬎16
4/76 to ⬎4/76
ⱕ0.25 to ⬎2
ⱕ0.5 to ⬎2
S to RS to RS to RS to R

⬎512
⬎256
⬎256
16–64
0.5 to ⬎8
512 to ⬎512
ⱕ1 to 8
⬎64
⬎512
⬎512
⬎128
⬎64
ⱕ4 to 32
ⱕ8
ⱕ1
ⱕ2
64 to ⬎64

MIC range(␮g/ml)Group 2

1 (10.0)07 (70.0)*A
7 (70.0)**A
1 (10.0)
1 (10.0)*A
1 (10.0)*A
5(50.0)*A
4 (40.0)1(10.0)*A

10 (100)**A0000
10 (100)**A,*C

10 (100)
10 (100)
10 (100)

10 (100)

10 (100)
10 (100)
10 (100)
10 (100)**A,C

No. of resistantisolates (%)
(B)h

ⱕ2
ⱕ4
ⱖ16
ⱕ0.5/9.5 to ⬎4/76
ⱕ0.25 to 0.5
ⱕ0.5 to 1
S to RS to RS to R
S

⬎512
⬎256
⬎256
ⱕ2
ⱕ0.25 to 0.5
128 to ⬎512ⱕ1
⬎64
512 to ⬎512
⬎512
⬎128
ⱕ8 to ⬎64
ⱕ4 to ⬎32
ⱕ8
ⱕ1
ⱕ2
ⱕ8 to 32

MIC range(␮g/ml)Group 300
6 (100)3 (50)*A00
1 (16.6)**A
4 (66.6)*A
3 (50)
0

4 (66.6)**A
1 (16.6)0003 (50)*A,B

6 (100)6 (100)6 (100)6 (100)6 (100)6 (100)6 (100)0**B

No. of resistantisolates (%)(C)hAssessed by the broth microdilution or disk diffusion method for 41 K. pneumoniae and 6 K. oxytoca isolates producing CTX-M-2 and 2 K. pneumoniae isolates producing CTX-M-14 (group 1), 10 E. coli isolates producing CTX-M15 (group 2), and other Enterobacteriaceae isolates producing CTX-M-2 or CTX-M-14 (group 3; CTX-M-2: E. coli, n ⫽ 1; C. koseri, n ⫽ 2; E. aerogenes, n ⫽ 1; E. coli, CTX-M-14, n ⫽ 2). Abbreviations: CAZ/CLA, ceftazidime/clavlanicacid; CTX, cefotaxime; OTET, oxytetracycline; SXT, trimethoprim/sulfamethoxazol; CHL, chloramphenicol.
bBreakpoint for resistance in accordance with CLSI document M31-A3 (2008) (15) for veterinary pathogens.
c
Breakpoint for resistance in accordance with CLSI document M100-S21 (2011) (20) for isolates from human infections with Enterobacteriaceae.
dBreakpoint for resistance or intermediate is not deﬁned in CLSI documents M31-A3 (2008) (15) and M100-S21 (2011) (20).
eAntimicrobial agent approved for cattle in Japan; the other 17 antimicrobials in this table are unapproved for cattle.
fSusceptibilities were tested by disc diffusion method according to CLSI documents M31-A3 (2008) (15) and M100-S21 (2011) (20).
g
Isolates in the susceptible, intermediate, and resistant categories could not be differentiated.
hSigniﬁcant differences were determined by the ␹2 test for comparison with the group indicated by the capital letter (A, B, or C): *, P ⬍ 0.05; **, P ⬍ 0.01.

a

⬎512
⬎256
⬎256
ⱕ2
ⱕ0.25
64
ⱕ1
⬎64
512
⬎512
128
ⱕ8
ⱕ4
ⱕ8
ⱕ1
ⱕ2
ⱕ8

⬎512
⬎256
⬎256
ⱕ2 to 64
ⱕ0.25 to ⬎8
ⱕ16 to 256
ⱕ1 to ⬎8
ⱕ8 to ⬎64
64 to ⬎512
64 to ⬎512
16 to ⬎128
ⱕ8 to 64
ⱕ4 to ⬎32
ⱕ8 to ⬎32
ⱕ1
ⱕ2 to 4
ⱕ8 to ⬎64

Ampicillin
Cefazoline
Cefuroximee
Ceftazidime
CAZ/CLA
Cefotaxime
CTX/CLA
Cefpodoxime
Ceftriaxone
Ceftiofure
Cefquinomee
Cefepime
Cefmetazole
Moxalactam
Imipenem
Meropenem
Aztreoname

MIC50(␮g/ml)

MIC range(␮g/ml)Antimicrobial
agentaGroup 1TABLE 2 Antimicrobial susceptibilities of isolates producing CTX-M-type ESBL from mastitic cows

Ohnishi et al.

Journal of Clinical Microbiology

CTX-M Producers in Bovine Mastitis

lation rates found in our study and the French study (5) by thechi-square test using StatFlex version 6.0 (Artech Co., Ltd., Osaka,
Japan). Among human and animal isolates in Western European
countries and Japan, the most common CTX-M types were the
CTX-M-1 cluster (CTX-M-1/15/55) and CTX-M-9 cluster (CTXM-9/14/27) (2, 3, 4, 5, 6). Except for the dominance of CTX-M-2,
our results are similar to these previous reports. The blaTEM-1,
blaSHV-1, and blaSHV-11 genes detected in the present study encode
non-ESBL enzymes (21); however, it is not clear whether the SHV28/52/83/92/98/108/148 and OKP-A are ESBLs, because the kinetic parameters of their puriﬁed enzymes were not determined.
Isolates producing CTX-M exhibited high resistance to oxyimino-cephalosporins; however, they exhibited high rates of susceptibility to cefmetazole, moxalactam, imipenem, meropenem,
gentamicin, and amikacin. The isolates producing CTX-M-2 andCTX-M-14 showed high rates of susceptibility to ceftazidime andﬂuoroquinolones. In contrast, the CTX-M-15-producing E. coli
strains showed signiﬁcantly higher rates of resistance to ceftazidime, aztreonam, cefepime, SXT, oxytetracycline, ciproﬂoxacin,
levoﬂoxacin, cefoxitin, and kanamycin than CTX-M-2/14-producing Klebsiella spp. and/or other CTX-M-2/14-producing Enterobacteriaceae (P ⬍ 0.05) by the chi-square tests (Table 2). Our
results are consistent with a previous study (1), and the CTX-M
types other than CTX-M-15, CTX-M-16, and CTX-M-27 efﬁciently hydrolyze cefotaxime and ceftriaxone but not ceftazidime(1).
Molecular subtyping proﬁles. Random ampliﬁed polymorphic DNA (RAPD)-PCR analysis of the 41 CTX-M-2-producing
K. pneumoniae isolates was performed using the oligonucleotide
RAPD7 as previously described (22). Pulsed-ﬁeld gel electrophoresis (PFGE) of a total of 13 CTX-M-producing E. coli isolates was
conducted according to the PulseNet standardized laboratory
protocol (23) using XbaI (Roche Applied Science, Mannheim,
Germany) and the CHEF-DR III electrophoresis systems (BioRad, Hercules, CA). Dendrograms of RAPD patterns and PFGE
patterns were analyzed using BioNumerics software, version 5.1(Applied Maths, Austin, TX). Four CTX-M-15-producing E. coli
strains isolated from bovine feces on farm M in our previous study(7) were used for comparison with the E. coli isolates from mastitis
cases.
Multilocus sequence typing (MLST) of the 13 CTX-M-producing E. coli isolates was conducted according to standard protocols using the E. coli database on the MLST website. (http://mlst.ucc.ie/mlst/dbs/Ecoli). The 13 E. coli isolates were serotyped
according to O and H antigens using the pathogenic E. coli Seiken
set 1 and set 2 antisera, respectively (Denka Seiken, Tokyo, Japan).
The 41 CTX-M-2-producing K. pneumoniae isolates from 15
farms revealed 32 RAPD types. More than half of the strains wereisolated from 2 farms (F and M). The 18 isolates from farm F
revealed 16 RAPD types. There was not a predominant RAPD type
among the 41 isolates. However, two or three isolates each of K.
pneumoniae (MCK17/18/8, MCK25/26, and MCK31/32), whichwere isolated from 2 different cows on same farm (F or M),
showed closely related RAPD types (R25, R4, and R22, respectively) (Fig. 1).
The 13 E. coli isolates from 7 farms belonged to 10 STs andshowed 12 PFGE types. Two isolates each of E. coli (MCE1/3,
MCE4/6, and MCE9/10), which were isolated from 2 different
cows on same farm (D, M, or P), had the same ST and closely
related PFGE types (ST23/W, ST58/V1 and V2, and ST10/Z1 andZ2, respectively). There were not any closely related strains between the 5 mastitis and the 4 fecal E. coli CTX-M-15-producing
isolates from farm M (Fig. 2). Most of the E. coli isolates had
untypeable O and H antigens (OUT, HUT). Neither E. coli clone
ST131 (O25:H4) nor enterohemorrhagic E. coli O157, O26, orO111 or other serotypes commonly isolated from human infections (8) were detected from the 13 isolates.
The genetic diversity in the 18 K. pneumoniae isolates obtained
from bovine mastitis cases on farm F suggests that these were
opportunistic infections originating from a wide variety of environmental sources (11). However, the presence of some strains ofK. pneumoniae and E. coli showing closely related genotypes,
which were isolated from the different cows on the same farm,suggests a contagious infection or an infection from an environmental point source (11). Similar to our results, E. coli clones
ST10/23/58 producing CTX-M-14/1 have also been isolated frombovine mastitis in France (5). Consistent with this French study,
we detected no E. coli clone ST131 (O25:H4) producing CTX-M15/27. Thus, these results suggest that cattle, unlike humans, canines and felines, have little signiﬁcance as a source of this clone(2, 4). In contrast, recently, two enterohemorrhagic E. coli strains(with serotypes O111:H8 and O26:H11) belonging to the B1 phylogenetic group and carrying blaCTX-M-15/9 were isolated from diarrheic cattle in France (8).
In conclusion, the genes encoding CTX-M2/15/14 are present
at a low frequency in Enterobacteriaceae isolates causing bovine
mastitis on Japanese dairy farms. The ESBL producers were
dominated by CTX-M-2-producing K. pneumoniae and CTXM-15-producing E. coli strains which showed multidrug resistance to ceftazidime, aztreonam, and cefepime. There was not a
predominant clonal type, and even the 18 K. pneumoniae
strains isolated from a single farm showed clonal diversity bymolecular subtyping.
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19. Kojima A, Ishii Y, Ishihara K, Esaki H, Asai T, Oda C, Tamura Y,
Takahashi T, Yamaguchi K. 2005. Extended-spectrum-␤-lactamaseproducing Escherichia coli strains isolated from farm animals from 1999 to
2002: Report from the Japanese Veterinary Antimicrobial Resistance
Monitoring Program. Antimicrob. Agents Chemother. 49:3533–3537.
20. Clinical and Laboratory Standards Institute (CLSI). 2011. Performancestandards for antimicrobial susceptibility testing, 21st informational supplement, M100-S21. CLSI, Wayne, PA.
21. Paterson DL, Bonomo RA. 2005. Extended-spectrum ␤-lactamases: aclinical update. Clin. Microbiol. Rev. 18:657– 686.
22. Ben-Hamouda T, Foulon T, Ben-Cheikh-Masmoudi A, Fendri C, Belhadj O, Ben-Mahrez K. 2003. Molecular epidemiology of an outbreak ofmultiresistant Klebsiella pneumoniae in a Tunisian neonatal ward. J. Med.
Microbiol. 52:427– 433.
23. Centers for Disease Control and Prevention (CDC). 2004. One-day(24 –28 h) standardized laboratory protocol for molecular subtyping ofEscherichia coli O157:H7, nontyphoidal Salmonella serotypes, and Shigellasonnei by pulsed ﬁeld gel electrophoresis (PFGE). Sections 5.1, 5.2, 5.4.
CDC, Atlanta, GA.

jcm.asm.org

Journal of Clinical Microbiology

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Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Herd prevalence of Enterobacteriaceae producing CTX-Mtype and CMY-2 b-lactamases among Japanese dairy farms
M. Ohnishi1, A.T. Okatani2, H. Esaki3, K. Harada4, T. Sawada5, M. Murakami6, K. Marumo7, Y. Kato2,
R. Sato8, K. Shimura3, N. Hatanaka2 and T. Takahashi5
1 Veterinary Clinical Laboratory, Nemuro District Agricultural Mutual Aid Association, Nakashibetsu, Hokkaido, Japan
2 Laboratory of Veterinary Public Health II, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan
3 Department of Biotechnology, Research Institute for Animal Science in Biochemistry and Toxicology, Sagamihara, Kanagawa, Japan
4 Department of Veterinary Internal Medicine, Tottori University, Tottori-shi, Tottori, Japan
5 Laboratory of Veterinary Microbiology, School of Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino, Tokyo, Japan
6 Laboratory of Molecular Biology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan
7 Department of Microbiology and Immunology, Showa University School of Medicine, Shinagawa, Tokyo, Japan
8 Laboratory of Gynecology and Obstetrics, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan

Keywords
CMY-2 b-lactamase, CTX-M type ESBLs, dairy
cattle, Escherichia coli, multilocus sequencetyping, pulsed-ﬁeld gel electrophoresis.
Correspondence
Mamoru Ohnishi, Veterinary Clinical Laboratory, Nemuro District Agricultural Mutual Aid
Association, 11-5 Minami, 5 Nishi, Nakashibetsu-cho, Shibetsu-gun, Hokkaido 086-1105,
Japan.
E-mail: monishi@kind.ocn.ne.jp
2013/0028: received 6 January 2013, revised
11 March 2013 and accepted 27 March 2013
doi:10.1111/jam.12211

Abstract
Aims: To determine the herd prevalence of Enterobacteriaceae producing CTXM-type extended-spectrum b-lactamases (ESBLs) among 381 dairy farms inJapan.
Methods and Results: Between 2007 and 2009, we screened 897 faecal samples
using BTB lactose agar plates containing cefotaxime (2 lg ml1). Positive
isolates were tested using ESBL conﬁrmatory tests, PCR and sequencing for
CTX-M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing
CTX-M-15 (n = 7), CTX-M-2 (n = 12), CTX-M-14 (n = 3), CMY-2 (n = 2)
or CTX-M-15/2/14 and CMY-2 (n = 4) in bovine faeces was 28/897 (3 1%)
faecal samples. These genes had spread to Escherichia coli (n = 23) and three
genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381(5 2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessedby multilocus sequence typing and pulsed-ﬁeld gel electrophoresis. The
pandemic E. coli strain ST131 producing CTX-M-15 or CTX-M-27 was notdetected.
Conclusions: Three clusters of CTX-M (CTX-M-15, CTX-M-2, CTX-M-14)
had spread among Japanese dairy farms.
Signiﬁcance and Impact of the Study: This is the ﬁrst report on theprevalence of multidrug-resistant CTX-M-15–producing E. coli among Japanese
dairy farms.

Introduction
Since 2000, Escherichia coli and other Enterobacteriaceae
species producing CTX-M-type extended-spectrum b-lactamases (ESBLs) (CTX-M) have been commonly isolated
from community-acquired extraintestinal infections inhumans and their companion animals (Harada et al.
2012; Kuroda et al. 2012) and faeces of food-producing
animals worldwide (Carattoli 2008). The blaCTX-M-related
genes were transferred separately to genetic mobilization

282

units (i.e. plasmids) from chromosomes of different Kluyvera species (i.e. K. ascorbata, K. georgiana, K. cryocrescens) that live in water, soil, human and animal intestinal
tract; and thereby, CTX-M has derived in ﬁve CTX-M
clusters (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 andCTX-M-25) from base sequence homology. This has been
facilitated by genetic mobilization units such as insertion
sequences (i.e. ISEcp1 or ISCR1) (Bonnet 2004; Canton
et al. 2012). CTX-M confers resistance against penicillins,
oxyimino-cephalosporins (i.e. cefuroxime, cefotaxime,

Journal of Applied Microbiology 115, 282--289 © 2013 The Society for Applied Microbiology453

M. Ohnishi et al.

ceftriaxone, cefpodoxime, ceftazidime, ceftiofur, cefquinome) and monobactams (Bonnet 2004).
Plasmidic CMY-2, derived from the AmpC b-lactamaseof Citrobacter freundii, became the dominant AmpC
b-lactamase among Enterobacteriaceae, especially Salmonella enterica, in humans and animals throughout theworld (Tamang et al. 2012). CMY-type b-lactamases are
cephamycinases that confer resistance to cephamycins
and oxyimino-cephalosporins. Recently, a high incidenceof blaCTX-M-positive E. coli was found in retail chicken
meat. (Overdevest et al. 2011). However, few studies havereported the prevalence of Enterobacteriaceae producing
CTX-M and CMY on dairy farms (Hartmann et al.
2012). The aims of the present study were to determine
the herd prevalence of Enterobacteriaceae producing CTXM and CMY among numerous dairy farms in Japan andto evaluate the genetic relatedness among E. coli isolates
derived from dairy cattle by serotyping, multilocus
sequence typing (MLST) and pulsed-ﬁeld gel electrophoresis (PFGE).
Materials and methods
Faecal samples
A total of 897 faecal samples were obtained from each of
897 cattle affected by diarrhoea (caused by i.e. colibacillosis, rotavirus infection, cryptosporidiosis, coccidiosis, parasitic gastroenteritis) or dictyocauliasis; the 897 cattle
consisted of 550 calves (<12 months old), 80 heifers(  12 months old to ﬁrst parturition) and 267 cows.
Faecal samples were submitted daily for clinical diagnostic tests to the clinical laboratory of the Nemuro District
Agricultural Mutual Aid Association by nine-member veterinary clinics. The faecal samples were obtained from a
total of 381 dairy farms localized in Nemuro Subprefecture of Hokkaido Prefecture, Japan; 475 samples wereobtained from 244 dairy farms between May and December in 2007 and 422 samples from 214 dairy farms
between July and December in 2009.
Screening, identiﬁcation, PCR and sequence typing for
ESBLs and AmpC producers
The faecal samples were cultured as the ﬁrst screening for
ESBL producers on the day they were submitted to thelaboratory. Approximately, 50 lg faeces was streaked
directly onto a BTB-lactose agar plate containing
2 lg ml1 cefotaxime and 8 lg ml1 vancomycin (Shiraki et al. 2004) and then incubated at 35°C for 40 h.
A total of 250 isolates were tested using the Clinicaland Laboratory Standards Institute (CLSI) combination
disc ESBL conﬁrmatory tests (CLSI 2008) and a

Dairy herd prevalence of CTX-M in Japan

chromogenic oxyimino-cephalosporins hydrolysis test
(Cica-b-test I; Kanto Chemical, Tokyo, Japan) to detect
ESBLs, plasmidic AmpC b-lactamases and metallo-b-lactamases. All positive isolates were screened for metallob-lactamases by the sodium mercaptoacetic acid (SMA)
double-disc synergy test using two Kirby–Bauer discs
containing ceftazidime and one disc containing SMA(Eiken Chemical, Tokyo, Japan). Enterobacteriaceae isolates were identiﬁed using the ID 32 E API system(Sysmex bioMerieux, Tokyo, Japan).
All isolates that were positive for the ESBL conﬁrmatory tests and/or the Cica-b-test were analysed by multiplex polymerase chain reaction (PCR) for the presence of
blaCTX-M genes (Xu et al. 2007) and plasmid-mediated
AmpC b-lactamase genes (i.e. CMY, ACC, FOX, MOX,
DHA, CIT and EBC groups) (Perez-Perez and Hanson
2002). CTX-M-positive isolates were analysed by bidirectional sequencing using group-speciﬁc PCR primers for
blaCTX-M-1 group (Mena et al. 2006), blaCTX-M-2 group andblaCTX-M-9 group (Kojima et al. 2005). The AmpC-positive
isolates were analysed using type-speciﬁc PCR primers(Kojima et al. 2005). The blaTEM and blaSHV genes wereanalysed using primers (Kojima et al. 2005) and bidirectionally sequenced (Table S1).
Antimicrobial susceptibility testing
The minimum inhibitory concentrations (MICs) of 23
antimicrobial agents were determined for isolates positive
for CTX-M and/or AmpC genes by broth microdilution
using a customer-designed, commercially prepared microtiter panel (Opt Panel MP; Kyokuto Pharmaceutical,
Tokyo, Japan), and additional susceptibility test forcefoxitin, kanamycin, chloramphenicol and levoﬂoxacin
was performed by disk diffusion according to the CLSI
guidelines (CLSI 2008, 2011).
The breakpoints for veterinary pathogens were used for
12 antimicrobial agents (CLSI 2008), and the breakpoints
for human Enterobacteriaceae isolates were used for theother 12 antimicrobial agents (CLSI 2011). Escherichia
coli ATCC25922 and Pseudomonas aeruginosa ATCC27853
were used as quality-control strains.
Serotyping, MLST and PFGE of Escherichia coli
The 23 E. coli isolates were serotyped according to O andH antigens using the pathogenic E. coli antisera ‘SEIKEN’
Set 1 for 50 O antigens and Set 2 for 22 H antigens(Denka Seiken, Tokyo, Japan) according to the manufacturer’s instructions.
We carried out the ampliﬁcation and sequencing ofseven housekeeping genes and determined the allelicproﬁle and sequence types (STs) using the protocols andJournal of Applied Microbiology 115, 282--289 © 2013 The Society for Applied Microbiology

454

283

Dairy herd prevalence of CTX-M in Japan

M. Ohnishi et al.

database on the E. coli MLST website (http://mlst.ucc.ie/
mlst/dbs/Ecoli).
Pulsed-ﬁeld gel electrophoresis was conducted according the PulseNet standardized laboratory protocol (CDC2004). The DNA restriction fragments digested with Xba
I or Bln I (Roche Applied Science, Mannheim, Germany)
were separated using the CHEF-DR III electrophoresis
systems (Bio-Rad, Hercules, CA, USA). Dendrogram of
the combined PFGE patterns of Xba I and Bln I was constructed using BioNumerics software version 5.1 (Applied
Maths, Austin, TX, USA) with Dice coefﬁcient and theunweighted pair group method, and the similarity of theaverage from the two experiments. PFGE types weredistinguished at a cut-off of  90% similarity.

n = 3; C. freundii, n = 1; Enterobacter cloacae, n = 1)
were isolated from 28 faecal samples obtained from 17
calves, one heifer and 10 cows from 20 dairy farms
between 2007 and 2009. The other two isolates identiﬁed
as Stenotrophomonas maltophilia or Burkholderia cepacia
were negative for CTX-M, AmpC, TEM and SHV genes.
Seven E. coli isolates (25 0%) harboured blaCTX-M-15, 12
isolates (42 9%) (E. coli, n = 7; K. pneumoniae, n = 3; E.
cloacae, n = 1; C. freundii, n = 1) harboured blaCTX-M-2
and three E. coli isolates (10 7%) harboured blaCTX-M-14.
Two E. coli isolates (7 1%) harboured blaCMY-2 and fourE. coli isolates (14 3%) harboured blaCMY-2 and blaCTX-M-2
or blaCTX-M-15 or blaCTX-M-14. In addition to CTX-M andCMY-2, 12 E. coli isolates (42 9%) and one C. freundii
isolate (3 6%) harboured blaTEM-1 or blaSHV-11 and one
K. pneumoniae isolate (3 6%) harboured blaTEM-1 andblaSHV-11. Four (50%) of eight CTX-M-15–producing
E. coli isolates also harboured blaTEM-1 (Tables 1 and 2).
The incidence of Enterobacteriaceae producing CTX-M
and/or CMY-2 in bovine faeces was 28 (3 1%) of the 897
faecal samples, and the incidence of E. coli producing
these b-lactamases was 23 of 897 (2 6%). The incidenceof Enterobacteriaceae was 17 (3 1%) of the 550 faecal
samples from calves and 10 (3 7%) of the 267 faecal sam-Results
Incidence and herd prevalence of CTX-M and CMY-2
producers
A total of 30 isolates were positive for the ESBL conﬁrmatory tests and/or the Cica-b-test. However, no isolate
was SMA test-positive. Twenty-eight CTX-M and/or
CMY-2 producers (E. coli, n = 23; Klebsiella pneumoniae,

Table 1 ST and ST complex (multilocus sequence typing), pulsed-ﬁeld gel electrophoresis (PFGE) and serotype of 23 b-lactamase–producing
Escherichia coli isolates obtained from faeces

IsolateYear, Farm, Cattle

CTX-M, CMY

FE1
FE2
FE3
FE4
FE5
FE6
FE7
FE8
FE9
FE10
FE11
FE12
FE13
FE14
FE15
FE16
FE17
FE18
FE19
FE20
FE21
FE22
FE23

07, IA, Calf1
07, IB, Cow1
07, M, Cow2
07, M, Cow3
07, M, Cow407, IC, Calf2
07, IC, Calf3
07, ID, Calf4
07, ID, Calf6
07, IF, Cow507, IG, Cow607, IH, Calf7
09, IJ, Heifer1
09, IL, Calf909, IM, Calf10
09, M, Calf11
09, IN, Calf12
09, II, Calf13
09, IP, Cow909, IN, Calf14
09, IQ, Cow10
09, IR, Calf16
09, IS, Calf17

CTX-M-2
CTX-M-2
CTX-M-15
CTX-M-15
CTX-M-15
CTX-M-2
CTX-M-2
CTX-M-14
CTX-M-14
CMY-2
CTX-M-15, CMY-2
CTX-M-15
CTX-M-15
CMY-2
CTX-M-14
CTX-M-15
CTX-M-2
CTX-M-2
CTX-M-2, CMY-2
CTX-M-14, CMY-2
CTX-M-2
CTX-M-2, CMY-2
CTX-M-15

TEM
SHV
TEM-1
TEM-1
TEM-1

TEM-1
TEM-1
TEM-1

TEM-1
TEM-1
TEM-1
SHV-11
SHV-11
TEM-1

ST (ST complex)

PFGE typeO-, H-serotype

58 (155)
10 (10)1167 ()†540 ()1167 ()
10 (10)10 (10)69 (69)10 (10)1284 ()744 ()394 (394)
1266 ()2438 ()57 (350)2325 ()2324 ()2437 ()44 (10)88 (23)48 (10)46 (46)744 ()

O
E
K
U
K
G
G
L
C
D
H
S
R
A
J
M
P
T
I
B
Q
N
F

OUT*: HUT*
O8: H7
OUT: H19
OUT: HUT
O28ac: HUT
OUT: H51
OUT: HUT
O15: HUT
OUT: H9
OUT: HUT
OUT: HUT
O44: H18
OUT: H34
OUT: H16
OUT: H6
OUT: HUT
OUT: HUT
O164: HUT
OUT: HUT
OUT: HUT
O124: H4
OUT: HUT
OUT: HUT*O-antigen untypable: H-antigen untypable.
†ST complex undeﬁned.

284

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M. Ohnishi et al.

Dairy herd prevalence of CTX-M in Japan

Table 2 Origins of CTX-M, TEM and SHV type b-lactamase–producing Enterobacteriaceae isolates other than Escherichia coli
IsolateIsolation year, Farm, Cattle

Bacterial species

CTX-M

TEM, SHV

FK1
FK2
FK3
FCf1
FEn1

07, IE, Calf5
09, II, Calf8
09, IK, Cow7
09, IO, Cow809, II, Calf15

Klebsiella pneumoniae
K. pneumoniae
K. pneumoniae
Citrobacter freundiiEnterobacter cloacae

CTX-M-2
CTX-M-2
CTX-M-2
CTX-M-2
CTX-M-2

TEM-1, SHV-11

ples from cows; however, this difference was not signiﬁcant (P > 0 05, chi-square test). The herd prevalence of
these Enterobacteriaceae was 20 (5 2%) of the 381 dairy
farms. Four E. coli isolates from three cows and one calf
on farm M harboured blaCTX-M-15. (Tables 1 and 2).
Antimicrobial susceptibility and antimicrobial use
Isolates producing CTX-M exhibited high resistance tooxyimino-cephalosporins. However, isolates producing
CTX-M-2 and CTX-M-14 were highly susceptible toceftazidime, cefmetazole, moxalactam, carbapenems, gentamicin, amikacin and ﬂuoroquinolones. MICs for ceftazidime, cefmetazole, enroﬂoxacin and ciproﬂoxacin werehigher for the E. coli isolates producing CTX-M-15 andCMY-2 than for the isolates producing CTX-M-2 andCTX-M-14 (Table 3). Most of isolates producing
CTX-M, especially CTX-M-15, exhibited resistance tokanamycin,
oxytetracycline,
chloramphenicol
andtrimethoprim-sulfamethoxazole (Table 3).
According to the clinical charts and the purchase volume of antimicrobial agents provided by the member
veterinary clinics, subcutaneous injections of enroﬂoxacin(4 mg kg1 body weight) were administered to one cow
and one calf with diarrhoea once daily for 2–4 days. The
other 26 calves, heifer and cows were not treated withoxyimino-cephalosporins or ﬂuoroquinolones. Between
2006 and 2010, the member veterinary clinics used 2016
daily doses of ceftiofur and 1408 daily doses of enroﬂoxacin per year for approximately 130 000 cows and heifersof 1000 dairy farms in Nemuro Subprefecture. Daily
doses were based on the weight of an adult cow. Neither cefquinome nor the other ﬂuoroquinolones were
used.
MLST, serotyping and PFGE of 23 Escherichia coli
isolates
The 23 E. coli isolates were grouped into 18 STs(Table 1). The eight CTX-M-15–producing E. coli isolates
belonged to six STs and were non-ST131. Four isolates
belonged to ST10, and two isolates belonged to ST44 andST48 [ST 10 complex (STC10)]. Five isolates belonged to

TEM-1

ST46 (STC46), ST57 (STC350), ST58 (STC155), ST69
(STC69) and ST88 (STC 23) (Table 1). Most of theE. coli isolates were O-antigen untypable (OUT), but theserotypes of six isolates were O8:H7, O15:H-antigen
untypable (HUT), O28ac:HUT, O44:H18, O124:H4 andO164:HUT (Table 1).
Pulsed-ﬁeld gel electrophoresis analysis with Xba I and
Bln I divided the 23 E. coli isolates into 21 genetic proﬁles. Nineteen of the genetic proﬁles consisted of a single
strain. Isolates FE03 and FE05, isolated from two different cows from farm M, showed PFGE type K, ST1167
and blaCTX-M-15; and FE06 and FE07, isolated from two
different calves from farm IC, showed PFGE type G,
ST10 and blaCTX-M-2, but showed distinct serotypes. Conversely, isolates FE2 and FE9, isolated from two different
cows from different farms, showed the ST10 but differedon PFGE type and serotype. The isolates FE11 and FE23,
isolated from two different cows from different farms,
showed ST744 but differed on PFGE type (Table 1,
Fig 1).
Discussion
The incidence and herd prevalence of ESBL-positive
E. coli among dairy farms in the present study were lower
than those of French cattle (Hartmann et al. 2012). In
agreement with Japanese studies of broiler chickens(Hiroi et al. 2012), three CTX-M clusters (CTX-M-15,
CTX-M-2 and CTX-M-14) were detected in the present
study. Among human and animal isolates in Western
European countries, Japan, China, Taiwan and South
Korea, the most common CTX-M types were the CTXM-1 cluster (CTX-M-1, CTX-M-3, CTX-M-15, CTXM-35, CTX-M-55) and CTX-M-9 cluster (CTX-M-9,
CTX-M-14, CTX-M-27); however, CTX-M-15 predominated among human isolates in India (Carattoli 2008;
Hawkey 2008; Suzuki et al. 2009; Overdevest et al. 2011;
Harada et al. 2012; Kuroda et al. 2012; Tamang et al.
2012). Except for the dominance of CTX-M-2, our results
are similar to these previous reports other than the
Indian study.
Most of the CTX-M-15 producers also harboured
blaTEM-1 and were resistant to ceftazidime andJournal of Applied Microbiology 115, 282--289 © 2013 The Society for Applied Microbiology

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285

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457

256 to >512>256
64 to >256
16–64
0 5 to >832 to >512
1 to 864 to >64
64 to >51264 to >512
16 to >128
8 to >64
4 to 32
8
1
2
8 to >64
2 to >16
4 to 8>16
0 5/9 5 to >4/76
0 25 to >2
0 5 to >2
S to RS to RS to RS to R

Ampicillin†
Cefazolin†
Cefuroxime†
CAZ
CAZ/CLACTX
CTX/CLA
Cefpodoxime
Ceftriaxone
Ceftiofur†
Cefquinome†
Cefepime
Cefmetazole
Moxalactam
Imipenem
Meropenem
Aztreonam
Gentamicin†
Amikacin
OTET†
SXT†
Enroﬂoxacin†
Ciproﬂoxacin
Cefoxitin¶
KAN†,¶
CHL¶
LVX¶
>512
>256
>256
4–32
05
64 to >512
1 to 8>64>512
512 to >512
32 to >128
8 to >64
4 to 32
8 to 32
1
2 to 416 to >64
2 to 44 to 8>16
0 5/9 5 to >4/76
0 25
05
S to RS to RS to R
S

>512
>256
>256
2 to 4
0 25 to 1
128–5121>64
512 to >512
>512
64 to >128
16 to >64481
2
82 to >16
44 to >16
0 5/9 5 to >4/76
0 25
05
S
S to RS to IS

E. coli CTX-M-14
n=3
>512
>256
>256
32–64
>8
512 to >512
8 to >8>64
512 to >512
512 to >512
128 to >128
64 to >6432
8 to 161
2
64 to >64
2 to 16
4 to >16>16>4/76
0 5 to >2
0 5 to >2R
S to RS to RS to RE. coli CMY-2
and CTX-M- 2/15/14
n=4
>512
>256
>256
16–64
8 to >864 to >512>8>64
64 to >512
128 to >512
32 to >128
8 to >6432
8
1
2
8 to >64
>16
4
>16
>4/76
2
>2
R
R
S or R
S or RE. coli
CMY-2
n=2
512 to >512
>256
>256
2 to 32
0 25 to >8
16 to >512
1 to >8>64
128 to >512
256 to >512
16 to >128
8 to >64
4 to >32
8 to >32
1
2
8 to >642
4 to 88 to >16
0 5/9 5 to >4/76
0 25
05
S to RS to RS to RS

Klebsiella pneumoniae, n = 3
Citrobacter freundii, n = 1
Enterobacter cloacae, n = 1;
CTX-M-2

CAZ, ceftazidime; CLA, clavulanic acid; CTX, cefotaxime; OTET, oxytetracycline; SXT, trimethoprim-sulfamethoxazole; KAN, Kanamycin; CHL, chloramphenicol; LVX, levoﬂoxacin.
*Breakpoint for resistance in accordance with CLSI document M31-A3 (2008) for veterinary pathogens.
†Antimicrobial agent approved for cattle in Japan; the other 17 antimicrobials in this table are unapproved for cattle.
‡Breakpoint for resistance in accordance with CLSI document M100-S21 (2011) for isolates from human infections with Enterobacteriaceae.
§Breakpoint for resistance is not deﬁned in CLSI documents M31-A3 (2008) and M100-S21 (2011).
¶Susceptibilities were tested by disk diffusion method according to CLSI documents M31-A3 (2008) and M100-S21 (2011).

E. coli CTX-M-15
n=7Antimicrobial
agent

E. coli
CTX-M-2
n=7

8
 4‡
8
-§
 32‡
 64‡
 64‡
 16
 4‡
 16‡
 16
 64
 16
 4/76
2
 4‡
14 mm‡
13 mm
12 mm
13 mm‡

 4‡

 32
 32
 32‡
 16‡

Breakpoint*lg ml1; mm

Table 3 Antimicrobial susceptibilities of 23 bovine faecal Escherichia coli isolates and ﬁve Enterobacteriaceae isolates other than E. coli that produce CTX-M type and/or CMY-2 b-lactamases, asassessed by broth microdilution or disk diffusion method

Dairy herd prevalence of CTX-M in Japan
M. Ohnishi et al.

Journal of Applied Microbiology 115, 282--289 © 2013 The Society for Applied Microbiology

M. Ohnishi et al.

Dairy herd prevalence of CTX-M in Japan

100

90

80

70

60

50

E coli Blnl+E coli Xbal
Composite Xbal Blnl

E coli Xbal

E coli Blnl

60·6

66·8
58
62·5
73
69·1
100

61

Isolate,

PFGE
type

FE14

A

FE20

B

FE09

C

FE10

D

FE02

E

FE23

F

FE06

G

FE07

G

FE11

H

55
74·5
64·8

52·8

100
66·8

52
57·4

66·9

49·7

60·6
46·6

57

53

FE19

I

FE15

J

FE03

K

FE05

K

FE08

L

FE16

M

FE22

N

FE01

O

FE17

P

FE21

Q

FE13

R

FE12

S

FE18

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Figure 1 Pulsed-ﬁeld gel electrophoresis (PFGE) types using Xbl 1 and Bln-I enzymes of 23 Escherichia coli isolates producing CTX-M and CMY-2
from faeces. PFGE analysis with Xba I and Bln I divided the 23 E. coli isolates into 21 genetic proﬁles. Nineteen of the genetic proﬁles consistedof a single strain. Dendrogram of the combined PFGE patterns of Xba I and Bln I was constructed with Dice coefﬁcient and the unweighted pairgroup method, and the similarity of the average from the two experiments. PFGE types were distinguished at a cut-off of  90% similarity.

ﬂuoroquinolones. CTX-M types other than CTX-M-15,
CTX-M-16 and CTX-M-27 efﬁciently hydrolyse cefotaxime and ceftriaxone but not ceftazidime (Bonnet 2004).
The blaTEM-1 and blaSHV-11 genes detected in the present
study encode non-ESBL enzymes (Paterson and Bonomo2005).
Annual ceftiofur and enroﬂoxacin use was low in thelarge dairy cattle population evaluated by our clinics, andtheir use does not appear to be a major risk factor for
ESBL-producing Enterobacteriaceae infections. An earlier
human study (Colodner et al. 2004) identiﬁed the previous use of ﬂuoroquinolones, penicillins and second- andthird-generation cephalosporins as risk factors for ESBLproducing E. coli and K. pneumoniae infections. Thus,
the spread of blaCTX-M and blaCMY-2 may be due in partto the frequent use of mastitis preparations consisting ofﬁrst- and second-generation cephalosporins in member
veterinary clinics in Nemuro Subprefecture and using
waste milk mixed with cephalosporins to feed calves. Previous studies (Lowrance et al. 2007; Daniels et al. 2009)
revealed that ceftiofur use had a transient effect on theselection of ceftiofur resistance in commensal E. coli atthe individual calf level. However, most CTX-M-15 producers are resistant to both oxyimino-cephalosporins andﬂuoroquinolones.
Consistent with our ﬁndings, the E. coli clones ST10,
ST44, ST48, ST57, ST58, ST69 and ST88, producing
CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14, CTX-M-15,
SHV-5 and SHV-12 were isolated from chicken caeca in
the UK (Randall et al. 2011), and retail chicken meat in

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Dairy herd prevalence of CTX-M in Japan

M. Ohnishi et al.

the Netherlands (Overdevest et al. 2011). The clonal
diversity of STs and PFGE types observed in our study
among bovine E. coli strains harbouring the same type of
blaCTX-M is in agreement with a recent canine study(Tamang et al. 2012). Both PFGE and MLST were unable
to differentiate isolates FCE03 and FCE05 and isolates
FCE06 and FCE07, which differed according to serotype,indicating that the two clusters of two isolates each wereclosely related (Table 1, Fig 1). However, four ST10 isolates showed three distinct PFGE types, and two ST744
isolates showed two distinct PFGE types. It should be
noted that PFGE showed a greater discriminatory ability
than MLST in the present study, differentiating strainsfrom different farms and strains of different serotypes. In
general, housekeeping genes have low mutation rates, but
PFGE is thought to easily detect mutations in E. coli.
In this study, we did not detect the CTX-M-15–producing E. coli ST131 (O25:H4), which is a worldwide
pandemic multidrug-resistant strain that infects humans(Kuroda et al. 2012), nor epidemic E. coli strains ST38
(O86:H18) or ST131 (O25:H4), which produce the CTXM-9 group of ESBLs that infect humans, canines andfelines in Japan (Suzuki et al. 2009; Harada et al. 2012).
Madec et al. (2012) revealed that bla CTX-M-15–carrying
plasmids from cattle-derived non-ST131 E. coli isolates
were highly similar to those found in human-derived
ST131 E. coli isolates.
Recently, a low incidence of blaCTX-M-positive E. coli
was reported in retail beef (Overdevest et al. 2011), suggesting that bovine blaCTX-M-positive E. coli may pose a
relatively low risk to public health.
In conclusion, the incidence of E. coli and three generaof Enterobacteriaceae producing CTX-M-15, CTX-M-2,
CTX-M-14 or CMY-2 in bovine faeces was 3 1% of the897 faecal samples. The herd prevalence was 5 2% of the381 dairy farms in Nemuro Subprefecture, Japan. The 23
E. coli isolates showed clonal diversity, as assessed by
MLST analysis and PFGE typing. The eight E. coli isolates
producing CTX-M-15 belonged to six STs and were nonST131; most were resistant to ceftazidime and ﬂuoroquinolones. The present results indicate that dairy cattle area potential reservoir of ESBL-producing bacteria. Thus,
routine monitoring for CTX-M (especially CTX-M-15)
and CMY-2 producers derived from faeces and mastitis
milk and the prudent use of antimicrobial agents arenecessary to prevent their clonal spread.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1 PCR primers used for detection and sequencing of CTX-M, CMY, AmpC, TEM, and SHV typeb-lactamases.

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DOI 10.1007/s10528-012-9555-8

Expression of Activin Receptor-like Kinase 7 in Adipose
Tissues
Masaru Murakami • Mitsuyuki Shirai • Ryo Ooishi • Asako Tsuburaya •Kumiko Asai • Osamu Hashimoto • Kenji Ogawa • Yoshii Nishino •Masayuki Funaba

Received: 2 December 2011 / Accepted: 11 September 2012 / Published online: 21 December 2012
 Springer Science+Business Media New York 2012

Abstract The tissue distribution of activin receptor-like kinase 7 (Alk7) expression, the signaling ability of Alk7 variants, and Alk7 expression in response to b3adrenergic receptor activation were examined. Expression levels of Alk7 varied
greatly among tissues but were highest in white adipose tissue and brown adipose
tissue. In addition to full-length Alk7 (Alk7-v1), Alk7-v3, an Alk7 variant, wasexpressed in adipose tissues, brain, and ovary. Nodal transmits signals via Alk7 incooperation with its coreceptor, Cripto. Evaluation of the ability of Alk7 variants toconfer Nodal signaling using luciferase-based reporter assays showed that Alk7-v3
does not transmit Nodal-Cripto-mediated signals. Expression of Alk7 was downM. Murakami (&) R. Ooishi A. Tsuburaya K. AsaiLaboratory of Molecular Biology, School of Veterinary Medicine, Azabu University,
Sagamihara 252-5201, Japane-mail: murakami@azabu-u.ac.jp
M. Shirai
Laboratory of Veterinary Pharmacology, School of Veterinary Medicine,
Azabu University, Sagamihara 252-5201, Japan
O. Hashimoto
Laboratory of Experimental Animal Science, School of Veterinary Medicine,
Kitasato University, Towada 034-8628, JapanK. Ogawa
Chemical Genomics Research Group, ASI, Riken, Wako 351-0198, Japan
Y. Nishino
Department of Animal Medical Sciences, Faculty of Life Sciences, Kyoto Sangyo University,
Kyoto 603-8555, Japan
M. Funaba (&)
Division of Applied Biosciences, Kyoto University Graduate School of Agriculture,
Kyoto 606-8502, Japane-mail: mfunaba@kais.kyoto-u.ac.jp

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regulated in brown but not in white adipose tissue treated with CL316,243, ab3-adrenergic receptor agonist. These results suggest involvement of Alk7 in modulationof metabolism in the adipose tissues in response to b3-adrenergic receptor activation.
Keywords

Alk7 Tgf-b family Variants Adipose tissue

Introduction
Transforming growth factor-b (Tgf-b) family members, such as Tgf-bs, activins,
and bone morphogenetic proteins, regulate a variety of cellular functions and play
important roles in cell differentiation, adhesion, migration, and apoptosis. Tgf-bfamily members transmit signals through speciﬁc heteromeric complexes with type
I receptors, called activin receptor-like kinases (Alks), and type II receptors; bothcomplexes have intracellular serine/threonine kinase domains. Type II receptor
kinases transphosphorylate and activate type I receptors and subsequently
phosphorylate receptor-regulated (R)-Smad on carboxy-terminal serines; that is,
Smad1, 5, and 8 are R-Smads regulated by bone morphogenetic proteins (BRSmads) and Smad2 and 3 are R-Smads regulated by activin and Tgf-b (AR-Smads).
Phosphorylated R-Smads form complexes with Smad4 and accumulate in the
nucleus, where they interact with transcriptional factors to regulate target genes(Feng and Derynck 2005; Miyazono et al. 2010).
One of the type I receptors, Alk7, was initially cloned from rats as an orphan
receptor (Lorentzon et al. 1996; Rydén et al. 1996; Tsuchida et al. 1996).
Subsequently, Nodal, activin AB, activin B, Gdf-1, and Gdf-3, which are all membersof the Tgf-b family, were identiﬁed as ligands of Alk7 (Reissmann et al. 2001;
Tsuchida et al. 2004; Andersson et al. 2006, 2008). Unlike the other Alks, theexpression pattern of Alk7 was tissue-restricted; it was predominantly expressed in thecentral nervous system, as determined using Northern blotting, in situ hybridization,
and RNase protection assays (Lorentzon et al. 1996; Rydén et al. 1996; Tsuchida et al.
1996). In addition, some reports have shown high levels of Alk7 expression in rat
kidneys (Rydén et al. 1996; Tsuchida et al. 1996), although other reports haveobserved no expression (Kang and Reddi 1996; Lorentzon et al. 1996). Furthermore,signiﬁcant expression of Alk7 was observed in pancreas, prostate, ovary, placenta,
white adipose tissue, and brown adipose tissue (Kang and Reddi 1996; Rydén et al.
1996; Watanabe et al. 1999; Roberts et al. 2003; Carlsson et al. 2009). In particular,
Carlsson et al. (2009) reported relatively higher expression of Alk7 in human adipose
tissues, although the expression was largely shown using data from the GEO database(http://www.ncbi.nlm.nih.gov/geo/). Currently, there are no well-controlled studies
that quantitatively compare Alk7 expression in different tissues.
In the human placenta, there are Alk7 variants that are generated by alternative
splicing (Roberts et al. 2003). In addition to the full-length Alk7 (Alk7-v1) consistingof 9 exons, three variants were identiﬁed that use an alternative ﬁrst exon, Ia (Alk7-v2),
lack exon III (Alk7-v3), or lack exons III and IV (Alk7-v4). As determined from theamino acid sequence, Alk7-v2 is a variant of Alk7 that is truncated at the extracellulardomain including the signal peptide, and Alk7-v3 and Alk7-v4 are expected to yield

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secreted forms of Alk7 (Roberts et al. 2003). The ability of these variants to confer
signals is unknown. The present study demonstrates the tissue distribution of murine
Alk7, the signaling capacity of the variant Alk7, and expression level of Alk7 in whiteand brown adipose tissues of mice treated with CL316,243, a b3-adrenergic receptor
agonist (Yoshida et al. 1994).

Materials and Methods
Animals
Animal experiments were approved by the Azabu University Animal ExperimentCommittee. Of the 28 C57BL/6 mice used, tissue distribution of Alk7 was examinedin 14 mice aged 4–9 weeks, and 14 male mice aged 8 weeks were subcutaneously
injected daily with 0.1 mg/kg CL316,243 (n = 6) or saline (n = 8) for 14 days.
RNA Isolation, RT-PCR, and Quantitative Real-Time RT-PCR
The isolation of total RNA from tissues, RT-PCR, and quantitative real-time RT-PCR(qRT-PCR) were performed as previously described (Murakami et al. 2007, 2009).
The relative expression level of Alk7 was evaluated by qRT-PCR. The Alk7 andb-actin mRNA levels were quantiﬁed by the standard curve method. The Alk7
mRNA level was expressed relative to b-actin expression. The PCR primers toevaluate Alk7 were 50 -tgggaaatagctcgaaggtg-30 and 50 -gtaaggcaactggtactcctcaa-30 ;these primers detect both Alk7-v1 and Alk7-v3. The PCR primers for b-actin werepreviously described (Murakami et al. 2009).
Expression of Alk7 variants was evaluated by RT-PCR. The PCR was performed
using cDNA synthesized from 5 ng total RNA in a total volume of 25 ll containing 19
Ex-Taq buffer with 2.0 mM MgCl2, 0.2 mM of each dNTP, 0.2 lM of each primer,and 1.25 U Ex-Taq HS DNA polymerase (Takara Bio, Otsu, Japan). The PCR primers
were 50 -tctgggaccccgaagccttgcacc-30 (F41) and 50 -ccagccctaagtcagctatggcac-30(R1111). The thermal cycling parameters consisted of an initial denaturation at
95 C for 3 min; followed by 38 cycles for Alk7 or 30 cycles for b-actin ofdenaturation at 95 C for 30 s, annealing at 58 C for 30 s, and extension at 72 C for
45 s; with an additional ﬁnal extension step of 72 C for 5 min. PCR products wereseparated on 1.2 % agarose gels in 0.759 TAE and visualized with ethidium bromide.
The band intensity was quantiﬁed by an ImageJ version 1.46p program (Rasband2012). As a negative control, an RT-PCR without cDNA was carried out.
Cloning Analysis
Full-length Alk7 was isolated from cDNA prepared from mouse brown adipose tissue.
The cDNA was ampliﬁed by PCR using DNA polymerase PrimeStar (Takara Bio),
which has high ﬁdelity, and PCR primers 50 -ttGGATCCatgaccccagcgcgcggctccg-30
and 50 -tttCTCGAGagctttacagtcttccttgacacaca-30 , with the indicated BamHI and XhoI
sites (capital letters). The thermal cycling parameters consisted of an initial

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denaturation at 95 C for 3 min; followed by 32 cycles of denaturation at 95 C for
30 s, annealing at 60 C for 5 s, and extension at 72 C for 80 s; with a ﬁnal extension
at 72 C for 5 min. The PCR products were puriﬁed using the Wizard SV Gel and PCR
Clean-Up System (Promega, Madison, WI), digested with BamHI and XhoI, cloned
into an HA-tagged pcDNA vector (Inohara et al. 1998), and then transformed into
Escherichia coli JM109. For 31 colonies, the nucleotide sequence of the insert wasdetermined to identify the Alk7 variant. Also, 511 independent colonies were
examined to determine the Alk7 variant by PCR ampliﬁcation using primers of
50 -ccggaactaaatgctcaggtctt-30 and R1111 and subsequent agarose electrophoresis.
Plasmids and Reporter Assay
CAGA-luc, an activin/Tgf-b-responsive reporter gene (Dennler et al. 1998), waskindly provided by Dr. P. ten Dijke. To prepare the Nodal and Cripto expression
plasmids of Alk7, we ampliﬁed the coding regions of the genes using PCR andcloned them into an HA-tagged pcDNA vector (Inohara et al. 1998). All resulting
constructs were veriﬁed by nucleotide sequencing.
Luciferase assays were conducted as previously described (Murakami et al.
2009). HepG2 cells were transiently transfected with the indicated expression
vectors, reporter construct, and a plasmid expressing b-galactosidase (pCMV-bGal).
The amount of DNA used in each experiment was supplemented with empty
pcDNA3 so that equal amounts of DNA were transfected. Cells were harvested 40 hafter transfection. Luciferase activity was normalized to b-galactosidase activity,
and the luciferase activity in cells transfected with empty vector was set to 1.

Results and Discussion
The tissue distribution of Alk7 was quantiﬁed by qRT-PCR (Fig. 1). Because b-actin
was appropriate as a reference gene to compare expression levels among tissues(Ooishi et al. 2012), gene transcript levels of Alk7 were calculated as the ratio to thatof b-actin. As expected, signiﬁcant expression of Alk7 was detected in the brain. In
addition, signiﬁcant expression was detected in white and brown adipose tissues.
Especially, expression in the adipose tissues was more than three-fold that in thebrain; this is partly consistent with the results of Carlsson et al. (2009), whichshowed higher expression in white adipose tissue. Similar results were obtained onrelatively higher expression in the adipose tissues, when it was corrected byexpression of Hprt1, another gene frequently used as a reference gene (data notshown). It is possible, however, that the expression level of Alk7 is higher in thespeciﬁc brain area than in the adipose tissues. Alk7 was expressed in neurons in thebrain (Rydén et al. 1996), and the brain Alk7 mRNA was localized to restricted
areas of the hippocampus, telencephalon, the basal ganglia, the thalamus, thehypothalamus, and cerebellar cortex (Lorentzon et al. 1996; Rydén et al. 1996;
Tsuchida et al. 1996).

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5

6*

Alk7
*
6

4
3
2
4
6 5

1
0

6 6
6 6 6 6 6 6 6

6 6 6 4

WAT
BAT
pancreas
brain
prostate
spinal cordovary
testisskeletal muscle
uterusbladderkidneysmooth muscle
heart
lung
spleenliver
placenta

relative mRNA level

Fig. 1 Tissue distribution of
Alk7. Alk7 expression in 18
types of tissues was measured by
qRT-PCR, as a ratio to b-actin
expression. Data are shown as
the mean ± SE. The number
above each bar indicates thenumber of samples analyzed forthat tissue. WAT, white adipose
tissue. BAT, brown adipose
tissue. Statistical differences
between tissues were determinedby one-way ANOVA, followedby Scheffe’s test. *Signiﬁcantly
different from the other tissues
at P \ 0.01

Biochem Genet (2013) 51:202–210

Expression in the pancreas and prostate was comparable to that in the brain,
which is consistent with previous results showing high expression in the rat
pancreas (Watanabe et al. 1999) and prostate (Kang and Reddi 1996). Expression of
Alk7 in the kidney of adult mice was relatively weak; the gene transcript level was
0.03 of the expression in the brain. The ﬁndings in the previous studies arecontroversial. Signiﬁcant expression of Alk7 was detected in the kidney of adult rats
(Tsuchida et al. 1996; Rydén et al. 1996), whereas no expression was detected inneonatal (Rydén et al. 1996) or fetal rats (Lorentzon et al. 1996). In addition to theeffects of growth and developmental stage on expression of Alk7 in the kidney, thismay reﬂect species differences. There may also be species-speciﬁc differences in
Alk7 expression in the placenta. Abundant expression of Alk7 could be detected
in human placental tissue (Roberts et al. 2003). By contrast, expression of Alk7 inmouse placenta was much lower, less than 0.01 of the expression in the brain.
Variants of Alk7, which are generated by alternative splicing, were isolated fromhuman placenta (Roberts et al. 2003). At ﬁrst, we explored expression of Alk7
variants in brown adipose tissue. Alk7 cDNAs were cloned by RT-PCR using
primers targeting the translation initiation site and the ﬁnal amino acid of full-length
Alk7. Of the 31 independent colonies we analyzed, nucleotide sequencing indicatedthat plasmids from 20 independent colonies encoded full-length Alk7 (Alk7-v1).
Plasmids from 10 colonies encoded an Alk7 variant corresponding to human Alk7-v3(Roberts et al. 2003), which lacks a part of the extracellular region, the wholetransmembrane spanning region, and a portion of the kinase domain. In addition, aplasmid encoding a novel variant of Alk7 lacking exons III and V was isolated fromone colony; this variant was named Alk7-v5. We also examined expression of the
Alk7 variant corresponding to human Alk7-v2 (Roberts et al. 2003) using a PCR
primer set targeting exon Ia, but no signiﬁcant band was detected (data not shown).
To compare expression levels among Alk7 variants, we performed PCR using
primers that detect all three variants (Alk7-v1, Alk7-v3, and Alk7-v5). Bands
corresponding to Alk7-v1 and Alk7-v3 but not Alk7-v5 were clearly detected in brown
and white adipose tissues, brain, and ovary; the ratio of Alk7-v1 to Alk7-v3 was not

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207

WAT
brain
ovary

cDNA:

BAT

greatly different among tissues (Fig. 2). In addition, analyses of 511 independentcolonies of E. coli transformed with plasmid with ampliﬁed Alk7 gene in brown
adipose tissue indicated an Alk7-v1 : Alk7-v3 ratio of about 2.7:1, and no colony was
detected for Alk7-v5 (Fig. 2). We concluded that Alk7-v5 is an artifact of PCR.
Given the signiﬁcant expression of Alk7-v3, we evaluated the ability of Alk7-v3
to confer Nodal-induced transcriptional activation in HepG2 cells by a luciferasebased reporter assay using CAGA-luc, a representative reporter to evaluate Alk4-,5-, and 7-mediated signaling (Dennler et al. 1998; Tojo et al. 2005). The expressionof Cripto, a protein that acts as a coreceptor for Nodal signaling (Whitman 2001),
was required for transcriptional activation of CAGA-luc by Nodal (Fig. 3).
Expression of Alk7-v1 but not Alk7-v3 enhanced transcription of CAGA-luc
induced by Nodal and Cripto. These results suggest that Alk7-v3 is not involved in
canonical Alk7-mediated signaling. It is also possible that these variants regulate thepathway differently from the Tgf-b family members. Currently, the role of serine/
threonine phosphorylation of extracellular molecules is not known; future studies
are needed to clarify the biological activities of the soluble serine/threonine kinases
Alk7-v3, especially in adipose tissues.
To gain insight on the role of Alk7 in adipose tissues, CL316,243, a b3adrenergic receptor agonist (Yoshida et al. 1994), was administered to C57BL/6
mice. It is known that the treatment induces emergence of brown adipocytes inwhite adipose tissue (Barbatelli et al. 2010) and activation of brown adipose tissue,
as evidenced by up-regulation of Ucp1 (Cinti et al. 2002), a brown adipocytespeciﬁc gene (Cannon and Nedergaard 2004). Expression of Alk7 was signiﬁcantly
lower in the interscapular brown adipose tissue treated with CL316,243 than in thecontrol brown adipose tissue; to the contrary, Alk7 expression in the inguinal white
adipose tissue was comparable between groups (Fig. 4). Decrease in Alk7
expression in brown adipose tissue may reﬂect down-regulation to reduce thesignal transduction via Alk7, resulting from the prolonged activation of b3adrenergic receptor. Previous studies revealed that Alk7 is expressed in fetal rat
brown adipocytes (Lorentzon et al. 1996). Thus, the regulatory changes in Alk7
expression in brown adipose tissue imply the involvement of Alk7 in b3-adrenergic
receptor-mediated modulation of brown adipocyte function.

Alk7β-actinv1
v3

Alk7-v1
Alk7-v3
Total

Colonies

(%)

371
140
511

(72.6)(72 6)(27.4)(100)

Alk7-v1/Alk7-v3: 1.
1 6 1.
1 3 1.
1 0 1.1
11

Fig. 2 Expression of Alk7 variants. (Left) RT-PCR using primers that detect Alk7-v1, Alk7-v3, and Alk7v5 (i.e., F41 and R1111) in four tissues, brown adipose tissue (BAT), white adipose tissue (WAT), brain,
and ovary. The PCR products were electrophoresed and stained by ethidium bromide. The band intensity
was measured, and the ratio of Alk7-v1 to Alk7-v3 was calculated. A representative result is shown below
each lane. (Right) Escherichia coli was transformed with plasmid ligated Alk7 ampliﬁed by PCR as atemplate of cDNA originated from brown adipose tissue. A total of 511 independent colonies wasanalyzed to identify the Alk7 variant

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Biochem Genet (2013) 51:202–210

CAGA-luc
luciferase activity

10.0

*

Vector
Nodal

7.5
5.0
2.5
0.0

-

Cripto:

-

+

-

Alk7:

- +

+
v1

v3

Fig. 3 Nodal-induced and Alk7-mediated transcription. HepG2 cells were transiently transfected with
CAGA-luc and the indicated expression plasmids along with CMV-b-galactosidase to adjust fortransfection efﬁciency. Luciferase activity was normalized to b-galactosidase activity, and the luciferase
activity in cells transfected with empty vector was set to 1. Data are shown as the mean ± SE. Statisticaldifferences between both Nodal and Cripto transfected groups were determined by one-way ANOVA,
followed by Scheffe’s test. *Signiﬁcantly different from the other groups at P \ 0.01

The present study examined comprehensive expression of Alk7 in tissues andsignaling of Alk7 variants. We show that (1) Alk7 expression in mice is higher in the
adipose tissues than in the brain, (2) the Alk7-v3 variant is expressed in adipose
tissues, brain, ovary, and possibly elsewhere, (3) only Alk7-v1 transmits NodalCripto signaling, and (4) Alk7 expression is down-regulated in activated brown

Alk7
control
CL316,243

1.0

0.0

*
ing WAT

0.5

iBAT

relative mRNA level

1.5

Fig. 4 Expression of Alk7 in brown and white adipose tissue treated with CL316,243. C57BL/6 mice
were treated with CL316,243 (n = 6) or saline (n = 8) for 14 days. Interscapular brown adipose tissue(iBAT) and inguinal white adipose tissue (ing WAT) were collected, and expression of Alk7 was
examined by qRT-PCR. The expression was normalized to b-actin expression, and the expression in thecontrol group in each fat depot was set to 1. Data are shown as the mean ± SE. Statistical differences
between groups were determined by one-way ANOVA. *Signiﬁcantly different from the other groups at
P \ 0.01

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209

adipose tissue. Phenotypic analyses of Alk7-null mice and Nodal-null mice veriﬁedthat Alk7 is not an essential mediator of Nodal signaling during mesoderm
formation and left–right patterning (Brennan et al. 2001, 2002; Jörnvall et al. 2004).
In addition, Alk7-null mice exhibited no histological abnormalities in the central
nervous system, including cerebella, cortices, and hippocampi (Jörnvall et al. 2004).
Alk7-null mice also exhibited an age-dependent syndrome involving progressive
hyperinsulinemia, reduced insulin sensitivity, liver steatosis, impaired glucose
tolerance, and pancreatic islet enlargement (Bertolino et al. 2008). In the study by
Bertolino et al. (2008), the aberrant glucose/insulin metabolism was proposed toresult from an Alk7 deﬁciency in the pancreas. In view of our data on the higher
expression of Alk7 in adipose tissues, however, the phenotypes of Alk7-null mice
may result from impaired production of adipokines in white adipocytes and energy
expenditure by brown adipocytes (Bertolino et al. 2008); these actually improveglucose metabolism through enhancing insulin sensitivity (Shimomura et al. 1999;
Kubota et al. 2007; Seale et al. 2011). The present study suggests the necessity offurther studies focusing on the role of Alk7 in adipose tissues.
Acknowledgments We thank Dr. Peter ten Dijke for providing plasmid. This work was supported by
Kakenhi from The Japan Society for the Promotion of Science and a research project grant awarded by
the Azabu University.

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Isolation of Microsporum gallinae from a fighting
cock (Gallus gallus domesticus) in Japan

A case of tinea corporis caused by Microsporum gallinae was found in 2011 in Okinawa,located in the southern part of Japan. The patient was a 96-year-old, otherwise healthy,
Japanese man, who had been working as a breeder of fighting cocks for more than
70 years. He was bitten on his right forearm by one of the cocks and a few weeks later,
two erythematous macules appeared on the right forearm, accompanied by a slight itchy
sensation. While the first isolate of this dermatophyte was recovered from the region by
Miyasato et al. in 2011, it was not obtained from the same fighting cock owned by thepatient. However, frequent exchanges of fighting cocks and special domestic breeds ofchickens related to fighting, mating, and/or bird fairs are common among the fans andbreeders. We investigated 238 chickens and 71 fighting cocks in Okinawa and in thesuburbs of Tokyo (Chiba, Tokyo, Ibaraki, and Sizuoka). One isolate of M. gallinae froma fighting cock in Chiba Prefecture in the Tokyo metropolitan area exhibited a different
genotype, with a single base difference from the patient isolate based on the internaltranscribed spacer 1-5.8s-ITS2 regions (ITS1-5.8S-ITS2) of the ribosomal RNA genesequence. The isolation of M. gallinae from a fighting cock on the mainland of Japan isthe first such finding in animals in our country.
Keywords

Microsporum gallinae, fighting cock, Gallus gallus domesticus

Introduction

often infected than hens [1], especially fighting cocks or
the oriental breed called ‘Shamo’ [6–9]. This fungal species also infects dogs [10–13], monkeys [14], cats [11,15],
squirrels [13], mice, canary, pigeon, and turkey [1], and has
been isolated from birds’ nests [16]. In addition, Microsporum gallinae is zoonotic causing ringworm in humans[13,17–27] and severe infections in immunocompromised
hosts [22,25]. These human cases involved patients who
had had contact with infected animals, especially cocks[17,27]. Based on 44 reports, it would appear that theendemic areas of M. gallinae include European, Middle
Eastern, and South American countries, in addition to theUSA [28], Sweden, France, Germany, Czechoslovakia,

Microsporum gallinae is a dermatophyte that causes favusof the comb in chickens and related species [1–5]. White
crusts or plaques containing fungal elements are associated with hyperkeratosis, and lesions may spread to theskin of the head and the neck with feather loss, although
the feathers are not invaded. Chicks and roosters are more
Received 19 March 2012; Received in final revised form 16 May 2012;
Accepted 7 June 2012.
Correspondence: Ayako Sano, University of the Ryukyus, Faculty ofAgriculture, Animal Sciences, Nakagusuku, Japan. E-mail: aya_grimalkin@
yahoo.co.jp© 2013 ISHAM

DOI: 10.3109/13693786.2012.701766

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Downloaded from http://mmy.oxfordjournals.org/ at Azabu University on April 22, 2016

MICHIKO MURATA*, HIDEO TAKAHASHI†,‡, SANA TAKAHASHI§,YOKO TAKAHASHI#, HIROJI CHIBANA#,YOSHITERU MURATA‡,^, KAZUTOSHI SUGIYAMA ⫹ , TAKASHI KANESHIMA$, SAYAKA YAMAGUCHI¶,HITONA MIYASATO¶, MASARU MURAKAMI*, RUI KANO∼, ATSUHIKO HASEGAWA∼, HIROSHI UEZATO¶,
ATSUSHI HOSOKAWA¶ & AYAKO SANO¥*Department of Molecular Biology, Faculty of Veterynary Medicine, Azabu University, Kanagawa, Japan, †A-land Oyumino Animal
Hospital, Chiba, Japan, ‡Committee of Infectious Diseases, Chiba Veterinary Medical Association, Chiba, Japan, §Faculty of Veterinary
Medicine, Azabu University, Kanagawa, Japan, #Medical Mycology Research Center, Chiba University, Chiba, Japan, ^Murata Animal
Hospital, Mobara, Chiba, Japan, ⫹ Sugiyama Veterinary Clinic, Shizuoka, Japan, $Ryukyu Animal Clinic Center,Tomigusuku, Okinawa,
Japan, ¶Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan, ∼Department of Pathology,
School of Veterinary Medicine, Nihon University, Fujisawa, Kanagawa, Japan, and ¥Department of Animal Sciences,
Faculty of Agriculture, University of the Ryukyus, Okinawa, Japan

Microsporum gallinae from a fighting cock145

The present isolate was crossed with; (1) Arthroderma
grubyi isolates IFM 48209, IFM 49901, and IFM 49902,(2) M. gallinae isolate IFM 56900 isolated from the firstclinical case in Okinawa, Japan [27] and (3) A. simii
isolates IFM 49897 and 49898, on salt-added 1/10 diluted
Sabouraud dextrose agar plates at 25°C for 8 weeks [28].
In addition, A. grubyi isolates IFM 48209, IFM 49901, andIFM 49902 were crossed with each other to observe theirsexual function.

Spain, Germany, Pakistan, Iran, Nigeria, Puerto Rico,
Venezuela, and Japan [27].
In the first Japanese human case, infection may havebeen due to infected animals, i.e., cocks [17,27], although
we could not isolate M. gallinae from the animal maintained by the patient. During a preliminary survey, welearned from a breeder that exchanges of birds and special
breeds of chickens related to fighting, mating, and/or bird
fairs are common among breeders throughout Japan. We
conducted the present study to isolate M. gallinae fromfowls in Japan.

Molecular biological identification

Animals
We examined 238 chickens and 71 fighting cocks in Japan invarious prefectures, but the specific prefectures are not mentioned to comply with privacy policies of the funding agency.
The research was supported by Grant-in-Aid for Scientific
Research© by the Japan Society for the Promotion of Science(JSPS), No. 23580419 and approved by the ethics committee
for animal welfare of the University of the Ryukyus.
Isolation
Eighteen-millimeter-wide transparent Scotch tape (Sumitomo 3A, Tokyo, Japan) was cut into strips approximately
10 cm in length and both 1-cm ends were folded for handling. The adhesive surface (approximately 6 cm in length)
was placed on the comb and gently rubbed with both thumband index fingers to collect scales. The tape was incubatedat 42°C for 4 h to remove mites and then applied to thesurface of BBL Mycosel Agar (Becton Dickinson [BD];Sparks, MD, USA) plates supplemented with chloramphenicol (100 mg/liter, Wako Pure Chemical Industries,
Ltd., Osaka, Japan) in duplicate, and incubated at 35°C for
up to 28 days.
A white cottony colony with strawberry-colored pigmentation diffusing into the medium was transferred onto
potato dextrose agar (Difco Potato Dextrose Agar; PDA,
BD) slants and incubated at 25°C for further mycologicaland molecular biological analysis.
Mycological studies
Colonies grown on Sabouraud dextrose agar (SDA) containing 2% dextrose (Wako Pure Chemical Industries,Ltd.), 1% neopeptone (Difco, BD), and 1.5% Bacto Agar
(Difco, BD) and PDA plates incubated at 25°C wereobserved for 21 days. The isolate was cultured on SDA andPDA blocks at 25°C for 28 days for light microscopic
observation after staining with lactophenol cotton blue.
© 2013 ISHAM, Medical Mycology, 51, 144–149

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Sequences of the ITS1-5.8S-ITS2 region of the rRNA gene
were obtained by routine methods [29]. Briefly, DNA wasextracted with a DEXPAT kit (TaKaRa, Ohtsu, Japan)
using a modified procedure. Approximately 100 μl of fungal cells from cultures incubated at 25°C for 1 month on
PDA slants was placed in a sterilized microtube (1.5 ml)
to which 0.5 ml of DEXPAT solution was added, and the
mixture homogenized with a plastic pestle. The tube wasincubated at 100°C for 10 min, and then centrifuged at
13,200 g for 10 min, and the supernatants were used as
DNA samples. Although this type of kit is designed forextracting DNA from paraffin-embedded tissue samples,
we routinely use it for the isolation of genes from fungal
cultures because of its convenience [30].
We mixed 2.5 μl of DNA extract with Ready-To-Go
PCR beads (Amersham Pharmacia Tokyo, Japan), 2.5 μlof 10 pM of the primers ITS-5 (5⬘- GGA AGT AAA AGT
CGT AAC AAG G-3⬘) and ITS-4 (5⬘- TCC TCC GCT TATTGA TAT GC-3⬘) [30], and 17.5 μl of distilled water. The
reaction mixture was subjected to 1 cycle of denaturation
at 95°C for 4 min, 30 cycles of amplification at 94°C for
1 min, 50°C for 1 min, and 72°C for 2 min, and a final
extension cycle at 72°C for 10 min in a PCR Thermal
Cycler MP (TaKaRa).
PCR products were visualized by electrophoresis on
1.0% agarose gels in 1 ⫻ TBE buffer (0.04 M Tris-boric
acid, 0.001 M EDTA [pH 8.0]) followed by ethidium bromide staining. PCR samples were purified using a PCR
purification kit (QIAquick, Qiagen Co. Ltd., Tokyo, Japan),and labeled with BigDye Terminator Ver. 1.1 (Applied
Biosystems, Foster City, CA, USA) following the manufacturer’s protocol. The labeled samples were directly
sequenced on an ABI PRISM 3100 sequencer (Applied
Biosystems) using the primers ITS-5, ITS-4, ITS-2 (5⬘GCT GCG TTC TTC ATC GAT GC-3⬘), and ITS3 (5⬘GCA TCG ATG AAG AAC GCA GC-3⬘) [30]. DNA
sequences amplified by at least two primer pairs werealigned using GENETEX-MAC genetic information processing software (Software Development Co., Ltd., Tokyo,
Japan). These procedures were performed twice on boththe present isolate and M. gallinae IFM 56900 isolatedMaterials and methods

146

Murata et al.

Table 1The ITS rDNA sequences of Microsporum gallinae and related species in the GenBank database.

Strain name

GenBank
Accession no.

Country and host

Arthroderma grubyi

CBS 244.66
CBS 243.66

AJ000613
AJ000612*

USA Man, scalp
USA, Montana ringworm infection in dog

IFM 48209
IFM 49901
IFM 49902
CBS 243.66

AB698453
AB698454
AB698455
AJ970147**

France, man, skin lesion
USA, ATCC 14422 obtained by mating
Canada, ATCC 14423 obtained by mating
USA, Montana ringworm infection in dog

CHUS95902
CBS 300.52
ATCC 12108
IFM 56900
IHEM 13565
IHEM 13563
IHEM 13564
IHEM 13373
778
IFM 60027
IFM 58803

EF581136
AJ000620
EF631610
AB455805
FJ356083
FJ428241
FJ428242
FJ416304
JN134141
AB698457
AB667976

Spain
Unknown
Puerto Rico, child’s scalp
Japan, tinea corporis
Israel, chicken
Israel, monkey
Israel, unknown (maybe human)Iran, unknown (maybe human)Iran, unknown (maybe human)
Israel, unknown (maybe human)
Japan, fighting cock. Present isolate

Microsporum vanbreuseghemii
Microsporum gallinae

Remarks
Deposited by M. Elfarin July 1997

Deposited by Y. Graser
in November 2005

* and ** derived from isolate CBS 243.66 showed one base difference. CBS, The Centraalbureau voor Schimmelcultures, The Netherlands. IFM, Medical
Mycology Research Center, Chiba University, Japan. CHUS, University of Santiago, Spain. ATCC, American Type Culture Collection, USA. IHEM,
BCCM/IHEM, Scientific Institute of Public Health Section Mycology and Aerobiology, Belgium.

from the first clinical case [27], to confirm differences inthe sequences. Sequences were analyzed using a BLAST
search (http:// www.ncbi.nlm.nih.gov/BLAST/Blast.cgi)
and closely related sequences were obtained.
Seventeen nucleotide sequences from M. gallinae andits teleomorph species A. grubyi deposited in GenBank(Table 1) were aligned, together with the present nucleotide sequences, using the computer program ClustalX(Version 1.8) [31] followed by manual adjustments with atext editor. Phylogenetic analyses were performed with
PAUP v4.0b10 [32] using a heuristic search for maximum
parsimony trees. Base-pair composition and transition

and/or transversion patterns of the dataset were estimatedby PAUP v4.0b10. Bootstrap values were calculated
over 1000 replicates to assess branch topology. Only one
phylogenetic tree was obtained from the analysis andwas drawn by Tree View PPC [33] (Roderic D. M. Page,
Glasgow, Scotland, UK, 1998; http://taxonomy.zoology.
gla.ac.uk/rod/treeview.html). Clades were supported bybootstrap values above 50.

Results
One M. gallinae isolate was obtained from a 10-monthold male fighting cock from Chiba Prefecture. The combof the cock was of the cushion type and showed whitescaling (Fig. 1). The primary isolate appeared as a slightly
yellowish white, cottony colony approximately 1.2 cm indiameter after incubation at 35°C for 2 weeks on a Mycosel agar plate, and showed slight strawberry-red pigment
diffused into the medium (data not shown). A colony of
the isolate grown on SDA at 25°C for 21 days was whiteand cottony, 5.7 cm in diameter, with slight strawberryred pigment diffused into the medium. Those on PDA
under the same culture conditions were pinkish-grey witha felt-like texture on the surface, 3.7 cm in diameter, andshowed vibrant strawerry-red pigment diffused into themedium (Fig. 2).
Septate hyphae with small numbers of macroconidia
consisting of 4–6 cells (4–7 ⫻ 12–18 μm) and ovoid topyriform, single-celled microconidia (1–2 μm in diameter)

Fig. 1 Microsporum gallinae-positive cock showing white scaling on
the comb.

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Species

Microsporum gallinae from a fighting cock147

Fig. 2 Colony of isolate IFM 58803 on SDA (a) and PDA (b) cultured at 25°C for 21 days.

Recently, it has become clear that M. gallinae is not
restricted to tropical and subtropical regions, but may
be present in temperate areas, as revealed by records
of infection and isolation in European countries [17,27].
Our isolation of M. gallinae from a fighting cock, following the human isolate reported by Miysasto et al.
[27], indicates that Japan is one of the endemic areas.
Interestingly, the sequence of the ITS1-5.8S-ITS2 regionof the rRNA gene derived from the present isolate wasdifferent from that of the human isolate in Okinawa prefecture, suggesting that at least two different genotypes ofM. gallinae exist in Japan as confirmed by duplicate
sequence analysis. In addition, phylogenetic analyses by
the maximum parsimony method indicated that M. gallinae
and A. grubyi were different species, although there areinsufficient sequence data to confirm this at present. Furthermore, we could not correlate actual mating ability
with genotype due to infertility of the tested A. grubyi
isolates.
The immigration route of M. gallinae to Japan is unclear
as only two isolates are known, i.e., from the clinical casein 2011 and the one described in this report.

Fig. 3 (a) Septate hyphae with a few macroconidia consisting of 4 to 6 cells, and (b) ovoid to pyriform single-celled microconidia attached to thehyphae at right angles on an SDA block cultured at 25°C for 28 days. Bars indicate 10 μm.

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Discussion

attached to the hyphae at right angles were observed on the
SDA block incubnated at 25°C for 28 days. Hyphae werepredominant on the PDA block with a few microconidia(Fig. 3).
Gymnothecia were not produced by any crosses. Crosses
among A. grubyi were also negative.
The sequence of the ITS1-5.8S-ITS2 region of therRNA gene obtained from the present isolate consisted of
597 base pairs, and showed 99% identity to GenBank
database (http://www.ncbi.nlm.nih.gov/nuccore) accession
number AB455805, derived from a clinical M. gallinae
isolate (IFM 56900) in Japan [27]. The present isolate
was deposited in the National BioResource Project culturecollection at the Medical Mycology Research Center,
Chiba University, 260-8673 Chiba, Japan as IFM 58803.
The sequence of the ITS1-5.8S-ITS2 region of the rRNA
gene was deposited in the GenBank database with accession number AB667976.
There were two genotype clusters of the ITS1-5.8SITS2 region of the rRNA gene based on the maximum
parsimony tree (Fig. 4). One cluster mainly consisted of
M. gallinae and the other comprised A. grubyi. The present
sequence was located in the same cluster as those derived
from a Japanese clinical isolate of M. gallinae.

148

Murata et al.

Declaration of interest: The corresponding author, Ayako
Sano declared the no conflict of interest submitted to the
University of the Ryukyus No. H24-763.

Neither human nor poultry infections had been reported
prior to the first human case of M. gallinae infection. However, M. gallinae infection in fighting cocks is mild, andinfections in humans closely resemble other dermatophytoses (such as M. canis infection). Therefore, many humancases and animal infections were likely ignored [27].
In conclusion, Japan is an endemic area of M. gallinae
infection, based on a human case [27] and the present
isolate from a fighting cock.

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Acknowledgements
This study was supported in part by the Special Research
Fund for Emerging and Re-emerging Infections of the
Ministry of Health, Welfare and Labor (Grant No. H-21Shinkou-004), and by a Grant-in-Aid for Scientific
Research© by the Japan Society for the Promotion of Science (JSPS), No. 23580419. We express our thanks to Drs
Takako Murano (Chiba Prefecture), Keiji Takahashi (Chiba
Prefecture), Tadao Ikeda (Association Pasteur Japon),
Eichiro Matayoshi (Okinawa Veterinary Medical Association), Taketoshi Kawamitsu (Okinawa Veterinary Medical
Association), Takako Okubo (Ibaraki Prefecture), Masami
Sudo (Ibaraki Prefecture), Tomo Inomata (Azabu University), Shigeyuki Mastui (Shizuoka Prefecture), Moriji
Iketani (Shizuoka Prefecture), Yoshimi Imura (Universityof the Ryukyus), and Morihiko Hirakawa (University of the
Ryukyus) for sample collection. We thank Ms Kyoko Yarita,
Akiko Takayama and Yuki Hanami (Medical Mycology
Research Center, Chiba University) for technical support.

© 2013 ISHAM, Medical Mycology, 51, 144–149

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Downloaded from http://mmy.oxfordjournals.org/ at Azabu University on April 22, 2016

Fig. 4 The exclusive most-parsimonious tree obtained from heuristic searches based on ITS1-5.8S-ITS2 rRNA gene sequences using 546 bases.
Bootstrap support value above 50% is indicated at the node. The bar indicates 1 base difference. Data are shown with accession numbers and fungal
species as MG, Microsporum gallinae; AG, Arthroderma grubyi; and MV, Microsporum vanbreuseghemii. CI, consistency index. RI, retention index.
RC, rescaled consistency index. HI, homoplasy index.

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32 Swofford DL. PAUP*. Phylogenetic Analysis Using Parsimony (*andother methods) (version 4.0). Sunderland, MA: Sinauer Associates,
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Downloaded from http://mmy.oxfordjournals.org/ at Azabu University on April 22, 201613 Georg LK, Ajello L, Friedman L, Brinkman SA. A new species ofMicrosporum pathogenic to man and animals. Sabouraudia 1962; 1:
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Veterinary Microbiology 161 (2012) 196–205

Contents lists available at SciVerse ScienceDirect

Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Distribution and molecular characterization of Porphyromonas gulae
carrying a new ﬁmA genotype
Yoshie Yamasaki a,b, Ryota Nomura a, Kazuhiko Nakano a,*, Hiroaki Inaba c, Masae Kuboniwa d,
Norihiko Hirai e, Mitsuyuki Shirai e, Yukio Kato f, Masaru Murakami g, Shuhei Naka a,
Soichi Iwai h, Michiyo Matsumoto-Nakano b, Takashi Ooshima a,
Atsuo Amano d, Fumitoshi Asai eaDepartment of Pediatric Dentistry, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japan
Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8556, Japanc
Department of Oral Frontier Biology, Center for Oral Science, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japand
Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japane
Department of Pharmacology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252-5201, Japanf
Department of Veterinary Public Health II, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252-5201, Japang
Department of Molecular Biology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 252-5201, Japanh
Department of Oral and Maxillofacial Surgey, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japanb

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 14 May 2012
Received in revised form 14 July 2012
Accepted 16 July 2012

Porphyromonas gulae is a gram-negative black-pigmented anaerobe which is known to be apathogen for periodontitis in dogs. Approximately 41 kDa ﬁlamentous appendages on thecell surface (FimA) encoded by the ﬁmA gene are regarded as important factors associatedwith periodontitis. The ﬁmA genotype was classiﬁed into two major types and strains intype B were shown to be more virulent than those in type A. In the present study, wecharacterized a strain with a novel ﬁmA genotype and designated it as type C. The putative
amino acid sequence was shown to be similar to the genotype IV ﬁmA of Porphyromonas
gingivalis, a major pathogen of human periodontitis. Analyses using an oral squamous cell
carcinoma cell line derived from tongue primary lesions revealed that the type C strain
inhibited proliferation and scratch closure more than genotype A and B strains. In addition,
experiments using a mouse abscess model demonstrated that the type C strain could
induce much higher systemic inﬂammation when compared with strains of the othergenotypes. Furthermore, molecular analyses of oral swab specimens collected from dogs
demonstrated that the detection frequencies of P. gulae and the genotype C in the
periodontitis group were signiﬁcantly higher than those in the periodontally healthygroup. These results suggest that FimA of P. gulae is diverse with the virulence of genotype
C strains the highest and that molecular identiﬁcation of genotype C P. gulae could be apossible useful marker for identifying dogs at high risk of developing periodontitis.
ß 2012 Elsevier B.V. All rights reserved.

Keywords:
Fimbriae
Genotype
PCR
Periodontitis
Porphyromonas gulae
Risk marker

1. Introduction
Periodontal diseases are initiated following gingivitis
with localized inﬂammation without the destruction of theperiodontal tissues (cementum, periodontal ligament andalveolar bone) (Pihlstrom et al., 2005). Without any
intervention, this localized inﬂammation generally progresses to periodontitis, especially in elder subjects and* Corresponding author at: Department of Pediatric Dentistry, Osaka
University Graduate School of Dentistry, 1-8 Yamada-oka, Suita, Osaka
565-0871, Japan. Tel.: +81 6 6879 2963; fax: +81 6 6879 2965.
E-mail address: nakano@dent.osaka-u.ac.jp (K. Nakano).
0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetmic.2012.07.026

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Y. Yamasaki et al. / Veterinary Microbiology 161 (2012) 196–205

197

and designated it as genotype C. The purpose of the present
study was to compare the properties of each FimA genotype
relative to its virulence potential in periodontitis. In
addition, the distribution of the each group was analyzed
focusing on the clinical conditions of the dogs sampled.

those with systemic disorders. The symptoms of periodontitis are generally irreversible and include severe
bleeding, pus discharge and mobility of the affected toothand ultimately leads to tooth loss. The distribution of theperiodontitis-related bacterial species has been investigated in the oral swab specimens taken from Japanese dogs
and it was observed that Porphyromonas gulae, a gramnegative black-pigmented anaerobe, was one of thespecies frequently detected (Kato et al., 2011).
The cell surface 41 kDa ﬁmbrillin (FimA), a subunit ofﬁmbriae, was characterized in P. gulae and is known to be
one of the major factors for periodontitis in dogs (Hamada
et al., 2008). Furthermore, P. gulae invasion into gingival
epithelial cells has also been demonstrated. In our recent
study, FimA of P. gulae could be classiﬁed into two major
genotypes, and analyses of the putative amino acidsequences of FimA in many clinical strains revealed thatthe type A FimA is speciﬁc for P. gulae, and the amino acidsequence of type B FimA is more closely related to that ofgenotype III FimA of Porphyromonas gingivalis, a major
pathogen of human periodontitis (Nomura et al., 2012). On
the other hand, there are several P. gulae-positive specimens which are negative for both types A and B, indicating
the presence of P. gulae strains without FimA or those withadditional ﬁmA genotypes (Nomura et al., 2012).
In the present study, we successfully characterized a new
genotype for ﬁmA genes in P. gulae encoding a novel FimA

2. Materials and methods
2.1. Bacterial and cell culture conditions
Table 1 lists the P. gulae strains analyzed in the present
study, among which all except for one strain (D049) werepreviously reported (Kato et al., 2011; Nomura et al., 2012).
Strain D049 was isolated from an oral swab specimen froma dog and conﬁrmed to be P. gulae by a molecular biologicalmethod described previously (Kato et al., 2011). In
addition, 11 P. gingivalis strains listed in Table 1 were also
used (Amano et al., 1999; Nakagawa et al., 2000, 2002). P.
gingivalis and P. gulae strains were cultured in mhTS broth[Tripticase soy broth (Becton, Dickinson & Co, Franklin
Lakes, NJ, USA) with hemin (50 mg/ml) and menadione(5 mg/ml)] under anaerobic conditions.
The SAS cells, an oral squamous cell carcinoma cell line,
were obtained from Japanese Collection of Research
Biosources (Tokyo, Japan). Cells were cultured in RPMI
1640 medium (Wako, Osaka, Japan) supplemented with
10% fetal bovine serum at 37 8C in 5% CO2.

Table 1
P. gulae and P. gingivalis strains used in the present study.
SpeciesName

ﬁmA types

Length of ﬁmA gene (bp)

Accession numbers of ﬁmA gene

References

P. gulae

ATCC 51700a
D024
D025
D028
D034
D035
D036
D042
D043
D060
D066
D067
D068

A
A
A
A
A
A
A
A
A
A
A
A
A

1152
1152
1152
1152
1152
1152
1152
1152
1152
1152
1152
1152
1152

AB297918
AB663087
AB663088
AB663089
AB663090
AB663091
AB663092
AB663093
AB663094
AB663095
AB663096
AB663097
AB663098

Hamada et al. (2008)
Nomura et al. (2012)
Nomura et al. (2012)
Nomura et al. (2012)
Nomura et al. (2012)
Nomura et al. (2012)
Nomura et al. (2012)
Kato et al. (2011)
Kato et al. (2011)
Kato et al. (2011)
Kato et al. (2011)
Kato et al. (2011)
Kato et al. (2011)

B43
D040a
D044
D052
D053
D077

B
B
B
B
B
B

972b
1161
1161
1161
1161
1161

CS228034
AB663099
AB663100
AB663101
AB663102
AB663103

Dreier et al. (2005)
Kato et al. (2011)
Kato et al. (2011)
Kato et al. (2011)
Kato et al. (2011)
Nomura et al. (2012)

D049a

C

1167

AB679295

This study

381
ATCC 33277
BH18/10
HW24D1OMZ314aOMZ409ATCC 49417
6/26
HG564
HNA99
HG1691

I
I
I
II
II
II
II
III
IV
V
Ib

1044
1044
1044
1047
1044
1047
1053
1062
1083
1104
1044

D17794
D17795
D17796
D17797
D17798D17799D17800
D17801
D17802
AB027294
AB058848

Amano et al. (1999)
Amano et al. (1999)
Amano et al. (1999)
Amano et al. (1999)
Amano et al. (1999)
Amano et al. (1999)
Amano et al. (1999)
Amano et al. (1999)
Amano et al. (1999)
Nakagawa et al. (2000)
Nakagawa et al. (2002)

P. gingivalisabStrains analyzed in the mouse abscess model.
Only partial sequences corresponding to ﬁmA of P. gulae are available.

480

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Y. Yamasaki et al. / Veterinary Microbiology 161 (2012) 196–205

2.2. Sequence of the ﬁmA genes(http://taxonomy.zoologygla.ac.uk/rod/treeview.html).
Since the sequence from D049 was distinct from thoseof other strains, we designated it as a novel genotype(type C).

The nucleotide alignment of the entire length of the
ﬁmA gene was determined by the method described
previously with some modiﬁcations as follows (Nomura
et al., 2012). PCR was performed with genomic DNA
extracted from P. gulae D049 using six types of P. gingivalis
ﬁmA speciﬁc sets of primers listed in Table 2 (Amano et al.,
1999; Nakagawa et al., 2000, 2002). Since the tested P.
gulae strains showed positive reaction for P. gingivalis typeIV ﬁmA, we designed the primer sets (HG564-F and
HG564-R) based on the adjacent sequence of HG564 (typeIV). Next, PCR was performed using these primers toamplify the entire length of the ﬁmA gene of P. gulae, which
was separated by electrophoresis on a 0.7% agarose gel
and the ampliﬁed DNA fragments were extracted from thegel using a QIAEX gel extraction kit (Qiagen, Düsseldorf,
Germany). The DNA was then directly cloned into a pGEMT Easy vector (Promega, Madison, WI, USA). The nucleotide sequence was determined by the dye-terminator
reaction with a DNA sequencing system (373-18 DNA
sequencer; Applied Biosystems, Foster City, CA, USA) andan ABI PRISM kit. Data analyses were performed using
Gene Works software (IntelliGenetics, Mountain View,
CA, USA). The putative amino acid sequences of the FimAof types A and B P. gulae strains and those of P. gingivalis
strains with ﬁmA genotypes I through V and Ib listed inTable 1 were compared using the neighbor-joining
method to construct a phylogenetic tree using CLUSTALW (DNA Databank of Japan) and Tree View software

2.3. Construction of a PCR method to classify ﬁmA groups
We previously developed a molecular biologicalmethod to classify type A and type B P. gulae strains(Nomura et al., 2012). In the present study, the entire ﬁmAsequence of D049 was compared to those of genotypes A
and B to ﬁnd speciﬁc regions for Type C. A primer set
speciﬁc for genotype C (Pguﬁm-CF and Pguﬁm-CR; Table 2)
was then constructed. The ampliﬁed fragment wasestimated to be 631 bp. The speciﬁcities of these primers
were tested by the program Amplify (Engels, 1993), basedon information on the DNA sequences of P. gulae strainsobtained in the present study as well as that of P. gingivalis
in the GenBank database (http://www.ddbj.nig.ac.jp/). The
speciﬁcity of the primers was also conﬁrmed using the
GenBank database. PCR ampliﬁcation was performed in a
total volume of 20 ml with 1 ml of template solution and Ex
Taq polymerase (Takara Bio. Inc., Otsu, Japan) according to
the supplier’s instructions. The PCR ampliﬁcation reaction
was performed in a thermal cycler (iCycler; Bio-Rad,
Hercules, CA, USA) with the following cycling parameters:
an initial denaturation at 95 8C for 4 min and then 30 cycles
consisting of 95 8C for 30 s, 60 8C for 30 s, and 72 8C for 30 s,with a ﬁnal extension at 72 8C for 7 min. The PCR products
were subjected to electrophoresis in a 1.5% agarose

Table 2
PCR primers used in the present study.
Speciﬁc primer set
Detection of P. gulae
P. gulae 16S rRNA

Determination of ﬁmA alignment
HG564-Fa
HG564-Ra
Speciﬁcation of ﬁmA typeType A ﬁmA
Pguﬁm-AFa
Pguﬁm-ARa
Type B ﬁmA
Pguﬁm-BFa
Pguﬁm-BRa
Type C ﬁmA
Pguﬁm-CFa
Pguﬁm-CRa
Type I ﬁmA
Type II ﬁmA
Type III ﬁmA
Type IV ﬁmA
Type V ﬁmA
Type Ib ﬁmAaSequence (50 –30 )References

TTG CTT GGT TGC ATG ATC GG
GCT TAT TCT TAC GGT ACA TTC ACA

Kato et al. (2011)

GAT TTG CTG CTC TTG CTA TGA CAG CTT GTA
TTT AGT CGT TTG ACG GGT CGA TTA CCA AGTThis study

TGA GAA TAT CAA ATG TGG TGC AGG CTC ACG
CTT GCC TGC CTT CAA AAC GAT TGC TTT TGG

Nomura et al. (2012)

TAA GAT TGA AGT GAA GAT GAG CGA TTC TTA TGTATT TCC TCA GAA CTC AAA GGA GTA CCA TCA

Nomura et al. (2012)

CGA TTA TGA CCT TGT CGG TAA GAG CTT GGA
TGT GGC TTC GTT GTC GCA GAA TCC GGC ATG
CTG TGT GTT TAT GGC AAA CTT C
AAC CCC GCT CCC TGT ATT CCG A
ACA ACT ATA CTT ATG ACA ATG GAAC CCC GCT CCC TGT ATT CCG A
ATT ACA CCT ACA CAG GTG AGG C
AAC CCC GCT CCC TGT ATT CCG A
CTA TTC AGG TGC TAT TAC CCA A
AAC CCC GCT CCC TGT ATT CCG A
AAC AAC AGT CTC CTT GAC AGT G
TAT TGG GGG TCG AAC GTT ACT GTCCAG CAG AGC CAA AAA CAA TCGTGT CAG ATA ATT AGC GTC TGC

This study

Names of primers are indicated.

481

Amano et al. (1999)
Amano et al. (1999)
Amano et al. (1999)
Amano et al. (1999)
Nakagawa et al. (2000)
Nakagawa et al. (2002)

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2.6. Distribution of P. gulae ﬁmA genotypes in oral swab
specimens

gel–Tris–acetate–EDTA buffer. The gel was stained with
0.5 mg ethidium bromide per ml and photographed under
UV illumination. A 100 bp DNA ladder (New England
BioLabs, Beverly, MA, USA) was used as the molecular size
standard. The sensitivity of the PCR assay was determinedby using titrated cultures of D049.

All study protocols were approved by the Animal
Research Committee of Azabu University, the Ethics
Committees of Osaka University Graduate School of
Dentistry and Okayama University Graduate School of
Medicine, Dentistry and Pharmaceutical Sciences. Prior
to collection of the specimens, the owners of all dogs
were informed of the protocols of the present study andgave approval for their participation. Oral swab specimens were collected from one speciﬁc location, thegingival margin of the mandibular left canine in the oralcavity of 139 dogs using swabs (Seed-Swab1 g-1, Eiken
Chemical Co. Ltd., Tokyo, Japan, or 2), as described
previously (Kato et al., 2011). The ages of the dogs ranged
from 0 to 17 years and mean age was 7.72. These dogs
came to the clinic for health examinations, vaccinations,coxarthropathy, external otitis, external disc disease,
trauma, or diarrhea. All of the dogs were reported to be
kept indoors.
The periodontal condition of each dog was evaluatedby measuring several parameters of a representative
tooth (mandibular left canine) as follows. The periodontal
pocket depth was measured to the nearest millimeteraround the circumference of each tooth from the gingival
margin to the deepest probing point, using a round-ended
probe tip 0.4 mm in diameter. The maximum values wererecorded as the values for periodontal pocket depth ofeach tooth. The periodontal pocket depths of 3 mm or lessfor large dogs, 2 mm or less for medium and small dogs
were regarded as healthy. In addition, other criteria, suchas no bleeding on probing, no pus discharge and no toothmobility were also observed in healthy subjects. Whenobserving the deeper periodontal pockets and bleeding on
probing, the dogs were diagnosed for gingivitis or
periodontitis. Periodontitis was diagnosed by pathological mobility due to obvious destruction of periodontal
tissues, such as alveolar bone. The bacterial DNA of eachspecimen was extracted and PCR was performed using P.
gulae-speciﬁc sets of primers, as described above. The
ﬁmA genotype was determined for P. gulae-positive
specimens using speciﬁc primer sets for types A, B andC (Table 2).

2.4. In vitro assays using cultured cells
Infected or control SAS cells were incubated with theTetraColor ONE Cell Proliferation Assay System (SEIKAGAKU, Tokyo, Japan) according to the instructions provided by the manufacturer. Data were calculated as therelative ratio of infected/uninfected (no infection) animals
and expressed as the means  SD from three independent
experiments. As for the scratch assays, conﬂuent cellmonolayers, grown on gelatin-coated 6 well dishes, were
scraped using a plastic tip to form a scratch wound as
described previously (Inaba et al., 2004). The exposed
surfaces were then re-coated with gelatin dissolved in RPMI
1640 at 37 8C for 1 h. The SAS cells were mixed with bacterial
strains at a MOI of 10 and 100 for 2 h and then incubated withmetronidazole (200 mg/ml) and gentamicin (300 mg/ml) for
12 h. The rate of scratch closure was determined using ImageJsoftware as described previously (Rincon et al., 2003).
2.5. Mouse abscess modelAll animal procedures and protocols were reviewed andapproved by the Institutional Animal Care and Use
Committee of Osaka University Graduate School of
Dentistry prior to the experiments. The virulence of P.
gulae D049 was evaluated using a mouse abscess model
developed for evaluation of P. gingivalis strains with somemodiﬁcations as follows (Nakano et al., 2004; Nomura
et al., 2012). As a reference, P. gulae ATCC 51700 (type A),
D040 (type B) and P. gingivalis OMZ314 (type II) were also
analyzed. Fifty female BALB/c mice (5 weeks old), whichwere randomly divided into 5 groups, were maintained inhorizontal-ﬂow cabinets and provided with sterile foodand water ad libitum. At 40 days of age, a single site
approximately 1 cm lateral from the midline on the dorsal
surface was depilated, and 0.1 ml of bacterial suspension,1  109 colony-forming units (CFU) of a test strain or PBS(control group), was injected subcutaneously. For quantitative evaluation of the infectious inﬂammatory change,
serum C-reactive protein (CRP) was measured. However,
90% of all mice in the group infected with D049 died by day
4. Therefore, the present experiment was terminated onthat day although the original model evaluated inﬂammatory changes for 2 weeks. Blood specimens (0.1 ml)
were collected from an orbital vein at 0, 1, 2 and 4 days
after bacterial infection and then centrifuged at 3000 rpm
for 10 min to separate the serum. CRP concentrations insera were calorimetrically quantiﬁed using a commercial
kit (MOUSE C-REACTIVE PROTEIN (CRP) ELISA TEST KIT;
Life Diagnostics, Inc., West Chester, PA, USA) according to
the supplier’s instructions. The mice were also monitored
for signs and symptoms of infection, i.e. rufﬂed hair,abscess formation and emergence of erosion, as described
previously.

2.7. Statistical analyses
Statistical analyses were performed using the computational software package StatView 5.0 (SAS Institute Inc.,
Cary, NC, USA) and Prism 4 (GraphPad Software Inc., La
Jolla, CA, USA). Intergroup differences in in vitro assays andanimal experiments were estimated using Bonferroni’s
method after an analysis of variance (ANOVA). Fisher’s
protected least-signiﬁcant difference test was utilized tocompare the detection frequencies of each ﬁmA genotype
in each gingival condition. Odds ratio (OR) and 95%
conﬁdence interval (CI95) values were calculated todetermine any signiﬁcant association regarding each ﬁmAgenotype in dogs with each periodontal condition. A P
value of less than 0.05 was considered statisticallysigniﬁcant.

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Fig. 1. Comparison of the deduced amino acid sequences of FimAs encoded by type A, B and C ﬁmA genes of P. gulae. Single and double dots indicate similar
and highly similar amino acid residues, respectively. Dashes indicate the gaps when multiple alignments were performed. The arrow indicates the cleavage
site estimated by the SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/). The alignment was made using the CLUSTAL W program of the DNA Data
Bank of Japan.

3. Results

3.2. Effects of P. gulae infection in SAS cells

3.1. Molecular biological properties of type C FimA

P. gulae ATCC 51700 (type A) and D049 (type C)
caused the cells to change into a contracted and roundedmorphology, which was similar to that produced by P.
gingivalis OMZ314 at a MOI 100 (Fig. 3A). In aproliferation assay, D049 (type C), but not types A andB strains, inhibited cell proliferation at a MOI 10(Fig. 3B). In addition, D049 showed the maximum
inhibition of cell proliferation and the other strains,except for D066 (type A), slowed proliferation at a MOI
100 (Fig. 3C). As for scratch closure assays, the migration
rate of D049 was 42.4%, which was the lowest rate
among the strains tested at a MOI 10 (Fig. 3D). The
closure rate of D049-infected cells decreased to 31.4% at
a MOI 100, although the rates for the other strains wereshown to be similar to D049 or slightly decreased (datanot shown).

The entire length of the ﬁmA gene of P. gulae strain D049
is 1167 bp, which was deposited in the DNA Database ofJapan under accession no. AB679295. Fig. 1 shows the
deduced amino acid sequence of strains ATCC 51700 (typeA) and D077 (type B) as compared to that of D049 (type C).
The FimA in D049 and that in P. gulae types A and B strains
ranged from 49 to 50% identical, which was almost similar
to those of types I, II, III, and Ib FimA in P. gingivalis. On theother hand, the identity of type C FimA with that in P.
gingivalis HNA99 was 59%. In contrast, the FimA of D049
and that of P. gingivalis HG564 (type IV) showed 92%
identity. Fig. 2 shows a phylogenetic tree comprised of the
FimA of P. gingivalis types I through V and Ib as well as P.
gulae types A, B and C. Type C FimA was shown to be themost closely related to genotype IV P. gingivalis.

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201

Fig. 2. Evolutionary relationship based on the synonymous site variation in the putative amino acid sequences of FimA encoded by ﬁmA genes of P. gulae andP. gingivalis. The neighbor-joining method was used to construct the phylogenetic tree using CLUSTAL W (DNA Databank of Japan) and Tree View software(http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Pgu and Pgi indicate P. gulae and P. gingivalis, respectively. The roman numerals following Pgi
indicate the ﬁmA genotypes of P. gingivalis. The numbers shown in the tree are bootstrap values.

3.3. Mouse abscess model

In addition, the sensitivity was shown to be approximately
10–100 CFU per reaction (data not shown).
Fig. 5B shows representative results for the analyses ofclinical specimens. P. gulae was detected in 110 out of 139(79.1%) oral swab specimens (Table 3). The detection rate
for P. gulae in the periodontitis group was 100%, which wassigniﬁcantly higher than that in healthy (63.6%) andgingivitis (77.7%) groups (P < 0.001 and P < 0.05, respectively). Single and multiples genotypes were identiﬁed in P.
gulae-positive specimens and there were a few untypeable
specimens in each group. The numbers of the specimens
containing multiple genotypes were higher in the gingivitis and periodontitis groups than that of the periodontally healthy group (P < 0.05 and P < 0.01, respectively).
The numbers of subjects with multiple genotypes or type B
in the gingivitis and periodontitis groups and those ofmultiple types or type C in the periodontitis group werehigher than those of the healthy group (P < 0.01 each).
The OR of P. gulae detection for periodontitis was 27.55(CI95; 1.48–514.10). In addition, those with multiple
genotypes, multiple genotypes or single genotypes B andC were 8.23 (CI95; 1.89–35.83), 8.44 (CI95; 2.12–33.61) and12.75 (CI95; 3.06–53.19), respectively. Furthermore, the ORof multiple types or single type B detection for gingivitis
was 4.50 (CI95; 1.42–14.30).

Subcutaneous injections of D049 (type C) caused
drastic weakened systemic conditions in the mice on
Day 1 and the formation of erosions was observed on Day 2
although similar lesions were not observed in the mice
injected with types A and B strains (Fig. 4). The serum CRP
values in mice infected with D049 on Days 1 and 2 weresigniﬁcantly higher than those injected with the otherstrains (data not shown). On Day 4, 90% of all mice injectedwith D049 died.
3.4. Clinical specimens
139 specimens were divided into periodontally healthy(n = 22), gingivitis (n = 94) and periodontitis (n = 23) groups
based on the clinical conditions of the dogs from which thespecimens were isolated. The ages of the healthy group(mean 5.05) were signiﬁcantly lower than those for thegingivitis (7.97) and periodontitis (10.57) groups (P < 0.01
and P < 0.001, respectively). Comparing the periodontitis
and gingivitis groups, ages of the periodontitis group weresigniﬁcantly higher (P < 0.05).
The PCR system for identiﬁcation of type C ﬁmA wasdeveloped based on the speciﬁc nucleotide alignment oftype C ﬁmA as compared to those of types A and B ﬁmA(Fig. 5A). The speciﬁcity of this PCR system was conﬁrmed
using the strains listed in Table 1, which demonstrated thatprimers designed for type C ﬁmA genes of P. gulae did not
generate positive bands from the P. gingivalis strains tested.

4. Discussion
The present study characterizes a novel ﬁmA genotypeof P. gulae. We previously isolated many P. gulae strains

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Fig. 3. Effects of P. gulae strains infection in cultured cells. (A) Light microscopy images showing morphology of the cells infected with P. gulae at a MOI of 10
and 100 for 24 h. Control cells were uninfected. (B and C) Cell proliferation assay measured by tetrazolium following infection with P. gulae strains at a MOIof 10 (B) and 100 (C). Data are expressed as relative ratio infected/uninfected (no infection) and are means  SD from three independent experiments
analyzed with a t-test (*P < 0.05, **P < 0.01). (D) In vitro scratch assay in the cells infected with P. gulae at a MOI of 10. Bars show the scratched wound regions at
0 h. The rates of wound closure are determined from assays and are means  SD from three independent experiments.

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203

Fig. 5. Construction of the primers speciﬁc for type C of P. gulae. (A) The
position of the primers for ﬁmA genotypes A, B and C are illustrated, (B)analyses of clinical specimens for determination of ﬁmA types. Lanes: M;
molecular size marker (100-bp DNA ladder), A; ATCC 51700, B; D077, C;
D049, W, sterile water. 1–10; clinical specimens.

the genotype C strain D049 caused the maximum
inhibition of proliferation. It was reported that oral
bacteria can inhibit cell migration (Inaba et al., 2004,
2005) and the in vitro scratch assay is effective incharacterizing the properties of cell proliferation andmigration (Liang et al., 2007). Thus, using the cell
migration assay, we also demonstrated that D049 inhibited cell migration the most. These results support the
proposal that type C ﬁmbriae might be one of theimportant factors for the impairment of cellular function
caused by P. gulae. However, further analyses, such as
evaluation of multiple type C strains or strains withreplacement of type C ﬁmA with other genotype ﬁmA genes
and vice versa should be carried out to conﬁrm thishypothesis.
A previous investigation using the mouse abscessmodel showed that strains with type B ﬁmA caused more
severe systemic inﬂammation than those with type A ﬁmA(Nomura et al., 2012). The present study clearly showedthat the properties inducing systemic inﬂammation by
D049 (genotype C) were strongest compared to other P.
gulae strains in the mouse model. When we developed amouse model for evaluating the properties of P. gingivalis
involved in inducing systemic inﬂammation, we found thatstrain OMZ314 (type II ﬁmA) possessed the highest
potential (Nakano et al., 2004). In fact, subcutaneous
injection of OMZ314 resulted in the death of mice underFig. 4. Macroscopic observation of a representative mouse in the abscessmodel experiment. (A) Dorsal view, (B) ventral view and (C) magniﬁcationof the erosion area. Arrows indicate the areas of erosion.

from Japanese dogs, almost all of which were classiﬁed into
types A or B ﬁmA (Kato et al., 2011). Only a single strain(D049) was shown to be of a non-A and non-B FimA
genotype, which is designated as type C in the present
study. Type A FimA was shown to be P. gulae-speciﬁc andshares 73% amino acid homology with P. gulae type B and P.
gingivalis type III FimA (Nomura et al., 2012). On the other
hand, the present study revealed that the similarity of type
C FimA and type IV FimA of P. gingivalis was very high.
Relative to the virulence of P. gingivalis, types II/IV/Ib wasshown to be more virulent than types I/III/V (Kuboniwa
et al., 2010). Thus, we speculated that the virulence of type
C would be the highest among all ﬁmA types of P. gulae.
Initially, we used cultured cells to perform in vitro
analyses. To begin the process of characterizing the
response of SAS cells to infection with P. gulae strains,the infected cells were observed by microscopy and foundto be altered in the morphology. Next, we analyzed theinhibition of cell proliferation, which demonstrated that

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Table 3Distribution frequency of ﬁmA genotypes in P. gulae-positive specimens in periodontally healthy, as well as dogs with gingivitis and periodontitis.
Healthy (n = 22)

Gingivitis (n = 94)

Periodontitis (n = 23)

Age [mean  standard deviation (range)]

5.05  4.29 (0–12)

7.97  3.91 (1–17)**

10.57  3.58 (2–16)***,#

P. gulae-positive

14 (63.6%)

73 (77.7%)

23 (100%)**#

Single typeABC

9 (40.9%)
7 (31.8%)
1 (4.5%)1 (4.5%)36 (38.3%)
20 (21.3%)
13 (13.8%)
3 (3.2%)10 (43.5%)
4 (17.4%)
2 (8.7%)4 (17.4%)

Multiple types
A and B
A and C
B and C
A, B and C

3 (13.6%)
0 (0%)
1 (4.5%)2 (9.1%)0 (0%)

34 (36.2%)*
4 (4.3%)16 (17.0%)
4 (4.3%)10 (10.6%)

13 (56.5%)**4 (17.4%)
8 (34.8%)
1 (4.3%)0 (0%)

Untypeable

2 (9.1%)3 (3.2%)0 (0%)

Single A type or multiple types
Single B type or multiple types
Single C type or multiple types

10 (45.5%)
4 (18.2%)
4 (18.2%)

54 (57.4%)
47 (50.0%)**37 (39.4%)

17 (73.9%)
15 (65.2%)**17 (73.9%)*** P < 0.05 against healthy group.
** P < 0.01 against healthy group.
*** P < 0.001 against healthy group.
#P < 0.05 against gingivitis group.

could be one possible risk marker for periodontitis. The
predominant cultural subgingival ﬂora in dogs shows great
similarity to the subgingival bacteria from humans at thegenus level, but distinct differences at the species level(Dahlén et al., 2012). In the present study, the statisticalanalyses indicated that P. gulae detection itself could be
one possible risk factor for development of periodontitis.
However, there are other species possibly associated withperiodontitis. Further studies should focus on the analysesof the other species related to periodontitis in dogs.
It should be noted that there exist specimens which arenegative for genotypes A, B and C ﬁmA in P. gulae-positive
specimens. This ﬁnding indicates the presence of P. gulae
strains without FimA or those with additional ﬁmAgenotypes. Further studies are required to elucidate theﬁmA genotypes of these specimens. However, we speculatethat the existence of unknown type(s) would not inﬂuence
the results obtained in the present study since the numberof such specimens are extremely low. Thus, we can designa system for identiﬁcation of dogs at high risk fordeveloping periodontitis without elucidating the detailsof the untypeable specimens.
Prediction of the risk for developing future periodontitis
would be clinically beneﬁcial. However, it was reported
that the oral ﬂora in health and periodontitis is highly
diverse and that it contains high proportions of uncultured
and potentially novel species (Riggio et al., 2011). It should
be noted that the present study demonstrated that speciﬁc
FimA genotypes of P. gulae could be one possible risk factor.
Speciﬁcally, three factors, such as the presence of P. gulae,
detection of multiple ﬁmA genotypes with single type B or
type C detection were suggested. The molecular biologicalanalyses of the specimens taken from the dogs enable theidentiﬁcation of high risk dogs, who should be provided
with intensive care for periodontitis. In addition, we
propose that the dogs designated as risk subjects in healthy

the conditions tested. Since we attempted to develop amouse model to evaluate systemic virulence over prolonged periods, we modiﬁed the mouse model so that all
mice survived even in the group infected with OMZ314.
Thus, it was very surprising that erosion was identiﬁed inalmost all of the mice with subcutaneous injections of
D049 even on Day 1, and dramatically weakened systemic
conditions were observed in almost all of the mice leading
to death by Day 4. Thus, we demonstrated that strain D049
could cause extremely severe systemic inﬂammation.
We are interested in applying the results obtained in
the present study for clinical purposes. Therefore, we plan
to develop a system for identifying high risk subjects forperiodontitis. As a ﬁrst step, the periodontal condition of
the mandibular left canine was evaluated as a representative tooth for several reasons. First, it is relatively easy tovisually inspect this region without general anesthesia. In
addition, traction of the frenulum close to the mandibularcanine can produce wide views of the area. For clinicalevaluation, we can further simplify the criteria fordiagnosis based on our current knowledge of periodontitis
in humans. Periodontal pocket depth, bleeding on probing
and tooth mobility were used as major criteria fordiagnosis of gingivitis and periodontitis together withother evaluations such as pus discharge.
The distribution of P. gulae in the dogs in the periodontitis group was signiﬁcantly higher than those of thehealthy and gingivitis groups. The detection rates of P.
gulae in Brazilian dogs with and without periodontitis
were reported to be 92% and 56%, respectively (Senhorinho
et al., 2011), which was almost similar to those of Japanese
dogs analyzed in the present study. As for humans, thedistribution rates of P. gingivalis in Japanese patients withperiodontitis and periodontally healthy subjects werereported to be 87.1%, and 36.8%, respectively (Amano et al.,2000), indicating that the presence of P. gingivalis itself

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Liang, C.C., Park, A.Y., Guan, J.L., 2007. In vitro scratch assay: a convenientand inexpensive method for analysis of cell migration in vitro. Nat.
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and/or gingivitis groups should be carefully followed up for
prevention of the onset of subsequent periodontitis.
Further studies should also focus on large-scale analyses
to identify the geographic speciﬁcity of the presence of the
ﬁmA genotypes. Such worldwide surveys could elucidate
the validity of this method for identiﬁcation of high risk
subjects for periodontitis in dogs.
Acknowledgements
This study was supported by a Grant-in-Aid for
Scientiﬁc Research for Challenging Exploratory Research
No. 23658256 from the Japan Society for Promotion of
Science, and was supported by the Promotion and Mutual
Aid Corporation of Private Schools of Japan and a Grant-inAid for Matching Fund Subsidy for Private Universities. We
also thank Prof. Howard K. Kuramitsu (State University of
New York at Buffalo) for his editing this manuscript.
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Experimental and Toxicologic Pathology
journal homepage: www.elsevier.de/etp

Short communication

Detection of Bcl-2 mRNA and its product in the glomerular podocytes of thenormal rat kidney
Atsuko Haishima a , Masaru Murakami b , Teruo Ikeda c , Kaoru Inoue a ,Junichi Kamiie d , Kinji Shirota a,d,∗aResearch Institute of Biosciences, Azabu University, Sagamihara, Kanagawa, Japan
Laboratory of Molecular Biology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japanc
Laboratory of Veterinary Immunology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, JapandLaboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japanba r t i c l e

i n f oArticle history:
Received 4 June 2010
Accepted 7 December 2010
Keywords:
Podocyte apoptosis
Bcl-2
Laser microdissection
Glomerulus
Kidneya b s t r a c t
Podocyte apoptosis underlies podocytepenia leading to glomerulosclerosis. An apoptosis inhibitory protein Bcl-2 is expressed in the podocytes in the early stage of nephrogenesis and downregulated in thematuring stage of human fetal kidneys. Recent studies reported changed localization and expression of
Bcl-2 in the renal glomeruli under the pathologic conditions. This study aimed to conﬁrm in situ localization of Bcl-2 mRNA and its product in the glomeruli, and to demonstrate the local expression of Bcl-2
mRNA in normal rat glomeruli. Parafﬁn sections of the kidneys from normal male Wistar rats were
immunostained by anti-Bcl-2 monoclonal antibody. The localization of Bcl-2 mRNA in the glomeruli wasevaluated by in situ hybridization. The glomeruli were dissected from frozen sections of the kidneys withthe laser microdissection (LMD) system. Total RNA extracted from 10, 100 or 200 dissected glomeruli
was used for reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. Bcl-2 mRNAand its product were detected in the podocytes but barely in the mesangial cells. In RT-PCR, the speciﬁcsized bands of Bcl-2 from 100 or 200 dissected glomeruli were clearly observed. Real-time PCR for Bcl-2
showed that cDNA from 100 or 200 dissected glomeruli became ampliﬁed at 36 or 33cycles, respectively.
Bcl-2 is expressed in the glomerular podocytes of the normal rat kidney and quantitative analysis of Bcl-2
mRNA in the renal glomeruli is possible using the LMD technique.
© 2010 Elsevier GmbH. All rights reserved.

1. Introduction
Apoptosis is a physiologic form of cell death, occurs in the individual cell in an endogenously programmed pattern (White, 1996;
Tsujimoto and Shimizu, 2000). It can be triggered by a variety ofphysiologic stimuli, however the pathologic factors also affect theapoptotic pathways and then contribute to the induction or progression of many diseases (White, 1996; Shinoura et al., 1999;
Yoshimura et al., 1999; Tsujimoto and Shimizu, 2000). Among thefactors affecting apoptotic pathways, Bcl-2 is a protein encoded by
bcl-2 proto-oncogene and one of the molecular members of the cell
survival factors in the Bcl-2 family (Yang and Korsmeyer, 1996;
Tsujimoto and Shimizu, 2000). This protein is an important regulator of apoptosis, and can block programmed cell death. On theother hand, Bax, one of the Bcl-2 family molecules regulates the Bcl2 protein activity and induces apoptosis. Therefore, the Bcl-2/Bax∗ Corresponding author at: 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa
252-2501, Japan. Tel.: +81 42 7692093; fax: +81 42 7692093.
E-mail address: shirota@azabu-u.ac.jp (K. Shirota).

ratio is critical for the entry into apoptosis (White, 1996; Yang andKorsmeyer, 1996).
Recently, the importance of apoptosis has been emphasizedin the progression of glomerular diseases (Sugiyama et al., 1997;
Yoshimura et al., 1999; Barisoni and Mundel, 2003). In particular,
podocyte apoptosis has been suggested to be an important causeof podocytepenia leading to glomeruloscrelosis (Yoshimura et al.,
1999; Barisoni and Mundel, 2003). Podocytes are highly differentiated cells and lack proliferative activity under normal conditions.
Their cellular foot processes are anchored to the outer surfaceof glomerular basement membrane (GBM) and keep structural
integrity of the glomeruli. Adjacent foot processes are connected bya modiﬁed intercellular junction called as the slit diaphragm, which
has a crucial role in selective ﬁltration barrier in the glomeruli(Asanuma and Mundel, 2003). Therefore, detachment and loss of
podocytes from the glomerular tufts consistently result in adhesionof denuded GBM to Bowman’s capsule.
The changed localization of Bcl-2 or Bax proteins in therenal glomeruli has been observed both in experimental settings(Sugiyama et al., 1997; Wang et al., 2001) and in human glomerulardiseases (Nakopoulou et al., 1996; Uda et al., 1998). In particular, the0940-2993/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.etp.2010.12.006

Please cite this article in press as: Haishima A, et al. Detection of Bcl-2 mRNA and its product in the glomerular podocytes of the normal
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expression of Bcl-2 increases in the proliferating cells in glomerulonephritis, suggesting that this protein is involved in pathologic
cellular proliferation (Takemura et al., 1995; Sugiyama et al., 1997;
Uda et al., 1998). On the other hand, a recent clinico-pathologicalstudy indicated that downregulation of Bcl-2 protein expression in
the renal glomeruli was associated with indices of poor prognosis
in human IgA nephritis (Qiu et al., 2004). Therefore, qualitative andquantitative analysis of the local expression of gene and productof Bcl-2 in the renal glomeruli will be very important to evaluate
the role of the apoptosis-relating protein in the pathogenesis ofglomerular injury and to access the inﬂuence of cellular stresses
such as chemicals on the podocytes.
In the rat, the expression of Bcl-2 mRNA was detected in the
podocytes of the renal glomeruli of puromycin aminonucleosidetreated and non-treated animals, and a focal increase of the signals
was demonstrated by in situ hybridization (Wang et al., 2001). However, no signiﬁcant change in glomerular Bcl-2 mRNA was revealed
by reverse transcription-polymerase chain reaction (RT-PCR) using
glomeruli isolated by the mesh-sieving method. Recently, we founda high expression of Bcl-2 protein in the glomerular podocytes of
the normal rat kidney by immunohistochemistry using a monoclonal anti-human bcl-2 antibody.
For further conﬁrmation of the localization of Bcl-2 in the
podocytes, detection of its gene expression in the glomeruli might
be required. We reported the utility of the laser microdissection(LMD) system for the analysis of the local gene expression in therenal glomeruli (Inoue et al., 2003) as well as in the ovarian follicles (Sakurada et al., 2006). In the present study, we conﬁrmed theexpression of Bcl-2 mRNA in the renal glomeruli dissected by LMD
system. We also conﬁrmed by in situ hybridization and immunohistochemistry the in situ localization of Bcl-2 mRNA and its productin the glomerular podocytes of the normal rat kidney.
2. Materials and methods
All procedures in this study were in accordance with theguidelines approved by the Animal Research Committee of Azabu
University.

Sense and antisense RNA probes labeled with digoxigenin (DIG)
were synthesized using T7 and SP6 RNA polymerase (MAXscriptTM
in vitro Transcription Kit, Ambion, USA), respectively. The ISH
was performed under the standard protocols using an ISH kit(PanPath B. V., Amsterdam) on 4% paraformaldehyde (PFA)-ﬁxed,
parafﬁn-embedded tissues. The parafﬁn sections were dewaxed
and air-dried and pretreated with 0.01 N HCl. After being washed intris (hydroxymethyl) aminomethane-buffered saline (TBS), the sections were dehydrated by ethanol and air-dried. Hybridization was
performed with a probe concentration of 0.6 ␮g/ml in a hybridization buffer (Nippon Gene Corp., Tokyo) at 50 ◦ C overnight. Afterhybridization, the sections were washed with TBS, and then incubated with alkaline phosphatase-conjugated anti-digoxigenin (Fabfragment) (PanPath B. V., Amsterdam). After being washed in TBS,a mixture of nitroblue tetrazolium solution and 5-bromo, 4-chloro,
3-indolyphosphate solution was applied to the sections for color
development.
Observation of the sections of immunohistochemistry and in situ
hybridization was performed with a differential interference contrast microscope.
2.3. Laser microdissection
A part of the renal cortex specimens was quickly frozen in liquid nitrogen, cut into 7 ␮m sections by a cryostat microtome, andmounted on a glass slide cover with a 2.5 ␮m thin laser pressure
catapulting membrane (PEN foil; Leica Microsystems, Wetzlar).
They were ﬁxed with 70% ethanol, gently washed twice with
diethylpyrocarbonate (DEPC)-treated water, and thoroughly airdried. Thereafter, the sections were stained with 0.05% toluidine
blue (TB) solution, pH 4.1 (Wako Pure Chemical Industries, Ltd.,
Osaka), and then the TB solution was rinsed out with the DEPCtreated water, and the sections were air-dried. The renal glomeruli
were randomly dissected from frozen sections with LMD systems(Leica Laser Microdissection System, Leica Microsystems). Ten, 100
or 200 glomeruli dissected from the frozen sections were collected
immediately into a microcentrifuge tube cap ﬁlled with lysis buffer.
2.4. RT-PCR

2.1. Kidney specimens and immunohistochemistry
Two 8-week-old normal male Wistar rats (Charles River Japan,
Inc., Kanagawa) were euthanized by bleeding under anesthesia with intraperitoneal administration of pentobarbital sodium(60 mg/kg body weight), and their kidneys were collected. Parts ofkidneys were ﬁxed in 10% formalin (Masked Formalin, Japan TannerCorp., Osaka) and embedded in parafﬁn after dehydration througha graded series of ethanol. Four-␮m sections were cut and used for
immunohistochemical detection of Bcl-2 by the streptavidin-biotin
peroxidase method using a commercial kit (Histoﬁne kit, Nichirei
Corp., Tokyo). After deparafﬁnization and treatment in 3% H2 O2 inmethanol, the sections were incubated with normal rabbit serum
and then with anti-human bcl-2 monoclonal antibody (Clone;124,pre-diluted, Nichirei Corp.). The immunoreaction was visualized bya diaminobenzidine-hydrogen peroxide solution, and the sections
were counterstained with methyl green.
2.2. In situ hybridization
The Bcl-2 mRNA was localized by in situ hybridization(ISH). A 368 bp fragment of the rat Bcl-2 cDNA was ampliﬁed from reverse-transcribed total RNA of the rat kidney cortex
using the following primers: upper, TAATACGACTCACTATAGGCGGGAGATCGTGATGAAGTA; lower, ATTTAGGTGACACTATAGAGAAGGGCGTCAGGTGC (Wang et al., 2001). The plasmid vector wasinserted the Bcl-2 fragment and incubated with restriction enzyme.

To see speciﬁc size bands of Bcl-2 and glyceraldehyde-3phosphate dehydrogenase (GAPDH) by electrophoresis, routine
RT-PCR using the cDNA from dissected glomeruli was performed
by Takara Thermal Cycler MP (Takara Shuzo, Kyoto). The total
RNA was extracted from dissected glomeruli with RNeasy Mini
Kit (Qiagen, Valencia, CA), and DNase digestion was performed
with the RNase-free DNase set (Qiagen). First-strand cDNA wasmade from total RNA using Sensicript (Qiagen) with oligo (dT)
15primer (Promega, Madison, WI). The sequences of primers wereas follows: upper, 5 -CGGGAGATCGTGATGAAGTA-3 ; down, 5 GGTAGCGACGAGAGAAGTCA-3 (Wang et al., 2001) and the GAPDH
primers as follows: upper, 5 -TCCCTCAAGATTGTCAGCAA-3 ; lower,
5 -AGATCCACAACGGATACATT-3 (Ando et al., 1995). The PCR was
performed with Takara TaqTM (Takara Shuzo, Kyoto) under the following conditions: denature at 94 ◦ C for 5 min; and then 40 cycles
of denature at 94 ◦ C for 1 min; annealing at 60 ◦ C for 1 min; extension at 72 ◦ C for 2 min; and ﬁnally extension at 72 ◦ C for 7 min.
Four ␮l of the PCR products were separated by electrophoresis in a
2% agarose gel and stained with ethidium bromide. The ampliﬁed
products were veriﬁed to be a single band of 284 bp (Bcl-2) and308 bp (GAPDH) in size.
2.5. Real-time PCR
The total RNA was extracted from dissected glomeruli with the
RNeasy Mini Kit (Qiagen), and DNase digestion was performed with

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Fig. 1. Immunohistochemical staining of Bcl-2 in the renal glomerulus of the normal rat. Brown color indicates positive staining of Bcl-2 mostly in the podocytes.
Bar = 50 ␮m.

the RNase-free DNase set (Qiagen). First-strand cDNA was madefrom total RNA using Sensicript (Qiagen) with oligo (dT) 15 primer(Promega). Real-time PCR was performed using the ABI Prism7700
Sequence Detection System (Applied Biosystems, Foster City, CA).
For quantitative analysis of Bcl-2 expression, the TaqMan Bcl-2(20X Pre-Developed TaqMan Assay Reagents, Applied Biosystems),
and the TaqMan GAPDH (TaqMan Rodent GAPDH Control Reagents,
Applied Biosystems) as an endogenous standard were ampliﬁed
with each cDNA from dissected glomeruli using the universal PCR
mastermix (Applied Biosystems). The reaction conditions were as
follows: 2 min at 50 ◦ C for warming up, samples were denatured
at 95 ◦ C for 10 min, then 40 cycles of 95 ◦ C for 15 s and at 60 ◦ C for
1 min.
3. Results
3.1. Immunohistochemistry and in situ hybridization
Immunohistochemistry clearly demonstrated that the Bcl-2
protein was localized in the podocytes but barely in the mesangial
cells in the glomeruli (Fig. 1). Also, in situ hybridization revealed
the limited localization of Bcl-2 mRNA in the glomerular podocytes(Fig. 2). Sense RNA probe used for negative control did not yielded
any signals in the renal tissues.

3

Fig. 2. In situ hybridization for Bcl-2 mRNA in the renal glomerulus of the normal
rat. Arrows indicate positive signals in the podocytes. Bar = 50 ␮m.

(Wang et al., 2001) but we demonstrated local expression of Bcl-2
mRNA in the glomeruli by RT-PCR. Moreover, the real-time PCR for
Bcl-2 revealed exponential ampliﬁcation plots of cDNA, suggestingthat quantitative evaluation of local Bcl-2 gene expression could bepossible using dissected glomeruli by the LMD system. We did notprecisely evaluate the difference in the results of PCR when cDNAfrom 10, 100 and 200 dissected glomeruli was ampliﬁed. However,
the ampliﬁcation plots of cDNA of the Bcl-2 indicated that 200 dissected glomeruli would be ideal for quantitative analysis of its local
gene expression.
In human kidneys, Bcl-2 is not present in the podocytes in thematuring stage of nephrogenesis (Nagata et al., 1998) and also Bcl2 mRNA was barely detectable in the normal human glomeruli(Qiu et al., 2004). There has been limited information on therole of Bcl-2 in the pathogenesis of glomerular injury. In humanglomerular diseases such as lupus nephritis, IgA nephropathy, focal
glomerulosclerosis and mesangial proliferative glomerulonephritis, increased expression of Bcl-2 in the glomeruli might contributeto the maintenance of hypercellularity by promoting cellular proliferation and by suppressing the function of Bax (Takemura et al.,
1995; Uda et al., 1998). Nakopoulou et al. (1996) also reported

3.2. Bcl-2 gene expression in the glomeruli
RT-PCR revealed that local expression of Bcl-2 mRNA was distinctly detected in 100 and 200 dissected glomeruli at 284 bp(speciﬁc size band of Bcl-2) but was not detected in 10 dissected
glomeruli (Fig. 3). In real-time PCR, the cDNA of Bcl-2 and GAPDH
derived from 200 dissected glomeruli began ampliﬁed at 33 and28 cycles, respectively. It was also possible to detect exponential ampliﬁcation of these cDNA from 100 dissected glomeruli that
became ampliﬁed at over 36 (Bcl-2) and 30 (GAPDH) cycles. The
cDNA of GAPDH from 10 dissected glomeruli was exponentially
ampliﬁed, however that of Bcl-2 was not ampliﬁed (Fig. 4).
4. Discussion
We conﬁrmed distinct in situ localization of Bcl-2 mRNA andits product in the glomerular podocytes of the normal rat kidney
in this study. The localization of Bcl-2 mRNA in the rat glomerular
podocytes was previously demonstrated by in situ hybridization

Fig. 3. The result of RT-PCR for GAPDH (a) and Bcl-2 (b) using total RNA from 10(lane 2), 100 (lane 3) or 200 (lane 4) dissected glomeruli by laser microdissection
system. Lane1: molecular weight marker.

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Fig. 4. Ampliﬁcation curves of cDNA in real-time PCR of GAPDH (a) and Bcl-2 (b) using total RNA derived from 10, 100 or 200 dissected glomeruli. The ampliﬁcation curves
expressed by four different colors in each chart are of duplication samples from two normal rats.

restricted expression of Bcl-2 in the glomerular epithelial cells near
intraglomerular ﬁbrotic lesions and early adhesion and cellularcrescents. In the rat model of focal segmental glomerulosclerosis, a focal increase in the expression of Bcl-2 mRNA in therenal glomeruli evaluated by in situ hybridization was suspected
to be due to proliferated mesangial cells (Wang et al., 2001).
More recently, Qiu et al. (2004) showed that Bcl-2 expression
was upregulated in the early stage of IgA nephropathy and was
downregulated in the advanced and end-stage of the disease. They
revealed that Bcl-2 downregulation was closely associated with thedevelopment of progressive glomerular injury as well as being aclinical prognostic indicator of poor renal prognosis in human IgA
nephropathy. These reports indicate that Bcl-2 may be closely associated with the pathogenesis of glomerulonephritis and that its rolein glomerular injury might depend on the types of the injury andcourse of the diseases.
The podocytes are highly differentiated and growth-arrested
cells (Nagata et al., 1998; Barisoni and Mundel, 2003). Therefore
loss of these cells results in podocytepenia and underlies glomerulosclerosis, representing irreversible glomerular injury (Barisoni
and Mundel, 2003) A recent experimental study has shown that
Bcl-2 protects podocytes from apoptosis (Wada et al., 2005) Constant expression of Bcl-2 in the podocytes in the normal kidney
as shown in this study may contribute to long-life of these cells
as a cell survival factor. Therefore, quantitative analysis of local
gene expression of Bcl-2 in the glomeruli under pathologic conditions caused by various factors such as immune mechanisms,
chemical toxicity, ageing and others must be important for eval-uating or monitoring podocyte injury and for understanding thepathogenesis of progressive glomerular injury.
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NOTE

Pathology

Deep Granulomatous Dermatitis of the Fin Caused by Fusarium solani in a False
Killer Whale (Pseudorca crassidens)
Miyuu TANAKA1), Takeshi IZAWA1), Mitsuru KUWAMURA1), Tatsuko NAKAO2), Yuko MAEZONO2) , Shu ITO2),
Michiko MURATA 3), Masaru MURAKAMI3), Ayako SANO4) and Jyoji YAMATE1)*
1)Veterinary Pathology, Graduate School of Life and Environmental Science, Osaka Prefecture University, 1–58 Rinkuu ourai Kita,

Izumisano, Osaka 598–8531, Japan
2)Adventureworld AWS Co. Ltd., Nishimurogun, Shirahama-cho, Katada, Wakayama 649–2201, Japan
3)School of Veterinary Medicine, Azabu University, 1–17–71 Fuchinobe, Sagamihara, Kanagawa 229– 8501, Japan4)Department of Subtropical Agro-Production Sciences, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara,

Okinawa 903–0213, Japan(Received 13 September 2011/Accepted 16 December 2011/Published online in J-STAGE 28 December 2011)
ABSTRACT. A 10-year-old female false killer whale (Pseudorca crassidens-

Fusarium solani
Fusarium
mon in that the lesions spread deeper into the skin.

Fusarium solani

KEY WORDS:

J. Vet. Med. Sci. 74(6): 779–782, 2012

FusariumF. solani and F. oxysporum

associated with contamination of contact lens solution has

Fusarium spp. possess viru-

In marine mammals, Fusarium spp. are most likely opmised immune systems presumably due to stress or illness

suppress both humoral and cellular immunity and may

Fusarium-induced der-humans and animals such as turtles, sharks, dolphins andsea lions, Atlantic white sided dolphins, harbor seals andIn humans, fusariosis tends to develop in patients who
refractory fever, skin lesions at infected sites and sino-

F. solani in a false

particularly with predominance in the extremities. The-

killer whale.
A 10-year-old female false killer whale (Pseudorca
crassidens-

Fusarium keratitis-*C ORRESPONDENCE TO: Y AMATE
Graduate School of Life and Environmental Science, Osaka

Besides the skin lesions, severe accumulation of foamy

e-mail: yamate@vet.osakafu-u.ac.jp

rumen, injured head skin and cerebral subdural hemor-

501

780

M. TANAKA ET AL.

Aspergillus spp. In the cere-

Candidations of 4 μm thick were cut and stained with hematoxylin
periodic acid-Schiff (PAS) reaction and Grocott’s methenatry, sections from skin lesions and ulcerated rumen lesions
Candida spp. or Aspergillus spp. (provided by Dr. M. Kubo,

penia in the lymph nodes and spleen. F. solani were not
Samples of the kidney, spleen, heart, pancreas, lymph
node, cerebrum, cerebellum and skin lesions were collected-

Corp., Glostrup, Denmark, 1:200), rabbit anti-CD20 (Ther1:1,000) for 16 hr at 4°C. Bound antibodies were detected-consisted of 4 to 6 cells and chlamydospores were detected in the micro culture on PDA. DNA was extracted fromthe isolates and tissue samples. The internal transcribed

F. solanisearches. As a result, F. solani-

into the blood vessels nor thrombus was found.
-

F. solani. Fusariuminduced dermatitis in marine mammals has been reported

isms were surrounded by intensely eosinophilic amorphous
oid or club-shaped intensely eosinophilic materials around-

Aspergillus spp. and Candida spp.
---

ported in association with mycetoma due to F. moniliforme
Fusarium spp. infection in marine mammals
F. so-

GMS stain and PAS reaction, and were immunopositive for

lani

502

781

sions spread diffusely into the deep dermis (arrows). Bar=1 cm.
μm.
μm.

μm.
μm.
phocytes (6b) are observed. Bar=200 μm.

small in number. Similar immunohistochemical reactivities

lular and humoral immunities.
-

-

503

have aspirated sea water and presumably died in a drown-

782

M. TANAKA ET AL.
lani
scalloped hammerhead sharks (Sphyrna lewini
Wildl. Dis. 31

J.
Clin.

matous dermatitis caused by F. solani and candidiasis increased immunocompetence.
tous dermatitis caused by F. solani
nomenon.

Microbiol. Infect. 10
my sperm whale, and two harbor seals. J. Am. Vet. Med. Assoc.
208: 727–729.
review. Can. Vet. J. 41
nomenon. J. Cutan. Pathol. 35: 979–988.

-

Kiyota, E., Sakashita, N., Araki, N. and Takeya, M. 2006.
Fusarium moniliforme, aEur. J. Epi-

phenotype. J. Histochem. Cytochem. 54: 763–771.

demiol. 1
caused by Fusarium solani
carreta L.). J. Clin. Microbiol. 35

Caretta

Genese, C. A., Alfonso, E. C., Jones, D. B., Srinivasan, A.,
Fusarium keratitis associated with use of a contact lens solution.
JAMA 296
4. Cocuroccia, B., Gaido, J., Gubinelli, E., Annessi, G. and Gicaused by a Fusarium species infection in a renal transplantpatient. J. Clin. Microbiol. 41
Fusarium so-

Boness, D. J. and Whitla, J. C. 1981. Cyclic dermatitis associated with Fusarium sp infection in pinnipeds. J. Am. Vet. Med.
Assoc. 179: 1198–1202.
Fusarium species. Clin.
Microbiol. Rev. 7
13. Nucci, M. and Anaissie, E. 2007. Fusarium infections in immunocompromised patients. Clin. Microbiol. Rev. 20704.
merbell, R. C., Padhye, A. A. and Geiser, D. M. 2006. Membersof the Fusarium solani species complex that cause infections
in both humans and plants are common in the environment. J.
Clin. Microbiol. 44: 2186–2190.

504

505

506

507

508

509

510

511

Biochimica et Biophysica Acta 1820 (2012) 76–83

Contents lists available at SciVerse ScienceDirect

Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen

Microphthalmia-associated transcription factor is required for mature
myotube formation
Ryo Ooishi a, Mitsuyuki Shirai b, Masayuki Funaba c,⁎, Masaru Murakami a,⁎⁎abcLaboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara 252-5201, Japan
Laboratory of Veterinary Pharmacology, Azabu University School of Veterinary Medicine, Sagamihara 252-5201, Japan
Division of Applied Biosciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japana r t i c l e

i n f oArticle history:
Received 15 May 2011
Received in revised form 29 October 2011
Accepted 10 November 2011
Available online 20 November 2011
Keywords:
Mitf
Myogenesis
Integrin
p21
Myotubea b s t r a c t
Background: The roles of microphthalmia-associated transcription factor (Mitf) in the skeletal muscle andduring myogenesis are unclear.
Methods: Expression of Mitf in mouse tissues and during myogenesis was evaluated. Effects of Mitf knockdown on myogenesis and gene expression related to myogenesis were subsequently explored. Furthermore,
effects of p21, a cyclin-dependent kinase inhibitor, and integrin α9 (Itga9) were examined.
Results: Mitf was highly expressed in the skeletal muscle; Mitf-A and -J were expressed. Mitf expression increased after differentiation stimulation in C2C12 myogenic cells. Down-regulation of Mitf expression bytransfection of siRNA for common Mitf inhibited myotube formation, which was reproduced by Mitf-A knockdown. Morphometric analyses indicated that both multinucleated cell number and the proportion of myotubes with more than 6 nuclei were decreased in Mitf-knockdown cells, suggesting that Mitf is required for
not only the formation of nascent myotubes but also their maturation. Searching for genes positively regulated by Mitf revealed p21 and Itga9; decreasing Mitf expression inhibited up-regulation of p21 expression after
differentiation stimulation and blocked the induction of Itga9 expression in response to differentiation.
Knockdown of p21 decreased the number of multinucleated cells, whereas Itga9 knockdown did not affectthe myotube number. Both p21 knockdown and Itga9 knockdown decreased the proportion of myotubes
with more than 6 nuclei.
General signiﬁcance: Mitf positively regulates skeletal muscle formation; Mitf is signiﬁcantly expressed
during myogenesis, and is required for efﬁcient myotube formation through expression of p21 andItga9.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
Skeletal muscle formation consists of a complex set of differentiation
steps: commitment of mesenchymal stem cells to myoblast lineage
cells, progression of differentiation with the expression of muscle-cellspeciﬁc proteins, and fusion of myoblasts into multinucleated myotubes.
Mammalian myotube formation occurs in two phases [1,2]. In the ﬁrst
phase, differentiated myoblasts fuse together to form small myotubes;
to accomplish this process, proliferating myoblasts exit cell cycle, andsome myoblasts undergo apoptosis. In the second phase, additional myoblasts subsequently fuse with myotubes to form large myotubes. Although the roles of myogenic regulatory factors (MRFs), including Myf5,
Myod, Myogenin and Mrf4, in myogenic differentiation are unquestionable [3–5], a number of factors, such as secreted proteins, membrane proteins and transcriptional regulators, are also involved in myogenesis [2].
⁎ Correspondence to: Tel.: + 81 75 753 6055.
⁎⁎ Correspondence to: Tel.: + 81 42 769 1624.
E-mail addresses: mfunaba@kais.kyoto-u.ac.jp (M. Funaba),murakami@azabu-u.ac.jp (M. Murakami).

Microphthalmia-associated transcription factor (Mitf) is a memberof the basic helix–loop–helix leucine zipper (bHLH-LZ) family of transcription factors [6–8]. Expression levels of Mitf, evaluated by Western
blotting and Northern blotting, vary among tissues; it is highly
expressed in melanocytes, mast cells, osteoclasts, and the heart[9–11]. Thus, the roles of Mitf have been mainly examined in thesecells [11–14], and Mitf activities in other tissues are largely unknown.
Of the Mitf variants that are not the result of genetic mutation, nine
Mitf isoforms have been identiﬁed in mice that differ in their transcriptional initiation site: Mitf-A, -B, -C, -D, -E, -H, -J, -M and -mc. The
Mitf variants contain an isoform-speciﬁc ﬁrst exon, while exons 2 to
9 of all Mitf isoforms examined to date are identical. Mitf isoforms
are expressed in a cell type-speciﬁc manner, and their transcriptional
activities are slightly but signiﬁcantly different depending on the target gene [15–19]. In addition, two types of Mitf mRNAs with or without an 18-base insert (exon 6a) are generated by alternative use of
the two acceptor sites located at the 5′ end of exon 6 in Mitf-A, -H, -J,-M and -mc [6,9,20,21]. In the present study, we show the relatively
higher expression of Mitf-A in the skeletal muscle, and present the potential role of Mitf in the progression of myogenesis.

0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2011.11.005

512

R. Ooishi et al. / Biochimica et Biophysica Acta 1820 (2012) 76–83

2. Materials and methods
2.1. Animals and cell culture
Nine C57BL/6 mice aged 7–8 wk were used to examine the tissue
distribution of Mitf. The experiment was approved by Azabu University Animal Experiment Committee. C2C12 myoblasts were obtained
from the American Type Culture Collection (Rockville, MD). Cells
were cultured in growth medium, i.e., Dulbecco's modiﬁed Eagle's
medium (DMEM) with heat-inactivated 10% fetal bovine serum(FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, at 37 °C
under a humidiﬁed 5% CO2 atmosphere. To induce differentiationfrom myoblasts to myotubes, the medium was replaced at conﬂuence(day 0) with differentiation medium consisting of DMEM with 2%
horse serum supplementing the antibiotics. To isolate myotubes,
cells on day 7 were trypsinized for a short time under a microscope
until detachment of multinucleated cells (~2 min), followed by centrifugation to obtain a myotube-rich fraction.

2.2. RT-PCR, restriction fragment length polymorphism and quantitative
RT-PCR
Total RNA isolation from tissues and cells, RT-PCR and restriction
fragment length polymorphism (RFLP) were performed as described

77

previously [21]. Quantitative RT-PCR (qRT-PCR) was carried out asdescribed previously [22]. The used PCR primers in qRT-PCR are presented in Table 1. To compare tissue Mitf expression, the appropriate
corrected gene was chosen using a mouse housekeeping gene primerset (TaKaRa, Otsu, Japan). The relative mRNA level was expressed as aratio with β-actin to evaluate tissue distribution and Gapdh was used
as a reference gene to examine regulatory expression in C2C12 cells.

2.3. Western blotting
To examine expression of Mitf in the skeletal muscle, thigh muscle
and heart as a positive control were homogenized in RIPA buffer.
After 30 min on ice, the debris was removed by centrifugation at
2500 ×g for 2 min at 4 °C. After centrifugation at 12,000 ×g for
5 min at 4 °C, the supernatant was recovered and the protein concentration was measured by the bicinchoninic acid method [23]. Fifty micrograms of the protein was loaded on 10% SDS-polyacrylamide gel.
Western blotting was performed as described previously [24,25]. Expression of Mitf and myosin heavy chain (Myhc) was examined byuse of mouse monoclonal antibodies against Mitf (X1405M; Exalpha
Biologicals) and Myhc (MY-32; Sigma), respectively. There are two
types of muscle ﬁber, i.e., fast-twitch and slow-twitch; fast-twitch
muscle ﬁbers express Myhc2a, Myhc2b and Myhc2x, whereas slowtwitch muscle ﬁbers do Myhc1 [26]. According to the manufacturer's

Table 1
Oligonucleotide PCR primers for qRT-PCR.
Oligonucleotide

Mitf
Common Mitf
Mitf-A
Mitf-H
Mitf-J
Mitf-M
Myogenic regulatory factors
Myf5
Myod1
Myogenin
Mrf4
Myosin heavy chains
Myhc2aMyhc2b
Myhc2xTarget candidates
Adam12
Calpastatin
Caveolin3
Ccna2
Cdh15
Ctsb
Itga3
Itga4
Itga5
Itga6
Itga9
Itgb1
Myof
p21
p57
Ptger4Rb1
v-Cam1
Vcl
Reference genes
β-actin
Gapdh

GenBank
accession
number5′-primer

3′-primer

5′-GCCTTGCAAATGGCAAATAC-3′5′-GAGGAGTTTCACGAAGAACC-3′5′-GAGGAGTTTCACGAAGAACC-3′5′-CCGTGTCTCTGGGCATCTGAAG-3′
5′-ATGCTGGAAATGCTAGAATACAG-3′

5′-GCTGGACAGGAGTTGCTGAT-3′
5′-GCTGGCGTAGCAAGATGCGTGA-3′5′-GCTGGCGTAGCAAGATGCGTGA-3′5′-GCTGGCGTAGCAAGATGCGTGA-3′5′-CATACCTGGGCACTCACTCTC-3′

NM_001113198
NM_001178049
AY632575
NM_008601

5′-GCCAGTTCTCCCCTTCTGAGTA-3′5′-GTGGCAGCGAGCACTA-3′5′-AGAAGCGCAGGCTCAAGAAA-3′5′-GGGCCTCGTGATAACTGCT-3′5′-TGGTCCCCAAACTCATCCTCT-3′5′-GGGCCGCTGTAATCCATC-3′
5′-ATCTCCACTTTAGGCAGCCG-3′5′-AAGAAAGGCGCTGAAGACTG-3′

NM_008656
NM_010866
NM_031189
NM_008657

5′-AAAGCTCCAAGGACCCTCTT-3′5′-CCGAGCAAGAGCTACTGGA-3′
5′-TCGCTGGCTTTGAGATCTTT-3′5′-AGCTCATGACTGCTGAACTCAC-3′5′-TGTTGATGAGGCTGGTGTTC-3′
5′-CGAACATGTGGTGGTTGAAG-3′

NM_001039545
NM_010855
NM_030679

5′-CAGAGCATCCCAGCCAAG-3′5′-CCTTCAGCTGGTGGAGAGAG-3′5′-CACAAGGCTCTGATCGCCTC-3′
5′-CTTGGCTGCACCAACAGTAA-3′5′-TGACATTGCCAACTTCATCAG-3′5′-AAGCTGTGTGGCACTGTCCTG-3′
5′-CATCAACATGGAGAACAAGACC-3′5′-ACCAGACCTGCGAACAGC-3′
5′-TACTCTGTGGCTGTGGGTGA-3′5′-GGTTGAGAGGCCATGAAAAG-3′5′-TGATGCCAACGTGTCCTTTA-3′5′-ATGCAGGTTGCGGTTTGT-3′5′-CATTACTGGCTTCTAAGCTGTCG-3′5′-TGGGCCCGGAACATCTC-3′
5′-CAGGACGAGAATCAAGAGCA-3′
5′-TCCAGATGGTCATCTTACTCAT-3′
5′-GCTGCAAATACAGAGACACAAGCAG-3′5′-TGATTGGGAGAGACAAAGCA-3′5′-GATAGCTGCTCTGACCTCTAA-3′

5′-CAGGCTGAGGATCAGGTCTC-3′
5′-GCTGGTGTGAATGGTTTCTG-3′
5′-TCCGTGTGCTCTTCGGTCA-3′
5′-CAAACTCAGTTCTCCCAAAAACA-3′5′-GATGAGAGCTGTGTCGTAGGGAG-3′5′-GATCTATGTCCTCACCGAACGC-3′
5′-CCAACCACAGCTCAATCTCA-3′
5′-CCCCCAGCCACTGGTTAT-3′
5′-GCCATTAAGGACGGTGACAT-3′5′-TTCTTTTGTTCTACACGGACGA-3′5′-GAAATGCCCATCTCCTCCT-3′5′-CATCCGTGGAAAACACCAG-3′5′-AAATTTACTCCACCAGTCAACG-3′5′-TGCGCTTGGAGTGATAGAAA-3′5′-TGCGCTTGGCGAAGAAGT-3′5′-TAACTGGTTAATGAACACTCGCA-3′5′-CGGAGATATGCTAGACGGTACACTT-3′5′-AACAACCGAATCCCCAACT-3′5′-TAGTGCCGTCGCCACTTGTTT-3′

NM_007400
NM_009817
NM_007617
NM_009828
NM_007662
NM_007798
NM_013565
NM_010576
NM_010577
NM_008397
NM_133721
NM_010578
NM_001099634
NM_007669
NM_009876
NM_001136079
NM_009029
NM_011693
NM_009502

5′-CTAAGGCCAACCGTGAAAAG-3′5′-CGTGTTCCTACCCCCAATGT-3′5′-ACCAGAGGCATACAGGGACA-3′
5′-TGTCATCATACTTGGCAGGTTTCT-3′

X03672
NM_008084

513

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manual, this antibody for Myhc recognizes fast-type muscle ﬁbers.
After incubation of the membranes with ECL Advance reagent (GEHealthcare), the chemiluminescent signals were exposed to X-rayﬁlm. Subsequently, antibodies as well as the detection reagentswere stripped and reprobed with anti-α-tubulin (ab11304; Abcam)
or anti-β-actin antibody (AC-15; Abcam).

2.4. Immunohistochemistry
C2C12 cells were grown on glass chamber slides coated with type I
collagen (Thermo Fisher Scientiﬁc) and were ﬁxed with 3% paraformaldehyde in PBS for 30 min, followed by treatment with 1% Triton
X-100 in PBS for 10 min. After washing with PBS, cells were incubatedwith 3% H2O2 to remove endogenous peroxidase. Subsequently, cells
were treated with 1.5% normal goat serum in PBS for 20 min toblock nonspeciﬁc reactions. Diluted mouse monoclonal antibody
against Mitf (X1405M; Exalpha Biologicals) with 0.1% bovine serum
albumin in PBS at 1:80 was used to identify Mitf expression at theprotein level. After incubation with the primary antibody for 2 h atroom temperature, Mitf-positive cells were visualized using VECTASTAIN Elite ABC kit (Vector Laboratories) and 3,3′-diaminobenzidine(DAB; Dojindo) according to the manufacturer's protocol.

2.5. siRNA transfection
To target the expression of common Mitf, Mitf-A or Itga9 and green
ﬂuorescent protein (GFP) controls, oligonucleotides for the respective
genes were synthesized by BONAC corporation (Kurume, Japan) as
follows: 5′-GAAACUUGAUCGACCUCUACA-3′ and 5′-UAGAGGUCGAUCAAGUUUCCA-3′ for common Mitf, 5′-GGAGUCAUGCAGUCCGAAUTT3′ and 5′-AUUCGGACUGCAUGACUCCTT-3′ for Mitf-A, 5′-CCUUAGUGCUCUUCCGAAAGA-3′ and 5′-UUUCGGAAGAGCACUAAGGUU-3′ for
Itga9, and 5′-GGUUACGUCCAGGAGCGCATT-3′ and 5′-UGCGCUCCUGGACGUAGCCTT-3′ for GFP. The siRNA for p21 was purchased (sc29428; Santa Cruz Biotechnology). An equal amount of the oligonucleotide was mixed for each gene to prepare siRNA. Thirty picomolesof siRNA was transfected into C2C12 cells seeded at a density of
1.5×10 4 cells in 24-well plates every 48 h. siRNA was transfectedusing Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol. Antibiotics were not supplemented to the culture
media in siRNA transfection experiments.

3. Results
3.1. Expression of Mitf in tissues and during myogenesis
Tissue distribution of Mitf was quantiﬁed by qRT-PCR. First, the expressions of 12 genes, Atp5f1, B2m, β-actin, Gapdh, Hprt1, Pgk1, Ppia,
RpLp1, Rps18, Tbp, Tfrc, and Ywhaz, were quantiﬁed for evaluation as
appropriate corrected genes; quantitative PCR using cDNA preparedfrom equal amounts of total RNA revealed that β-actin expression
was the most stable among the tested tissues, which were evaluatedby “geNorm” (http://medgen.ugent.be/~jvdesomp/genorm/) (datanot shown). Thus, the gene transcript level of Mitf was corrected forthat of β-actin (Fig. 1A). Expression of Mitf was highest in the heart,as expected. Although Mitf is recognized as a tissue-restricted transcription factor [6–8], its expression was detected in a wide varietyof tissues. In particular, expression in the skeletal muscle was relatively higher. Previous studies indicated Mitf expression in the skeletal muscle [9,27], but the role of Mitf is not known. Thus, wecharacterized the expression and function of Mitf in the skeletal muscle and during myogenesis in detail. Examining Mitf isoforms differing in transcriptional initiation site in the skeletal muscle indicatedthat more than 99% of Mitf was Mitf-A, and that Mitf-H and -J werealso expressed signiﬁcantly (Fig. 1B).
In a C2C12 myotube differentiation model, C2C12 myoblasts fuse
to form multinucleated myotube cells upon serum starvation on day
0 [28]. To evaluate gene expression in C2C12 cells, we chose Gapdh instead of β-actin as a reference gene; qPCR using cDNA prepared fromequal amount of total RNA indicated gradual decrease in β-actin expression with progression of myogenesis, whereas expression of
Gapdh was relatively constant (Supplementary Fig. 1). Total Mitf expression level increased up to day 8 and then reached a plateau(Fig. 1C). Similar to the expression pattern in the skeletal muscle,
Mitf-A was the main isoform (59–76% of total Mitf). In addition,
Mitf-J but not -H was expressed. The expression pattern of Mitf isoform was constant during myogenesis; the ratio of Mitf-A to Mitf-J
was 1.5 to 3.2:1. Expression of Mitf in the skeletal muscle andC2C12 cells was also veriﬁed at the protein level by Western blot analyses (Fig. 1D). In addition, consistent with the changes at the mRNAlevel, expression of Mitf protein was increased after differentiation
stimulation (Fig. 1E). Furthermore, Mitf was exclusively localized in
the nucleus of C2C12 cells (Fig. 1F). We also examined the ratio ofgene transcripts of Mitf with exon 6a to that of Mitf without exon
6a by PCR-RFLP analyses; the ratios of Mitf-A and Mitf-J were relatively constant during myogenesis (Fig. 1G).

2.6. Morphological analysis
Cells were ﬁxed with methanol for 2 min and subsequently
stained with Giemsa staining for 20 min. Cells with more than 3 nuclei were judged as myotubes. Eight views per treatment in an experiment were analyzed. Experiments were repeated two or three times,
and similar results were obtained.

2.7. BrdU incorporation
Proliferation of C2C12 cells was measured by a BrdU cell proliferation assay (Cell Proliferation ELISA, BrdU (colorimetric); Roche)
according to the manufacturer's protocol.

2.8. Statistical analysis
Data are presented as the mean ± SE. Comparisons between
groups were conducted using Student's t-test. Results were considered signiﬁcant at P b 0.05.

3.2. Disruption of Mitf expression inhibits myotube formation by blocking
increased expression of p21 and Itga9 induction
To clarify the roles of Mitf in myogenesis, we evaluated effects of decreased expression of Mitf, which was achieved by transfection withsiRNA for Mitf (Supplementary Fig. 2A, lanes 2 and 3, and B). Transfectionof siRNA for common Mitf effectively decreased the emergence of multinucleated myotubes on day 6, as compared with GFP (Fig. 2A). Knockdown of Mitf-A also blocked the formation of thick myotubes (Fig. 2B,
Supplementary Fig. 2C). Morphological analyses on day 6 indicated thatthe number of multinucleated cells, which were deﬁned as cells withmore than 3 nuclei, was signiﬁcantly decreased by transfection of siRNAfor Mitf (Fig. 2C). We also evaluated the differentiated myotubes in detail;knockdown of Mitf increased the proportion of myotubes with 3 to 5 nuclei, but decreased that of myotubes with more than 6 nuclei (Fig. 2D).
Similar results were also obtained in C2C12 cells transfected with siRNAfor Mitf-A (Supplementary Fig. 3). Furthermore, Mitf knockdown decreased the expression of Myhc, a myotube-speciﬁc protein [3–5], inC2C12 cells on day 8 (Fig. 2E, lanes 1 and 2). These results suggest that
Mitf is required not only for myoblast–myoblast fusion but also for514

R. Ooishi et al. / Biochimica et Biophysica Acta 1820 (2012) 76–83

A

79

B

8

Mitf-A
Mitf-H
Mitf-JJ
Mitf

100

6

75

4

50

2

25

0

0

C 7.5

D

E

Mitf-A
Mitf-J

5.0
2.5
0.0

F

G
Mitf -A

Mitf -J-

Mitf
Prim. AbDay : 0 2 4 6 8 10 12 14
RT-PCR
RFLP(6a+6b):(6b) 1.30 1.20 1.22 1.38 1.29 1.33 1.29 1.40

6a+6b
6b

RT-PCR
RFLP(6a+6b):(6b) 1.67 1.88 2.00 1.80 2.75 2.17 2.50 1.67

6a+6b
6b

Fig. 1. Mitf expression is highly expressed in the skeletal muscle and increases with progression of myogenesis in C2C12 cells. (A) Mitf expression in various tissues was measured
by qRT-PCR. Mitf expression was expressed as a ratio to β-actin expression. Data are shown as the mean ± SE. (B) Expression of Mitf isoform differing with transcriptional initiationsite in the skeletal muscle was identiﬁed by qRT-PCR using isoform-speciﬁc 5′-primer and 3′-primer spanning the common Mitf. The percentage of the expression of each Mitf isoform to that of total Mitf was calculated. Data are shown as the mean ± SE. (C) C2C12 myoblasts were cultured to conﬂuence (day 0) in growth medium, followed by culture indifferentiation medium. Time-course changes in expression of Mitf isoform differing with transcriptional initiation site during myogenesis were examined in C2C12 cells. Mitf expression was expressed as a ratio to Gapdh expression. Data are shown as the mean ± SE (n = 2). (D) Expression of Mitf in the skeletal muscle. Protein extracted from heart, thighmuscle and C2C12 cells was subjected to Western blot analyses to examine Mitf expression at the protein level. A representative result of Western blot is shown. In the heart, Mitf-H
as well as Mitf-A is mainly expressed shown in (B). Note that calculated molecular weight of Mitf-H and Mitf-A is 56.8 k and 58.6 k, respectively. (E) Time-course changes in expression of Mitf protein were examined during myogenesis of C2C12 cells. A representative result of Western blot is shown. (F) Immunolocalization of Mitf in C2C12 cells. C2C12
myoblasts were ﬁxed and reacted with (right) or without (left) mouse monoclonal anti-Mitf antibody. Subsequently, cells were reacted with anti-mouse IgG antibody, and the antibody was visualized. A representative cell staining is shown. (G) The ratio of exon 6a-containing Mitf isoforms (exon 6a + 6b) to non-exon 6a-containing isoforms (exon 6b) was
evaluated in C2C12 cells. PCR products of Mitf-A and -J were digested by Hinf I, and DNA fragments with or without exon 6a were separated by PAGE. A photograph of a representative gel stained with ethidium bromide is shown.

maturation of nascent myotubes. We also examined effects of forced Mitf
expression on myotube formation; the expression did not enhance myogenesis (data not shown).
Molecular bases of decreased myotube formation by Mitf knockdown were then explored. Myogenesis is regulated through modulation of the expression and activity of MRFs [29,30]; however, genetranscript levels of Myf5, Myod, Myogenin and Mrf4 were unchanged
by Mitf knockdown, suggesting that impairment of myogenesis
resulting from Mitf knockdown is not due to altered expression of
MRFs (Fig. 3A–D).
Other than MRFs, several molecules related to cell attachment andfusion, such as Adam12, Calpastatin, Caveolin3, Cdh15, Ctsb, Itga3,
Itga4, Itga6, Itga9, Itgb1, Myof, and v-Cam1 are known to regulate myotube differentiation [2,31–34]. In addition, the cDNA microarraydatabase (http://www.ncbi.nlm.nih.gov/geo/) indicated that the expressions of Ccna2, Itga5, p21, p57, Ptger4, Rb1, and Vcl increased
with differentiation. In view of Mitf as a transcription factor, we
expected that Mitf is involved in myogenesis through transcriptional
activation of these myogenesis-regulating genes. Thus, the effects ofdecrease in Mitf expression on the expression level of these genes
were explored. We found that gene transcript levels of p21 andItga9 after differentiation stimulation were decreased by transfectionof siRNA for common Mitf; expression of p21 was increased in response to differentiation stimulation, and Mitf knockdown blocked

515

the increased expression of p21 (Fig. 3E). Signiﬁcant expression of
Itga9 was detected after day 4, and the expression level was decreased by transfection of siRNA for Mitf (Fig. 3F). Decreased expression of p21 and Itga9 was also detected in Mitf-A-knockdown cells(Supplementary Fig. 4). Gene transcript levels of the other genes
were unaffected by Mitf knockdown (Supplementary Fig. 5).
In a C2C12 myogenesis cell model, not all myoblasts not fuse into
myotubes; co-existence of proliferating myoblasts and myotubes is
detected after differentiation stimulation [35–37]. We separated the
myotube-rich fraction by limited trypsinization to explore cells affected by Mitf knockdown. Although Myhc2a expression was not signiﬁcantly different between the myotube fraction and the residual
cell fraction in control cells (Fig. 4A), Myhc2b and Myhc2x were predominantly expressed in the myotube fraction as expected (Fig. 4B
and C). Expression of Myhc2x in the myotube fraction was signiﬁcantly lower in Mitf-knockdown cells than in control cells, whereas that of
Myhc2a in the myotube fraction was higher in the Mitf-knockdown
cells. The Myhc2b expression in the myotube fraction was comparable
between in control cells and in Mitf-knockdown cells. Consideringthat Myhc2x was the major isoform of type 2 Myhc (>93%)(Fig. 4D), total expression of Myhc constituting of fast-twitch myoﬁbers was largely limited to the myotube fraction, and was decreasedby Mitf knockdown; the latter was consistent with the results on
Myhc expression at the protein level (Fig. 2E).

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A

B

control
controlsi-Mitf

2

0

6

4

D

C
controlsi-Mitf

50

E
**

80

relative number ofmultinucleated cell

relative number ofmultinucleated cell100

si-Mitf-A**

controlsi-Mitfsi-Itga9Day:si-Mitf

controlsi-Mitf60Myhc
β-actin

40

1 2 3

*

20

0

*

0

3-5

6-10

11<

nuclear number
Fig. 2. Mitf expression is required for efﬁcient myotube formation. C2C12 cells were transfected with siRNA for GFP as a control, common Mitf or Mitf-A every 2 days after day − 2.
(A) Effect of Mitf knockdown on myogenesis was evaluated. Representative phase-contrast images on days 0, 2, 4, and 6 are presented. (B) Role of Mitf-A was examined. Representative cells stained by Giemsa solution on day 6 are presented. (C and D) Myotube formation was evaluated in Mitf-knockdown C2C12 cells. Cells were transfected withsiRNA for GFP as a control or Mitf, and morphology was evaluated on day 6. After staining with Giemsa solution, cell number and the number of nuclei were calculated. (C)Ratio of multinucleated cell number to total cell number was calculated, and the ratio of cells transfected with siRNA for GFP was set to 1. (D) Number of cells with 3 to 5, 6 to
10 or > 11 nuclei was counted, and percentage to total multinucleated cells is shown. Data are shown as the mean ± SE (n = 6). * and **: P b 0.05 and P b 0.01, respectively, as compared to control. (E) Expression of Myhc in response to transfection of siRNA for GFP as a control, Mitf or Itga9 in C2C12 cells on day 8 was examined by Western blot analyses.
Subsequently, the membranes were reprobed with anti-β-actin antibody.

The extent of the decreased expression of Mitf resulting fromknockdown of Mitf was comparable between the myotube fraction
and the residual cell fraction, indicating effective inhibition of Mitf expression in both myoblasts and myotubes (Fig. 5A). Expression of p21
was signiﬁcantly higher in the myotube fraction than in the residual
cell fraction (Fig. 5B). Knockdown of Mitf equally decreased the expression of between the two fractions. Expression of Itga9 was alsosigniﬁcantly higher in the myotube fraction (Fig. 5C). Similar to theexpression of p21, Mitf knockdown down-regulated the expressionof Itga9 in both fractions.

1.0

0.5

0.0

Day: 0D

2

4

relative mRNA level

controlsi-Mitf

1.0
0.5

2

Day: 0

2

E

1.5

0.0

controlsi-Mitf

0.5

0.0

6

Mrf4

Day: 0

1.0

4

6

4

controlsi-Mitf

4
2
0

Day: 02

4

6controlsi-Mitf

30
20
10

Day: 0F

p21
6Myogenin

0

6

2

4

6

Itga9

relative mRNA level

controlsi-Mitf

relative mRNA level

relative mRNA level

1.5

C

Myod

relative mRNA level

B

Myf5

relative mRNA level

A

Cell cycle exit in response to differentiation stimulation is a prerequisite for myotube formation [2]; p21, a cyclin-dependent kinase
inhibitor inducing cell cycle exit, plays a role in post-mitotic myogenesis, although it also maintains anti-apoptotic states in differentiatedmyocytes [1]. To link the decreased Mitf-induced inhibition of myotube formation and blockage of p21 induction, effects of the decreased
Mitf expression on BrdU uptake were next examined; BrdU uptake
was signiﬁcantly increased by transfection of siRNA for Mitf on days
4–6 (Fig. 6A). Knockdown of p21, however, did not affect BrdU uptake(Fig. 6B). Similar to Mitf knockdown, decreased expression of p21

2.0

controlsi-Mitf

1.5
1.0
0.5
0.0

Day: 0

2

4

6

Fig. 3. p21 and Itga9 are targets of Mitf for progression of myogenesis. C2C12 cells were transfected with siRNA for GFP as a control or common Mitf every 2 days after day − 2.
Gene expression of Myf5 (A), Myod (B), Myogenin (C), Mrf4 (D), p21 (E) and Itga9 (F) was quantiﬁed by qRT-PCR. The expression was normalized to Gapdh expression. The
expression in cells on day − 2 was set to 1 for Myf5, Myod, Myogenin, Mrf4 and p21, and that in cells transfected with siRNA for GFP on day 4 was set to 1 for Itga9. Data areshown as the mean ± SE (n = 2).

516

R. Ooishi et al. / Biochimica et Biophysica Acta 1820 (2012) 76–83

3

Residual
Myotube

b

2
1

c c

0

4. Discussion

Myhc2b

a

5.0

a

2.5

b

b

0.0

dsRNA: control si-Mitf

C

D

3

Myhc2aMyhc2b
Myhc2x100

Residual
Myotubeb2
1

relative expression levelto total Myhc expression

relative mRNA level

dsRNA: control si-Mitf

Myhc2xacd0Here, we demonstrate that 1) an isoform of Mitf mRNA is abundant in the skeletal muscle, 2) Mitf-A expression increased with progression of myogenesis, 3) knockdown of Mitf mRNA disrupts
myotube formation through inhibition of myoblast–myoblast fusionand subsequent myoblast–myotube fusion, but does not affect the expression of MRFs, and 4) expression of p21, a cyclin-dependent kinase
inhibitor, and Itga9 is regulated by Mitf in a stage-dependent manner
for appropriate myogenesis. The present results indicate a novel roleof a tissue-restricted transcription factor Mitf in the regulation of
myogenesis, which is probably mediated by an MRF-independent
pathway.
Mitf regulates myogenesis in multiple steps. Expression of p21 andItga9 was modulated by decreased expression of Mitf; downregulation of p21 expression resulting from knockdown of Mitf
mRNA was limited to post-differentiation, and signiﬁcant Itga9 expression was detected just after differentiation stimulation. Considering that signiﬁcant expression of Mitf was detected prior todifferentiation stimulation, the gene expression of p21 and Itga9
could not be regulated by Mitf alone, but in concert with Mitf andas yet unidentiﬁed factor(s). Transfection of siRNA for Mitf resulted
in down-regulated expression of Mitf both in myoblasts and myotubes. Thus, the precise mechanism of how Mitf regulates not only
p21 in myoblasts and myotubes but also Itga9 expression in myotubes
remains unclear. Since Mitf binds to the E-box (CAnnTG) located in
the 5′-untranslated region and activates transcription [6–8], Mitf
may directly regulate transcription of p21 and Itga9. Alternatively, it
is possible that a factor regulated by Mitf in myoblasts acts as a regulator to induce p21 or Itga9 in myotubes.
A target gene of Mitf was Itga9. Among members of the integrinfamily, Itga3, 4, 6 and 9 have been suggested to regulate myogenesis[2,31,33,34]. Transcription of Itga4 was activated through Mitf binding to the E-box located in the 5′-untranslated region in mast cells[38]. In the present study, however, Itga4 mRNA was not decreased
but rather increased by transfection of siRNA for Mitf in C2C12 cells(Supplementary Fig. 5H), suggesting that transcription by Mitf isregulated in a cell context-dependent manner. In addition, Mitf
knockdown did not affect the expression level of Itga3 and 6 (Supplementary Fig. 5G and J). In a study using a human mononucleatedmyogenic precursor cell culture system, addition of anti-INTEGRIN
α9β1 antibody to the culture medium decreased the proportion of
nuclei included in large myotubes (≥5 nuclei), whereas the proportion of nuclei incorporated in small myotubes (2–4 nuclei) was
unchanged [33]. In the present study using a murine myogenesis
model, transfection with siRNA for Itga9 decreased the proportion oflarge myotubes (≥6 nuclei) and increased that of small myotubes(3–5 nuclei), but did not affect the number of multinucleated cells.
Taking these results together, Itga9 is suggested to promote the

Residual
Myotube50

0dsRNA: control si-Mitf

dsRNA: control si-Mitf

Fig. 4. Knockdown of Mitf in myoblasts and myotubes down-regulates expression of
Myhc2x. C2C12 cells were transfected with siRNA for GFP as a control or common
Mitf every 2 days after day − 2. On day 7, multinucleated myotubes were separated
from the residual cells by limited trypsinization. Expression of Myhc2a (A), Myhc2b(B) and Myhc2x (C) was quantiﬁed by qRT-PCR, and normalized to Gapdh expression.
The expression in residual cells after limited trypsinization in the control group was
set to 1. (D) The percentage of the expression of each Myhc isoform to that of total
type II Myhc was calculated. Data are shown as the mean ± SE (n = 3). a, b, c, d:
Means that do not have a common letter on the bar differ signiﬁcantly (P b 0.05).

inhibited emergence of maturated and thick myotubes (Fig. 6C). In
addition, it signiﬁcantly decreased the number of multinucleatedcells (Fig. 6D), and increased and decreased the proportion of myotubes with 3 to 5 nuclei and those with more than 6 nuclei, respectively (Fig. 6E).
We next explored the effects of Itga9 knockdown on myotube formation. The morphology of cells transfected with siRNA for Itga9 indicated that, compared to control cells, thinner myotubes were evident
by Itga9 knockdown, suggesting that Itga9 is required for myotube
maturation (Fig. 7A), which was veriﬁed by morphometry. The multinucleated cell number itself was not affected by knockdown of Itga9(Fig. 7B), but the proportion of the number of myotubes with 3–5 nuclei in a cell to that of total myotubes was signiﬁcantly increased, andthat with more than 6 nuclei was decreased (Fig. 7C). Consistent withthe results, knockdown of Itga9 gene resulted in the decreased expression of Myhc (Fig. 2E, lanes 1 and 3).

1.0

a

B

Mitf
a
Residual
Myotube

0.5

b b
0.0

dsRNA: control si-Mitf

relative mRNA level

relative mRNA level

A

C

p21
2.5

a

Residual
Myotube

2.0
1.5
1.0

b
d

0.5

c

0.0

dsRNA: control si-Mitf

relative mRNA levela4

relative mRNA levelB

Myhc2arelative mRNA level

A

81

Itga9
a

4

Residual
Myotube

3
2

b
1

c

b

0

dsRNA: control si-Mitf

Fig. 5. Knockdown of Mitf in myoblasts and myotubes down-regulates expression of p21 and Itga9. C2C12 cells were transfected with siRNA for GFP as a control or common Mitf
every 2 days after day − 2. On day 7, multinucleated myotubes were separated from the residual cells by limited trypsinization. Expression of Mitf (A), p21 (B), and Itga9 (C) was
quantiﬁed by qRT-PCR, and normalized to Gapdh expression. The expression in residual cells after limited trypsinization in the control group was set to 1. Data are shown as themean ± SE (n = 3). a, b, c, d: Means that do not have a common letter on the bar differ signiﬁcantly (P b 0.05).

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A

B

BrdU uptake

arbitrary unit

arbitrary unit

200

controlsi-Mitf

100
75
50

**

25

C

BrdU uptake**

controlsi-p21150

controlsi-p21

100
50
0

0

2

4

Day:

6

4

6

days post-differentiation

E
100

50

relative number ofmultinucleated cell

relative number ofmultinucleated cellD
controlsi-p21

**

0

75

**

controlsi-p21

50

*

25

0

*
3-5

6-10

11<

nuclear number
Fig. 6. p21 is involved in Mitf-mediated myogenesis. (A and B) Role of Mitf and p21 in BrdU uptake in C2C12 cells was evaluated. (A) Cells were transfected with siRNA for GFP as acontrol or common Mitf every 2 days after day −2, and BrdU uptake was examined on day −1, 0, 4 and 6. BrdU uptake in control cells on day − 1 was set to 100. (B) Cells weretransfected with siRNA for GFP or p21 every 2 days after day 2, and BrdU uptake was examined on days 4 and 6. Data are shown as the mean ± SE (n = 3–4). **: P b 0.01, as compared
to control. (C–E) Role of p21 in myogenesis was evaluated. Cells were transfected with siRNA for GFP or p21 every 2 days after day 2. (C) Representative cells stained by Giemsa
solution on day 6 are shown. (D and E) Myotube formation was evaluated in p21-knockdown C2C12 cells. After staining cells with Giemsa solution on day 6, cell number andthe number of nuclei were calculated. (D) Ratio of multinucleated cell number to total cell number was calculated, and the ratio of cells transfected with siRNA for GFP was set
to 1. (E) Number of cells with 3 to 5, 6 to 10 or > 11 nuclei was counted, and percentage to total multinucleated cells is shown. Data are shown as the mean ± SE (n = 4). * and**: P b 0.05 and P b 0.01, respectively, as compared to control.

growth of preformed myotubes rather than the formation of nascent
myotubes during myogenesis.
Effects of knockdown of Mitf mRNA partly overlapped but weredistinct from those of Itga9 knockdown; the number of multinucleated cells was decreased by Mitf knockdown. This suggests that
Mitf is involved in both myoblast–myoblast fusion and subsequent
maturation of nascent myotubes. It is possible that Mitf regulates
myoblast–myoblast fusion through the increase in p21 expression;
regulatory expression of p21 by Mitf has also been shown in melanocytes [39]. Cell cycle inhibition and apoptosis of myoblasts precede to
myoblast–myoblast fusion, and both processes are necessary for thecell fusion [1]. p21 is principally involved as a molecule to establish
post-mitotic and apoptosis-resistant states [1]; BrdU uptake was notdetected in p21-positive myoblasts [40], and forced expression of
p21 but not inactive p21 inhibited apoptosis of C2C12 myoblastsC

controlsi-Itga9

relative number ofmultinucleated cellB
controlsi-Itga9

100

50

80

relative number ofmultinucleated cellA[41]. In fact, p21 knockdown decreased the myotube number and increased proportion of small myotubes, suggesting that p21 expression is required for efﬁcient myoblast–myoblast fusion.
Knockdown of p21 did not increase BrdU uptake on days 4 and 6;
it may be offset effects of the cell growth inhibition and the antiapoptotic activity. In view of signiﬁcant inhibition of BrdU uptake in
Mitf-knockdown cells on days 4 and 6, Mitf regulates expression
and activity of additional molecule(s) unidentiﬁed in this study.
Bharti et al. [19] revealed that Mitf-A and -J are expressed throughout eye development in both retina and pigment epithelium, whereas
expression of Mitf-D and -M was limited to pigment epithelium. In
addition, Mitf-H was preferentially found in pigment epithelium
with temporal expression proﬁle. Unlike the eye development, theproportion of expressed Mitf isoforms, i.e., Mitf-A and Mitf-J was almost constant during myogenesis of C2C12 cells, although total Mitf

controlsi-Itga9

60
40
20
0

0

**

**
**
3-5
11<
6-10
nuclear number

Fig. 7. Itga9 are involved in Mitf-mediated myogenesis. (A–C) Role of Itga9 in myogenesis was evaluated. Cells were transfected with siRNA for GFP or Itga9 every 2 days after day−2. (A) Representative cells stained by Giemsa solution on day 6 are shown. (B and C) Myotube formation was evaluated in Itga9-knockdown C2C12 cells. After staining cells with
Giemsa solution on day 6, cell number and the number of nuclei were calculated. (B) Ratio of multinucleated cell number to total cell number was calculated, and the ratio of cells
transfected with siRNA for GFP was set to 1. (C) Number of cells with 3 to 5, 6 to 10 or >11 nuclei was counted, and percentage to total multinucleated cells is shown. Data areshown as the mean ± SE (n = 4). **: P b 0.01, as compared to control.

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expression was increased with progression of myogenesis (Fig. 1C).
Thus, expression of Mitf isoforms is tissue-speciﬁc and stagedependent. In view of different activity as transcription factors between the isoforms [15–18], regulation on the use of alternative promoter must be clariﬁed in future studies.
Mitf encoded by the mutant mi allele deletes one of four consecutive arginines in the basic domain, and acts as a dominant-negative
mutant [9,27,42]. The mi/mi mutant mice exhibited the pleiotropic effects of microphthalmia, depletion of pigment in both hair and eyes,
and osteopetrosis resulting from defects of osteoclastogenesis [9,14].
In addition to these phenotype alterations, Katayama et al. [43] recently showed that masseter muscle development was disrupted inmi/mi mutant mice; they speculated that the disorder is a secondary
event related to abnormal tooth formation related to osteoclast dysfunction; however, in view of the present results showing direct inhibition of myogenesis by decreased expression of Mitf, muscle
development itself may be disturbed in mi/mi mice.
The present study indicates signiﬁcant expression of Mitf in theskeletal muscle from re-evaluation of tissue distribution of Mitf bysensitive qRT-PCR analyses. Evaluation of Mitf activity clariﬁes itsrole as a positive regulator during myogenesis. Because Mitf isexpressed also in other tissues with a variation of expression level, future studies should be directed to elucidate the role of Mitf in as yet
uncharacterized tissues.
Supplementary materials related to this article can be found online at doi:10.1016/j.bbagen.2011.11.005.
Acknowledgements
This work was supported by Kakenhi from the Japan Society for
the Promotion of Science, and a research project grant awarded by
the Azabu University.
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Biochimica et Biophysica Acta 1810 (2011) 1127–1135

Contents lists available at SciVerse ScienceDirect

Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen

Endogenous Bmp4 in myoblasts is required for myotube formation in C2C12 cells
Takenao Umemoto a, Yuuma Furutani a, Masaru Murakami b, Tohru Matsui a, Masayuki Funaba a,⁎ab

Division of Applied Biosciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan
Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara 229-5201, Japana r t i c l e

i n f oArticle history:
Received 16 March 2011
Received in revised form 9 August 2011
Accepted 15 September 2011
Available online 22 September 2011
Keywords:
Bmp4
Myotube
Myogenesis
Myogenina b s t r a c t
Background: Our previous study revealed the indispensable activity of endogenous bone morphogenetic protein(Bmp) prior to differentiation induction of C2C12 myoblasts for myogenesis. Here we investigated the Bmp isoform
responsible for endogenous Bmp activity during differentiation and its role in myogenesis.
Methods: Gene expression of Bmp4 during myogenesis was evaluated in C2C12 cells. Effects of inhibition of the Bmp
pathway on myogenesis were examined. Cells expressing Bmp4 and regulation of Bmp4 expression in myoblasts
were explored.
Results: The expression of Bmp4 increased with the progression of myogenesis, although the extent of the increase after differentiation induction was smaller than that before the induction. Down-regulation of Bmp signal
components including Bmp4, Bmpr2, and Alk2/3 inhibited the emergence of positive cells for myosin heavy chain
II. The treatments also decreased the Myogenin expression. Treatment with cytosine arabinoside decreased theexpression of Bmp4. Also, Bmp4 expression was also lower in isolated myotubes than in residual cells. Expressionof Rgm c was higher in the myotube fraction. Transcription of Bmp4 was repressed by the conditioned medium of
mixed cells consisting of myoblasts and myotubes.
Conclusion: Bmp4 expressed in myoblasts has a positive role in myotube formation/maturation through myogenin
expression. The presence of myotubes inhibits Bmp4 expression in proliferating myoblasts through transcriptional
regulation, although the expression is intrinsically increased with time of culture.
General signiﬁcance: Taken previous results on involvement of Bmp in the commitment of osteoblasts and adipocytes with the present results together, Bmp may act as a general promoter of mesenchymal cell differentiation.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
Skeletal muscle is a highly specialized tissue composed of postmitotic, multinucleated muscle ﬁbers, which contract to generate force
and movement. Skeletal muscle is also critical to maintain metabolic
health through glucose uptake and insulin sensitivity [1,2]. In addition, contracting skeletal muscle has been recently suggested to play arole as an endocrine organ producing various cytokines [3–5]. Skeletal
muscle formation consists of a complex set of differentiation steps: commitment of mesenchymal stem cells to myoblast lineage cells, progression of differentiation with the expression of muscle-cell-speciﬁc
proteins, and fusion of myoblasts into multinucleated myotubes. Myogenic differentiation is principally governed by activities of the MyoD
family basic helix–loop–helix (bHLH) transcription factors, also known
as myogenic regulatory factors (MRFs), i.e., Myod, Myf5, Myogenin
and Mrf4. MRFs form a complex with E proteins, another class of bHLH
transcription factors, such as E12 and E47, and stimulate the transcription of skeletal muscle-speciﬁc genes through binding to E-box⁎ Corresponding author at: Division of Applied Biosciences, Kyoto University
Graduate School of Agriculture, Kitashirakawa Oiwakecho, Kyoto 606-8502, Japan.
Tel.: + 81 75 753 6055; fax: + 81 75 753 6344.
E-mail address: mfunaba@kais.kyoto-u.ac.jp (M. Funaba).
0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2011.09.008

520(CANNTG) in the regulatory region [6–8]. To accomplish appropriate
myogenesis, the activities of MRFs must be strictly regulated.
Bone morphogenetic proteins (Bmps), which were originally isolated as bone-inducing proteins [9], potently regulate various biological processes, including hematopoietic and neuronal development,
iron metabolism, and vascular homeostasis [10]. The diverse effectsof Bmp are mostly elicited through the phosphorylation and activationof Bmp-regulated Smad (BR-Smad), i.e., Smad1, Smad5 and Smad8, atthe carboxyl-terminal serines [10]. Phosphorylated BR-Smad forms
complexes with Smad4, which accumulate in the nucleus where
they participate in transcriptional regulation of the target genes.
Previous studies revealed that Bmp acted as a negative regulatorof myogenesis. Addition of Bmp to the culture medium of myogenic
cells led to differentiation into osteoblast lineage cells [11–15]. Thistransdifferentiation was related to gene induction of the inhibitor of
DNA binding 1 (Id1) [11]. Id1 heterodimerized with E proteins,which blocked the formation of active complex consisting of E proteins and Myod [16,17]. In addition, Id1 expression accelerated thedegradation of myogenin [18]. As compared with the well-known effects of exogenous Bmp, information on the activity and role of endogenous Bmp is limited.
Recently, we revealed the importance of endogenous Bmp activity
in undifferentiated myoblasts for myogenesis in a C2C12 myoblast

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differentiation model [19]; in this model, mononucleated myoblasts
are fused to multinucleated myotubes upon reduced serum orserum starvation [20]. Endogenous Bmp activity, which was monitored by the phosphorylation level of BR-Smad, was higher in thestage prior to differentiation induction, and inhibition of the Bmp
pathway prior to differentiation induction down-regulated the expression of Myf5 and Myod, leading to impaired myotube formation[19]. The role of endogenous Bmp sharply contrasted to previous
knowledge on the action of exogenously treated Bmp for myogenesis[11–15]; however, the level of phosphorylated BR-Smad remained after
differentiation induction, although it was lower than before differentiation. This situation encouraged us to explore further the signiﬁcance ofendogenous Bmp activity during myotube differentiation. The present
study reveals that Bmp4 expression in proliferating myoblasts after differentiation induction is required for the progression of myogenesis tomaintain Myogenin expression, and that the expression is negatively
regulated by the presence of myotubes.
2. Materials and methods
2.1. Materials
The following reagents were purchased: recombinant Bmp4 wasfrom R&D Systems (Minneapolis, MN, USA); cytosine arabinoside(cytosine-1-β-D(+)-arabinofuranocide) was from Wako (Tokyo,
Japan); rabbit polyclonal antibody against Myogenin (ab11986),
rabbit monoclonal antibody against Smad1 (ab33902) and β-actin(AC-15) were from Abcam (Cambridge, MA, USA); mouse monoclonal antibody against myosin heavy chain (MyHC) (MY-32) wasfrom Sigma (St. Louis, MO, USA); rabbit polyclonal antibody against
phospho-Smad1 (Ser463/Ser465) / Smad5 (Ser463/Ser465)/ Smad8(Ser426/Ser428) (#9511) was from Cell Signaling Technology (Danvers, MA, USA); Alexa Fluoro 488 was from Invitrogen (Carlsbad, CA,USA).
2.2. Cell culture
C2C12 myoblasts were cultured in growth medium, i.e., Dulbecco's
modiﬁed Eagle's medium (DMEM) with heat-inactivated 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, at
37 °C under a humidiﬁed 5% CO2 atmosphere. To induce differentiationfrom myoblasts to myotubes, the medium was replaced at conﬂuence(day 0) with differentiation medium consisting of DMEM with 2%
horse serum supplemented the antibiotics. Alternatively, the differentiation was induced by culture with serum-free DMEM supplemented
the antibiotics to collect conditioned medium. To examine the effects
of removing proliferating myoblasts, cytosine arabinoside was added
to the culture medium at the ﬁnal concentration of 10 μM or 20 μM fordays 4 to 7 or days 11 to 14, and cells were recovered on day 7 or day
14, respectively. To isolate myotubes, cells on day 10 were trypsinized
for a short time under the microscope until detachment of multinucleated cells (~2 min), followed by centrifugation to obtain a myotuberich fraction. Collection of conditioned medium was done for days 4–6,
days 6–8 and days 8–10. The conditioned medium was concentrated,
and the solvent was changed to 20 mM HEPES (pH 7.5), 150 mM NaCl,
1 mM PMSF, 1% aprotinin by means of Centriprep-10 (Millipore, Bedford, MA), and protein concentrations were measured by the bicinchoninic acid method [21].
2.3. qRT-PCR
RNA isolation and qRT-PCR were carried out as previously described[22]. The following oligonucleotides were used as PCR primers: 5′ACTCCCTTACGTCCATCGTG-3′ and 5′-CAGGACAGCCCCACTTAAAA-3′ forMyogenin (Genbank accession number: NM_031189.2), 5′-CAGCTACAAACCCAAGCAAG-3′ and 5′-AGGCATCCACGTTTGCTC-3′ for Mrf4

(Genbank accession number: NM_008657.2), 5′-GCCTGGGCTTACCTCTCTATCAC-3′ and 5′-CTTCTCAGACTTCCGCAGGAA-3′ for myosin heavy
chain (Myhc) I (Genbank accession number: BC158018), 5′-CCTGAGCAAGAAGCTGAGGA-3′ and 5′-GGTCATTCCACGCGTACAG-3′ for Noggin(Genbank accession number: NM_008711), 5′-ACCGCCCTACACCTAGTCTTC-3′ and 5′-CATACCCATCCATCCAGCTC-3′ for repulsive guidance
molecule (Rgm) a (Genbank accession number: NM_177740.5), 5′ACCTTTCGGTTCAAGTGACG-3′ and 5′-TCACAGCTTGGTACACCTTCTG-3′
for Rgm b (Genbank accession number: NM_178615.3), and 5′-GGCAATCATGGAGAAAGAGATG-3′ and 5′-TTTCTCTGGGTACTTCTGTGATGT-3′ for
Rgm c (Genbank accession number: NM_027126.4). The other PCR
primers used in this study were previously described [22,23]. The Ct
value was determined, and the abundance of gene transcripts was calculated from the Ct value using Hprt1 as the corrected gene.
2.4. Measurement of DNA content
To examine time-course changes in DNA content during myotube
differentiation, recovered cells were resuspended in hypertonic buffer(200 mM phosphate buffer (pH 7.4), 2 M NaCl, 2 mM Na3VO4, 1 mM
PMSF, 1% aprotinin), followed by lysis by ultrasonication. DNA content
was measured by the method of Labarca and Paigen [24].
2.5. Double-stranded RNAi transfection
To target the expression of Bmp4, Bmpr2, Alk2 or Alk3 and green
ﬂuorescent protein (GFP) control, double-stranded (ds) RNAi of therespective genes was synthesized by Samchully Pharmaceuticals(Seoul, Korea). The coding sequences to inhibit the expression of
Bmp4, four oligonucleotides, were designed: 5′-GUUGAAAAAUUAUCAGGAGAU-3′ (set 1), 5′-CUCCUGAUAAUUUUUCAACAC-3′ (set 1),
and 5′-CAGACUAGUCCAUCACAAUGU-3′ (set 2), and 5′-AUUGUGAUGGACUAGUCUGGU-3′ (set 2). Two oligonucleotides of each set
were mixed, and 1:1 mixtures of dsRNAi were prepared. The other
dsRNAi used in this study were described previously [23]. Overall, 60
pmol dsRNAi in a 12-well plate was transfected using Lipofectamine
RNAiMAX (Invitrogen), according to the manufacturer's protocol.
2.6. Immunoﬂuorescence staining
Immunoﬂuorescence staining for Myhc was performed as described
previously [19]. Nuclei were also stained by 1 μg/ml 4′,6-diamidino-2phenylindole (DAPI). The number of nuclei was calculated by Image J(http://rsbweb.nih.gov/ij/). Fusion index was calculated as a ratio of thenumber of nuclei incorporated in myotubes to the number of total nuclei[25].
2.7. Western blotting
To evaluate roles of endogenous Bmp activity in myogenin expression, dsRNAi for Bmp signal components was introduced in cells for
48 h. In addition, BR-Smad phosphorylation was evaluated in cells
transfected with dsRNAi for Bmp4 and treated with recombinant
Bmp4. Western blotting was performed as described previously [19].
2.8. Reporter assays
The 5′-ﬂanking regions of Bmp4 (nucleotides (nt) −2372 to +115, relative to the transcriptional initiation site at +1) was isolatedby PCR using mouse genomic DNA. The DNA fragment was cloned
into a region upstream of the luciferase gene in pGL4-Basic (Promega,
Madison, WI, USA) using the Bgl II and Hind III sites; it was veriﬁed by
DNA sequencing. Luciferase-based reporter assays were conducted as
described previously [23]. C2C12 myoblasts were seeded on 24-wellplates, and transiently transfected with Bmp4-luc by use of Lipofectamine LTX reagent (Invitrogen). A Renilla reniformis luciferase vector

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driven by thymidine kinase promoter (Promega) was co-transfected
to serve as an internal control for transfection efﬁciency. At 24 hafter the transfection, C2C12 cells on day −3, day 0, day 3 or day 6,
which were recovered by use of cell dissociation buffer (Cell Dissociation Solution (1 ×) Non-enzymatic, Sigma) and resuspended in thegrowth medium for cells on day − 3 or the differentiation medium
for cells on day 0, day 3 and day 6, were added for 24 h. Alternatively,
at 24 h after the transfection, 50 μg of protein prepared from the conditioned medium was added to the culture medium for 24 h. Fireﬂy
luciferase activity was normalized to Renilla luciferase activity, andthe luciferase activity in the cell lysate cultured with growth medium
and without addition of cells was set to 1.
2.9. Statistical analyses
Data are expressed as the mean±SEM. Differences between treatments were examined by Student's t-test. Differences of Pb 0.05 wereconsidered signiﬁcant.
3. Results
3.1. Bmp4 expression increases with progression of myogenic differentiation
Changes in gene expression with myogenesis were examined(Fig. 1). Expression of MyoD family members, such as Myf5 (Fig. 1A)

0
8

1

0

12

0

day

F

Myhc I
7500
5000
2500
0
0

4

8

12

day

relative mRNA level

I

8

1000
500
0

12

0

day

relative mRNA level

relative mRNA level

E

4

G

Myhc IIb
150000100000
50000
0
0

4

8

4

8

Mrf4

D

3
2
1
0
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12

12

20
10
0
0

4

8

day

day

H2

Bmp4

30

4

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12

day

day

relative mRNA level42

relative mRNA level

1

Myogenin

C 1500relative mRNA level

relative mRNA level

relative mRNA level

2

0

Myod

B

and Myod (Fig. 1B), which are necessary for myotube differentiation[26], was highest on day 0. The expression level was decreased within
2 days after differentiation induction, and remained lower on day 14.
Expression of Myogenin was extremely low prior to differentiation induction, and rapidly increased after differentiation induction. The expression of Myogenin reached a peak on day 2 (Fig. 1C). Expression of
Mrf4 peaked on day 8 (Fig. 1D). Expression of Myhc I (Fig. 1E) andMyhc IIb (Fig. 1F), which are myocyte (myotube)-speciﬁc genes,
was increased in response to differentiation induction within 2 daysfor Myhc I and 4 days for Myhc IIb, and reached a peak on day 6–
8 for Myhc I and on day 6–10 for Myhc IIb. We examined changes inthe expression of several Bmp isoforms during myogenesis; expression of Bmp2 and Bmp7 was negligible ([22], data not shown). We
also searched for the expression of the Bmp subfamily belonging to
the Tgf-β family in a cDNA microarray database (http://www.ncbi.
nlm.nih.gov/geo/) thoroughly, and found no signiﬁcant expressionof Bmp2, Bmp5, Bmp6, Bmp7, Bmp8a, Bmp8b, Bmp9, Bmp10 and
Bmp15 (data not shown). Gdf5 elicits their signal via BR-Smad [13],but the expression of Gdf5 could not be detected. Only Bmp4 was signiﬁcantly expressed in undifferentiated myoblasts. Bmp4 expression
was increased 7.8-fold for 2 days before the differentiation induction.
The expression was still increased after the differentiation induction,
but the extent of the increase was smaller, i.e., 3.1-fold increase for
14 days after the differentiation induction. As the results, the levelon day 14 was more than 20-fold higher than that on day − 2

relative mRNA level

Myf5

A

1129

12

Tgf- 1

1

0
0

4

8

12

day

Noggin
2

1

0
0

4

8

12

day
Fig. 1. Time-course changes in expression of molecules related to myogenesis, Bmp4, Tgf-β1 and Noggin in C2C12 cells. C2C12 myoblasts were cultured to reach conﬂuence (day 0)
in growth medium, followed by culture in differentiation medium. Expression of the indicated gene was quantiﬁed by qRT-PCR, and normalized to Hprt1 expression. The expression
on day − 2 was set to 1. Data are shown as the mean ± SE (n = 4).

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T. Umemoto et al. / Biochimica et Biophysica Acta 1810 (2011) 1127–1135(Fig. 1G). This expression pattern contrasted to that of the structurally
related Tgf-β (Fig. 1H); Tgf-β1 expression was relatively constantduring myogenesis. Noggin acts as antagonists of the Bmp pathway[27]. Noggin expression was highest on day 0, and decreased after differentiation induction (Fig. 1I); the time-course changes in Noggin expression resembled to those in BR-Smad phosphorylation [19],suggesting the ﬁne regulation of endogenous Bmp activity before
the differentiation.
Cell morphology showed mononuclear myoblasts on day 0, theemergence of multinucleated myotubes within 4 days after differentiation induction, and an increase in the number of myotubes by day 14(Fig. 2A); however, mononucleated cells were observed even on day
14, indicating that not all myoblasts fuse to and differentiate into
myotubes (Fig. 2A). Cell number as well as DNA content also increased after differentiation induction (Fig. 2B and C). Cell growthduring myogenic differentiation was basically consistent with theprevious results, in which cell proliferation was observed on days
3–7 [28–30]. The present results indicate the co-existence of myoblasts and myotubes even by culture in differentiation medium for
14 days.

components of the Bmp pathway (Fig. 3A–E). Down-regulation of
the Bmp4 pathway also decreased gene transcript levels of Myogenin
but not Myf5 and Myod (Fig. 4). These results suggest that endogenous Bmp4 produced in proliferating myoblasts positively regulates
myogenesis through Myogenin expression.
Although Bmp4 has been previously recognized only as a secreted
growth factor, a recent study revealed a variant of Bmp4 localized in
the nucleus [31]. We examined whether down-regulated Myogenin expression in Bmp4-knockdown C2C12 cells is recovered by simultaneous
treatment with recombinant Bmp4. The used recombinant Bmp4 ranged
over the recoverable concentrations on the decreased BR-Smad phosphorylation resulting from the Bmp4 knockdown (Fig. 5A). The additional
Bmp4 did not effectively recover Bmp4 knockdown-induced downregulation of Myogenin (Fig. 5B). We also examined effects of the higher
concentrations of Bmp4, but could not detect up-regulation of Myogenin
expression in any concentrations of Bmp4 (data not shown).
3.3. Expression of Bmp4 in myoblasts is modulated by the presence of
myotubes
Co-existence of myoblasts and myotubes in a culture dish after differentiation induction suggests that increased expression of Bmp4
with culture time does not necessarily reﬂect its expression in myotubes. Thus, we tried to identify cells expressing Bmp4 predominantly
by two approaches: Bmp4 expression was examined in cells treatedwith cytosine arabinoside and in isolated myotubes. Cytosine arabinoside is incorporated into replicating DNA in the S-phase and damages
DNA [30,32], leading to elimination of proliferating cells. Treatment
with cytosine arabinoside for days 4–7 and days 11–14 efﬁciently decreased the expression of Bmp4 but not Tgf-β1 (Fig. 6A and B). Proliferating myoblasts express MRFs, such as Myf5 and Myod [29], and theexpression of these genes decreased as expected (Fig. 6C and D).

3.2. Bmp4 is required for efﬁcient myotube formation
Effects of inhibition of the Bmp4 pathway were examined to elucidate the role of Bmp4 in myogenesis; we evaluated effects of transfection of dsRNAi for Bmp4, the type II receptor Bmpr2, and the type I
receptors Alk2 and Alk3. Alk6 is another type I receptor for Bmp, but
the expression level in C2C12 cells was much lower (data notshown). Knockdown of genes for Bmp4, Bmpr2 or Alk2/3 induced
the emergence of thin Myhc-positive cells (Fig. 3A). In fact, the fusionindex, the ratio of the number of nuclei in Myhc-positive cells to thenumber of total nuclei, was decreased by down-regulation of

A

B

day 0

day 2

day 4

day 6

day 8

day 10day 12day 14cell number

C

400 m

75

g / well

150

x104 / well

DNA content

100
50
0

50

25

0
0

4

8
day

12

0

4

8

12

day

Fig. 2. Time-course changes in morphology, cell number and DNA content in C2C12 cells. C2C12 myoblasts were cultured to reach conﬂuence (day 0) in growth medium, followedby culture in differentiation medium. (A) Representative morphology observed by phase-contrast microscope is shown. (B and C) Time-course changes in cell number (B) and DNA
content (C) were examined. Data are shown as the mean ± SE (n = 6).

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T. Umemoto et al. / Biochimica et Biophysica Acta 1810 (2011) 1127–1135

1131

A
dsRNAi:

GFP

Bmp4

Bmpr2

Alk2/3fusion index

arbitrary unit

1.0

***

0.5

0.25

dsRNAi:

*

0.50

0.25

0.00

dsRNAi:

1.00

0.75

0.50

**

0.25

0.00

dsRNAi:Alk2/3

**
0.00

Bmp4

0.00

dsRNAi:

GFP

0.25

0.50

0.75

Alk3

GFP

0.50

0.75

1.00

relative mRNA level

0.75

1.00

1.25

Alk2/31.00

1.25

relative mRNA level

relative mRNA level

relative mRNA level

1.25

E

Alk2

GFP

1.25

D

Bmpr2

Bmpr2

1.50

C

Bmp4

GFP

B

GFP
Bmp4
Bmpr2
Alk2/30.0

dsRNAi:

1.0

0.5

*

Alk2/3

Bmpr2

DdsRNAi:
Myogenin-actin*dsRNAi:Alk2/30.0
Bmpr2

relative mRNA level

Myogenin
1.5

GFP

dsRNAi:Alk2/30.0
Bmpr2

Alk2/3

Bmpr2

dsRNAi:

GFP

0.0

0.5

GFP

0.5

1.0

Bmp4

relative mRNA level

1.0

C

Myod
1.5

Bmp4

B

Myf5
1.5

Bmp4

relative mRNA level

A

GFP
Bmp4

Fig. 3. Myotube differentiation in C2C12 cells down-regulated expression of the Bmp signal components. C2C12 myoblasts were cultured to reach conﬂuence (day 0) in growth
medium, followed by culture in differentiation medium. On day 2, cells were transfected with the indicated dsRNAi. (A) On day 8, myotube formation was examined by immunoﬂuorescent analyses using anti-Myhc antibody. Representative immunostaining images are shown. Fusion index was calculated as a ratio of nuclei incorporated in myotubes relative to the total nuclei, and the index in cells treated with dsRNAi for GFP was set to 1 (n = 3). (B–E) On day 6, the gene transcript level of Bmp4 (B), Bmpr2 (C), Alk2 (D), and Alk3
(E) was quantiﬁed by qRT-PCR. The gene expression was normalized to Hprt1 expression. The expression in cells treated with dsRNAi for GFP was set to 1. Data are shown as themean ± SE (n = 4). * and **: P b 0.05 and P b 0.01, respectively, as compared to control (GFP).

Fig. 4. Expression of MRFs in C2C12 cells down-regulated expression of the Bmp signal components. C2C12 myoblasts were cultured to reach conﬂuence (day 0) in growth medium,
followed by culture in differentiation medium. On day 4, cells were transfected with the indicated dsRNAi. On day 6, gene transcript level of Myf5 (A), Myod (B), and Myogenin (C)was quantiﬁed by qRT-PCR. The gene expression was normalized to Hprt1 expression. The expression in cells treated with dsRNAi for GFP was set to 1. Data are shown as themean ± SE (n = 4). *P b 0.05, as compared to control (GFP). (D) On day 0, cells were transfected with the indicated dsRNAi. On day 2, Myogenin expression was examined byWestern blot analyses. After stripping the antibodies and the detection reagents, the membranes were reprobed with anti-β-actin antibody.

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T. Umemoto et al. / Biochimica et Biophysica Acta 1810 (2011) 1127–1135

A
dsRNAi for Bmp4:
Bmp4:

+

--

pSmad1/5/8
Smad1

B

Myogeninrelative mRNA level

1.00
0.75
0.50

** ** ** ** ** ** ** **

0.25
0.00

Bmp4:

-

dsRNAi for Bmp4:-+

Fig. 5. Phosphorylation of BR-Smad and Myogenin expression in C2C12 cells down-regulated Bmp4 and treated with Bmp4. C2C12 myoblasts were cultured to reach conﬂuence (day 0) in growth medium, followed by culture in differentiation medium. Cells
were transfected with or without dsRNAi for Bmp4 on day 1. At 12 h after the transfection, cells were treated with various concentrations of Bmp4 (16 fM to 1.25 nM) for
36 h. (A) Phosphorylated Smad1/5/8 was evaluated by Western blot analyses. After
stripping the antibodies and the detection reagents, the membranes were reprobedwith anti-Smad1 antibody. (B) Gene transcript level of Myogenin was quantiﬁed by
qRT-PCR. The gene expression was normalized to Hprt1 expression. The expression incells treated without dsRNAi for Bmp4 was set to 1. Data are shown as the mean ± SE
(n = 4). **P b 0.01, as compared to expression in cells treated without dsRNAi for Bmp4.

We next separated the myotube-rich fraction by limited trypsinization. The expression level of Bmp4 but not Tgf-β1 was lower in isolated myotubes than in residual cells (Fig. 7A and B). Expectedly,
Myhc IIb expression was predominantly detected in isolated myotubes (Fig. 7C), indicating the minimal presence of myotubes in the

0.5

0.0

Day:

7

relative mRNA level

C

**

Myf5

D

*

0.0

Day:

7

**

14

-Cyt (10 M)

1.0

Cyt (20 M)

0.5

0.0

Day:

14

1.0

0.5

Myogenesis is a complex biological process, and is principally regulated by the expression and activity of MRFs [6–8,26]. The present study
reveals that: (1) proliferating myoblasts express Bmp4; (2) Bmp4 expression in myoblasts is required for myotube formation/maturation
through Myogenin expression in myotubes; and (3) possibly myotubes
secrete factors down-regulating Bmp4 expression in myoblasts. Recently, we revealed that inhibition of endogenous Bmp activity prior to differentiation induction blocked myotube formation in C2C12 cells
through down-regulation of Myf5 and Myod expression [19]. In

Tgf- 1

relative mRNA level

1.0

****

4. Discussion

B

Bmp4

relative mRNA level

relative mRNA levelA

residual cell fraction. Consistent with previous results [29,33], Myogenin expression was also largely limited in the myotube fraction(Fig. 7D). Taking these results with results treated with cytosine arabinoside together, Bmp4 is expressed in proliferating myoblasts in thepresence of myotubes.
The expression of receptors for the Bmp pathway was also examined.
Bmp receptors were comparatively expressed in both cell fractions(Fig. 7E–G). Members of the Rgm family: Rgm a; Rgm b, also known as
Dragon; and Rgm c, also known as Hemojuvelin, are co-receptors of
Bmp2 and Bmp4, and enhance Bmp signaling [34–36]. Expression of
Rgm c was detected predominantly in the myotube fraction (Fig. 7H–J).
We next explored regulation of Bmp4 expression in myoblasts. At
ﬁrst, effects of the presence of myoblasts or mixed cells consisting ofmyoblasts and myotubes on Bmp4 transcription were examined. Reporter assays using Bmp4 promoter indicated that the presence of proliferating myoblasts increased expression of Bmp4-luc (Fig. 8A). By
contrast, the presence of cells after differentiation induction signiﬁcantly
decreased expression of Bmp4-luc. Addition of the conditioned medium
prepared from cells on days 4–6, days 6–8 or days 8–10 also decreased
Bmp4-luc (Fig. 8B). Consistent with the results, the conditioned medium
signiﬁcantly down-regulated Bmp4 expression (Fig. 8C) but not Tgf-β1
expression (Fig. 8D). These results suggest that Bmp4 expression in
myoblasts is positively regulated in the presence of proliferating myoblasts and negatively regulated in the presence of myotubes at the transcription level.

7

14

Myod
1.0

**

0.5

0.0

Day:

7

14

Fig. 6. Effects of cytosine arabinoside on expression of Bmp4, Tgf-β1, Myf5 and Myod in C2C12 cells. C2C12 myoblasts were cultured to reach conﬂuence (day 0) in growth medium,
followed by culture in differentiation medium. On days 4–7 and days 11–14, cytosine arabinoside (Cyt) at the indicated concentration was added to the differentiation medium.
Gene expression of Bmp4 (A), Tgf-β1 (B), Myf5 (C) and Myod (D) was quantiﬁed by qRT-PCR, and normalized to Hprt1 expression. The expression in cells treated without cytosine
arabinoside on each sampling day was set to 1. Data are shown as the mean ± SE (n = 4). * and **: P b 0.05 and P b 0.01, respectively, as compared to expression in cells treated without cytosine arabinoside.

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T. Umemoto et al. / Biochimica et Biophysica Acta 1810 (2011) 1127–1135

0.75
0.50

*

0.25

0.75
0.50
0.25

**

10

5
4
3
2
1

0.00

T

Bmpr2

H

1.00

1.00

0.50
0.25

R

T

I

R

0

T

J

Rgm b
1.25

0.75
0.50
0.25
0.00

0

T

Rgm a
1.25

0.75

R

R

T

1.00
0.75
0.50
0.25
0.00

R

R

Alk3

1.25

1.25

1.00

1.00

0.75
0.50
0.25
0.00

R

T

0.75
0.50
0.25
0.00

R

T

**

4

2

0

T

T

F

Alk2

Rgm c
6

relative mRNA level

R

1.25

0.00

**

20

E

7
6

relative mRNA level

G30

D Myogenin

relative mRNA level

1.00

Myhc IIb

relative mRNA level

1.00

relative mRNA level

1.25

C

relative mRNA level

Tgf- 1

1.25

0.00

relative mRNA levelBrelative mRNA level

Bmp4

relative mRNA level

relative mRNA level

A

1133

R

T

Fig. 7. Gene expression of Bmp4, Tgf-β1, Myhc IIb, Myogenin, Alk2, Alk3, Bmpr2, Rgm a, Rgm b and Rgm c in C2C12 cells after limited trypsinization. C2C12 myoblasts were cultured
to reach conﬂuence (day 0) in growth medium, followed by culture in differentiation medium. On day 10, multinucleated myotubes (T) were separated from the residual cells (R)by limited trypsinization. Expression of Bmp4 (A), Tgf-β1 (B), Myhc IIb (C), Myogenin (D), Alk2 (E), Alk3 (F), Bmpr2 (G), Rgm a (H), Rgm b (I) and Rgm c (J) was quantiﬁed by qRTPCR, and normalized to Hprt1 expression. The expression in residual cells after limited trypsinization was set to 1. Data are shown as the mean ± SE (n = 4). * and **: P b 0.05 and P
b 0.01, respectively, as compared to expression in the residual cells.

addition to the importance of Bmp activity in myoblasts before differentiation induction, the present results indicate the positive role of endogenous Bmp4 after differentiation induction in myogenesis. The role ofBmp4 produced in myoblasts as an accelerator of myogenesis and theinhibition of the Bmp4 expression by differentiated myotubes suggest
the reﬁned regulation of myogenesis through feedback loop mediatedby Bmp4.
Bmps in fetal bovine serum in the culture medium were possibly responsible for the high endogenous Bmp activity prior to differentiationinduction [19]. By contrast, Bmp4 produced in myoblasts was responsible for sustained endogenous Bmp activity after differentiation induction. Thus, differentiation induction changes the source of endogenous
Bmp activity. Endogenous Bmp activity, however, plays a positive rolein the progression of myogenesis, irrespectively of the differentiation
stages.
Expression of Rgm c, which is a co-receptor of Bmp to enhance responsiveness to Bmp [36], was predominantly detected in myotubes.
In C2C12 myotubes, soluble Rgm c was also produced by release fromthe cell membrane [37]; this molecular form of Rgm c inhibited thefunction of membrane-bound Rgm c [38]. However, the formationof soluble Rgm c was only partial, and was regulated by the iron status but not by the Bmp pathway [37]. Thus, we consider that myotubes efﬁciently receive Bmp4 produced by proliferating myoblasts
through expressing Rgm c.
The present results suggest that effects of endogenous Bmp4 on
myogenesis could not be explained by Bmp4 produced and secreted
from myoblasts; knockdown of Bmp4 inhibited myogenesis throughdown-regulation of Myogenin expression, but simultaneous526

administration of recombinant Bmp4 did not recover the expressionof Myogenin. As described above, in addition to secreted form of
Bmp4, there is a nuclear variant of Bmp4, which results from difference of translational initiation codon [31]. Since expression of this variant was detected in BALB/3T3 ﬁbroblasts, C3H10T1/2 mesenchymal
stem cells, and RCS chondrosarcoma cells, the two forms of Bmp4
would not be expressed in limited cells (Felin et al., 2010). Thus, it ispossible that nuclear form of Bmp4 is required for the progression of
myogenesis. In view of impaired myotube formation and decreased
expression of Myogenin also in cells down-regulated Bmp receptors,
both secreted form and nuclear form of Bmp4 may be indispensablefor efﬁcient myogenesis.
Expression of Bmp4 in myoblasts was stimulated by the presenceof proliferating myoblasts and inhibited by the presence of myotubes.
These results partly explain remarkable increase with time in Bmp4
expression before the differentiation and the slow increase after thedifferentiation; expression of Bmp4 in myoblasts is intrinsically increased with progression of myogenesis, and factors being responsible for the increase should be clariﬁed in future studies. Also, futurestudies should be directed to clarify soluble factor(s) produced inmyotubes being responsible for the repression of Bmp4 transcription
in proliferating myoblasts.
The necessity of endogenous Bmp4 activity for myogenesis contrasts
to the well-known effects of exogenous Bmp on myogenesis; Bmp4 addition to low serum-containing media in C2C12 myoblasts suppressed
myogenesis, and induced transdifferentiation into osteoblast lineage
cells [11–15]. Myoblasts responded to exogenous Bmp4, whereas myotubes responded to endogenous Bmp4; difference of the target cells

T. Umemoto et al. / Biochimica et Biophysica Acta 1810 (2011) 1127–1135

B

1

0
-

-3

0

-

6

0.5

References
0.0

conditioned
medium:

-

ia
t

io
n

3

re

di
ffe

Bmp4
1.00
0.75
0.50
0.25

Tgf- 1
2.0
1.5
1.0
0.5

-

day 6-8

conditioned
medium:day 8-10

day 8-10

day 6-8

day 4-6-

day 4-6

0.0

0.00

conditioned
medium:D
relative mRNA level

relative mRNA level

C

Acknowledgments
This work was supported by Kakenhi from the Japan Society for
the Promotion of Science, by a research project grant awarded by
the Azabu University, and by Core Stage Backup Grants from Kyoto
University.

nt

gr

cell:
medium:1.0

day 6-82

Bmp4-lucday 8-10

relative luciferase activity

Bmp4-lucowthrelative luciferase activityA

day 4-6

1134

Fig. 8. Regulatory expression of Bmp4 in myoblasts by the presence of myotubes andby conditioned medium from myoblasts and myotubes. (A and B) C2C12 myoblasts
were transiently transfected with Bmp4-luc and Renilla-luc. (A) At 24 h after the transfection, C2C12 cells on day − 3, day 0, day 3 or day 6 were added for 24 h. (B) At 24 hafter the transfection, 50 μg of protein prepared from the conditioned medium wasadded to the culture medium of the indicated culture period for 24 h. Fireﬂy luciferase
activity was normalized to Renilla luciferase activity, and the luciferase activity in thecell lysate cultured with growth medium and without addition of cells (A) and in
those treated without the conditioned medium (B) was set to 1. Data are shown as
the mean ± SE (n = 3 or 4). (C and D) C2C12 myoblasts were cultured in the presenceor absence of the conditioned medium for 48 h. Gene expression of Bmp4 (C) and
Tgf-β1 (D) was quantiﬁed by qRT-PCR, and normalized to Hprt1 expression. The expression in cells treated without the conditioned medium was set to 1. Data areshown as the mean ± SE (n = 3). * and **: P b 0.05 and P b 0.01, respectively, as compared to expression in cells cultured with the same medium alone (A) and to expression in cells treated without the conditioned medium (B and C). ††: P b 0.01, as
compared to expression in cells treated with cells on day − 3.

may be related to the disparate roles. Alternatively, as described above,the difference of molecular form between exogenous Bmp4, i.e., secreted
Bmp4, and endogenous Bmp4, i.e., secreted and nuclear Bmp4, may be involved in the differential results. Furthermore, optimal Bmp activity islikely to be indispensable for myotube differentiation. Suppression of endogenous Bmp activity prior to differentiation induction inhibited myotube formation as described above [19], but increased Bmp activity by
the addition of Bmp2 during this period also decreased the formation ofmultinucleated myotubes [19].
The present study revealed that the endogenous Bmp pathway
unexpectedly regulates myogenesis positively. Myoblasts, osteoblasts
and adipocytes are derived from common mesenchymal stem cells[39]. Involvement of Bmp in the commitment of osteoblasts and adipocytes is well known [39]. The present results in conjunction with
our previous results [19] indicate that the differentiation and maturation of committed myoblast-lineage cells are positively regulated byendogenous Bmp at various differentiation stages. Thus, Bmp may
act as a general promoter of mesenchymal cell differentiation andmaturation, and contribute to the growth and development of thewhole body.

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International Journal of Systematic and Evolutionary Microbiology (2011), 61, 2610–2615

DOI 10.1099/ijs.0.024521-0

Bifidobacterium kashiwanohense sp. nov., isolated
from healthy infant faeces
Hidetoshi Morita,1 Akiyo Nakano,1 Hiromi Onoda,1 Hidehiro Toh,2
Kenshiro Oshima,3 Hideto Takami,4 Masaru Murakami,1 Shinji Fukuda,5, 6
Tatsuya Takizawa,1 Tomomi Kuwahara,7 Hiroshi Ohno,5, 6 Soichi Tanabe8
and Masahira Hattori3
Correspondence
Hidetoshi Morita

1

morita@azabu-u.ac.jp

2

School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara,
Kanagawa 229-8501, Japan
Advanced Science Institute, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045,
Japan

3

Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa,
Chiba 277-8562, Japan

4

Microbial Genome Research Group, Japan Agency of Marine-Earth Science and Technology,
2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan

5

Laboratory for Epithelial Immunobiology, RIKEN Research Center for Allergy and Immunology,
1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, Japan

6

Graduate School of Nanobioscience, Yokohama City University, 1-7-29 Suehiro, Tsurumi,
Yokohama, Kanagawa 230-0045, Japan

7

Department of Molecular Bacteriology, Institute of Health Biosciences, University of Tokushima,
Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan

8

Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama,
Higashi-Hiroshima, Hiroshima 739-8528, Japan

Strains HM2-1 and HM2-2T were isolated from the faeces of a healthy infant and werecharacterized by determining their phenotypic and biochemical features and phylogenetic
positions based on partial 16S rRNA gene sequence analysis. They were Gram-positive,
obligately anaerobic, non-spore-forming, non-gas-producing, and catalase-negative non-motile
rods. They did not grow at 15 or 45 6C in anaerobic bacterial culture medium, and their DNA
G+C content was in the range 56–59 mol%. In enzyme activity tests, strains HM2-1 and HM2-2T
were positive for a/b-galactosidases and a/b-glucosidases but negative for b-glucuronidase andcystine arylamidase. An analysis of the cell-wall composition of strains HM2-1 and HM2-2T revealed
the presence of glutamic acid, alanine and lysine. The presence of fructose-6-phosphate
phosphoketolase shows that isolates HM2-1 and HM2-2T are members of the genus
Bifidobacterium. These two isolates belong to the same species of the genus Bifidobacterium.
Strain HM2-2T was found to be related to Bifidobacterium catenulatum JCM 1194T (97.4 % 16SrRNA gene sequence identity: 1480/1520 bp), Bifidobacterium pseudocatenulatum JCM 1200T(97.2 %: 1472/1514 bp), Bifidobacterium dentium ATCC 27534T (96.7 %: 1459/1509 bp) and
Bifidobacterium angulatum ATCC 27535T (96.5 %: 1462/1515 bp). The predominant cellular fatty
acids of strains HM2-1 and HM2-2T were 16 : 0 and 18 : 1v9c, with proportions greater than 18 %of the total. Phylogenetic analyses involving phenotypic characterization, DNA–DNA hybridizationand partial 16S rRNA gene sequencing proves that the strains represent a novel species of thegenus Bifidobacterium, for which the name Bifidobacterium kashiwanohense sp. nov. is proposed.
The type strain is HM2-2T (5JCM 15439T 5DSM 21854T).

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and partial hsp60 gene sequences of strain HM2-1 are AB491757 and AB578933,
respectively. Those for strain HM2-2T are AB425276.2 and AB491759.2, respectively.
Four supplementary figures are available with the online version of this paper.

2610

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Bifidobacterium kashiwanohense sp. nov.

The genus Bifidobacterium represents one of the bacterialgroups within the class Actinobacteria and is prevalent
within the gastrointestinal tracts of humans and animals(Mikkelsen et al., 2003). At the time of writing, the genus
Bifidobacterium is represented by over 30 species andsubspecies, and the type strains of Bifidobacterium adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B.
dentium, B. longum subsp. infantis, B. longum subsp. longum,
B. pseudocatenulatum and B. scardovii were isolated fromhuman specimens (Scardovi, 1986; Hoyles et al., 2002;
Sakata et al., 2002). The bifidobacteria have not been foundto cause human diseases; instead, they are considered to beprobiotic micro-organisms (Fuller, 1991; Ventura et al.,2009; Whorwell et al., 2006; Brenner et al., 2009).
During our studies, two isolates, HM2-1 and HM2-2T,from faeces of a healthy infant were not identified to thespecies level. Our results show that these isolates represent
a novel Bifidobacterium species, for which the name
Bifidobacterium kashiwanohense sp. nov. is proposed.
We isolated 51 strains of putative bifidobacteria inhabiting
the faeces of a healthy infant (male; 1.5 years old) in 2008.
Blood liver (BL) agar plates (Eiken Chemical) and anaerobic
bacterial culture medium (ABCM broth; Eiken Chemical)
were used for cell cultures. All 51 isolates were anaerobicallycultured at 37 uC in ABCM broth and on BL agar plates for
24 and 72 h, respectively. Of the 51 isolates, Bifidobacterium
longum subsp. longum (20 strains), B. longum subsp. infantis(2 strains), B. bifidum (6 strains) and B. breve (3 strains)
were identified, whereas the other isolates (20 strains) were
not identified to the species level. Of the 20 other isolates,
the unidentified strains HM2-1 and HM2-2T formed a
subcluster in the genus Bifidobacterium in the taxonomic
study. Strains HM2-1 and HM2-2T were found to be Grampositive, obligately anaerobic, non-spore-forming, non-gasproducing and catalase-negative non-motile rods. Table 1
shows the major characteristics of isolates HM2-1 andHM2-2T, and the related strains B. catenulatum JCM 1194T,
B. pseudocatenulatum JCM 1200T, B. dentium JCM 1195T
and B. angulatum JCM 7096T. The results were recorded
after 48 h at 37 uC. The isomer of lactic acid produced from
D-glucose was determined by using an F-kit [D(2)-lactic
acid/L(+)-lactic acid; Roche]. Other biochemical tests on
motility, growth at a fixed temperature and gas production from D-glucose were performed using the methods
described by Mitsuoka (1969). The optimal temperature for
growth of strains HM2-1, HM2-2T, JCM 7096T and JCM
1194T was 37 uC; no growth was observed at 15 or 45 uC in
ABCM broth. Although all strains grew at pH 7.2 andpH 8.0, no growth was observed at pH 4.5. B. pseudocatenulatum JCM 1200T grew weakly at 45 uC and pH 4.5. B.
dentium JCM 1195T grew at pH 4.5. Acid production fromcarbohydrates in ABCM broth is shown in Table 1. For thedetermination of phenotypic characteristics, API ZYM
tests (bioMérieux) were performed in duplicate using themethods recommended by the manufacturer. Cells werecollected from each ABCM culture by centrifugation at
12 000 r.p.m. for 5 min, inoculated onto the test strips andhttp://ijs.sgmjournals.org

Table 1. Physiological characteristics of isolates HM2-1 andHM2-2T and the type strains of closely related species of thegenus Bifidobacterium
Strains: 1, HM2-1; 2, HM2-2T; 3, B. catenulatum JCM 1194T; 4, B.
pseudocatenulatum JCM 1200T; 5, B. dentium JCM 1195T; 6, B.
angulatum JCM 7096T. +, Positive; –, negative; W, weakly positive.
All strains were positive for the following characteristics: fermentationof glucose, fructose, mannose, maltose and sucrose; growth in ABCM
broth at 37 uC, at intial pH 7.2 and 8.0, and under anaerobic
conditions; no growth in ABCM broth at 15 uC under anaerobic
conditions. All strains produced L-lactic acid and did not produce gas
from D-glucose. All strains produced acid from D-xylose, D-ribose, Larabinose, D-glucose, D-galactose, sucrose, maltose, lactose, melibiose,raffinose and salicin. None of the strains produced acid from glycerol
or rhamnose. All strains possessed leucine arylamidase, a-galactosidase, b-galactosidase, a-glucosidase and b-glucosidase; none possessed alkaline phosphatase, esterase (C4), lipase (C14), valine
arylamidase, trypsin, a-chymotrypsin, naphthol-AS-BI-phosphohydrolase, N-acetyl-b-glucosaminidase, a-mannosidase or a-fucosidase.
DNA G+C contents were determined by HPLC.
Characteristic
Growth at:
45 uC
Initial pH 4.5
Acid from:
D-Mannose
Cellobiose
Trehalose
Melezitose
DextrinStarchInulinD-Mannitol
D-Sorbitol
D-Fructose
API ZYM
Esterase lipase (C8)
Cystine arylamidase
Acid phosphataseb-Glucuronidase
DNA G+C content(mol%)

1

2

3

2
2

2
2

2
2

+
+
2
2

+
2
2

5

6

2
+

2
2

2
+
2
2

+
+
+

2
+
+

2
2
2
+
+

+
+
+
2
+
+
2
2
+
+

2
2
2
2
+
+
+
2
2
+

W

W

W

2
2
2
+
+

2

+
2
+
2
58.6

+
2
+
2
56.3

+
+
+
2
54.7

2
2
+
2
57.5

2
2
2
2
+ 2
+ 2
61.2 59.0

W

W

4
W
W

W

+
+
2
+
2
W

incubated for 4 h at 37 uC. Nineteen enzymic activities were
estimated by the API ZYM tests, and strains HM2-1 andHM2-2T showed 7 enzymic activities (Table 1). Enzyme
activity tests were positive for a/b-galactosidases and a/bglucosidases but negative for b-glucuronidase and cystine
arylamidase. Isolates HM2-1 and HM2-2T had fructose-6phosphate phosphoketolase activity like the reference strains(Biavati & Mattarelli, 1991; Gavini et al., 1991). An analysisof the cell-wall composition revealed the presence ofglutamic acid, alanine and lysine by ultraperformance liquid
chromatography according to the methods described by
Komagata & Suzuki (1987). The ACQUITY UPLC System(Waters) was used in this study. Whole-cell fatty acid methyl

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2611

H. Morita and others

esters were obtained by saponification, methylation andextraction followed by analysis using a standardized
Microbial Identification System (Microbial ID) (Tan et al.,2010). The Sherlock standard library MOORE5 5.00 made it
possible to standardize the bacterial fatty acid analysis(http://www.midi-inc.com) in Table 2.
The DNA G+C contents of strains HM2-1 and HM2-2T
were determined in three separate trials by high-performance liquid chromatography (Okamoto et al., 2008). The
DNA G+C contents of strains HM2-1 and HM2-2T were
58.6 and 56.3 mol%, respectively. These differ from thevalues obtained for B. catenulatum JCM 1194T and B.
dentium JCM 1195T, whose DNA G+C contents were 54.7
and 61.2 mol%, respectively (Scardovi & Crociani, 1974).
However, the DNA G+C content of strains HM2-1 andHM2-2T were similar to those of B. pseudocatenulatum
JCM 1200T (57.5 mol%) and B. angulatum JCM 7096T(59.0 mol%) (Scardovi & Crociani, 1974; Scardovi et al.,1979).
The 16S rRNA genes of strains HM2-1 and HM2-2T were
amplified by PCR using the universal primers 27F and
1492R (Weisburg et al., 1991). The closest known relativesof the isolate were determined by performing databasesearches, and the sequences of closely related species wereretrieved from public databases. Multiple alignments of thesequences were carried out using CLUSTAL W (Thompson
et al., 1994). We used the neighbour-joining method toTable 2. Cellular fatty acid contents (%) of isolates HM2-1 andHM2-2T and the type strains of closely related species of thegenus Bifidobacterium
Strains: 1, HM2-1; 2, HM2-2T; 3, B. catenulatum; 4, B.
pseudocatenulatum; 5, B. dentium; 6, B. angulatum. Data for reference
strains were obtained from the standard Library MOORE5 5.00 of the
Sherlock Microbial Identification System.
Fatty acid

1

2

3

4

5

6

10 : 012 : 011 : 0 DMA
14 : 014 : 0 DMA
16 : 1v9c
16 : 1v7c
16 : 0
16 : 0 DMA
16 : 1v7c DMA
17 : 0 anteiso
18 : 2v6,9c
18 : 1v9c
18 : 018 : 1v7c DMA
18 : 0 12-OH
18 : 1v9c DMA
18 : 0 DMA

1.6
3.8
0.6
6.8
2.7
3.4
0
30.3
0.9
0
0
0.8
18.1
6.1
0
0
19.1
0

2.1
8.1
0.8
15.9
3.3
6.3
0
26.8
0.7
0
0
1.1
19.4
2.5
0
0
9.1
0

0.7
1.3
0.7
5.3
3.5
2.2
1.8
25.7
0.5
1.1
0
0
18.3
3.3
0.6
1.5
23.3
0

1.2
2.9
0.5
10.5
2.7
2.2
3.9
13.7
0
0.5
0.7
0
33.6
2.3
0
0
12.2
0

0.7
1.2
0
5.1
2.9
1.8
1.1
25.8
0
0
0
0
22.8
6.9
0
0.6
23.2
0.6

1.5
2.7
0
8.4
2.1
2.6
2.6
18.1
0
0.6
0
0
33.5
3.7
0
0
14.3
0

2612

reconstruct a phylogenetic tree (Saitou & Nei, 1987) andestimated the robustness of the individual branches byperforming bootstrapping analysis with 1000 replicates(Felsenstein, 1985). Phylogenetic trees were also reconstructed using the maximum-likelihood (Cavalli-Sforza &Edwards, 1967) and maximum-parsimony (Kluge & Farris,1969) methods with PHYLIP version 3.66. The partial 16S
rRNA gene sequence of strain HM2-2T was consistent withthat of strain HM2-1. DNA–DNA hybridization was carriedout using the microdilution-well technique, with photobiotin for DNA labelling (Ezaki et al., 1989). Immobilized
DNA in microdilution wells was incubated with a photobiotin-labelled DNA probe prepared from each strain at
48 uC for 3 h in hybridization buffer containing 50 %formamide, after 30 min of incubation at 37 uC in
prehybridization buffer. The fluorescence intensity wasmeasured with a MicroPlate reader (PerkinElmer) at awavelength of 360 nm for excitation and 450 nm foremission. Values (%) of DNA–DNA hybridization werethe mean of the two trials in this study. Strains HM2-1 andHM2-2T shared high levels of DNA–DNA relatedness(92.1 %). We analysed the DNA profile by random amplified
polymorphic DNA (RAPD) PCR performed using therandom primers 103 (59-GTGACGCCGC-39), 127 (59ATCTGGCAGC-39) and 173 (59-CAGGCGGCGT-39)(University of British Columbia) to compare strains HM21 and HM2-2T. Their RAPD profiles (Supplementary Fig. S1
available in IJSEM Online) showed that they were different
strains of the same species, supported by phenotypic andbiochemical features and their phylogenetic position basedon partial 16S rRNA gene sequence and DNA–DNA
hybridization analyses.
Two organisms are considered to represent the same
species if their purified DNA exhibits greater than 70 %hybridization (Wayne et al., 1987). DNA–DNA hybridization analysis was carried out comparing isolates HM2-1
and HM2-2T and other related strains listed in Table 1
according to the method previously by Ezaki et al. (1989).
DNA–DNA relatedness values between strain HM2-2T
and B. catenulatum JCM 1194T were 8.1 %±1.6 % and14.6 %±2.4 % using the probes for HM2-2T and B.
catenulatum JCM 1194T, respectively. DNA–DNA relatedness values using the reference strains as DNA probes forstrain HM2-2T were as follows: HM2-2T and B. pseudocatenulatum JCM 1200T (6.5 %), HM2-2T and B. dentium
JCM 1195T (5.7 %), and HM2-2T and B. angulatum JCM
7096T (6.8 %).
We reconstructed a phylogenetic tree based on a total of 44
partial 16S rRNA gene sequences, including those of
members of the genus Bifidobacterium and related genera.
The tree was rooted using Actinomyces bovis (Fig. 1).
Strains HM2-1 and HM2-2T had 16S rRNA gene sequencesthat were similar to those of B. catenulatum, B. angulatumand B. dentium. Levels of similarity for the partial 16S
rRNA gene sequence of HM2-2T in relation to B.
catenulatum JCM 1194T, B. pseudocatenulatum JCM
1200T, B. dentium ATCC 27534T and B. angulatum

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Bifidobacterium kashiwanohense sp. nov.

Fig. 1. Phylogenetic relationship between isolates HM2-1 and HM2-2T, species of the genus Bifidobacterium and related
genera, determined by 16S rRNA gene sequencing. The tree was reconstructed using the neighbour-joining method.
Actinomyces bovis NCTC 11535T was used as an outgroup. Bootstrap percentages (based on 1000 replicates) are shown at
nodes; values lower than 50 % are not indicated. Accession numbers are given in parentheses. Bar, 10 % difference innucleotide sequence.

ATCC 27535T were 97.4 % (1480/1520 bp), 97.2 % (1472/
1514 bp), 96.7 % (1459/1509 bp) and 96.5 % (1462/1515bp), respectively. We obtained identical tree topologies in the
maximum-parsimony and maximum-likelihood analyses(Supplementary Figs S2 and S3).
Heat-shock protein (hsp60; also known as groEL, cpn60,
groES or dnaK) genes have also been adopted for taxonomic
studies of bifidobacterial species (Jian et al., 2001; Zhu et al.,
2003; Simpson et al., 2004; Delcenserie et al., 2005; Ventura
et al., 2005). The partial hsp60 gene sequence of strain HM22T was obtained by using the conditions and primers for
PCR amplification described by Jian et al. (2001) andOkamoto et al. (2008). We reconstructed a phylogenetic tree
http://ijs.sgmjournals.org

based on a total of 40 sequences of hsp60 genes, including
those of members of the genus Bifidobacterium and related
genera (Supplementary Fig. S4). Levels of similarity for thepartial hsp60 gene sequence of HM2-2T in relation to B.
catenulatum JCM 1194T, B. pseudocatenulatum JCM 1200T,
B. dentium ATCC 27534T and B. angulatum ATCC 27535T
were 96.1 % (567/590 bp), 90.8 % (536/590 bp), 92.0 %(544/591 bp) and 89.8 % (531/591 bp), respectively. The
16S rRNA and hsp60 gene sequences were well related anduseful for determining the phylogeny of strain HM2-2T.
Detailed characteristics of the novel isolates are provided in
the species description and in Table 1, and these characteristics were compared with those of the phylogenetic relatives

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H. Morita and others

B. catenulatum JCM 1194T, B. pseudocatenulatum JCM
1200T, B. angulatum JCM 7096T and B. dentium JCM 1195T.
The isolates were able to produce L(+)-lactic acid from Dglucose. Anaerobic conditions were essential for the growthof strains HM2-1 and HM2-2T. These two isolates could be
distinguished from B. pseudocatenulatum JCM 1200T, B.
angulatum JCM 7096T and B. dentium JCM 1195T by thepattern of acid production from D-mannose, cellobiose,trehalose, melezitose, dextrin, starch, inulin, D-mannitol, Dsorbitol and D-fructose. The cellular fatty acid profiles ofstrains HM2-1, HM2-2T and the related genera B. catenulatum, B. pseudocatenulatum, B. dentium and B. angulatum
are presented in Table 2. The predominant cellular fatty acidsof strains HM2-1 and HM2-2T were 16 : 0 and 18 : 1v9c, withproportions greater than 18 % of the total.
Experimental data suggest that isolates HM2-1 and HM22T can be assigned to the genus Bifidobacterium on the basisof their phylogenetic position; it can be concluded that these
two isolates might be considered as representing a single
novel species. Thus, these two isolates represent a novel
species, for which we propose the name Bifidobacterium
kashiwanohense sp. nov.
Description of Bifidobacterium kashiwanohense
sp. nov.
Bifidobacterium kashiwanohense (ka.shi.wa.no.hen9se. N.L.
neut. adj. kashiwanohense of Kashiwanoha in Japan, whichis the name of the area, University of Tokyo, where thisbacterium was originally isolated).
Cells are Gram-positive, non-motile, non-spore-forming
rods measuring 1.061.3 mm. Colonies on BL agar after
incubation under anaerobic conditions for 2 days at 37 uC
are beige, smooth and approximately 1.0 mm in diameter.
No growth is observed at 15 or 45 uC. Produces L(+)-lactic
acid from D-glucose. Produces acid from D-xylose, D-ribose,
D-mannose, cellobiose, D-sorbitol, L-arabinose, D-glucose,
D-fructose, D-galactose, sucrose, maltose, lactose, melibiose,raffinose and salicin, and, to a weaker extent, from dextrin.
Exhibits esterase lipase (C8), leucine arylamidase, acidphosphatase, a-galactosidase, b-galactosidase, a-glucosidase,b-glucosidase, and fructose-6-phosphate phosphoketolase
activities. The cell wall contains glutamic acid, alanine andlysine. The DNA G+C content is 56–59 mol%.
The type strain is HM2-2T (5JCM 15439T 5DSM 21854T),
isolated from faeces of a healthy infant (1.5 years old).

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2615

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535

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࢖࣑ࣝࢼ♫ࡢ &KLSDUUD\ ࢆ⏝࠸࡚  ୓ಶࡢ 613 ࢆ⥙⨶ⓗ࡟ゎᯒࡋࡓ࡜ࡇࢁࠊ≉Ⓨᛶ࡚ࢇ࠿ࢇ⨯ᝈ≟⩌࡛ኚ␗ࡢฟ⌧㢖ᗘࡢ㧗࠸ 613 ࢆ」ᩘぢฟࡋࡓ㸦ᅗ ࠊ⾲㸯㸧ࠋࡇࢀࡽࡢᗄࡘ࠿ࡣྠ୍ᰁⰍయୖࡢ୍㒊ࡢ㡿ᇦ࡟Ꮡᅾࡋࠊ㧗࠸࢜ࢵࢬẚࢆ♧ࡋࡓࠋࡇࡢ㡿ᇦ࡟ࡣ⬻ࡢᶵ⬟࡟㛵ࢃࡿ㑇ఏᏊࠊࣄࢺࡢ࡚ࢇ࠿ࢇࡢཎᅉ࡜࡞ࡿ㑇ఏᏊࠊࡉࡽ࡟≟ࡢ⑕ೃᛶ࡚ࢇ࠿
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࠸࡚ࡣࠊ≟ࡢ⬻⤌⧊࠿ࡽᢳฟࡋࡓ P51$ ࢆ⏝࠸࡚ 573&5 ࢆ⾜࠸ࠊ≟࡛Ⓨ⌧ࡋ࡚࠸ࡿࡇ࡜
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㸯.ㄽᩥⓎ⾲1) Ogawa M, Uchida K, Isobe K, Saito M, Harada T, Chambers JK, Nakayama H.: Globoid
cell leukodystrophy (Krabbe’s disease) in a Japanese domestic cat. Neuropathology
34,190-6, 2014.
2) Utsugi S, Saito M, Shelton D.: Resolution of polyneuropathy in a hypothyroid dog
following thyroid supplementation. J Am Anim Hosp Assoc 50,345-9, 2014.
3) Kurihara Y, Suzuki T, Sakaue M, Murayama O, Miyazaki Y, Onuki A, Aoki T, Saito M,
Fujii Y, Hisasue M, Tanaka K, Takizawa T.: Valproic acid, a histone deacetylase inhibitor,
decreases proliferation of and induces specific neurogenic differentiation of canine adipose
tissue-derived stem cells. J Vet Med Sci 76:15-23, 2014.
4) Tsuboi M, Uchida K, Ide T, Ogawa M, Inagaki T, Tamura S, Saito M, Chambers JK,
Nakayama H.: Pathological features of polyneuropathy in three dogs. J Vet Med Sci
75,327-35, 2013.
5) Mizukami K, Kawamichi T, Koie H, Tamura S, Matsunaga S, Imamoto S, Saito M,
Hasegawa D, Matsuki N, Tamahara S, Sato S, Yabuki A, Chang H, Yamato O.:Neuronal
ceroid lipofuscinosis in border collie dogs in Japan: Clinical and molecular epidemiologicalstudy (2000-2011). ScientificWorldJournal Volume 2012 Article ID 383174, 7 pages, 2012.
6) Ᏹὠᮌ┿୍ࠊ㰻⸨ᘺ௦Ꮚ㸸࣑࣑࢜࢟࢔/ࢽ࣮࣑ࣗࣟ࢜ࢺࢽ࢔ࡢ࣮ࣚࢡࢩ࣮ࣕࢸࣜ࢔ࡢ
1 ౛. ᪥⋇఍ㄅࠊ64, 56-60, 2011.
㸰㸬Ꮫ఍Ⓨ⾲
1) Nomura N, Saito M, Hasegawa D, Watanabe N. Uchida K, Okuno S, Nakai M, Orito K.:
Clinical efficacy and safety of zonisamide monotherapy in dogs with newly diagnosed
idiopathic epilepsy. 2015 ACVIM Forum, Indianapolis, June, 2015.
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Neuropathology 2014; 34, 190–196

doi:10.1111/neup.12076

Case Report

Globoid cell leukodystrophy (Krabbe’s disease)in a Japanese domestic cat
Mizue Ogawa,1 Kazuyuki Uchida,1 Kyoko Isobe,3 Miyoko Saito,2 Tomoyuki Harada,1James K. Chambers1 and Hiroyuki Nakayama1
1

Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Tokyo, Departments of 2Surgery II and 3Veterinary Teaching Hospital, School of Veterinary Medicine, Azabu University,
Kanagawa, Japan

A male Japanese domestic cat developed progressive limb
paralysis from 4 months of age. The cat showed visual
disorder, trismus and cognitive impairment and died at 9months of age. At necropsy, signiﬁcant discoloration of thewhite matter was observed throughout the brain and spinal
cord. Histologically, severe myelin loss and gliosis wereobserved, especially in the internal capsule and cerebellum. In the lesions, severe inﬁltration of macrophages
with broad cytoplasm ﬁlled with PAS-positive and nonmetachromatic granules (globoid cells) was evident. On
the basis of these ﬁndings, the case was diagnosed as feline
globoid cell leukodystrophy (Krabbe’s disease). Immunohistochemical observation indicated the involvement ofoxidative stress and small HSP in the disease.
Key words: cat, central nervous system, globoid cellleukodystrophy, Krabbe’s disease, macrophages.

INTRODUCTION
Globoid cell leukodystrophy (GLD; also known as
Krabbe’s disease) is an early-onset, rapidly progressive andfatal degenerative disease. The disease is pathologicallycharacterized by almost complete myelin loss, reactive
astrocytosis and the appearance of characteristic large
round cells called globoid cells in the white matter of the
CNS.1 Diseases identical to GLD have been reported inseveral animal species:2 dogs,3,4 sheep,5 rhesus monkeys6,7
and cats.8–10 GLD is deﬁned by the deﬁciency of a

Correspondence: Kazuyuki Uchida, DVM, PhD, Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657,
Japan. Email: auchidak@mail.ecc.u-tokyo.ac.jp
Received 7 August 2013; revised 29 August 2013 and accepted 24
September 2013; published online 8 November 2013.

© 2013 Japanese Society of Neuropathology

540

lysosomal enzyme, galactocerebrosidase (GALC), resulting in the accumulation of a cytotoxic metabolite,
psychosine.1 GALC-deﬁcient mice, such as Twitcher
mice,11,12 are being used as experimental GLD models.13
Besides GALC, the deﬁciency of saposin A, 14 one of thesphingolipid activator proteins, also causes an identicaldisease to GLD in mice15 and humans.10 While GALC
mutation was found in dogs16 and rhesus monkeys.17 To
date, there have been only three reports of the disease incats,8–10 indicating that the disease is extremely rare incats. These previous reports on feline GLD revealed
histopathological features characterized by symmetricalmyelin loss, astrocytosis and perivascular accumulation oflarge macrophages with intracytoplasmic deposits (globoid
cells).
The present report describes histopathological, immunohistochemical and ultrastructural features of GLD in ayoung cat. In addition, immunohistochemistry for nitricoxide (NOS) and two types of small HSP (sHSP) was
performed to assess the involvement of oxidative stressand/or sHSP, which are commonly upregulated in glial cells
in patients with neurodegenerative diseases.18

CLINICAL SUMMARY
A male Japanese domestic cat had exhibited hind limb
paresis since 4 months of age. Then the symptom progressed and clear tetraparesis developed. Although temporary recovery was achieved, loss of voluntary movement,
trismus, and visual and cognitive impairment wereobserved during the last 2 months before death at 9 monthsof age. No signiﬁcant ﬁndings were detected by X-ray
examination or hematologic and biochemical analyses. The
serum antibodies for feline immunodeﬁciency virus (FIV),
feline leukemia virus (FeLV), feline coronavirus, feline
parvovirus and Toxoplasma were all negative.

Krabbe’s disease in a cat

191

MATERIALS AND METHODSJapan) and embedded in Quetol651 resin (Nisshin EM).
Ultrathin sections were stained with uranyl acetate andlead citrate and examined with a Hitachi H-7500 transmission electron microscope (Hitachi High-Technologies,
Tokyo, Japan).

Tissue samples including the brain, spinal cord and brachial
plexus were ﬁxed in 10% neutral-buffered formalin, processed routinely and embedded in parafﬁn wax. Parafﬁnsections 4 μm thick were stained with HE. Some selected
sections were also subjected to PAS, toluidine blue
(TB, pH 7.0), LFB, Sudan-black and von-Kossa staining.
Immunohistochemistry was performed using the Envision
polymer method. The primary antibodies used are listed inTable 1. Deparafﬁnized sections were ﬁrst autoclaved at
120°C for 10 min in 10 mmol/L citrate buffer (pH 6.0) or
Target retrieval solution (pH 9.0), for antigen retrieval.
Then, the tissue sections were treated with 3% hydrogen
peroxide (H2O2)-methanol at room temperature for 5 min
and incubated in 8% skimmed milk–Tris-buffered salinewith 0.2% Tween 20 (TBST) at 37°C for 1 h to avoid nonspeciﬁc reactions. The sections were then incubated at 4°C
overnight with one of the primary antibodies. Afterwashing three times in TBST, the sections were incubatedwith Envision horseradish peroxidase (HRP) mouse or
rabbit polymer (Dako, Glostrup, Denmark) at 37°C for
40 min Then, the sections were washed with TBS and visualized with 0.05% 3-3’diaminobenzidine and 0.03% H2O2
in TBS. Counterstaining was performed with Mayer’s
hematoxylin.
For electron microscopic analysis, the formalin-ﬁxed
cerebellar white matter was ﬁxed again in 2%
glutaraldehyde in 0.1 mol/L phosphate buffer (PB) at 4°C
for 2 h, and post-ﬁxed in 1% OsO4 at 4°C for 2 h. The
samples were dehydrated in an ethanol series, replacedwith QY-1 solution (Nisshin EM Corporation, Tokyo,

PATHOLOGICAL FINDINGS
No marked gross lesions were detected in the CNS at
necropsy, although discoloration of the white matter wasobserved on the cut surface of the formalin-ﬁxed brain(Fig. 1) and spinal cord.
Histologically, thinning of the white matter wasobserved throughout the brain and spinal cord. LFBstaining (Figs 2,3a) and immunostaining for myelin basic
protein (MBP) revealed marked myelin loss in thewhite matter. Severe axonal degeneration characterized
by swollen and fragmented axons (Fig. 3b) was also
observed. Astrocytosis (Fig. 3c) and inﬁltration of ionized
calcium-binding adapter molecule 1 (Iba-1)-positive cells(Fig. 3d) were frequently observed in the lesions.
Oligodendrocyte transcription factor 2 (Olig2)-positive
oligodendrocytes slightly decreased in number. Theselesions were severe in the white matter of the internal
capsule, cerebellum, medulla and spinal cord, but relatively less severe in the white matter of the cerebrum. In
the deep white matter of the cerebellum, focal and symmetric necrosis was evident. In the necrotic areas, there
were many calciﬁed deposits forming psammoma bodies.
The bodies were stained with LFB, PAS and von Kossa(Fig. 4).

Table 1 Primary antibodies used
Antibody

Type

Dilution

SourceMarker for

Antigen retrieval

GFAP
NF
pNF-H&M
MBP
Olig2

p
m
m
p
p

1:1000pre-diluted
1:10001:200
1:500

Dako, Glostrup, Denmark
Dako
Millipore, MA, USA
Dako
Millipore

Astrocyte
Axon/neuron–
MyelinOligodendrocyte

CD3
CD20
Iba1

p
p
p

1:50
1:200
1:500

T cellB cellMicroglia/macrophage

HLA-DR
HSP27
αB-crystallin
iNOS
HO-1

m
p
m
p
p

1:100
1:100
1:10001:200
1:100

Macrophage––––

Citrate buffer, pH 6.0
Citrate buffer, pH 6.0
Citrate buffer, pH 6.0
Citrate buffer, pH 6.0
Citrate buffer, pH 6.0

SOD1
SOD2

p
p

1:200
1:200

Dako
Thermo Scientiﬁc, CA, USA
Wako Pure Chemical Industries,
Osaka, Japan
Dako
Cell Signaling Technology, MA, USA
Santa Cruz Biotechnology, CA, USA
Calbiochem, CA, USA
ENZO Life Sciences, Plymouth
Meeting, PA, USA
ENZO Life Sciences
ENZO Life Sciences

None
None
None
Citrate buffer, pH 6.0
Target retrieval solution(Dako), pH 9.0
Citrate buffer, pH 6.0
None
Citrate buffer, pH 6.0––

Citrate buffer, pH 6.0
Citrate buffer, pH 6.0

HLA-DR, humanleukocyte antigen type DR; HO-1, heme oxygenase-1; Iba1, ionized calcium-binding adapter molecule 1; iNOS, inducible nitricoxide synthase; m, mouse monoclonal; MBP, myelin basic protein; NF, neuroﬁlament; Olig2, oligodendrocyte transcription factor 2; p, rabbit
polyclonal; SOD, superoxide dismutase.

© 2013 Japanese Society of Neuropathology

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M Ogawa et al.

b
Fig. 1 Cut surfaces of the formalin-ﬁxed
brain. Severe discoloration throughout thewhite matter (arrowheads). (a) Cerebrum
and thalamus, hypothalamus level. (b)Frontal lobe. (c) Cerebellum and ventral
pons. Scale bar = 1 cm.

c

a

a

b
Fig. 2 The cerebellum. LFB-HE. (a) The present case. (b) A
normal adult cat. Almost complete myelin loss is observed in thepresent case (a), compared with the control (b). Scale bar = 1 cm.

In the gray matter throughout the brain and spinal cord,
prominent proliferation of gemistocytic astrocytes wasobserved. In the cerebellar cortex, there was moderate
loss of granule cells and Purkinje cells with proliferationof Bergmann’s glia. Neuroﬁlament- and phosphorylated neuroﬁlament-positive torpedoes were frequently
observed in the granular cell layer (Fig. 5).
A large number of macrophages with broad cytoplasm
and single or multiple eccentric nuclei (globoid cells) wereobserved in the lesions of the brain white matter, especiallyin the perivascular area (Fig. 6a). In the thoracic andlumbar cord, where myelin and axons were completely lost,
there were a few globoid cells. Immunohistochemically,
these globoid cells were strongly positive for Iba-1 (Fig. 6a,
insertion) and HLA type DR (HLA-DR). The globoid
cells had ﬁne granular or ﬁlamentous deposits in their cytoplasm, which were PAS-positive (Fig. 6b), Sudan-blacknegative and non-metachromatic by TB staining (Fig. 6c).
Ultrastructural examination indicated the detailed features of the cytoplasmic deposits: variably sized, andstraight and curved tubular structures (Fig. 6d). In thesame area, mild perivascular inﬁltration of CD3-positive
lymphocytes was also observed, whereas there was no inﬁltration of CD20-positive cells.
The results of immunohistochemistry also demonstrated that the cytoplasm of the globoid cells and of the
few reactive astrocytes was positive for inducible nitricoxide (iNOS) (Fig. 7). Superoxide dismutase (SOD)1
expression was seen in reactive astrocytes and degenerated
axons. Granules strongly positive for SOD2 were observedin cytoplasm of globoid cells, excluded by cytoplasmic
deposit. Reactive astrocytes were occasionally positive forheme oxygenase-1 (HO-1).
On the other hand, reactive astrocytes were mostly positive for αB-crystallin in a broad area of the CNS. HSP27positive astrocytes were rarely observed.
In the peripheral nerves including the brachial plexus,
trigeminal nerve and dorsal and ventral roots of the spinal
cord, various levels of myelin loss and axonal degeneration
were observed, while there were no globoid cells in thelesions. No signiﬁcant lesions were observed in the visceralorgans.

DISCUSSION
Considering the severe myelin loss throughout the CNS
and the appearance of characteristic globoid cells withintracellular PAS-positive and non-metachromatic deposits, the present case was diagnosed as feline GLD. In© 2013 Japanese Society of Neuropathology

542

Krabbe’s disease in a cat

a

193

b

c

d

Fig. 3 A lesion of the cerebellar white matter. (a) LFB-HE. Immunostain for (b) neuroﬁlament, (c) GFAP and (d) Iba-1. In addition to
myelin loss (a), severe axonal degeneration/loss (b) and astrogliosis (c) are observed in the area with macrophage inﬁltration (d). Scalebar = 50 μm.

Fig. 4 Psammoma body in the cerebellar
white matter. (b)–(e) are higher magniﬁcations of (a): (a) and (b) HE, (c) LFB-HE, (d)PAS, and (e) von Kossa. The eosinophilic
deposits were positive for LFB, PAS and vonKossa stains. Scale bar = 100 μm (a) and25 μm (b–e).

aaddition, the formation of torpedoes observed in thepresent case is also common in human GLD.19
As far as we know, there have been three reports on
spontaneous GLD in cats8–10 and the distribution of thelesions and histological ﬁndings in the present case resemble those of GLD in humans20 and in cats,9 indicating thatthe present feline case is included in the same disease

b

c

d

e

category. This is the ﬁrst report of GLD in a Japanese
domestic cat. In addition, despite the exact age of the catbeing unknown, the present case may be relatively oldcompared with the previous feline GLD cases, probably
because of effective care.
Psammoma body-like calcium deposits probably
contained decayed products of myelin because they

© 2013 Japanese Society of Neuropathology

543

194

a

a

c

M Ogawa et al.

b

Fig. 5 A torpedo in the cerebellum.
(a) HE. Immunostain for (b) neuroﬁlamentand (c) phosphorylated neuroﬁlament.
Neuroﬁlament- and phospho-neuroﬁlamentpositive torpedoes were frequently observed.
Scale bar = 25 μm.

cbFig. 6 Globoid cells in the cerebellar whitematter. (a) HE, (b) PAS, (c) toluidine blue(TB) and (d) ultrastructural observation ofa globoid cell. Globoid cells were roundshaped macrophages with broad cytoplasm(a). The deposits in the cytoplasm of a
globoid cell were PAS-positive (b) andnon-metachromatic with TB stain (c).
Ultrastructurally, ﬁne tubular structures witha diameter of 30–100 nm were accumulated
in the cytoplasm of a globoid cell (d). Scalebar = 25 μm (a), 50 μm (b, c) and 500 nm (d).

dwere LFB-positive. A slight decrease of olig2-positive
oligodendrocytes in spite of the severe myelin loss suggeststhat feline GLD may be a disease mostly caused by thedysfunction of myelination, maybe due to psychosine accumulation, rather than by the degeneration of mature
myelin. Biochemical analysis or analysis of GALC or thesaposin A gene was not performed because all tissues were
unfortunately formalin-ﬁxed at nectropsy because of thesuspicion of infectious diseases. To determine whether thisfeline disease is exactly the same as human GLD, gene
analysis should be performed.
The inﬁltration of CD3-positive T cells into the lesions
in the present case indicates the involvement of immunological responses in the pathogenicity of the feline disease,like in human GLD.19 However, the inﬁltration was notvery severe in view of the longer survival and/or because ofsteroid treatment.
The production of reactive oxygen species (ROS) was
observed in Twitcher mice, a GLD model, as a secondary
event following psychosine toxicity.21 The expression of
iNOS, which results in ROS production, was reported to be
distributed mostly in GFAP-positive astrocytes in human
GLD,21 and in both astrocytes and globoid cells of GLD ofrhesus macaques,7 as well as in the present case. On theother hand, in the present case, SOD2, a mitochondrial
antioxidant enzyme, was strongly expressed by globoid
cells themselves. SOD117 and HO-1,23 the molecules usuallyinduced by ROS, were also expressed by reactive© 2013 Japanese Society of Neuropathology544

Krabbe’s disease in a cat

Fig. 7 The cerebellum. Immunohistochemistry for inducible nitric oxide (iNOS),
SOD, heme oxygenase-1 (HO-1) and sHSPs.
(a) iNOS, (b) SOD1, (c) SOD2, (d) HO-1, (e)αB-crystallin and (f) HSP27. Positive staining
for iNOS and SOD2 was observed in thecytoplasm of globoid cells, not including ﬁlamentous deposit (a, c). SOD1 (b) and HO-1(d) expression was occasionally observed inreactive astrocytes. αB-crystallin expression
was observed in reactive astrocytes (e),whereas only few astrocytes were positive for
HSP27 (f). Scale bar = 50 μm.

195

a

b

c

d

e

f

astrocytes. Although it is interesting that the iNOS expression patterns vary among animals, it may be a common
accelerating factor for disease progression, not an exclusive
factor for the pathogenesis. The induction of the antioxidant enzymes may be a physiological consequence in therecovery of neuronal tissue following oxidative damage.
Increased expressions of αB-crystallin and HSP27
were reported in Rosenthal ﬁbers of Alexander’sdisease and in GFAP-positive gliosis lesions of humanneurodegenerative diseases,18 but the roles of these molecules in the pathogenicity of such neurodegenerative diseases are still controversial. The strong expressions of themolecules in the CNS of the present case suggest involvement of the molecules in disease progression also in feline
GLD.

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the sphingolipid activator protein (prosaposin) gene
results in a late-onset, chronic form of globoid cellleukodystrophy in the mouse. Hum Mol Genet 2001;
10 (11): 1191–1199.
16. Victoria T, Raﬁ MA, Wenger DA. Cloning of thecanine GALC cDNA and idntiﬁcation of the mutation
causing globoid cell leukodystrophy in West Highland
White and Cairn terriers. Genomics 1996; 33 (3):457–462.
17. Andersen JK. Oxidative stress in neurodegeneration: cause or consequence? Nat Med 2004; 10: S18–
S25.
18. Van Rijk A, Bloemendal H. Alpha-B-crystallinin neuropathology. Ophthalmologica 2000; 214 (1):
7–12.
19. Itoh M, Hayashi M, Fujioka Y, Nagashima K,
Morimatsu Y, Matsuyama H. Immunohistologicalstudy of globoid cell leukodystrophy. Brain Dev 2002;
24 (5): 284–290.
20. Percy AK, Odrezin GT, Knowles PD, Rouah E,
Armstrong DD. Globoid cell leukodystrophy: comparison of neuropathology with magnetic resonanceimaging. Acta Neuropathol 1994; 88 (1): 26–32.
21. Hawkins-Salsbury JA, Qin EY, Reddy AS, Vogler CA,
Sands MS. Oxidative stress as a therapeutic target ingloboid cell leukodystrophy. Exp Neurol 2012; 237 (2):444–452.
22. Giri S, Jatana M, Rattan R, Won JES, Singh I, Singh
AK. Galactosylsphingosine (psychosine)-induced
expression of cytokine-mediated inducible nitric oxide
synthases via AP-1 and C/EBP: implications for
Krabbe disease. FASEB J 2002; 16 (7): 661–672.
23. Schipper HM, Song W, Zukor H et al. Heme
oxygenase-1 and neurodegeneration: expanding frontiers of engagement. J Neurochem 2009; 110 (2): 469–
485.

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CASE REPORTS

Resolution of Polyneuropathy in a Hypothyroid
Dog Following Thyroid Supplementation
Shinichi Utsugi, DVM, Miyoko Saito, DVM, PhD, G. Diane Shelton, DVM, PhD, DACVIM

ABSTRACTAn 8 yr old male golden retriever was evaluated because of chronic, progressive, multiple neurologic signs. Physical examination showed marked obesity and facial swelling with a “tragic facial expression.” Neurologic evaluation revealed the dog
had multiple cranial nerve deﬁcits and lower motor neuron signs in the pelvic limbs. Serum biochemical analysis and thyroid
function tests were consistent with hypothyroidism. A biopsy from the common peroneal nerve revealed a loss of myelinatedﬁbers, inappropriately thin myelinated ﬁbers, and resolving subperineurial edema. The diagnosis of polyneuropathy associatedwith hypothyroidism was made. Levothyroxine therapy was initiated. Response to levothyroxine treatment was slow, with mostneurologic abnormalities persisting for .6 wk. However, the dog made a full neurologic recovery within 6 mo. Although theoccurrence of polyneuropathy in dogs resulting from hypothyroidism has been controversial, the study authors demonstrated
that hypothyroid polyneuropathy can occur in dogs as documented in humans. This is the ﬁrst report describing long-term
follow-up information together with detailed pathological features of hypothyroid polyneuropathy in a dog. In hypothyroid
polyneuropathy, the response to thyroid replacement may be slow, but a recovery can be expected if treatment is initiatedbefore peripheral nerve ﬁber loss becomes severe. (J Am Anim Hosp Assoc 2014; 50:345–349. DOI 10.5326/JAAHA-MS-6035)

Introduction

canine peripheral nerves may be relatively resistant to chronic

Hypothyroidism is the most common hormonal disease in dogs.1

thyroid hormone deﬁciency.8

Because thyroid hormone has a wide variety of physiologic effects,

In humans, it is known that hypothyroidism is an un-

this disease can cause a broad range of problems in many organs.

common cause of peripheral neuropathy, such as carpal tunnel

Reportedly, various neurologic signs resulting from either central

syndrome and some types of polyneuropathies.9 Although theor peripheral nervous system involvement, such as vestibular dys-pathogenesis of peripheral neuropathy due to hypothyroidism

function; facial paralysis; and reduced spinal reﬂexes, ataxia, andremains to be fully elucidated, it is generally understood thatparesis, are associated with hypothyroidism. However, a causal re-

hypothyroidism can cause mucous deposits and ﬂuid retention

lationship with hypothyroidism has been proven in only a limited

resulting in nerve damage through a compression mechanism.9

2–7

number of cases.

In this report, an older golden retriever with chronic,

The occurrence of hypothyroid polyneuropathy in dogs has

progressive, multiple neurologic signs was diagnosed with hy-

been controversial, and pathological ﬁndings have been mentioned

pothyroid polyneuropathy after complete diagnostic investi-

brieﬂy in only rare cases.2,3,5 In a recent study in which hypo-gations. To the authors’ knowledge, this is the ﬁrst reportthyroidism was experimentally induced in dogs, peripheral neu-

describing long-term follow-up information together with de-ropathy was not evident clinically, electrophysiologically, or

tailed pathological features of hypothyroid polyneuropathy of8histopathologically. Based on that study, it was suggested thata dog.

From the Department of Surgery II, School of Veterinary Medicine,
Azabu University, Kanagawa, Japan (S.U., M.S.); and Departmentof Pathology, University of California, San Diego, La Jolla, CA(G.D.S.).

ATP, adenosine triphosphate; PO, per os; T4, thyroxine

Correspondence: msaito@azabu-u.ac.jp (M.S.)ª 2014 by American Animal Hospital Association

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Case Reportapproach for whether levothyroxine treatment would have a pos-

An 8 yr old male golden retriever weighing 47.5 kg was referreditive effect on neurologic signs. The dog was anesthetized andto the Azabu University Veterinary Teaching Hospital for

MRI of the head and spine was performed 9 days after initial

evaluation of head tilt, ptosis, pelvic limb ataxia, and exercise

presentation. T1-weighted, T2-weighted, and ﬂuid-attenuatedintolerance. Clinical signs began 1 mo prior to evaluation andinversion recovery scans were performed in the transverse andhad progressively worsened. The owner also noticed that the dog

sagittal planes. Additionally, gadoteridolb at 0.2 mL/kg was ad-

was less active and appeared somewhat depressed, althoughministered IV, and a postcontrast transverse T1-weighted se-appetite and water consumption was normal. The dog’s vacci-

quence was obtained. MRI showed no abnormality. Following

nation status was current. On physical examination, the dog had

MRI, cerebrospinal ﬂuid was obtained from the cerebellomedul-

a rectal temperature of 38.28C, pulse rate of 96 beats/min, and re-lary cistern. No abnormalities were detected on cytologic analysis

spiratory rate of 24 breaths/min. The dog was obese (body condition

including cell counts and protein concentrations. Serologic eval-score 5 out of 5) and appeared to be moderately swollen in the faceuation for canine distemper virus immunoglobulin G antibodies

with a “tragic facial expression.” Mild otitis externa of both ears wasin the cerebrospinal ﬂuid and serum were negative. Polymerase

noted. Neurologic examination revealed ataxia of both pelvic limbs,

chain reaction examination of nasal, oral, and conjunctival epi-

but conscious proprioception and hopping reactions were normal inthelium, stool, and digital pads was performed and were negative

all limbs. Patellar and withdrawal reﬂexes were diminished in thefor the canine distemper virus. Toxoplasma gondii and Neospora

right pelvic limb. There was a right-sided head tilt together withcaninum immunoglobulin G antibodies were both negative in thea right-sided ptosis. Pain sensation was considered normal in allserum. Serum T4 was measured before anesthesia and was con-limbs. The remaining neurologic examinations did not show any

sidered still slightly high (94.36 nmol/L). A dose reduction ofabnormalities. The complete blood cell count and serum bio-

levothyroxine Na to 0.005 mg/kg PO q 12 hr was instructed.

chemical analysis (after fasting for 12 hr) were performed. Results

Hypercholesterolemia (19.89 mmol/L) and hypertriglyceridemiaof the complete blood cell count were unremarkable. Serum(1.36 mmol/L) were further improved.

biochemical analysis revealed hypercholesterolemia (46.1 mmol/L;

Five weeks after initial presentation, the dog was reevaluatedreference range, 2.77–8.13 mmol/L) and hypertriglyceridemia

at the hospital. The body weight decreased to 43.5 kg. Ataxia,(13.97 mmol/L; reference range, 0.19–1.15 mmol/L), with no otherexercise intolerance, and facial swelling were resolved, but otherabnormalities. Hypothyroidism was suspected, and thyroid func-

neurologic signs including diminished patellar and withdrawal

tion tests were performed. They revealed low serum thyroxine (T4)reﬂexes, head tilt, and ptosis did not show any apparent im-,5.13 nmol/L; (0.3 mg/dL; reference range, 18.8–61.54 nmol/L; 1.1–

provement. Serum creatine kinase was measured and was within

3.6 mg/dL), low serum free T4 [(3.86 pmol/L; 0.3 ng/dL; reference

normal limits (46.0 IU/L; reference range, 40–151 IU/L). The

range, 11.58–33.46 pmol/L; 0.9–2.6 ng/dL)], and an increased serum

dog was placed under general anesthesia for evaluation of lower

level of thyroid-stimulating hormone (1.6 ng/mL; reference range,

motor neuron dysfunction. Electromyography was within normal

0.08–0.32 ng/mL). Thoracic radiographs were interpreted as normal.

limits. Motor nerve conduction velocity in the right tibial nerve

On the basis of thyroid function test results, a diagnosis of

was within the reference range, but was polyphasic with a de-hypothyroidism was made. Differential diagnoses for neurologic

creased amplitude of the compound muscle action potential

signs included a brain stem tumor, encephalitis, and polyneuropathy(0.9 mV; reference range, 23.3 6 2.3 mV).10

due to variable causes, including hypothyroidism and possibly

Biopsy specimens were obtained from the right common

otitis media and/or interna. Levothyroxine Naa (0.02 mg/kg per os

peroneal nerve and were ﬁxed in neutral-buffered 10% formalin.

[PO] q 12 hr) was initiated.

Nerve specimens were plastic embedded and evaluated in 1 mm

Seven days after the ﬁrst presentation, the dog was reevaluatedsections (Figure 1). Subjectively, there was a mild (medium-sizeat the hospital. The dog was more active, but did not show any

fascicle) to moderate (smaller fascicle) loss of myelinated ﬁberschanges in other clinical signs. Serum T4 levels were measured andwithout obvious axonal degeneration (Figure 1A). Occasional

were high (130.77 nmol/L). Serum biochemical analysis revealed

large and small caliber ﬁbers showed inappropriately thin myelinsome improvement of hypercholesterolemia (33.39 mmol/L) andsheaths for the axon diameter (Figure 1B). Numerous collagenhypertriglyceridemia (2.05 mmol/L). The dose of levothyroxine

bundles consistent with resolving subperineurial edema were ev-

Na was decreased to 0.01 mg/kg PO q 12 hr.

ident in all fascicles (Figure 1A). To determine quantitatively if

At that time, the owner decided to have the dog undergoﬁber loss was selective to speciﬁc nerve ﬁber diameters, axonal size

further diagnostic testing rather than adopting a “wait and see”

frequency distribution was determined for the golden retriever

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Polyneuropathy Resolution Following Thyroid Supplementation

FIGURE 2 Histogram showing axonal size frequency distribu-

tion of myelinated nerve ﬁbers from the peroneal nerve of the dog ofthis report compared with peroneal nerves from three large-breed
control dogs $8 yr of age and no neurologic dysfunction. Loss ofmyelinated ﬁbers in the 3–7 mm range was demonstrated in the
hypothyroid dog. Bars, standard error of the mean for control dogs.

nerve ﬁber loss was limited to those with axon diameters in the
3–7 mm range.
Serum T4 was again reassessed and was within the normal
range (40.85 nmol/L). Serum biochemical analysis revealed further improvement in hypercholesterolemia (10.46 mmol/L) andhypertriglyceridemia (0.59 mmol/L). Although the neurologic
signs remained, biochemical abnormalities showed marked improvement and the levothyroxine Na dosage was not changed(i.e., 0.005 mg/kg PO q 12 hr).
Six weeks after the initial presentation, the withdrawal reﬂex
normalized but other neurologic abnormalities including diminished patellar reﬂex, head tilt, and ptosis persisted. The dog wasagain reevaluated at 6 mo following the initial presentation. The
owner reported that the dog showed steady improvement andbecame completely normal. The body weight increased to 45.9 kg.
The head tilt and ptosis resolved, and the patellar reﬂexes normalized. There were no abnormalities noted on neurologic examFIGURE 1

ination at this time.

A: Low-power, resin-embedded, toluidine blue-

stained 1 mm section from the peroneal nerve biopsy showing two
nerve fascicles. Resolving subperineurial edema is present in both

Discussion

fascicles. Although axonal degeneration was not noted, nerve ﬁber

The polyneuropathy documented in this report is most likely

loss was suspected indicating previous degeneration. B: Higher-powera result of hypothyroidism because the dog had clinical andimage of the larger nerve fascicle highlighting edema (*) and nerve

biochemical abnormalities consistent with hypothyroidism, otherﬁbers with inappropriately thin myelin sheaths for the axon diameter

causes of neurologic signs such as intracranial disorders were(arrows). Bar represents 50 mm.

ruled out, and lastly, complete recovery of neurologic abnormalities
was achieved following supplementation with levothyroxine Na
alone.

of this report and compared with three large-breed dogs with-In a recent study in which hypothyroidism was experi-

out neurologic abnormalities that were used as controls foranother study (Figure 2).

11mentally induced in young adult beagles, peripheral neuropathy

Compared with the control group,

JAAHA.ORG

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was not evident 18 mo following induction.8 However, older dogs

axonal degeneration were noted on histopathological examinationand dogs with a larger body size (like the dog described herein)of peripheral nerves in two dogs with signs of polyneuropathy andwere not studied. Peripheral neuropathy associated with hypo-

reduced T4 levels.14 However, both dogs were diagnosed withthyroidism may target only some dogs, and the authors speculate

neoplasia and thyroid-stimulating hormone concentration wasthat age and body size may play a role in disease development.

not measured. Therefore, it cannot be ruled out that poly-

The pathophysiology of hypothyroidism-induced peripheral

neuropathy in those cases was a paraneoplastic syndrome or theneuropathy in adults has not been clariﬁed; however, mucinous

cause of reduced T4 levels was euthyroid sickness.

deposits around peripheral nerves (i.e., mechanical compression

Pathological changes in peripheral nerves in the dog de-and entrapment of nerves by localized myxedema) is considered toscribed herein included myelinated ﬁber loss accompanied by

be the main cause of hypothyroidism-associated mononeuropathy

resolving subperineurial edema. To the authors’ knowledge, edema-

9and multiple mononeuropathy. In diffuse polyneuropathy asso-

tous changes within nerves in dogs with this disease have notciated with hypothyroidism, direct metabolic changes due tobeen previously reported, but are consistent with pathologicala decreased thyroid hormone level in either Schwann cells or

ﬁndings in human hypothyroid polyneuropathy.15 Although the9,12neurons may be considered.

For example, it has been dem-cause of edema was not investigated in this case, epi- and peri-

onstrated in rat studies that thyroidectomy results in slowed ax-

neurial inﬁltration by edematous protein assumed to be a meta-

onal transport.13 Because thyroid hormone acts on mitochondria

chromatic mucoid material, probably mucopolysaccharide-protein

directly and increases adenosine triphosphate (ATP) production

complexes, has also been observed in humans.15 Perineurial de-and ATPase activity by promoting cellular respiration, reduced

position of that substance has not been reported in hypothyroid

thyroid hormone levels induce ATP deﬁciency and reducedogs, but subcutaneous hyaluronic acid deposition manifesting as

ATPase activity levels in nerve cell bodies. That results in re-skin myxedema has been described.16 Hyaluronic acid is a muco-

1

duced Na /potassium pump activity and impairment of axonaltransport.

polysaccharide and its deposition may cause edema because it ismarkedly hygroscopic. Furthermore, myelinated ﬁber loss ap-

13

Motor nerve conduction velocity was not decreased in theparent in the current case is a common pathological ﬁnding in

dog described herein. That ﬁnding is not unexpected given thathuman hypothyroid neuropathy.9 In this dog, edematous de-the largest caliber fastest conducting nerve ﬁbers were not de-position resulting in compression of nerve ﬁbers may have oc-

creased (Figure 2). However, a markedly decreased amplitude

curred due to hypothyroidism.

of the compound muscle action potential and temporal dis-

In canine hypothyroid polyneuropathy, both spinal andpersion were noted. Those ﬁndings may reﬂect loss of 3–7 mm

cranial nerves may be impaired.5 However, there are reports innerve ﬁbers and the presence of the inappropriately thin mye-

which various cranial nerve abnormalities were induced by cere-

linated ﬁbers identiﬁed by peripheral nerve biopsy in the dog ofbral infarction due to the cerebral artery atherosclerosis.17,18 Be-

this report. Regarding electrophysiological abnormalities of spon-

cause a necropsy was not performed in this dog, it cannot betaneously occurring hypothyroidism in dogs, variable changes

deﬁnitively concluded that the cranial nerve abnormalities were(including reduced motor-nerve conduction velocity and electro-

part of a polyneuropathy and not a central manifestation. How-

myographic abnormalities, such as ﬁbrillation potentials, positive

ever, the neurologic history of this dog (i.e., chronic onset andsharp waves, and complex repetitive discharges) have been re-progression) was not suggestive of a vascular disorder. Further-ported.

2,3Abnormalities on electromyography were not noted in

more, abnormalities such as necrotic lesions were not noted on

the dog in this report. Although muscle biopsies were not per-

MRI. Cranial nerve signs slowly improved and resolved along

formed, the absence of both muscle atrophy and hypertrophy

with spinal nerve signs following only hormone replacement

clinically together with the depressed spinal reﬂexes suggest thattherapy.
Most dogs with hypothyroid myopathy rapidly respond to

hypothyroid myopathy was unlikely.
There is only limited information concerning pathologicalthyroid replacement therapy, and clinical normalization is ob-changes in peripheral nerves in dogs with hypothyroidism. Pe-

tained within 2–8 wk.2,3 In contrast, the response of peripheral

ripheral nerve biopsy abnormalities included myelin irregulari-neuropathy to thyroid replacement therapy is not clear and it isties, intercalated internodes, internodal globules, and axonal

said that responses to treatment may be poor.19 The dog in thisnecrosis.2,3,5 Neurogenic atrophy was often seen on muscle biopsy

report required 6 wk to observe the ﬁrst improvement of neuro-of dogs showing clinical signs of polyneuropathy.2,3 Segmental

logic signs after initiation of thyroid replacement. The remaining

demyelination and marked myelinated ﬁber loss accompanied bysigns slowly improved, and the dog was neurologically normal on

348

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50:5 Sep/Oct 2014550

Polyneuropathy Resolution Following Thyroid Supplementation

follow up at 6 mo. It has been pointed out that relatively early

4. Higgins MA, Rossmeisl JH Jr, Panciera DL. Hypothyroid-associatedcentral vestibular disease in 10 dogs: 1999–2005. J Vet Intern Med
2006;20(6):1363–9.
5. Dewey CW, Shelton GD, Bailey CS. Neuromuscular dysfunction
in ﬁve dogs with acquired myasthenia gravis and presumptive
hypothyroidism. Prog Vet Neurol 1995;6(4):117–23.
6. Bichsel P, Jacobs G, Oliver JE Jr. Neurologic manifestations
associated with hypothyroidism in four dogs. J Am Vet Med Assoc
1988;192(12):1745–7.
7. Bruchim Y, Kushnir A, Shamir MH. L-thyroxine responsive
cricopharyngeal achalasia associated with hypothyroidism in a dog.
J Small Anim Pract 2005;46(11):553–4.
8. Rossmeisl JH Jr. Resistance of the peripheral nervous system to theeffects of chronic canine hypothyroidism. J Vet Intern Med 2010;24(4):875–81.
9. Pollard JD. Neuropathy in diseases of the thyroid and pituitary
glands. In: Dyck PJ, Thomas PK, eds. Peripheral neuropathy. 4th ed.
Philadelphia (PA): Elsevier Saunders; 2005:2039–49.
10. Walker TL, Redding RW, Braund KG. Motor nerve conduction velocity and latency in the dog. Am J Vet Res 1979;40(10):1433–9.
11. Shelton GD, Johnson GC, O’Brien DP, et al. Degenerative myelopathy
associated with a missense mutation in the superoxide dismutase 1(SOD1) gene progresses to peripheral neuropathy in Pembroke Welsh
corgis and boxers. J Neurol Sci 2012;318(1-2):55–64.
12. Shirabe T, Tawara S, Terao A, et al. Myxoedematous polyneuropathy: a light and electron microscopic study of the peripheral
nerve and muscle. J Neurol Neurosurg Psychiatry 1975;38(3):241–7.
13. Sidenius P, Nagel P, Larsen JR, et al. Axonal transport of slow
component a in sciatic nerves of hypo- and hyperthyroid rats.
J Neurochem 1987;49(6):1790–5.
14. Dyer KR, Duncan ID, Hammang JP, et al. Peripheral neuropathy
in two dogs: correlation between clinical, electrophysiological andpathological ﬁndings. J Small Anim Pract 1986;27:133–46.
15. Ropper AH, Samuels MA. Diseases of the peripheral nerves. In:
Ropper AH, Samuels MA, eds. Adams and Victor’s principles ofneurology. 9th ed. New York: McGraw-Hill Medical; 2009:1251–325.
16. Doliger S, Delverdier M, Moré J, et al. Histochemical study of cutaneous mucins in hypothyroid dogs. Vet Pathol 1995;32(6):628–34.
17. Patterson JS, Rusley MS, Zachary JF. Neurologic manifestations of
cerebrovascular atherosclerosis associated with primary hypothyroidism in a dog. J Am Vet Med Assoc 1985;186(5):499–503.
18. Blois SL, Poma R, Stalker MJ, et al. A case of primary hypothyroidism causing central nervous system atherosclerosis in a dog.
Can Vet J 2008;49(8):789–92.
19. Platt SR. Neuromuscular complications in endocrine and metabolic
disorders. Vet Clin North Am Small Anim Pract 2002;32(1):125–46.
20. Quattrini A, Nemni R, Marchettini P, et al. Effect of hypothyroidism
on rat peripheral nervous system. Neuroreport 1993;4(5):499–502.

impairments caused by hypothyroidism are mainly metabolic
functional abnormalities, rather than structural changes in peripheral nerves.20 Hence, early recovery by thyroid replacement
can be expected at this stage. In contrast, when either apparent
structural changes of nerves occur or there is nerve ﬁber loss
resulting from axonal degeneration, a prolonged time may berequired to recover or recovery may be incomplete. Therefore,
establishing an early and accurate diagnosis and initiating appropriate treatment are necessary when hypothyroid neuropathy
is suspected.

Conclusion
The study authors demonstrated that hypothyroid polyneuropathy
can occur in dogs as described in humans. Pathological changes
in peripheral nerves included myelinated ﬁber loss accompanied
by subperineurial edema. The dog made a full recovery with only
thyroid supplementation. However, response to the treatment
was slow and most neurologic abnormalities persisted for .6 wk.
When apparent structural changes of nerves occur, a prolonged
time may be required to recover or recovery may be incomplete.
Therefore, establishing an early and accurate diagnosis andinitiating appropriate treatment are both necessary when hypothyroid neuropathy is suspected.
This research was partially supported by a research project grantawarded by the Azabu University.
FOOTNOTESa
Soloxine; Virbac, Fort Worth, TX
bProHance; Eisai, Tokyo, Japan
REFERENCES
1. Ferguson DC. Testing for hypothyroidism in dogs. Vet Clin NorthAm Small Anim Pract 2007;37(4):647–69, v.
2. Indrieri RJ, Whalen LR, Cardinet GH, et al. Neuromuscularabnormalities associated with hypothyroidism and lymphocytic
thyroiditis in three dogs. J Am Vet Med Assoc 1987;190(5):544–8.
3. Jaggy A, Oliver JE, Ferguson DC, et al. Neurological manifestationsof hypothyroidism: a retrospective study of 29 dogs. J Vet Intern Med
1994;8(5):328–36.

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FULL PAPER

Surgery

Valproic Acid, a Histone Deacetylase Inhibitor, Decreases Proliferation of and Induces

Yasuhiro KURIHARA1), Takehito SUZUKI1), Motoharu SAKAUE1), Ohoshi MURAYAMA2), Yoko MIYAZAKI1),
Atsushi ONUKI1), Takuma AOKI1), Miyoko SAITO1), Yoko FUJII1), Masaharu HISASUE1), Kazuaki TANAKA1) and
Tatsuya TAKIZAWA1)*
1)Graduate School of Veterinary Medicine, Azabu University, Fuchinobe, Chuo-ku, Sagamihara 252–5201, Japan
2)School of Life and Environmental Science, Azabu University, Fuchinobe, Chuo-ku, Sagamihara 252–5201, Japan(Received 30 April 2013/Accepted 7 August 2013/Published online in J-STAGE 27 August 2013)
ABSTRACT. Adipose tissue-derived stem cells (ADSCs) isolated from adult tissue have pluripotent differentiation and self-renewal capability.

The tissue source of ADSCs can be obtained in large quantities and with low risks, thus highlighting the advantages of ADSCs in clinicalapplications. Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to affect ADSC differentiation inmice and rats; however, few studies have been performed on dogs. We aimed to examine the in vitro effect of VPA on canine ADSCs.
Three days of pretreatment with VPA decreased the proliferation of ADSCs in a dose-dependent manner; VPA concentrations of 4 mM andabove inhibited the proliferation of ADSCs. In parallel, VPA increased p16 and p21 mRNA expression, suggesting that VPA attenuatedthe proliferative activity of ADSCs by activating p16 and p21. Furthermore, the effects of VPA on adipogenic, osteogenic or neurogenic
differentiation were investigated morphologically. VPA pretreatment markedly promoted neurogenic differentiation, but suppressed theNSE, TUBB3 and MAP2, a decrease in the adipogenic marker, LPL, but noof neurogenic differentiation of canine ADSCs and is a useful tool for studying the interaction between chromatin structure and cell fate
determination.
KEY WORDS: adipose tissue-derived stem cell, cell proliferation, histone deacetylase inhibitor, pluripotency, valproic acid.
doi: 10.1292/jvms.13-0219; J. Vet. Med. Sci. 76(1): 15–23, 2014

Spinal cord injury (SCI) often occurs in dogs, due to motor vehicle accidents or intervertebral disease (IVDD). Most
canine patients suffer from sustained incontinence and lossof walking ability, and the prognosis of severe SCI cases ispoor. Unfortunately, no effective drug or surgical therapy has
been established for severe SCI cases, and there is a needfor new therapeutic approaches. One possibility is stem celltransplantation therapy, which is used as a radical cure treatment for refractory SCI [2, 14, 25, 26].
Adipose tissue-derived stem cells (ADSCs) are isolated
from the stromal vascular fraction of adipose tissues [8, 24,39]. ADSCs, similar to pluripotent adult mesenchymal stem
cells, can differentiate into mesenchymal lineage cells, suchas adipocytes, osteocytes, chondrocytes and myocytes [36,38]. ADSCs have characteristics similar to those of bonemarrow-derived mesenchymal stem cells (BMSCs), including gene expression and differentiation potential [1, 3, 5, 17,
19, 22, 33, 35, 37, 39]. Unlike BMSCs, however, the tissuesource of ADSCs can be obtained in large quantities and withlow risks [11]. Therefore, it is reasonable that ADSCs will be
the preferred adult stem cells for future clinical applications[24]. Moreover, the dog has been found to be a good animalmodel of human disease [32], and thus, the study of canine
ADSCs is particularly useful. Stem cells derived from bonemarrow and from olfactory ensheathing glia (OEG) havebeen studied for spinal cord regenerative therapy in dogs [6,14, 16, 25, 26, 31]. However, only a few studies have been
performed on the differentiation of canine ADSCs, except
for a comparative study showing that BMSCs and ADSCs
could be differentiated into neurospheres and neuron-likecells in dogs [2].
Valproic acid (VPA), a widely used antiepileptic and anticonvulsant drug, is an inhibitor of class 1 histone deacetylases (HDACs) [7, 27]. Histone acetylation correlates withterminal tails through acetylation or deacetylation can alter
the interaction between histones and DNA, affecting theregulation of gene expression [9, 12, 13, 18, 34]. Therefore,
HDAC inhibitors have been a useful tool for studying the*C ORRESPONDENCE TO: TAKIZAWA, T., Graduate School of Veterinary
Medicine, Azabu University, Fuchinobe, Chuo-ku, Sagamihara
252–5201, Japan.
e-mail: takizawa@azabu-u.ac.jp©2014 The Japanese Society of Veterinary Science
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-ncnd) License <http://creativecommons.org/licenses/by-nc-nd/3.0/>.

of hippocampal neural progenitors into neurons, but inhibit
their glial differentiation in adult rats [12].
In the present study, we have investigated the effects
of VPA on the proliferation and differentiation of canine
ADSCs isolated from subcutaneous adipose tissue in the

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Table 1.

Primers used in RT-PCR and real-time PCR
Product
length (bp)

Gene
p16
p21

FABP4
LPL
BMP2
Dlx5
COL1A1
NSE
TUBB3
MAP2
NEFH
GFAP
GAPDH

ForwardCGATCCAGGTCATGATGATGG

Reverse

ACCACCAGCGTGTCCAGGAA

ForwardCATCCCTCATGGCAGCAAG

ReverseAGGCAGGGAGACCTTGGACA

ForwardACACGATGCTGGCGTCCTTGATG

ReverseTGGCTCCATGAAGTCACCAAAGG

Forward

ATCAGTGTAAACGGGGATGTG

ReverseGACTTTTCTGTCATCCGCAGTA

ForwardACACATTCACAAGAGGGTCACC

ReverseCTCTGCAATCACACGGATGGC

ForwardCACTAACCACGCCATTGTTCA

ReverseACAACCCTCCACAACCATGTC

ForwardTGCTCTCCTACCTCGGCTTC

ReverseTTGCCATTCACCATCCTCAC

ForwardGTAGACACCACCCTCAAGAGC

ReverseTTCCAGTCGGAGTGGCACATC

ForwardGACCAACCCAAAGCGTATTGA

ReverseGCAATGAACGTGTCCTCAGTC

ForwardAGCCAAGTTCTGGGAAGTCA

Reverse

CCCACTCTGACCAAAGATGAA

ForwardAGAGGAGGTGTCTGCAAGGA

ReverseGTGATGGAGGTGGAGAAGGA

ForwardCTCAAAGGCACCAAGGACTC

ReverseCAAAGCCAATCCGACATTCT

ForwardAGATCCACGATGAGGAGGTG

ReverseTCTTAGGGCTGCTGTGAGGT

ForwardGCTGAACGGGAAGCTCACTG

ReverseCGTCGAAGGTGGAAGAGTGG

145
208
119
117
134
163
224
119
180
238
161
244
104
221

contaminating blood cells and local anesthetics. The tissue
was minced into small pieces and then incubated in a solution containing 0.05% collagenase type IA (Sigma-Aldrich,
St. Louis, MO, U.S.A.) at 37°C for 1 hr with vigorous
shaking. The top lipid layer was removed, and the remaining liquid portion was centrifuged at 200 × g for 10 min.

inguinal region.
MATERIALS AND METHODS
Animals: Subcutaneous adipose tissue was obtained fromthe inguinal region of 8 healthy laboratory beagles (age, 1–2
years) (Kitayama Labes, Ina, Japan). All animals were anesthetized with propofol, before tissue samples were obtained.
rane (2.0%) in oxygen. At the end of each experiment, theanimals were euthanized by additional doses of anesthesia(pentobarbital, 100 mg/kg). The protocol of this study wasapproved by the Committee for Animal Experimentation at
Azabu University.
Isolation and culture of canine ADSCs: Adipose tissue
samples were processed for ADSC isolation as described

medium (DMEM, Nissui, Tokyo, Japan) supplemented with
10% newborn bovine serum (NBS, Invitrogen, Carlsbad,
CA, U.S.A.) and spread in 100-mm collagen type I-coateddishes (Iwaki, Tokyo, Japan) at a density of 1 × 106 cells
per dish. Cells were maintained in growth medium (DMEM
supplemented with 10% NBS, penicillin [100 U/ml] andstreptomycin [0.25 μg/ml]) at 37°C and 5% CO2. After 24
hr, the unattached cells were removed by washing with PBS.
Canine ADSCs from passages 1–3 were used, and no difference was observed in ADSCs between these passages.
Measurement of proliferation potential: The effects of
VPA or valpromide (VPM; Wako Pure Chemical Ind.,
Osaka, Japan), an analogue of VPA with no HDAC inhibi-

adipose tissue removed was extensively washed with sterile
phosphate-buffered saline (PBS) containing penicillin (100U/ml) and streptomycin (0.25 μg/ml) in order to remove

553

PLURIPOTENCY OF ADIPOSE STEM CELLS FROM DOGS

tory activity, on ADSC proliferation were measured usinga 3-(4, 5-dimethyl-thiazol-2-yl)-2, 3-diphenyltetrazolium
bromide (MTT) assay kit (Roche Applied Science, Basel,
Switzerland) according to the manufacturer’s instructions.

17

Reverse transcription-PCR and real-time PCR: Total
RNA was extracted using ISOGEN (NIPPON GENE, Tokyo,
Japan) and reverse-transcribed to single-strand cDNA using
oligo-dT primer and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. PCR
was performed using Taq DNA polymerase (KAPA Biosys-

density of 1 × 104 per well and cultured in growth medium
for 48 hr. VPA or VPM (0–8 mM) was then added to themedium, and cultures were incubated for 3 days. Subsequently, 10 μl of MTT stock solution was added, and theplates were further incubated for 4 hr at 37°C. Diluted HCl(100 μl) was then added to solubilize the formazan crystals,
and the absorbance of each well at 570 nm was measured
with a microplate reader LS-PLATE manager 2004 (Wako
Pure Chemical Ind.); the average of measurements of 6 wells
per sample has been presented.
In vitro differentiation assay: In vitro assay of cell differentiation into adipogenic, osteogenic and neurogenic
lineages was performed as described previously [4, 29, 39]each cycle consisted of the following steps: denaturation for
10 sec at 98°C, annealing for 30 sec at 53–65°C and a 30sec elongation at 72°C (Table 1). Reaction products were
electrophoresed on a 2.0% agarose gel and visualized withethidium bromide. Real-time PCR of the mRNAs for p16,
p21 and GAPDH was performed using an ABI PRISM 7500
Sequence Detection System (Applied Biosystems Japan,
Tokyo, Japan) according to the manufacturer’s instructions.
Analysis of the results was carried out using ABI PRISM
7500 Dissociation Curve Software v 1.0 (Applied Biosystems Japan). The relative amount of mRNA was normalized
to that of GAPDH.
Statistical analysis: Results are expressed as the mean
± standard error. Multiple comparisons were done with theTurkey–Kramer test after one-way analysis of variance. A
p35-mm dishes at a density of 1 × 105 cells per dish. The
cells were incubated on glass coverslips in growth medium
containing 4 mM VPA for 3 days and then transferred toadipogenic induction medium (DMEM supplemented with
10% FBS, 1 μM dexamethasone, 10 μM insulin and 0.5 mM
isobutylmethylxanthine) or to osteogenic induction medium(DMEM supplemented with 10% FBS, 0.1 μM dexamethasone, 50 μM L-ascorbate-2-phosphte and 10 mM glycerophosphate) for 14 days [39] and then to neurogenic induction
medium (DMEM supplemented with100 μM dibutyryl cyclic
adenosine monophosphate and 125 μM isobutylmethylxanthine) for 2 hr [23, 24]. Intracellular lipid accumulation, as
an indicator of adipogenic differentiation, was visualized by

RESULTS
VPA induces acetylation of histone H3 and decreased cellproliferation
acetylation of histone H3, we examined the expression ofcence staining. Minimal HDAC1 and acetylation of histone

calcium deposition. Neurogenic differentiation was assessed

the morphology of ADSCs and increased the expression of
HDAC1 and the acetylation of histone H3 in ADSCs after 3
days of treatment (Fig. 1A and 1B). In contrast, VPM did not

were purchased from Wako Pure Chemical Ind.
: Immunocytochemical
analyses of HDAC1, acetylation of histone H3 (ace H3),
cubated in growth medium for 3 days as described above.
Some cultures were processed for neurogenic induction. At

HDAC1 and histone H3 staining in ADSCs.
liferation of ADSCs in a dose-dependent manner: about 20%(2 mM VPA), 40% (4 mM VPA) and 80% (8 mM VPA) of
the control group (Fig. 1C). However, VPM treatment did
not substantially affect ADSC proliferation. No dead cells
were observed in any VPA-treated groups by phase-contrast
microscopy.
VPA induces upregulation of cyclin-dependent kinase
inhibitors: To assess the effect of VPA on the expression of
cyclin-dependent kinase (CDK) inhibitors, we examined theexpression of these genes by real time-PCR. The expression
levels of p16
treated with 4 mM VPA (4.6 fold vs. control); however, p16
mRNA expression levels did not change in the cells treatedwith 8 mM VPA (2.1 fold vs. control; Fig. 2A). p21 mRNA

3.7% formaldehyde for 15 min at 4°C. After PBS washes,
the cells were permeabilized with 0.2% Triton X-100 for 10
min at room temperature. The cells were then incubated withan anti-HDAC1 antibody (sc-7872, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), an anti-histone H3 (acetyl
K9) antibody (Novus Biologicals, Littleton, CO, U.S.A.),
U.K.) or an anti-NSE antibody (PA1-46203, Thermo ScienAfter further PBS washes, cells were incubated with secondary antibody (Cy3-conjugated goat anti-rabbit IgG or FITCconjugated goat anti-mouse IgG, Jackson ImmunoResearch,West Grove, PA, U.S.A.) for 30 min at room temperature.
The cells were then washed with PBS and counterstainedwith 8 mM VPA (Fig. 2B). The expression levels of p21
mRNA were approximately 2.6 fold (4 mM VPA) and 3.0
fold (8 mM VPA) of that of the control group.

554

18

Y. KURIHAR A ET AL.

Fig. 1. Effects of valproic acid on histone deacetylase 1 expression, histone H3 acetylation and cell proliferation. Canine adipose tissue-derived stem cells (ADSCs) were treated with valproic acid (VPA) or valpromide (VPM). (A) ADSCs were then

HDAC1 (A) and acetylation of histone H3 (B). Scale bar, 50 μm. (C) Cell proliferation was measured by MTT assay andexpressed as percentage of the negative control (DMSO). Data represent the means ± S.E. (% of control) of 4 independent
experiments; each measurement was the average for 6 wells. *P

VPA suppresses adipogenic and osteogenic differentiation: To assess the effect of VPA on the pluripotency of
ADSCs, we investigated whether VPA treatment alters thedifferentiation of ADSCs into adipogenic and osteogenic
lineages using an in vitro differentiation assay. Oil red O
staining revealed that ADSCs that differentiated into theadipogenic lineage accumulated lipid droplets in the cytosol, as compared to undifferentiated cells, which did notaccumulate lipid droplets (Fig. 3A). VPA pretreatment fol-

affected by VPA pretreatment (Fig. 4B).
VPA promotes neurogenic differentiation: We further
examined the effect of VPA on the neurogenic lineage ining revealed that ADSCs that differentiated into neurogenic
cells had typical neuron-like cell protrusions and higher

accumulation of lipid droplets. RT-PCR analysis showed thatthe mRNA expression levels of adipogenic markers, peroxi), fatty acidbinding protein 4 (FABP4) and lipoprotein lipase (LPL) were
elevated by adipogenic induction (Fig. 3B). On the other
hand, VPA pretreatment followed by adipogenic induction
LPL mRNA expression level, inparallel with the decreased accumulation of lipid droplets.
Alizarin red S staining revealed that ADSCs differentiatedinto osteogenic lineage cells with accumulated calcium deposition, as compared with the undifferentiated cells, which
demonstrated no calcium deposition (Fig. 4). VPA pretreat-).
mRNA levels of neurogenic markers, viz., NSE, TUBB3
and microtubule-associated protein 2 (MAP2), were also
elevated by neurogenic induction, but mRNA expression of
the glial cell marker, GFAP, was not observed in any groups(Fig. 5B). Pretreatment with VPA followed by neurogenic
induction increased the expression of NSE, TUBB3 andNEFH), as
MAP2compared to that in the neurogenic induction group. VPA

positive cells also stained for NSE. VPA pretreatment fol-cells, even without neurogenic induction. Furthermore, VPA
elevated the mRNA expression levels of neurogenic markers
in ADSCs with and without neurogenic induction.

calcium deposition (Fig. 4A). mRNA expression levels of
osteogenic markers, viz., bone morphogenetic protein 2(BMP2) and distal-less homeobox 5 (DLX5), were also el-

DISCUSSION-

555

PLURIPOTENCY OF ADIPOSE STEM CELLS FROM DOGS

19

ogy of canine ADSCs and markedly induced their expressionof HDAC1 and acetylation of histone H3. In contrast, VPM,
an analogue of VPA with no HDAC inhibitory activity, dideffects on HDAC1 and histone H3, indicating that the H3
acetylation was increased by VPA. These observations supet al. [20]. Thus, our results clearly
indicated that VPA induced H3 acetylation by reducing
HDAC1 activity in canine ADSCs.
pendent decrease in the proliferation of ADSCs, suggestingthat VPA suppresses ADSC proliferation through acetylationof histone H3. p21 and p16, well-known CDK inhibitors,
regulate cell cycle arrest. VPA induces expression of these
CDK inhibitors in human ADSCs and mesenchymal stem
mRNA expression levels of p16 at 4 mM and of p21 at 8mM without inducing cell death. p21 is also a well-known
HDAC-inhibitor responsive gene that is upregulated byhyperacetylation of histones H3 and H4 [10, 20, 28]. In
acetylation was markedly increased by VPA and that p21
viability was further reduced by 8 mM VPA treatment, again
et al. [20] who reported that
VPA causes cell cycle arrest through increased p21 expression in the absence of p16 mRNA expression in human ADSCs. Therefore, the inhibitory effect of VPA on proliferationof canine ADSCs was due to cell cycle arrest, although theunderlying mechanism needs to be further examined.
Furthermore, VPA promoted differentiation of approximately 90% of ADSCs into a neuronal cell lineage after 3
days of treatment. The differentiated cells have neuron-like
Pretreatment with VPA followed by neurogenic induction
also promoted mRNA expression of the neuronal markers
NSE, TUBB3, MAP2 and NEFH as compared to the pretreatment without VPA, suggesting that promotional effects of
VPA on neuronal differentiation of ADSCs were induced byupregulation of these genes. Previous reports have shownthat VPA promotes differentiation of neural stem cells into
neurons in adult rats [12, 15]. To our knowledge, the present
tiation of ADSCs.
Interestingly, VPA pretreatment in the absence of neurogenic induction caused moderate differentiation into neuronlike cells and also increased mRNA expression levels ofneurogenic markers in ADSCs. Thus, our data indicated that
VPA could induce neurogenic differentiation in the absenceof neurogenic induction. We also demonstrated that VPA
increased the acetylation of histone H3 that has been correlated with gene activation [12, 13]; thus, VPA caused gene
expression in part through H3 acetylation. In addition, we
showed that the neuron-like differentiated cells all stained
et al.
[21] recently reported that neuronal differentiation could
modulate gene transcription, translation and post-translational modulation of Ca2+ channels to change the Ca2+ ion

Fig. 2. Effects of valproic acid on cyclin-dependent kinase inhibitor expression. Adipose tissue-derived stem cells (ADSCs) weretreated with valproic acid (VPA) or valpromide (VPM). Total
RNA was extracted from ADSCs after 3 days of treatment with
VPA (4 or 8 mM) or VPM (8 mM). The relative expression of thecyclin-dependent kinase (CDK) inhibitors p16(A) and p21(B)dehydrogenase (GAPDH) was used as an internal standard.
Data are the means ± S.E. of 4–7 independent experiments. a, b:
P<0.05.

556

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Y. KURIHAR A ET AL.

Fig. 3. Valproic acid suppresses accumulation of lipid droplets. Adipose tissue-derived stem cells (ADSCs) werepretreated with valproic acid (VPA) for 3 days followed by adipogenic induction for 14 days. (A) Adipogenic differentiation was visualized by oil red O staining after 14 days of induction with adipogenic medium. Arrowheads
show cells that accumulated lipid droplets. Scale bar, 50 μm. (B) RT-PCR analysis of adipogenic markers,,
FABP4 and LPL, was performed using total RNA extracted from ADSCs after 14 days of adipogenic induction. AIM,
adipogenic induction medium.

Fig. 4. Valproic acid suppresses calcium deposition. Adipose tissue-derived stem cells (ADSCs) were pretreated withvalproic acid (VPA) for 3 days followed by osteogenic induction for 14 days. (A) Osteogenic differentiation was
evaluated by alizarin red S staining after 14 days of induction with osteogenic medium. Arrowheads show cells that
accumulated calcium in the cytosol. Scale bar, 200 μm. (B) RT-PCR analysis of osteogenic markers, BMP2, Dlx5
and COL1A1, was performed using total RNA extracted from ADSCs after 14 days of osteogenic induction. OIM,
osteogenic induction medium.

currents; our results suggested that neuronal differentiation
could modulate TUBB3 transcription and translation. In thepresent study, VPA promoted ADSCs differentiation into
neuronal cells in the absence of neurogenic induction factors
and induced acetylation of histone H3, indicating that VPA
is a useful tool for studying the interaction between chromatin structure and cell fate determination. Further studies
are needed to examine the molecular mechanism underlying
the neurogenic differentiation induced by VPA using canine

557

ADSCs.
In contrast, VPA suppressed the late stage of differentiation into adipogenic and osteogenic lineage cells. Two of the
3 adipogenic marker genes examined showed no reduction
after VPA pretreatment; however, lipid accumulation appeared to be suppressed, suggesting that the VPA inhibits
accumulation of lipid droplets rather than an inhibiting thewhole adipogenic differentiation process.
Similarly, osteogenic marker genes showed no changes

PLURIPOTENCY OF ADIPOSE STEM CELLS FROM DOGS

21

Fig. 5. Valproic acid promotes neurogenic differentiation. Adipose tissue-derived stem cells (ADSCs) were pretreatedwith valproic acid (VPA) for 3 days followed by neurogenic induction. (A) Neurogenic differentiation was assessed

Scale bar, 50 μm. (B) RT-PCR analysis of the neurogenic markers, NSE, TUBB3, MAP2 and NEFH and the glial
marker, GFAP, was performed using total RNA extracted from ADSCs after 2 hr of neurogenic induction. NIM, neurogenic induction medium.

after VPA treatment, but accumulation of calcium deposition
ing that VPA inhibits calcium deposition rather than inhibiting the osteogenic differentiation process as a whole. The
mechanism underlying the differential effects of VPA on thepluripotent capacity of ADSCs remains unclear. A previous
report has shown that VPA decreases adipogenic and neurogenic differentiation, but increases osteogenic differentiation in human ADSCs [20]. The reason for VPA acting as a
stimulator for differentiation of canine ADSCs and as a suppressor for that of human ADSCs is not immediately clear.
Therefore, chromatin structure and cell fate determinationneed to be further examined in relation to the pluripotency of
ADSCs, including this difference between humans and dogs.
In conclusion, pretreatment with VPA dose-dependently
decreased proliferation of canine ADSCs. In parallel with
its inhibitory effects, VPA increased p16 and p21 mRNA
expression, implying induction of cell cycle arrest throughactivation of p16 and p21. In addition, pretreatment with
VPA followed by adipogenic, osteogenic or neurogenic
induction markedly promoted in vitro neurogenic differentiation, but suppressed accumulation of lipid droplets andcalcium deposition. These in vitro
by pretreatment with VPA were associated with changes in
expression of relevant markers. These results suggested thatcanine ADSCs and is a useful tool for studying the interaction between chromatin structure and cell fate determination.
ACKNOWLEDGMENTS. This work was supported in partby the Science Research Promotion Fund of The Promotionand Mutual Aid Corporation for Private Schools of Japan.
This work was also supported in part by the MEXT Program558

for the Strategic Research Foundation at Private Universities, 2011–2015.
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FULL PAPER

Pathology

Pathological Features of Polyneuropathy in Three Dogs
Masaya TSUBOI1), Kazuyuki UCHIDA1)*, Tetsuya IDE1), Mizue OGAWA1), Takehiko INAGAKI2), Shinji TAMURA3),
Miyoko SAITO4), James K. CHAMBERS1) and Hiroyuki NAKAYAMA1)
1)Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1 –1–1 Yayoi, Bunkyo-

ku, Tokyo 113–8657, Japan2)Department of Neurology, Japan Animal Referral Medical Center, 2 –5–8 Kuji, Takatsu-ku, Kawasaki-shi, Kanagawa 213–0032, Japan
3)Tamura Animal Clinic, 7–16 Saeki-ku, Hiroshima-shi, Hiroshima 731-5100, Japan
4) Department of Surgery II, School of Veterinary Medicine, Azabu University, 1–17–71 Fuchinobe, Chuo-ku, Sagamihara-shi, Kanagawa

229–8501, Japan(Received 16 May 2012/Accepted 18 October 2012/Published online in J-STAGE 1 November 2012)
Canine polyneuropathy is a neurological disorder characterized by a dysfunction of multiple peripheral nerves. The etiology ofthe disease is diverse; it may occur in cases of infectious, immune-mediated, or hereditary conditions or in association with e ndocrinopathy,

ABSTRACT.

investigate pathological differences among three canine polyneuropathy cases with each presumably having a different etiology. Cases
included a 13-month-old female border collie (Dog No.1), a 21-month-old male chihuahua (Dog No.2) and an 11-year-old male beagle(Dog No.3). Clinical examinations revealed hindlimb ataxia and sensory loss in Dog No.1, forelimb paralysis and vertebral pain in Dog
No.2, and paddling-gait and hypothyroidism in Dog No.3. Histopathologically, axonal swelling and pale myelin were observed in Dog
No.1. Giant axons mimicking giant axonal neuropathy were obvious in Dog No.2. Dog No.3 showed atrophic axons and severe interstitial
edema. Distributions of peripheral nerve lesions coincided with respective clinical symptoms. According to their clinical and pathologicalfeatures, Dogs No.1 and No.2 were suspected of hereditary polyneuropathy, while Dog No.3 seemed to have hypothyroidism-associatedpolyneuropathy. As each case demonstrated unique pathological features, different pathogeneses of peripheral nerve dysfunction weresuggested.
KEY WORDS: canine, congenital, hypothyroidism, polyneuropathy.
doi: 10.1292/jvms.12-0224; J. Vet. Med. Sci. 75(3): 327–335, 2013

Polyneuropathy in dogs is a neurological disorder characterized by a dysfunction of multiple peripheral nerves. Initial
symptoms of the disease are a lack of coordination and in-hereditary, familial or breed-associated factors are related toevidence of denervation and decreased nerve conduction-

nerve dysfunctions are most predominant, and autonomic
nerves may also be affected. As autonomic dysfunction may
lead to laryngeal or pharyngeal paralysis, aspiration pneumonia is frequently diagnosed as a cause of death in canineof this disease through clinical symptoms.
The distribution of lesions (i.e. motor or sensory, distal orproximal, anterior or posterior and symmetric or asymmetric)

Several etiologies of polyneuropathy in dogs have beenpreviously proposed. In some cases, the disease occurs
in association with Neospora caninum
some immune-mediated diseases, such as systemic lupus

cording to lesion distribution, age onset, progression speed

canine-inherited polyneuropathy has been reported in 22*C ORRESPONDENCE TO: U CHIDA, K., Department of Veterinary Pa thology, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–8657,
Japan.
e-mail:auchidak@mail.ecc.u-tokyo.ac.jp

The aim of this study is to investigate pathological differences among three cases of canine polyneuropathy witheach presumably having a different etiology. Possible pathogenesis and respective etiologies of each case are discussed.

©2013 The Japanese Society of Veterinary Science

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μg/dl, and fT4: 0.08 ng/dl, relative to normal values of>0.6
ng/dl). Thus, the dog was diagnosed with hypothyroidism.
The dog was treated with thyroid hormone, but symptoms

MATERIALS AND METHODS
Cases: Three dogs were examined; Dog No.1 wasa 13-month-old female border collie, Dog No.2 was a
21-month-old male chihuahua and Dog No.3 was an
11-year-old male beagle. Clinical features of these cases are
summarized in Table 1.
Dog No.1 showed progressive hind limb ataxia at the ageof 3 months, and started chewing her front paw. Ataxia thenwalking at the age of 9. Using sensory nerve action potential
examination, the dog was further diagnosed with sensory
nerve predominant segmental demyelinative neuropathy at
the age of 10, and was treated with an immune suppressor
nally, the dog died of aspiration pneumonia at the age of 11.
: Antemor-in sciatic and femoral areas of both sides of the distal hindlimbs and in the radial area of both sides of the distal front
limbs. Magnetic resonance imaging (MRI) of the spinal

in Dog No.1. Peripheral nerves (including the right common
peroneal nerve and right caudal cutaneous antebrachial
nerve) and the skin of the dorsal part of the right front paw
were taken for biopsy.
Postmortem examination: Tissue samples were takenfrom the brain, spinal cord and nerve roots of the 3 dogs.
Other main organs including the heart, lungs, liver, spleen,kidneys, alimentary tract, pituitary gland, thyroid glands,
adrenal glands, ovaries and uterus were taken only from Dog
No.1, because autopsies of the other dogs were performedat the veterinary hospitals. The trigeminal ganglion, stellate
ganglion, brachial plexus, femoral obturator nerve, vagus
nerve, biceps femoris muscle, temporal muscle and lingual
muscle were also taken from Dog No.1. Sciatic and tibial
nerves were taken from Dog No.3. All tissue samples weredeveloped megaesophagus at the age of 12 months. The dog
suddenly died 1 month after the onset of megaesophagus.
Dog No.2 was presented to the veterinary hospital 2months prior to his death with a chief complaint of crying
when held in his owner’s arms. One month after the initial
symptom, the dog exhibited salivation and a pitter-patter
vertebral pain induced by manual compression. MRI andcomputerized tomography (CT) of the entire body showed
and anti-acetylcholine receptor antibody was not detected in
the serum. Tandem mass spectrometry used for the diagnosis
of inherited metabolic disorders was conducted, but no spe-

μm. Sections
toxylin-eosin (HE).
Immunohistochemistry: Primary antibodies used werewith corticosteroids and vitamin supplements, symptoms did
not improve. The forelimbs gradually became paralyzed,

sank on these limbs during the impact. Two years after thenary hospital with a chief complaint of a wobbling gait. The
dog exhibited bradycardia at the initial visit, and neurological examination revealed a reduced postural reaction. MRI

Dako, Glostrup, Denmark), rabbit polyclonal anti-peripheral
myelin protein 22 (PMP22, 1:400, Sigma, St. Louis, MO,
U.S.A.), rabbit polyclonal anti-periaxin (PRX, 1:250, Sigma)
and rabbit polyclonal anti-Myelin protein zero (MPZ, 1:100,
Abcam, Cambridge, UK). Immunohistochemistry was performed using Envision polymer reagent (Dako). Antigen
retrieval was performed by heating sections in an autoclave
at 121°C for 10 min in 10 mM citrate buffer (pH6.0), except2O2)-methanol at room
temperature for 5 min to block endogenous peroxidase, inage of 7 revealed low thyroxine (T4) and free T4 (fT4) concentrations (T4: 0.79 μg/dl, relative to normal values of>2.0

body at 4°C overnight. Sections were further incubated withdog exhibited epileptiform seizures and died of respiratory
failure.
Dog No.3 began to show a characteristic paddling gait at
the age of 3, wherein the distal palmar and plantar aspects ofTable 1.

Canine polyneuropathy cases examined

Case

Breed

Gender

Onset

Death

Major clinical signs

3m

1y1m

Hind limb ataxia, pain sensability loss, megaesophagus,
chewing front paws, and aspiration pneumonia

Dog No.1

Border collie

Dog No.2

Chihuahua

Male

1y7m

1y9m

Dog No.3

Beagle

Male

3y

11y

562

prolapse, ataxia, forelimb paralysis, and vertebral pain
Paddling-gait, bradycardia, hypothyroidism, walking

329

Envision horseradish peroxidase (HRP)-labeled mouse or
rabbit polymer reagent (Dako) at 37°C for 60 min. Reaction
products were visualized with 3.3’-diaminobenzidine (DAB,
2O2 in TBS. Sections were counterstained with Mayer’s hematoxylin. As acontrol, sections from dogs showing no clinical/pathologicalsigns of polyneuropathy were used.
Quantitative analysis
the lumber nerve root were used for quantitative analysis.
Microscopic pictures of 2 randomly selected-areas of dorsal
and ventral nerve roots were taken with a Nikon Digitaltion of × 200 with ACT-1 software (Nikon). The smallest
puted using Mac-Biophotonics ImageJ software. More thanwere more severe in sensory nerve roots than those in motorin any cases.
Distal peripheral nerves were also examined in Dogs
No.1 and No.3. In Dog No.1, lesions of the femoral obturator nerve and brachial plexus were more severe than thoseof nerve roots. Severe axonal swelling, mild axonal loss,
showed a severe decrease in axons and loss of myelin. Most
1J).
Minor lesions were observed in the central nervous system (CNS), such as focal neuronal loss in the cerebral cortex
of Dog No.2 and diffuse spheroid formation in the spinal
cord of Dog No.3 (Data not shown).
Quantitative analysis: The axonal diameter of nerve roots).
In control cases, the shape of the histogram for the motor
nerve root showed a “bimodal” pattern, which indicates theμm, andμsory nerve root was fewer than that in the motor nerve root

RESULTS: Antemortem peripheral nerve biopsy was conducted only in
Dog No.1. The right common peroneal nerve, right caudal
cutaneous antebrachial nerve, and peripheral nerves from thedorsal skin of the right front paw showed severe degeneration and denervation with both sensory and motor involve-

showed a “triangular” pattern.
In Dog No.1, a bimodal pattern in motor nerve roots was
still evident, but the distribution of the axonal diametertribution in the sensory nerve root showed a slight left shiftsory nerves shifted to the left, and giant axons more than 15μand 2K). In Dog No.3, the axonal distribution of both motorand sensory nerve roots markedly shifted to the left, and theregular “bimodal” pattern of the motor nerve root changed to

conducted only in Dog No.1. Neither Wallarian-like axonal
degeneration nor regenerative changes were observed in the: Details were available only
in Dog No.1. Severe emaciation, multiple ulcerations on thebilateral hind limb paws, swelling of systemic lymph nodes,
and aspiration pneumonia were observed. There were no
spinal cord of all three cases.
: Major pathological lesions were located at nerve roots and/or distal periph). Histopathological lesions and their distributions in peripheral nerves weredifferent among cases (Table 2).
In Dog No.1, lesions were predominant at lumbar andsacral ventral nerve roots, and the major lesion was axonal

Immunohistochemistry for myelin components: Among 3
peripheral myelin components examined, PMP22 and PRX
were strongly positive in the axonal surroundings. MPZ
expressions were observed in the axons of Dogs No.2 andNo.3 (data not shown).

usual at the central area of nerve roots. Axonal loss was notevident in any nerve roots of Dog No.1. Lesions in Dog No.2
were located mainly in cervical and thoracic nerve roots.
Almost all axons were lost and lesions were replaced by

DISCUSSION
Severe degenerative changes were broadly distributed in
the peripheral nervous system (PNS) of the three present
served in the CNS. Thus, the present cases were diagnosed
as canine polyneuropathy. Interestingly, the distributions
and pathological features of lesions were different among
the cases. This fact indicates different pathogeneses in therespective cases.

were asymmetric and randomly distributed. At the lumber
level, axonal and myelin loss was milder than that at thecervical level, but multiple axonal swellings were observedswollen axons were roughly distorted and were huge (10 to
30 μm as the shortest diameter). In Dog No.3, lesions were
found at the cervical and lumber level of motor and sensory
nerve roots, where diffuse axonal atrophy was evident. Se-

Dogs No.1 and No.2 were suspected of hereditary polyneuropathy. Both dogs were young at the onset of the disease
and took long-standing courses until death. Such features are

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M. TSUBOI ET AL.

HE stain. Bar=500 μμm. (D) Axons in Dog No.1 were severely swollen, and surroundingμμaxons and myelin than that in nerve roots. (J) Axons and myelin in the tibial nerve of Dog No.3 were severely lost, and mostlesions were replaced with connective tissue.

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Table 2.

Histological lesions in the cervical, thoracic, lumber and sacral nerve roots
Dog No.1
Motor

CervicalAxonal swelling
Axonal loss
Myelin loss
Edema

Dog No.2

Sensory++

SacralAxonal swelling
Axonal loss
Myelin loss
Edema

Sensory

Motor

Sensory

+++
+++
+
+++

+++
+++
++

++
++
++
++

+++
++
++
++

++
+++
++
++

+
+++
++
+

ND
ND
ND
ND
ND

ND
ND
ND
ND
ND

+
++
+
+

+++
+
++
+
+

+
++

++
++
+++
++

ND
ND
ND
ND
ND

ND
ND
ND
ND
ND

++
+
+

LumbarAxonal swelling
Axonal loss
Myelin loss
Edema

Motor

+

+
ThoracicAxonal swelling
Axonal loss
Myelin loss
Edema

Dog No.3

+++
++

+

+++
++

++

such as chemical or drug intoxication were rejected, becauseof their clinical histories. Infectious or autoimmune diseases

nerves, and more proximal regions, as in the present case,were not examined in previous reports.
The disorder in the border collie was previously called“sensory neuropathy”, because initial symptoms were preet al.

was not associated with the present cases. Endocrinopathy
was not detected through the antemortem blood test, andneoplasm hardly occurs at their ages. Therefore, breedassociated congenital factors are the most likely etiology inthe 2 dogs.
Breed-associated polyneuropathy in border collies has-

because motor nerves were also involved in his case. Lesionsof the present case support Harkin’s opinion, because bothmotor and sensory nerves were impaired.
Giant axons observed in the dorsal lumber root of Dog
No.2 were variable in size and had an irregular shape. They
seemed to be different from the axonal swelling observedin Dog No.1, but were similar to lesions observed in “gi-information concerning the familial history of the presentborder collie dog (Dog No.1), clinical signs were almost
set of disease occurred at 2 to 5 months old, and erosion in
antemortem biopsy (i.e. severe degeneration and denerva-

axons in Dog No.2 was different from that in previous GAN
cases. While the distribution of giant axons in GAN has been

Dog No.1 suffered border collie polyneuropathy.
In spite of clinical and antemortem pathological similarities, postmortem pathological features in Dog No.1 werequite different from those of previous reports. Axonal swelling and pale myelin seen in the present case have never been
mentioned in previous reports. This intriguing phenomenon
may be explained by a difference in examined regions between past and present cases; pathological features previ-

Moreover, hind limbs were mostly affected in GAN cases,
while forelimbs were mostly affected in Dog No.2. Combining these differences, we suggest that the present caseof Dog No.2 is a unique congenital polyneuropathy newly
emerged in the chihuahua.
Although peripheral nerves were mostly impaired in Dog
No.2, a minor lesion was also observed in the CNS; there
was focal neuron loss at the cerebral cortex. This pathologic

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M. TSUBOI ET AL.

nohistochemistry. Bar=20 μm. Axonal diameters were measured using Mac-Biophotonics ImageJ software. (G-L):
Axonal diameter distributions of motor (G-I) and sensory (J-L) nerve roots of Dogs No.1 (G and J; shown in dark bars),
No.2 (H and K; shown in dark bars), No.3 (I and L; shown in dark bars), and control dogs (G-L; shown in light bars),
respectively.

feature correlates with antemortem epileptic symptoms. On
another hand, there were some discrepancies between clinical and pathological appearances in Dog No.2; the dog clini-

mutations in the MPZ
wise, PRX and PMP22 protein construct peripheral myelin
sheaths, and genetic failures in these proteins are the causes

nal nerve, mesencephalon and facial nerve, but the CNS partof this neural circuit was not impaired in this dog. Thus, we
suspect that the clinical symptom seemed to be attributed tocranial nerve dysfunction. Nevertheless, we could not evaluate pathological changes in these cranial nerves, because wefailed to investigate them on necropsy.
In human CMT disease, more than 30 genes have beenamino acid sequences of these constructive proteins lead toa failure to interact properly with other myelin components,
and results in myelin disruption.
On another hand, the genetic background of canine hereditary polyneuropathy remains unclear in most breeds.
Recently, Drögemüller et al.
N-myc downstream regulated gene 1 (NDRG1) gene in greycally characterized canine model of human CMT disease, as
the NDRG1
disease type 4D. It may offer an opportunity to gain further

encode constructive proteins of peripheral myelin sheaths.
MPZ, for example, is an integral membrane glycoprotein
MPZ guides the wrapping process in Schwann cells and

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insight into the pathobiology and therapy of human NDRG1
associated CMT disease.
We probatively tried to investigate the genetic causesof the 2 present cases that were suspected of hereditarypolyneuropathies. Using immunohistochemical methods,
we evaluated PMP22, PRX and MPZ protein expressions
in intact peripheral nerves. Among them, PMP22 and PRX
were expressed normally in the peripheral myelin sheath inall cases. Therefore, genes encoding these proteins do notseem to be responsible for the onset of the disease. MPZ
expression in the myelin sheath was observed only in Dog
No.1, and only faint axonal expression was observed in Dogs

tive etiologies seemed different from each other. The casesof the border collie and chihuahua were suspected of inherited polyneuropathy, with each perhaps having different
genetic factors, while the case of the beagle was presumably
associated with hypothyroidism. This paper presents new
aspects of the respective polyneuropathies. In the case of
the border collie, the pathologic appearance of peripheral
nerves showed quite different features from that of previous reports. The case of the chihuahua indicates a newlyemerged hereditary polyneuropathy, the features of which
partly resemble GAN in German shepherd dogs. The caseof the beagle presented a novel pathologic feature of canine-

several samples from control cases. Therefore, we concluded
tion conditions, not MPZ gene mutations.
Contrary to Dogs No.1 and No.2, Dog No.3 was suspected to have acquired neuropathy with an association of
hypothyroidism. Clinical signs of tetraparesis and postural
reaction dysfunction in Dog No.3 were consistent with thoseof previous reports of canine hypothyroid polyneuropathy
mon endocrinopathy of dogs and has been blamed for gen-polyneuropathy.

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The Scientiﬁc World Journal
Volume 2012, Article ID 383174, 7 pages
doi:10.1100/2012/383174

The cientiﬁcWorldJOURNAL

Research Article
Neuronal Ceroid Lipofuscinosis in Border Collie Dogs in Japan:
Clinical and Molecular Epidemiological Study (2000–2011)
Keijiro Mizukami,1 Takuji Kawamichi,2 Hiroshi Koie,3 Shinji Tamura,4 Satoru Matsunaga,5Shigeki Imamoto,6 Miyoko Saito,7 Daisuke Hasegawa,8 Naoaki Matsuki,9 Satoshi Tamahara,9Shigenobu Sato,10 Akira Yabuki,1 Hye-Sook Chang,1 and Osamu Yamato1
1 Laboratory of Clinical Pathology, Department of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto,Kagoshima, Kagoshima 890-0065, Japan
2 Japan Border Collie Health Network, 1-14-8 Manabigaoka, Tarumi-ku, Hyogo, Kobe 655-0004, Japan
3 Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Kanagawa,Fujisawa 252-0880, Japan
4 Tamura Animal Clinic, 7-16 Yoshimien, Saeki-ku, Hiroshima, Hiroshima 731-5132, Japan
5 Japan Animal Referral Medical Center, 2-5-8 Kuji, Takatsu-ku, Kanagawa, Kawasaki 213-0032, Japan
6 Shinjo Animal Hospital, 104-1 Katsuragi, Nara, Katsuragi 639-2144, Japan
7 Laboratory of Veterinary Surgery II, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Kanagawa,

Sagamihara 229-8501, Japan
8 Department of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyounan-chou, Tokyo,

Musashino 180-8602, Japan
9 Department of Veterinary Clinical Pathobiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo,

1-1-1 Yayoi, Tokyo, Bunkyo-ku 113-8657, Japan
10 Sato Animal Hospital, 6-38-20 Goinishi, Chiba, Ichihara 290-0038, Japan

Correspondence should be addressed to Osamu Yamato, osam@vet.kagoshima-u.ac.jp
Received 12 April 2012; Accepted 3 May 2012
Academic Editors: M. F. Landoni and E. J. Thompson
Copyright © 2012 Keijiro Mizukami et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Neuronal ceroid lipofuscinosis (NCL) is an inherited, neurodegenerative lysosomal disease that causes premature death. The
present study describes the clinical and molecular epidemiologic ﬁndings of NCL in Border Collies in Japan for 12 years, between
2000 and 2011. The number of aﬀected dogs was surveyed, and their clinical characteristics were analyzed. In 4 kennels withaﬀected dogs, the dogs were genotyped. The genetic relationships of all aﬀected dogs and carriers identiﬁed were analyzed. The
survey revealed 27 aﬀected dogs, but there was a decreasing trend at the end of the study period. The clinical characteristics of theseaﬀected dogs were updated in detail. The genotyping survey demonstrated a high mutant allele frequency in examined kennels(34.8%). The pedigree analysis demonstrated that all aﬀected dogs and carriers in Japan are related to some presumptive carriers
imported from Oceania and having a common ancestor. The current high prevalence in Japan might be due to an overuse of thesecarriers by breeders without any knowledge of the disease. For NCL control and prevention, it is necessary to examine all breedingdogs, especially in kennels with a high prevalence. Such endeavors will reduce NCL prevalence and may already be contributing to
the recent decreasing trend in Japan.

1. Introduction

accumulation of ceroid- or lipofuscin-like autoﬂuorescent
lipopigments in neurons, retinal cells, and other visceral
cells throughout the body [1–4]. NCL shares certain clinicalfeatures in both human beings and animals, including
behavioral abnormalities, such as personality changes and

Neuronal ceroid lipofuscinosis (NCL) is a rare groupof inherited, neurodegenerative lysosomal storage diseases characterized histopathologically by the abnormal

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aggressiveness, mental retardation and/or dementia; motor
disturbances, such as ataxia and incoordination; visual problems leading to central and/or retinal blindness; premature
death, but these diﬀer in degree based on the causative gene,of which there are currently at least 8, all recessively inherited[4]. NCL has been described in several domestic species andoccurs most commonly in dogs [1, 2, 5].
NCL in Border Collies was ﬁrst identiﬁed in Australia in
the 1980s [6–8], and a sporadic case with the disease was also
reported in the USA in the 1990s [9]. A diagnosis of the ﬁrst
case in Japan was made in a Border Collie that was born in2000 [10]. The pathogenic mutation was reported in 2005 to
be a nonsense mutation (c.619C>T) in exon 4 in the canine
CLN5 gene [11], which enabled a DNA-based genotyping ofaﬀected dogs and carriers. Recently, several types of rapid
genotyping assays for this mutation were developed, andthe carrier frequency (8.1%) in Japan was determined by agenotyping survey using these assays, suggesting the mutant
allele frequency of NCL in Border Collies is high enough inJapan that measures to control and prevent the disease would
be warranted [5].
The present study describes the clinical and molecularepidemiologic ﬁndings of NCL in Border Collies in Japan for
12 years, between 2000 and 2011. This study also discusses
the control and prevention of the disease based on the resultsof these analyses.

Associates ﬁlter paper (FTA card, Whatman International).
Genotyping was performed as reported previously [5].

2.3. Pedigree Analysis. Pedigree analysis was performed toelucidate the genetic relationships of aﬀected and carrierdogs identiﬁed in Japan and to deduce the pathway oftransmission and distribution of the mutant allele. The
genetic relationships among aﬀected and carrier dogs foundin the present study were analyzed using the pedigree papers
issued by the Japan Kennel Club (http://www.jkc.or.jp/) andthe Kennel Club of Japan (http://www.kcj.gr.jp/). The pedigree information of carrier dogs identiﬁed in the previous
random survey in Japan [5] was analyzed and added to
the results of the present study. The pedigree informationof the dogs was used under the informed consent of
their owners. The ancestry in Oceania was traced using
pedigree information of carrier dogs provided by the New
South Wales Border Collie Club (http://www.mybcsite.com/
bccnswwebﬁles/bccnswframe.htm), which was also disclosed
via the website of the Japan Border Collie Health Network(http://www.jbchn.net/cl carrier.htm), a volunteer breeders’association for the healthy breeding of Border Collies. Pedigree information published in the Border Collie Database(http://db.kennel.dk/) was used to supplement the information about the ancestor dogs in Oceania.
All animals were cared for and were used in the experiments in accordance with the guidelines for proper conductof animal experiments issued by the Science Council ofJapan (http://www.scj.go.jp/en/animal/index.html). All experimental procedures using animals and their specimens
were performed in accordance with the guidelines regulating
animal use at Kagoshima University.

2. Materials and Methods
2.1. Dogs Aﬀected with NCL. The number of aﬀected dogs
was surveyed based on the records in our laboratory, whichhave been exclusively supporting the diagnosis of the diseasein Japan. Among 27 aﬀected dogs identiﬁed in the present
study, NCL was diagnosed deﬁnitively in 25 dogs by a genetic
test [5] using their specimens containing DNA, but in theremaining 2 dogs that died without any specimens before
the genetic test got available, NCL was strongly suspectedbased on their typical clinical history and blood relationship with molecularly deﬁned aﬀected littermates and/or
carrier parents. The clinical characteristics were analyzed
and summarized using information about all of the aﬀected
dogs, which was gleaned from interviews and questionnairesof their owners and veterinarians. Some of the aﬀected
dogs were examined using the following prediagnostic tests:
magnetic resonance image (MRI) scan in 7 dogs, including apreviously reported dog [10]; computed tomography (CT)scan in 2 dogs; ophthalmologic examination in 3 dogs.
The ﬁndings of these examinations were also analyzed andsummarized.

3. Results
3.1. The Number and Clinical Characteristics of Border Collies
with NCL. The survey revealed that there were 27 aﬀected
dogs (13 males and 14 females) in the 12 years from 2000
to 2011 in Japan (Table 1). The ﬁrst case, born in 2000, wasdiagnosed with NCL histopathologically [10], which was
conﬁrmed molecularly using the stored liver specimen. Since
this case, several aﬀected dogs from a single or a few litterswere diagnosed nearly every year up until recently in Japan.
However, currently there seems to be a decreasing trend
based on the observation that only 1 aﬀected dog was
identiﬁed since 2009.
The clinical features of the aﬀected dogs are summarized
and listed in Table 2. They were divided into 3 stages, thatis, early (15–20 months of age), middle (19–23 months ofage), and late-to-terminal stages (22–32 months of age).
The overlaps in age between adjacent stages were due toindividual variability in the onset and progression rate ofthe disease. The features at the early stage were mainly
behavioral problems that most owners of the dogs did notregard as pathological changes. The features at the middle stage included serious behavioral abnormalities, visual
impairments, and slight motor disorders, which prompted

2.2. Genotyping Survey. Border Collies belonging to 4 special
kennels (A, B, C, and D), which generated one or more
aﬀected dogs, were surveyed between 2008 and 2010 using
a genetic test with the breeders’ cooperation. The number ofdogs examined was 82, including 23, 20, 29, and 10 in kennels
A, B, C, and D, respectively. Whole blood or saliva specimens
were collected from these dogs using the Flinders Technology

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speciﬁc changes before 15 months of age, some of the owners
had an impression that their dogs had unusual characteristics
and behaviors and low learning ability in the puppy andjuvenile years. There was no abnormal change on generalclinicopathological examinations. No abnormal vacuoles in
the cytoplasm of leukocytes were observed in blood smear
examinations.

Table 1: Number of Border Collies with neuronal ceroid lipofuscinosis and the number of litters that included aﬀected dogs during
the 12 years between 2000 and 2011 in Japan.
Year of birth
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010–present∗
Total

Number of aﬀected dogs
(male and female)
2 (2, 0)6 (3, 3)0 (0, 0)4 (3, 1)4 (1, 3)2 (1, 1)1 (0, 1)2 (1, 1)5 (2, 3)
1 (0, 1)0 (0, 0)27 (13, 14)

Number of litters
1
3
0
3
2
2
1
2
4
1
0
19

3.2. MRI, CT, and Ophthalmologic Examinations. MRI,
CT, and ophthalmologic examinations were performed inaﬀected dogs in the middle-to-late stage (at approximately
2 years of age). The common ﬁndings of MR and CT images
were ventricular enlargement and well-demarcated cerebral
sulci (Figure 1). Dilation of cerebellar ﬁssures was also
observed at a sagittal section of the MRI scan (Figure 1(c)).
No other organic lesion was detected in MRI and CT scans.
In the aﬀected dogs examined ophthalmologically, slight narrowing of blood vessels in the retina was commonly observedin ophthalmoscopy (Figure 2(a)), but this ophthalmoscopicﬁnding was evaluated within the normal range by veterinary
ophthalmologists. An examination using a slit lamp detected
no abnormal ﬁnding, such as clouding of the cornea and lens(Figure 2(b)).

∗June 2012.

Table 2: Summary of clinical features in Border Collies with neuronal ceroid lipofuscinosis∗ .
Stage
Clinical signs(months of age)
Altered characteristics; disregard for owner’scommands; loss of interest in play and otherdogs; morbid fear of noise, humans, andEarlyunspeciﬁed things; hallucination; disorienta(15–20)
tion; biting; averse to going up and down stairs(especially down)Fly-biting behavior; continuous shaking of thehead; sudden halts during walks; uncooperativeness with other dogs; head tilt; chomping
without food; tooth grinding; leg jerking; visual
Middleimpairments (fear of darkness, unawareness(19–23)of things such as food, and frequently hitting obstacles); aggressiveness; excitation; staggering; falling; toileting accidents; myoclonus;
myoclonic seizure
Wandering; hair-pulling disorder; circadianrhythm disorder; acoustic and cutaneous hyperesthesia; cognitive and emotional impairments;
Late to terminal
blindness; dysmetria; gait deﬁciency; convulsive(22–32)
seizure; face and mouth tic; chewing diﬃculty;
lethargy; stupor; death (mean age 26.7 months,
ranging from 23 to 32 months)†

3.3. Genotyping Survey. The results of the genotyping survey
in the 4 speciﬁc kennels are shown in Table 3. The frequenciesof carriers (32.9%), aﬀected dogs (18.3%), and the mutant
allele (34.8%) were markedly high in the 4 kennels.
3.4. Pedigree Analysis. The pedigree analysis was performed
mainly using the pedigree information of the 27 aﬀected dogs
and 58 carriers identiﬁed so far in Japan (Figure 3). These
dogs were related to at least 13 possible carriers imported
from Oceania to Japan in the middle 1990s, which ultimately
revealed an ancestry from a male dog born in 1944 inAustralia through 9 carriers reported by the New South Wales
Border Collie Club. As a result, this analysis revealed thatall dogs carrying the mutant allele in Japan share a blood
relationship and a single common ancestor in Australia.
The pedigree analysis also revealed that breeding was
repeated using a small number of carriers and their oﬀspringin the 4 kennels surveyed in the present study. The breedersof these kennels were not aware of NCL in Border Collies
before the genotyping survey. As a result, these kennels had
markedly high frequencies of the mutant allele.

∗Data summarized from the information of 27 aﬀected dogs.

4. Discussion†Data from 17 aﬀected dogs that died naturally without euthanasia.

The present study revealed 27 Border Collies (13 males and
14 females) with NCL in Japan during 2000–2011 (Table 1),
although there might have been additional aﬀected dogs
elsewhere that had no opportunity to be diagnosed. Since
the ﬁrst aﬀected dog, born in 2000, was diagnosed in 2002[10], several aﬀected dogs from a single or a few littershave been diagnosed nearly every year up until recently, butcurrently there seems to be a decline in the frequency ofNCL. The decreasing trend may be due to the publicationof the pathogenic mutation in 2005 [11], the subsequent

the owners to consult veterinarians. The features at the lateto-terminal stage included serious motor, visual, psychointellectual, and vital dysfunctions due to a wide spectrum of
brain dysfunctions. The mean life span of the 23 aﬀected
dogs examined was 26.4 months of age. Of these, 17 diednaturally at a mean age of 26.7 months, ranging from 23 to
32 months. The other 6 dogs were euthanized at the terminal
stage an average of 25.6 months. Although there were few

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Table 3: Results of the genotyping survey carried out in 4 kennels that generated aﬀected dogs∗ .

Kennel
A
B
C
D
Total

Dogs examined
23
20
29
10
82

Number of
Carriers
9
11
6
1
27

Aﬀected dogs
8
2
2
3
15

Frequency (%)
Aﬀected dogs
34.8
10.0
6.7
30.0
18.3

Carriers
39.1
55.0
20.7
10.0
32.9

Mutant allele
54.3
37.5
17.2
35.0
34.8

∗These genotyping surveys were carried out between 2008 and 2010.

(a)

(b)

(c)

(d)

Figure 1: Representative magnetic resonance (MR) and computed tomography (CT) images of the brain of Border Collies with neuronal
ceroid lipofuscinosis. MR images were obtained using a 0.3-tesla system (AIRIS2-comfort, Hitachi Medical Corporation) in a 24-month-old
dog under general anesthesia. CT images were obtained using a multislice CT system (ECLOS, Hitachi Medical Corporation) in another24-month-old dog under general anesthesia. (a) MR T1-weighted image (TR/TE = 500/20 ms) of a transverse section at the level of thethalamus, (b) MR T2-weighted image (TR/TE = 4,000/120 ms) of a transverse section at the level of the thalamus, (c) MR T2-weighted
image of a sagittal section, and (d) CT image of a transverse section at the level of the thalamus. These images show enlarged ventricles anddilated cerebral and cerebellar sulci, suggesting forebrain atrophy.

development of a variety of genetic testing methods [5],and genotyping surveys in a random population of breeding
Border Collies [5] and in a few speciﬁc kennels that generated
aﬀected dog(s) (present study). These events may havecontributed to the prevention and control of the disease
through the education of related breeders and fanciers of
Border Collies.

Based on information from the owners and veterinariansof the aﬀected dogs, their characteristic clinical features were
determined (Table 2), although there were individual diﬀerences in the onset and progression rate of the disease andthe symptoms, as reported previously [6]. Mild clinical signs,
such as behavioral problems, begin at 15 months of age
at the earliest, but the onset is usually a few months later

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(a)

(b)

Figure 2: Photographs of ophthalmoscopic and slit-lamp examinations of the right eye in a 21-month-old Border Collie with neuronal
ceroid lipofuscinosis. (a) Slight narrowing of blood vessels in the retina is observed, but (b) there is no abnormal ﬁnding in the slit-lamp
examination.

Oceania
∗

∗

∗

∗

∗

∗

∗

∗

∗ Imported

∗
∗

∗

∗

∗

Japan

Kennel A
Kennel B

Kennel D

Kennel C

Affected
Carrier
Carrier reported by NSWBCC
Wildtype (clear)Not determined

Figure 3: Genetic relationship of aﬀected and carrier dogs identiﬁed in Japan between 2000 and 2011. The analysis was carried out using
pedigree papers issued by the Japan Kennel Club and the Kennel Club of Japan and information reported by the New South Wales Border
Collie Club (NSWBCC) and the Border Collie Database. The gray area indicates a border between Oceania and Japan. Dogs imported from
Oceania to Japan were marked with an asterisk (∗ ). All dogs carrying the mutant allele shared a common ancestor († ) born in 1944 inAustralia. Areas surrounded by a dashed line indicate the 4 kennels surveyed using a genotyping test.

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The Scientiﬁc World Journal(approximately 18 months of age) at the early stage. The
earliest age of onset in the present study is consistent withthat in a previous report [8]. Clinical signs observed at theearly stage (15–20 months of age) are mainly mild behavioral
abnormalities that the owners, without any knowledgeof NCL, hardly regard as pathological. Visual disorders
become unambiguous and behavioral abnormalities increase
in severity at the middle stage (19–23 months of age). Serious
clinical signs, such as convulsive seizure and motor disorders,appear at the late-to-terminal stage (older than 22 months ofage). The mean life span of aﬀected dogs was 26.7 monthsof age in the present study. The life span was 26.4 monthsof age in the dogs that died naturally, ranging from 23 to 32
months of age. This is longer than the 23.1-month life spanof aﬀected Border Collies in a previous report in Australia[7]. The diﬀerence could be attributed to the fact that many
Japanese pet owners feel that euthanasia is inhumane and do
not want to kill animals before they die naturally.
The characteristic clinical features determined in thepresent study are similar to those in previous reports [6, 7].
However, a previously reported clinical sign, mania [7],was not observed in the present study. In addition, someaﬀected dogs had unusual characteristics and behaviors andlow learning ability in the puppy and juvenile years, which
was not reported previously. These clinical characteristics,
updated in detail, will be of further help for the diagnosisof NCL in Border Collies.
In the MRI examination, ventricular enlargement anddilated cerebral and cerebellar sulci were common ﬁndings in aﬀected dogs (Figure 1). The CT examination also
demonstrated ventricular enlargement. These characteristic
ﬁndings suggest atrophy of the forebrain, which was similarly
observed in previous MRI [10] and CT examinations [9].
The changes suggesting brain atrophy developed as early as
the middle stage, when aﬀected dogs were usually referred
to animal hospitals. Similar changes have been observedin other types of NCL [12, 13] and other lysosomal
storage diseases, such as GM1 gangliosidosis [14] and GM2
gangliosidosis [15–17] in dogs and cats. The brain atrophy
usually develops as a speciﬁc and severe change even at the
middle stage in NCL but as a secondary and mild change
in the late-to-terminal stage in other lysosomal diseases.
Therefore, the atrophic changes would be useful as an
adjunct to the diagnosis of NCL. No other organic lesion was
detected in MRI scans in Border Collies with NCL, although
hyperintensity in T2-weighted images of the cerebral whitematter is observed in GM1 and GM2 gangliosidoses in dogs
and cats [14, 16–19]. Meningeal thickening, reported in
Chihuahuas with NCL [13], was not observed in Border
Collies with NCL.
Visual impairments appeared to various degrees inall aﬀected dogs, especially at the middle stage (Table 2).
Previous reports have not described any abnormal changes
on ophthalmoscopic examination [7, 10]. In the present
study, the aﬀected dogs examined had slight narrowing ofblood vessels in the retina (Figure 2(a)), but this ophthalmoscopic ﬁnding was considered within the normal range
by veterinary ophthalmologists (Figure 2). In humans with
NCL, there is a pronounced loss of photoreceptors in theend stage of the disease, but in NCL in English Settersonly minimal structural damage is observed in the retina[20]. In Border Collies with NCL, inclusions with variableultrastructure are common in all cells of the retina, but
the pigment accumulation does not damage the retinalarchitecture [8]. The retinal lesions in the Border Collies
are similar to those in the English Setters but are much lesssevere than in juvenile NCL in humans. The narrowing of
retinal blood vessels observed in aﬀected Border Collies may
be an indication of mild retinal degeneration, but this featurecannot be used as a valuable diagnostic for NCL.
Based on data of the pedigree analysis, aﬀected dogs andcarriers identiﬁed in Japan share a blood relationship anda single common ancestor born in 1944 in Australia via
some presumptive carriers in Oceania (Figure 3). Although
it is impossible to demonstrate whether this ancestor wasa founder of NCL in Border Collies, there is a possibility
that the mutant allele was transmitted from Oceania toJapan in this way mainly in the middle of the 1990s, when
the causative mutation had not been identiﬁed yet. The
recent increase in prevalence of the disease in Japan is
likely due to an overuse of imported carriers and theiroﬀspring as breeding dogs without any knowledge of thedisease. Hereafter, dogs that will be bred should be genotyped
beforehand.
The pedigree analysis also suggests that breeding has been
repeated using a small number of carriers and their oﬀspringin the 4 kennels that generated aﬀected dogs (Figure 3).
There was a trend toward inbreeding in these kennels. In
addition, the breeders of the kennels were not aware of thedisease until they were informed of the generation of aﬀected
dogs in their kennels. These issues may have caused thehigh frequencies of carriers (32.9%) and the mutant allele(34.8%) in the 4 kennels (Table 3), compared to the carrier(8.1%) and mutant allele frequencies (4.1%) in the random
population of Border Collies [5]. Fifteen (55.6%) of the 27
aﬀected dogs identiﬁed so far in Japan were generated inthese 4 kennels. It is thought that aﬀected dogs are generated
in a small proportion of kennels, which have a high frequencyof the mutant allele. Therefore, it is important for preventionof the disease to rapidly genotype all breeding dogs in kennels
that have had an opportunity to generate aﬀected dogs.
This type of examination helps the kennels not only stop
generating additional aﬀected dogs but also stop spreading
carriers to other kennels. In addition, a genotyping test using
specimens from a random population of breeding Border
Collies should be continued [5] to detect sporadic carriers
and prevent them from being used as breeding dogs. These
approaches would gradually decrease the number of dogs
carrying the mutant allele. These active and continuous
preventive measures may be necessary to eliminate NCL in
Border Collies.

Acknowledgments
The authors are grateful to Ms. Kasukawa (the ﬁrst aﬀected
dog’s owner), all other aﬀected dogs’ owners, and all
veterinarians associated with the dogs for providing their575

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7

specimens and information about them. The authors are also
grateful to all members of the Japan Border Collie Health
Network for their help with the collection of information
about aﬀected and carrier Border Collies. This study was
supported ﬁnancially by grants (nos. 14560258, 1680210,
20380173, 20-08112, and 21658109, O. Yamato) from the
Ministry of Education, Culture, Sports, Science and Technology of Japan.

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References[1] R. D. Jolly, D. N. Palmer, R. H. Studdert et al., “Canine ceroidipofuscinoses: a review and classiﬁcation,” Journal of Small
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[2] R. D. Jolly and D. N. Palmer, “The neuronal ceroid-lipofuscinoses (Batten disease): comparative aspects,” Neuropathology
and Applied Neurobiology, vol. 21, no. 1, pp. 50–60, 1995.
[3] S. L. Hofmann and L. Petronen, “The neuronal ceroid lipofuscinoses,” in The Metabolic and Molecular Bases of Inherited
Disease, C. Scriver, A. Beaudet, W. Sly, and D. Valle, Eds., pp.
3877–3894, McGraw-Hill, New York, NY, USA, 8th edition,
2001.
[4] A. Jalanko and T. Braulke, “Neuronal ceroid lipofuscinoses,”
Biochimica et Biophysica Acta, vol. 1793, no. 4, pp. 697–709,
2009.
[5] K. Mizukami, H.-S. Chang, A. Yabuki et al., “Novel rapid
genotyping assays for neuronal ceroid lipofuscinosis in Border
Collie dogs and high frequency of the mutant allele in Japan,”
Journal of Veterinary Diagnostic Investigation, vol. 23, no. 6, pp.
1131–1139, 2011.
[6] R. M. Taylor and B. R. H. Farrow, “Ceroid-lipofuscinosis in
Border Collie dogs,” Acta Neuropathologica, vol. 75, no. 6, pp.
627–631, 1988.
[7] V. P. Studdert and R. W. Mitten, “Clinical features of ceroid
lipofuscinosis in Border Collie dogs,” Australian Veterinary
Journal, vol. 68, no. 4, pp. 137–140, 1991.
[8] R. M. Taylor and B. R. H. Farrow, “Ceroid lipofuscinosis in
the Border Collie dog: retinal lesions in an animal model ofjuvenile Batten disease,” American Journal of Medical Genetics,
vol. 42, no. 4, pp. 622–627, 1992.
[9] J. N. Franks, C. W. Dewey, M. A. Walker, and R. W. Storts,“Computed tomographic ﬁndings of ceroid lipofuscinosis in adog,” Journal of the American Animal Hospital Association, vol.
35, no. 5, pp. 430–435, 1999.
[10] H. Koie, H. Shibuya, T. Sato et al., “Magnetic resonanceimaging of neuronal ceroid lipofuscinosis in a Border Collie,”
Journal of Veterinary Medical Science, vol. 66, no. 11, pp. 1453–
1456, 2004.
[11] S. A. Melville, C. L. Wilson, C. S. Chiang, V. P. Studdert, F.
Lingaas, and A. N. Wilton, “A mutation in canine CLN5
causes neuronal ceroid lipofuscinosis in Border Collie dogs,”
Genomics, vol. 86, no. 3, pp. 287–294, 2005.
[12] M. Kuwamura, R. Hattori, J. Yamate, T. Kotani, and K. Sasai,“Neuronal ceroid-lipofuscinosis and hydrocephalus in a chihuahua,” Journal of Small Animal Practice, vol. 44, no. 5, pp.
227–230, 2003.
[13] Y. Nakamoto, O. Yamato, K. Uchida et al., “Neuronal ceroidlipofuscinosis in longhaired chihuahuas: clinical, pathologic,
and MRI ﬁndings,” Journal of the American Animal Hospital
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[14] D. Hasegawa, O. Yamato, Y. Nakamoto et al., “Serial MRI
features of canine GM1 gangliosidosis: a possible imaging

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短

報

ミオキミア／ニューロミオトニアのヨークシャーテリアの 1 例
齋藤弥代子†

宇津木真一

麻布大学獣医学部（〒 229h8501

久末正晴

相模原市淵野辺 1h17h71）（2010 年 2 月 8 日受付・ 2010 年 9 月 24 日受理）要

約

8 歳齢の雌のヨークシャーテリアが呼吸促迫および四肢の硬直を主徴とする発作を主訴に来院した．身体一般および神経学的検査で，体幹部および四肢の筋肉に律動的で波状のミオキミアに特徴的な動きが観察された．筋電図ではミオ
キミア放電を認めた．脳脊髄液検査は単核細胞増多を示したが，脳の MRI では明らかな異常を認めなかった．これらの所見から炎症性脳疾患を伴うミオキミア／ニューロミオトニアと診断し，抗生剤とゾニサミド，プロカインアミドの投与を行った．初診から 5 カ月後の脳脊髄液検査は，正常に復していた．現在 11 カ月が経過し，ゾニサミドとプロカイン
アミドの投薬を継続中だが，呼吸促迫および四肢の硬直を示すニューロミオトニアは認められておらず，ミオキミアは頻度と程度が顕著に改善されている．―キーワード：犬，ミオキミア，ニューロミオトニア．
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筋の不随意的反復性収縮に伴い直上の皮膚が波打ち，た．発作が長時間に及ぶと全身を繰り返し大きくピクッ

まるで皮膚の下を虫がうごめくように見える臨床徴候とさせる（ミオクローヌス（MC）様）徴候に移行するは，ミオキミア（m y o k y m i a ： M K ）と呼ばれることがあるとのことだった．この発作以外にほぼ毎日間［1h4］．また，運動や興奮により誘発される発作性の全欠的に皮膚がピクピク動き頻繁にその部分を噛む動作が

身性筋硬直を特徴とする持続的筋収縮は，ニューロミオ

認められていた．この皮膚徴候の発現は前述の発作とほトニア（n e u r o m y o t o n i a ： N M T ）と呼ばれているぼ同時期の発症であった．身体一般検査では四肢の近位［1h6］．MK と NMT はともに末梢神経軸索の興奮性の筋の肥大が認められた．さらに体幹部および四肢の筋

亢進により生じ，同一症例にて MK と NMT がともに認に，律動的で波状のあたかも皮膚の下を小さな虫がうごめられることが多いため，同一の異常が背景にあると考めいているかのような MK に特徴的な動きが観察され，えられている［2, 4, 6, 7］．獣医療における MK/NMT

特に上腕と大腿外側面の筋で顕著であった．その他の身の報告は非常に少なく，8 例の犬と 1 例の猫での報告が体一般検査と神経学的検査に異常は認めなかった．全血あるのみである［6h9］．またわれわれの知るかぎり本邦

球算定と生化学的検査では CK の軽度上昇（177IU/l）における報告はない．今回，われわれは炎症性脳疾患に以外異常を呈さなかったが，総胆汁酸は空腹時 2 5 . 1

伴い MK/NMT を呈したヨークシャーテリア（YT）に

μmol/l，食後 2 時間 91.5μmol/l と高値を示した（表）．

遭遇し，診断と治療を行う機会を得たので，その臨床像

腹部単純 X 線で小肝を認めたが超音波検査では肝実質にを報告する．

異常はなく門脈体循環シャントを疑う所見も認めなかっ症

た．続いて全身麻酔下で神経系の精査を行ったが，全身例

麻酔中もさまざまな四肢や体幹の筋で前述と同様の波状

8 歳齢，体重 2.4kg，雌の YT が約 2 年前から月に 1，の細かい動きが時折観察された．頭部 MRI において明2 回発作を起こし，悪化傾向にあるとの主訴で麻布大学

らかな異常は認めなかった．大槽からの脳脊髄液（CSF）

附属動物病院神経科（以下当院）に紹介来院した．発作検査では，単核細胞増多症（総有核細胞数 49/μl（基準はおもに車での外出や来客，トリミング時に発現し，呼値 0 〜 5/μl），大単核球と小単核球各 50 ％）を呈した．

吸速迫から始まり四肢が硬直し，その後後肢が虚脱し起蛋白は 21.8mg/dl（基準値＜ 25mg/dl），糖は 71mg/dl

立不能に陥り重症時には横臥へと進行，それらが約 2 〜とともに正常であった．CSF あるいは血液にて，犬ジス

5 時間持続しこの間は呼びかけに反応するとのことだっ

テンパー，Toxoplasma gondii，Neospora caninum の† 連絡責任者：齋藤弥代子（麻布大学獣医学部外科学第二研究室）〒 229h8501 相模原市淵野辺 1h17h71 蕁 042h754h7111
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FAX 042h769h1639

E-mail : msaito@azabu-u.ac.jp

宇津木真一表項目

結
2

WBC（×10 /μl）
RBC（×104/μl）
PCV（％）
Hgb（g/dl）
MCV（fl）
MCH（pg）
MCHC（g/dl）
PLT（×104/μl）

果

118.0
700.0
48.6
16.6
69.3
23.8
34.3
54.6

項

齋藤弥代子

久末正晴

大学初診時血液検査結果

目結TP（g/dl）
Alb（g/dl）
Glu（mg/dl）
AST（IU/l）
ALT（IU/l）
ALP（IU/l）
Tbil（mg/dl）
Tchol（mg/dl）
BUN（mg/dl）
Cre（mg/dl）

果

7.9
4.2
116.0
26.0
38.0
337.0
0.4
269.0
15.0
0.4

項

目

結

果

Ca（mg/dl）
IP（mg/dl）
LDH（IU/l）
CK（IU/l）Na（mmol/l）K（mmol/l）
Cl（mmol/l）

11.8
2.7
68.0
177.0
143.0
4.1
103.0

総胆汁酸（空腹時）（μmol/l）
総胆汁酸（食後 2 時間）（μmol/l）

25.1
91.5

1.10s
1.20s
1.30s
1.40s
1.50s
1.60s
1.70s
1.80s

200μV

1.90s
図1

5 ms

左橈側手根屈筋の針筋電図検査で記録されたミオキミア放電の一部．
単一運動単位電位が短持続（約 20ms）の群をなして発射し，約 0.5 秒の静止期の後，同一の群放電の反復が認められた．

IgG 測定，犬ジステンパー抗原 PCR 検査，細菌・真菌として飼い主との相談の下合計 2 カ月間上記 2 種の抗生培養を行ったが，いずれも異常は認めなかった．脳波検剤投与を継続した．初診から 5 日目に筋硬直から開始し

査では左前頭・側頭葉優位に突発性発射（棘波）を多数

全身をピクッとする発作が半日持続したと連絡を受け認めた．針筋電図検査（EMG）では，左側橈側手根屈た．そこでゾニサミド（ZNS）（エクセグラン散 20 ％，筋から単一運動単位電位が 130 〜 250Hz の頻度で短持大日本住友製薬譁，大阪）5mg/kg 1 日 2 回の投与開始

続の群をなして発射し，短い静止期の後，同一の群放電を紹介元獣医師へ指示した．その後 3 カ月間 MK は改善が反復するという，ミオキミア放電に一致する所見が認なく持続したが NMT と MC 様徴候は認めなかった．しめられた（図 1）．また，左上腕三頭筋からは複合反復放かし初診から 110 日目に NMT が再発し，さらに 117 日電が得られたがその他の四肢，体幹，顔面の筋は異常を目の当院再診時にも院内で呼吸速迫および軽度の四肢硬呈さなかった．EMG 時に MK 様の動きが視認された筋直を示す NMT を認めその際発熱（39.5 ℃）も確認されはなかったが，左側橈側手根屈筋においては筋がピクピた．安静と冷却処置を施したところ発作は急速に終息しクと動く感触を触知することは可能であった．以上を総平熱に復した．同日からプロカインアミド（PA）（アミ

合して，本症例における筋の波状の動きは MK であり，

サリン錠，第一三共㈱，東京）17mg/kg 1 日 3 回の経

発作性の筋硬直は N M T の可能性が高いと判断した．

口投与を開始した．同日行った ZNS 血中濃度測定結果

CSF 所見と臨床所見から髄膜脳炎を始めとする炎症性は 7.6μg/dl（トラフ値）と低値を示し，粉末状 ZNS の脳疾患が疑われたため，感染性疾患の検査結果を待つ間投与不良が判明したため 10mg/kg 1 日 2 回への増量とクリンダマイシン（ダラシンカプセル，ファイザー譁，

錠剤への変更を指示した．145 日目に再度 CSF 検査を行

東京）10mg/kg 1 日 2 回およびドキシサイクリン（ビったところ細胞数は 4/μl と正常に復していた．NMT はブラマイシン錠，ファイザー譁，東京）5mg/kg 1 日 2

終息していたが M K は改善がなかったため，P A を回の経口投与を開始した．後日出た検査結果は感染性髄26mg/kg 1 日 3 回へ増量し，その後 MK の頻度と程度

膜脳炎を示唆するものではなく特定の免疫介在性脳炎をが顕著に減少した．現在初診から 11 カ月が経過し ZNS

裏付ける所見にも乏しかったため，原因不明の髄膜脳炎と PA 投薬を継続中だが，NMT と MC 様徴候は認めら
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0

30

60

90

120

150

180

210

330日

大学初診日
MKの頻度と程度
NMT
MC
クリンダマイシン
10mg/kg BID
ドキシサイクリン
5mg/kg BID
ゾニサミド
10mg/kg BID

5mg/kg BID
プロカインアミド

17mg/kg TID

26mg/kg TID

図 2 治療および MK/NMT，MC 徴候の経過（MK ：ミオキミア，NMT ：ニューロミオトニア，MC ：ミオクローヌス様の発作）

れていない．さらに飼い主の報告や院内での観察では，検出されることが多い［1h3, 5, 13, 14］．しかし獣医学

MK の律動的で波状の筋の動きが程度と頻度ともに初診領域では，MK/NMT の発症機序や併発誘因疾患につい時よりも明らかに減少した状態が継続している（図 2）．

てはまったく解明されていない．MK/NMT を呈した 1

考頭の猫での抗 VGKC 抗体測定の報告があるが陰性であ

察

った［6］
．われわれの症例では炎症性中枢疾患に一致す

本症例では体幹部および四肢の筋に律動的で波状の虫る CSF 有核細胞数増多が認められたが，顔面 MK の犬 1が這っているような動きが観察され，この動きは全身麻頭においても原因不明の CSF 有核細胞数増多が報告さ

酔中も消失しなかった．これらは犬猫における過去のれている［9］．人では，NMT を呈した抗 VGKC 抗体陽MK の報告と同一である［6, 8］．MK における EMG で性辺縁系脳炎の報告があるが［15］，一般的に本脳炎には，単一運動単位が 5 〜 400Hz の頻度で数秒間群をなおける NMT はまれであり，本脳炎と NMT との関連性して発射し短い静止期の後に同じ群放電を繰り返すミオ

は不明と結論付けられている［15, 16］．われわれの症例

キミア放電が認められるが［4, 6, 10］，われわれの症例では，人の抗 V G K C 抗体陽性辺縁系脳炎に類似したでも同様の EMG 所見が認められた．NMT は発作性の

MRI 所見や臨床所見は観察されなかった．われわれの全身性筋硬直を特徴とする持続的筋収縮であるが，スト症例における CSF 有核細胞数増多の原因は不明である

レスや興奮が NMT の引き金になる，発作中は意識が保が，炎症性脳疾患と MK/NMT が同一の免疫学的機序

たれる，高体温を示すなどの共通点が示されている［6h9］．

により発症した，あるいは炎症性脳疾患が先行感染としさらに NMT はわれわれの症例を含めすべての犬猫でて MK/NMT 発症に関与した可能性があるかもしれな

MK と併発している．NMT における EMG はニューロ

い．本症例ではさらに総胆汁酸の高値を認めたが飼い主

ミオトニー放電と呼ばれ MK と同様突発性に生じる運動の希望によりそれ以上の精査を行うことができなかっ

単位発射であるが，ミオキミア放電よりも持続時間が長た．過去の報告をみると，MK/NMT の犬において肝酵

く非律動性で典型例では電位の振幅が漸減する［7, 10］．

素の軽度上昇を認めるものがあるが MK/NMT との関

われわれの症例の EMG では，ニューロミオトニー放電

連性は言及されていない［6］．人では B 型肝炎とデルタは観察されなかった．MK/NMT はともに末梢神経軸索肝炎が併発した MK の報告が 1 例あるが，MK と肝炎との興奮性の亢進によって生じるが，この亢進は電位依存の関連は不明とされている［17］．人における MK/NMT

性カリウムチャネル（VGKC）機能不全が原因とされるの治療は併発誘因疾患の治療が主体をなす．MK/NMT［11］．人の先天性 MK では VGKC 遺伝子変異が報告さに対する治療として，カルバマゼピン，フェニトインなれている［12］．後天性の場合は，各種免疫介在性疾患

どの抗けいれん薬が人では使用され［3］，特に抗 VGKCとの併発を始め感染症との併発あるいは腫瘍随伴症候群

抗体陽性例において高い効果が認められているとしての発症が知られ，患者血清中に抗 VGKC 抗体が［18h20］．これらの薬剤の作用機序として，Na ＋チャネ

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with myokymia during acute cohinfection with
hepatitis B and delta viruses, J Med Virol, 25,245h247 (1988)［18］ Ishii A, Hayashi A, Ohkoshi N, Oguni E, Maeda M,
Ueda Y, Ishii K, Arasaki K, Mizusawa H, Shoji S :Clinical evaluation of plasma exchange and highdose intravenous immunoglobulin in a patient with
Isaacs’ syndrome, J Neurol Neurosurg Psychiatry,57, 840h842 (1994)［19］ 園田至人，有村公良： Isaacs 症候群と抗カリウムチャネ

考えられている［21］．犬ではカルバマゼピンやフェニ
トインは薬物代謝が速く血中濃度を維持できないため通常使用されない［22］．同様の作用機序を持つ PA 投与の効果が MK/NMT を呈した 1 頭の YT で報告されている［8］．その他免疫抑制量のプレドニゾロンやジアゼパム使用の報告があるが MK/NMT に効果は認められていない［6, 7］．本症例の長時間の NMT に続発した MC様徴候をわれわれが直接観察する機会はなかったが，MC 徴候は必ず NMT 発現時に認められたため，NMTの一徴候として生じた可能性が考えられる．しかし鑑別のために行った脳波上でてんかん性異常波を多数確認したため，MC 様徴候は炎症性脳疾患により生じたてんかん発作である可能性も考慮すべきと考えた．そのため
Na ＋チャネル不活性化による膜の安定化作用を有し理論
上 MK/NMT にも効果がある可能性のある抗てんかん薬，ZNS 投与をまず試みた．ZNS 投与のコンプライア
ンスは良好とはいえなかったが ZNS 開始から 3 カ月間
NMT の制御が可能であった．その後 NMT が再燃した
ため ZNS 増量とともに PA 投与を開始した．以降 NMTは認めておらず MK においても頻度や程度の改善が認められた．MK/NMT はまれな疾患であるが罹患動物のQOL を著しく落とし死亡例も報告されていることから見過ごしてはいけない疾患である．未解明な点が非常に多く今後は世界レベルでの症例の蓄積を行い獣医療における本疾患の発症機序，臨床像，さらに適切な治療法を明らかにする必要があると考えられた．
最後に，症例をご紹介頂いた八王子市ノア動物病院に深謝する．なお，本研究は科研費（20580359）の助成を一部受けたものである．

引

用

文

久末正晴

献

［ 1 ］ Gutmann L, Gutmann L : Myokymia and neuromyotonia 2004, J Neurol, 251, 138h142 (2004)［ 2 ］ Gutmann L, Libell D, Gutmann L : When is myokymia
neuromyotonia?, Muscle Nerve, 24, 151h153 (2001)［ 3 ］ Vincent A : Understanding neuromyotonia, Muscle
Nerve, 23, 655h657 (2000)［ 4 ］ Dewey CW : A Practical Guide to Canine and Feline
Neurology, 2nd, 482h485, Blackwell Publishing, Iowa
(2008)［ 5 ］ Gutmann L : facial and limb myokymia, Muscle
Nerve, 14, 1043h1049 (1991)［ 6 ］ Van Ham L, Bhatti S, Polis I, Fatzer R, Braund K,
Thoonen H : Continuous muscle fibre activity in six
dogs with episodic myokymia, stiffness and collapse,

ル抗体，Clinical Neuroscience，16，84h87（1998）［20］ Ramseyer JC : Isaacs’ syndrome, West J Med, 123,130 (1975)［21］ Bowman WC, Rand MJ : Textbook of pharmacology,
2nd ed, 31, Blackwell Scientific, Oxford (1980)［22］ Frey HH : Anticonvulsant drugs used in the treatment of epilepsy, Probl Vet Med, 1, 558h577 (1989)

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56 〜 60（2011）ミオキミア／ニューロミオトニアの犬の 1 例

Generalized Myokymia and Neuromyotonia in a Yorkshire Terrier
Shinichi UTSUGI ＊, Miyoko SAITO† and Masaharu HISASUE＊ School of Veterinary Medicine, Azabu University, 1h17h71 Fuchinobe, Sagamihara, 229h
8501, JapanSUMMARY
An eight-year-old female Yorkshire terrier presented with episodes of hyperpnea and stiffness of the limbs.
A physical and neurological examination revealed rhythmic, undulating movement across the muscles of thetrunk and limbs. This movement was characteristic of myokymia. An electromyogram demonstrated
myokymicdischarges. The cisternal cerebrospinal fluid (CSF) showed mononuclear pleocytosis, which wasindicative of inflammatory central nervous system disease. The MRI of the brain was unremarkable. A clinical diagnosis of myokymia/neuromyotonia with inflammatory brain disease was made, and the dog was treated with antibiotics, zonisamide and procainamide. A follow-up CSF analysis performed 5 months after the initial examination was normal. At present, 11 months after the diagnosis, the dog is still being treated with zonisamide and procainamide. No episodes of hyperpnea or stiffness of the limbs have been observed, and the
myokymia has decreased markedly in frequency and severity. ― Key words : dog, myokymia, neuromyotonia.
† Correspondence to : Miyoko SAITO (Department of Veterinary Surgery 蠡, Azabu University)
1h17h71 Fuchinobe, Sagamihara, 229h8501, JapanTEL 042h754h7111 FAX 042h769h1639 E-mail : msaito@azabu-u.ac.jp―――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――― J. Jpn. Vet. Med. Assoc., 64, 56 〜 60 (2011)

日獣会誌

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56 〜 60（2011）

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ࣛ࢖ࢻ㸦LPS㸧ࡢㄆ㆑࡟㛵ࢃࡿศᏊ࡛࠶ࡿࠋCD14 ࡢࣉ࣮ࣟࣔࢱ࣮㡿ᇦࡢኚ␗ࡣႍᜥࠊ࢔ࢺࣆ࣮
ᛶ⓶⭵⅖ࠊ㰯⭍࣏࣮ࣜࣉ࡜ࡢ㛵ಀᛶࡀே࡛᫂ࡽ࠿࡜࡞ࡗ࡚࠸ࡿࠋ≟ࡢ CD14 ࡢ㑇ఏᏊࡣࡲࡔࢡࣟ
࣮ࢽࣥࢢࡉࢀ࡚࠸࡞࠸ࠋ≟ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡟⑌ᝈཎᅉ㑇ఏᏊࡢゎᯒࡢࡓࡵࡢᇶ♏ⓗ࡞◊✲ࡢ㸯ࡘ࡜ࡋ࡚≟ࡢ CD14 ࡢ㑇ఏᏊࡣࢡ࣮ࣟࢽࣥࢢࢆ⾜࠺ࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋᮦᩱ࡜᪉ἲ ࣅ࣮ࢢࣝ≟࠿ࡽ cDNA ࢆྜᡂࡋࠊCD14 ࡢࢡ࣮ࣟࢽࣥࢢࢆ 5’-race ࠾ࡼࡧ 3’-race ἲࢆ⏝࠸࡚ᐇ᪋ࡋࡓࠋ㯞ᕸ኱Ꮫ⓶⭵⛉࡟᮶㝔ࡍࡿ⾑㣰᳨యࢆ㏙࡭ 500 ᳨య௨ୖ཰㞟ࡋࡓࠋ⤖ᯝ࡜⪃ᐹ
CD14 ࡢ㑇ఏᏊ㓄ิࡣࠊPubmed ࡟ᥖ㍕ࡉࢀ࡚࠸ࡿ᝟ሗ࡜␗࡞ࡗ࡚࠸ࡓ㸦ᅗ㸱㸧ࠋ≟ࡢ CD14
ࡣ 290 ಶࡢ࢔࣑ࣀ㓟࠿ࡽ࡞ࡾࠊ∵࡜ 75%ࠊ⨺࡜ 76%ࠊே࡜ 72%ࠊࣖࢠ࡜ 68%ࡢ┦ྠᛶ࡛࠶ࡗࡓࠋ
⾑㣰ࡼࡾ⢭〇ࡋࡓࢤࣀ࣒ DNA ࢆ⏝࠸࡚௒ᚋࠊ㑇ఏᏊከᆺࡢゎᯒࢆ㐍ࡵࡿࡇ࡜ࡀྍ⬟࡟࡞ࡗࡓࠋ
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1)

Kawarai S, Fujimoto A, Nozawa G, Kanemaki N, Madarame H, Shida T, Kiuchi A.: Evaluationof weekly bathing in allergic dogs with methicillin-resistant Staphylococcal colonization. Jpn J
Vet Res in press.

2)

Kawarai S, Hisasue M, Matsuura S, Ito T, Inoue Y, Neo S, Fujii Y, Madarame H, Shirota, K,
Tsuchiya R.: Canine pemphigus foliaceus with concurrent immune mediated thrombocytopenia. J
Am Anim Hosp Assoc 51:56-63, 2015

3)

Kawarai S, Matsuura M, Yamamoto S, Kiuchi A, Kanemaki N, Madarame H, Sirota K.: A case of
cutaneous sterile pyogranuloma/granuloma syndrome in a Maltese. J Am Anim Hosp Assoc585

50:278-283, 2014
4)

Kanemaki N, Tchedre KT, Imayasu M, Kawarai S, Sakaguchi M, Meguro A, Mizuki N.:
Identification of S1 RNA Binding Domain-1 SRBD1 as a Major Gene Determining Japanese
Shiba-Inu Dog Glaucoma. PLoS One 8:e74372, 2013

5)

Kaburaki Y, Fujimura T, Kurata K, Matsuda K, Toda M, Yasueda H, Chida K, Kawarai S,
Sakaguchi M.: Induction of Th1 immune responses to Japanese cedar pollen allergen in mice
immunized with Cry j 1 conjugated with CpG oligodeoxynucleotide. Comp Immunol Microbiol
Infect Dis 34,157-61, 2011

6)

Kawarai S, Sato K, Horiguchi A, Kurata K, Kiuchi A, Tsujimoto H, Sakaguchi M.: Potential
immunological adjuvant of ‘K’-type CpG-oligodeoxynucleotides enhanced the cell proliferation
and IL-6 mRNA expression in canine B cells. J Vet Med Sci 73,177-184, 2011Ꮫ఍Ⓨ⾲ᕝཎ஭᫴ᖹ㸸⓶⭵ឤᰁ⑕ࡢ᳨ᰝ࡜ゎ㔘࣭⥔ᣢࡢ௙᪉㸪᪥ᮏ⋇་ෆ⛉Ꮫ࢔࢝ࢹ࣑࣮➨ ᅇグᛕᏛ⾡኱఍ࠊᶓ὾ࠊ ᭶  ᖺ

2)

Kawarai S, Fujimoto A, Nozawa G, Kanemaki N, Madarame H, Shida T, Kiuchi A.: Decreasing
methicillin-resistant Staphylococcal colonization in allergic dog skin by shampoo. The Asian
Meeting of Animal Medicine Specialties, Bogor, Indonesia, Dec, 20133)ᮧୖᘯṇࠊ⣽ᕝ⪷▮ࠊᕝཎ஭᫴ᖹࠊᩬ┠ᗈ㑻: ῶឤస⒪ἲࢆᐇ᪋ࡋࡓ≟࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࡢ 2 ౛. ᪥ᮏᑠື≀⋇་Ꮫ఍ᮾ໭ᆅ༊኱఍ࠊ㒆ᒣࠊ10 ᭶ 2013 ᖺ 1㣤㔝⍞㈗ࠊすᮏඃᏊࠊ⸨ᮏ࠶ࡺࡳࠊ㔝⃝※ኴࠊᕝཎ஭᫴ᖹࠊಙ⏣༟⏨ࠊᮌෆ᫂⏨㸸≟ࡢ࢔ࣞࣝࢠ࣮ᛶ⓶⭵⅖ࡢ⑕≧⥔ᣢ࡟࠾ࡅࡿ࣐࢖ࢡࣟࣂࣈࣝࢆ⏝࠸ࡓࢩࣕࣥࣉ࣮⒪ἲࡢ᭷ຠᛶࡢ᳨ウ᪥ᮏື≀┳ㆤᏛ఍㛵すᆅ༊➨  ᅇ౛఍኱㜰ࠊ ᭶  ᖺ⣽ᕝ⪷▮ࠊᕝཎ஭᫴ᖹࠊὠஂ஭฼ᗈࠊஂᮎṇᬕࠊ༳∾ಙ⾜ࠊᩬ┠ᗈ㑻ࠊᅵᒇுࠊᑠ᪉᐀
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7)

Kawarai S, Takahashi R, Madarame H, Shirota K.: A case of granulomatous dermatitis in a dog.
The Asian Meeting of Animal Medicine Specialties. Thailand, May, 2011

586

Japanese Journal of Veterinary Research 64(2): 000‑000, 2016

NOTE

Evaluation of weekly bathing in allergic dogs withmethicillin-resistant Staphylococcal colonization
Shinpei Kawarai1,＊), Ayumi Fujimoto2), Genta Nozawa3),
Nobuyuki Kanemaki1), Hiroo Madarame1), Takuo Shida4) andAkio Kiuchi3)
1)

Veterinary Teaching Hospital Laboratory of Small Animal Clinics; Department of Veterinary
Medicine
2)
Laboratory of Internal Medicine II;
3)
Laboratory of Microbiology I; and4)
Laboratory of Veterinary Radiology, Azabu University, 1‑17‑71, Fuchinobe, Chuo-ku, Sagamihara,
Kanagawa 252‑5201, Japan
Received for publication, 00000 00, 0000; accepted, 00000 00, 0000

Abstract
We evaluated the efficacy of weekly bathing in reducing methicillin-resistant Staphylococcus(MRS) colonization in canine allergic dermatitis in a pilot clinical trial. Six dogs withallergic dermatitis controlled by prescription medications were treated with weekly
bathing for 1 month. The Canine Atopic Dermatitis Extent and Severity Index version 3(CADESI-03) and pruritus scores and frequency of mecA-positive Staphylococcus spp.
isolated from three body sites between weeks 0 and 4 were compared. There was no
significant difference in CADESI-03 scores with bathing, whereas the pruritus scores weresignificantly reduced (p ＜ 0.05). Furthermore, MRS frequency was decreased in four of thesix dogs (p ＜ 0.05). In conclusion, weekly bathing should be considered for reducing MRS
colonization in canine allergic dermatitis.
Key Words: allergic dermatitis, bathing, mecA

acquire genes that provide resistance toantimicrobial agents; one example is mecA foundin methicillin-resistant Staphylococcus spp.
(MRS)1). These resistance genes in pathogenic
Staphylococcus spp. constitute a risk for horizontal
gene transfer to commensals and transients2,4),
which is particularly problematic for methicillin-

Staphylococcus spp. are part of thecommensal flora of canine skin. In dogs withallergic skin conditions, such as canine atopic
dermatitis (CAD) and adverse food reactions(AFR), Staphylococcus spp. are common causative
pathogens isolated from skin lesion6,7,13). During
antibiotic therapy, Staphylococcus spp. frequently

Corresponding author: Shinpei Kawarai, Veterinary Teaching Hospital, Azabu University, 1‑17‑71 Fuchinobe,
Chuo, Sagamihara, Kanagawa 252‑5201, Japan
Phone: ＋81‑(0)42‑754‑7111. Fax: ＋81‑(0)42‑850‑2506. E-mail: kawarai@azabu-u.ac.jp
doi: 10.14943/jjvr.64.2.★★

587

Weekly bathing for allergic dog with MRS

atopic dermatitis. At each site, an area of 5 cm2
was swabbed and cultured for bacteria on blood
agar. Five, morphologically similar colonies
representing the majority from each agar plate
were selected. The expression of mecA of grampositive cocci was detected by polymerase chain
reaction (PCR), as described by Sasaki et al12).
The bacterial species of mecA-positive grampositive cocci were identified by sequencing 16SrRNA with species-specific primers using theMicroseq 500 PCR kit (Applied Biosystems; Foster
City, U.S.A.), according to the manufacturer’s
protocols12). The frequencies of mecA in isolated
Staphylococcus spp. from each body site at week 0
and 4 were compared. Changes in clinical scores
were not available to the microbiologists until
the end of the trial.
Changes in outcome measures between
weeks 0 and 4 were compared using statisticalanalysis. Wilcoxon rank sum test was used forclinical scores, and Mann-Whitney U test wasused for frequency of mecA-positive Staphylococcus
colonization using the Microsoft Excel programwith the Statcel software add-in (version 4; OMS
Publishing, Japan). Data were presented asmeans ± standard deviation, and p values of＜0.05 were considered statistically significant
for all analyses performed.
The changes in the frequency of mecApositive Staphylococcus colonization, CADESI-03
and pruritus scores are presented in Fig. 1.
Table 2 shows the PCR analysis for mecA. The
frequency of mecA-positive Staphylococcus spp.
isolated from all three body sites decreased infour dogs (cases 2, 4, 5, and 6). Specifically, thefrequency at week 4 was significantly lower thanthat at week 0 (0.8 ± 0.4 versus 2.0 ± 0.9,
respectively; p ＜ 0.05). At week 0, mecA-positive
Staphylococcus spp. were isolated from 12 out of a
total of 18 body sites (eleven S. pseudointermedius
and one S. aureus). In contrast, mecA-positive
Staphylococcus spp. were isolated from five bodysites (four S. pseudointermedius and one S.
epidermis) at the end of the trial at week 4. There
was no significant difference in the CADESI-03

resistant Staphylococcus pseudointermedius indogs because of the risk of colonization or mecA
gene transfer to owners3,6). In this clinical pilot
study, we evaluated the efficacy of weekly
bathing in decreasing MRS colonization in theskin of allergic dogs.
Six dogs with chronic, recurrent, and pruritic
skin symptoms that were diagnosed with CAD
and/or concurrent AFR based on Favrot’s criteria
for canine atopic dermatitis, serum allergenspecific immunoglobulin E (IgE) test, and foodelimination and provocation trials were enrolled(Table 1)5,9). The allergic skin conditions of alldogs were under control by prescription
medications and food avoidance. All six dogs wereroutinely bathed by their owners at a frequency
of once a week or once every other week. All dogs
had a history of superficial pyoderma due
to Staphylococcus spp., in which mecA-positive
Staphylococcus spp. was isolated from the skinsurface. After informed consent was obtained,owners were instructed to declare any unusual
events regarding their dogs such as the living
environment and diet during the study period.
The trial was conducted between September 2010
and December 2011. This trial was approved by
the ethical committee for clinical trials at theVeterinary Teaching Hospital of Azabu University
in Sagamihara, Kanagawa Prefecture, Japan.
The trial participants were bathed byveterinary technicians (VTs) once a week for 1
month. VTs photographed the dogs before theirweekly baths. The Canine Atopic Dermatitis
Extent and Severity Index version 3 (CADESI03), which has a maximum score of 1,240, wasused to evaluate skin symptoms8). Pruritus wasassessed daily by the owners on a scale of 0‑10
(0 ＝ no pruritus; 10 ＝ severe pruritus), and weekly
average scores were calculated. To evaluate changes
in clinical scores, the scores at the start (week 0)
and end (week 4) of the trial were compared.
The swab samples were collected from theskin surface before the baths. Three swab samples
were collected from three different affected bodysites that showed signs such as erythema and

588

589

Shiba Inu

Yorkshire
Terrier

Toy
Manchester
Terrier

Shiba Inu

Brussels
GriffonLabrador
Retriever

1

2

3

4

5

6

F

NM

NM

SF

SF

SF

Sex1)

8

8

10

9

6

10

Age
(year)

2

4

1

2

1

5

1st
onset
(year)All yearAll yearAll year

Autumn,WinterAll yearAll year

Seasonalityofsymptoms

Erythema, Papule,
Lichenification,
Hyperpigmentation,
Alopecia, Fistula

Erythema,Excoriation, Papule,
Scales,Lichenification,
Hyperpigmentation,
Alopecia, Fistula

Scales,Lichenification,
Hyperpigmentation,
Alopecia

Erythema, Scales,
Alopecia

Erythema,Excoriation, Papule,
Scales,Lichenification,
Hyperpigmentation,
Alopecia

Erythema, Scales,
Lichenification,
Hyperpigmentation,
Alopecia

Major Skin
symptoms

Muzzle, Face,
Ear, Axillae,
Ventral abdomen,
Interdigital skin

Muzzle, Face,
Dorsal trunk,
Ventral abdomen,
Interdigital skin

Muzzle,Periocular, Feet,
Axillae, Ventral
abdomenAxillae, Dorsal
trunk, Ventral
abdomenMuzzle, Ear,
Periocular, Feet,
Axillae, Dorsal
trunk, Ventral
abdomenMuzzle,Periocular, Feet,
Axillae, Ventral
abdomen, Prianal

Major Skin
lesions

A. fumigatus

A. siroD. pteronyssinus

A. siroD. farinae,
D. pteronyssinus

D. farinae,
D. pteronyssinus

Results of serum
allergen specific
Immunoglobulin
E testChicken,
PotatoJapanesesweetsChicken,Sardine,Sprout,Potato, Rice,
Wheat

Not
determined

ChickenNot
determined

Results offood
provocation
test1) F, female; SF, Spayed female; NM, Neutered male2) Ointment, Hand-mixed topical corticosteroid (0.12% betamethasone valeratel and 0.3% heparinoidm)
3) Chlorhexidine, Nolvasan, Fort dodge animal health, IA; Miconazole nitrate and Chlorhexidine, Malaseb, Fort dodge animal health, IA

BreedsCase
No.

Prednisolone,
Thyroxin,
Ointment

Ointment

Prednisolone,
Clemastine
fumarate,
Glycyrrhizinate,
Ointment

Prednisolone,
Ointment

Clemastine
fumarate,
Ointment

Clemastine
fumarate,
Glycyrrhizinate,
Ointment

Prescribed
medications2)

Chlorhexidine

Chlorhexidine

Miconazole
nitrate andChlorhexidine

Chlorhexidine

Chlorhexidine

Chlorhexidine

Prescribedshampoos3)

Table 1. Profile, skin symptoms, skin lesions, identified allergens, and prescribed medications and shampoos of six allergic dogs enrolled in
the trial

Shinpei Kawarai et al.

Weekly bathing for allergic dog with MRS

Fig. 1. Changes in the clinical scores and frequency of mecA-positive Staphylococcus colonization in six
dogs enrolled in the trial.

genes are increased with this approach1‑3,6,10,14), as
demonstrated by the colonization of healthy dogs
by mecA-positive S. pseudointermedius10. In thisstudy, S. epidermis, a typically non-pathogenic
spp. that was isolated from case 3 at week 4 wasfound to have mecA. The roles of S. epidermis asa reservoir and transmitter of linezolid resistancegenes were previously described2. Restoration ofcommensal skin flora and prevention of drug
resistance are considered as logical goals tocontrol allergies in dogs14). Thus, weekly bathing
following clinical cure with antibiotics should be
useful in preventing the risk for development ofmultidrug resistance in not only skin microflora
but also in other organs.
In the present study, we performed weekly
bathing only for enrolled dogs that were being
bathed two to four times a month by their owners
before the trial. The environment andhousekeeping conditions differed between thedogs. For example, the elderly owners may have
had trouble in efficiently bathing their dogs, andone major reason for the success of this trial
could be because of the experienced VTs whoscore between week 0 (178 ± 83) and week 4(166 ± 95); however, two of the six participant
dogs (cases 1 and 5) exhibited decreases of 侒25%
in CADESI-03 scores compared with those atweek 0. The pruritus score was significantly
lower at week 4 than at week 0 (3.8 ± 2.5 versus
4.3 ± 2.5, respectively; p ＜ 0.05). Cases 4 and 5
showed decreases of 侒25% in pruritus scores atweek 4 compared with those at week 0.
In this trial, we were successful in maintaining
allergic dermatitis under control using weekly
bathing without antibiotics. Four weeks after thetrial, the pruritus scores and MRS colonizationon the skin surface decreased. Previous studies
showed that transient infectious as well as
resident non-infectious Staphylococcus spp. were
present in the skin of atopic canines6,11,14). Whileaggressive antibiotic therapy can be useful forthe elimination of pathogenic Staphylococcus spp.
from skin lesions, long term antibiotic use arelikely to increase the risk for disruption of theskin and other organ-resident floras14). In addition,
the risk of both pathogenic and non-pathogenic
Staphylococcus spp. to acquire antibiotic resistance590

Shinpei Kawarai et al.

Table 2. Summary of Staphylococcus spp. and their mecA status in six dogs enrolled in the trial
At week 0

At week 4

Case
No.

Body sites where
swab samples
were collectedThe
results ofthe cultureThe
results ofmecA

Isolated
Staphylococcus spp.
by PCR

The
results ofthe cultureThe
results ofmecA

Isolated
Staphylococcus spp.
by PCR

1

Right axillae

Positive

Positive

S. peudointermedius

Positive

Positive

S. peudointermedius

Ventral abdomen

Negative

Positve

S. peudointermedius

2

3

4

5

6

Right feet

Negative

Right axillae

Positive

Negative
Negative
Positive

S. peudointermedius

Negative

Right ear

Positive

Positive

S. peudointermedius

Positive

Dorsal trunk

Positive

Positive

S. peudointermedius

Negative

Left Axillae

Negative

Dorsal trunk

Positive

Postitive

S. peudointermedius

Negative

Negative

Ventral abdomen

Negative

Positive

Positive

S. epidermidis

MuzzlePositive

Postitive

S. peudointermedius

Positive

Postitive

S. peudointermedius

Right Axillae

Positive

Postitive

S. peudointermedius

Negative

S. peudointermedius

Negative
Negative
Postitive

S. peudointermedius

Ventral abdomen

Negative

Right
Interdigital skin

Positive

Postitive

Negative

Dorsal trunk

Positive

Postitive

S. peudointermedius

Ventral abdomen

Positive

Postitive

S. aureus

Positive

Right
Interdigital skin

Positive

Postitive

S. peudointermedius

Negative

MuzzlePositive

Postitive

S. peudintermedius

Negative

Right Axillae

Negative

Negative

funded by the Azabu University Research Service
Division, by Grant-in-Aid for Young Scientists (B)by the Japan Society for the Promotion of Science(JSPS) (23780330), and by MEXT-Supported
Program for the Strategic Research Foundation
at Private Universities (2011‑2015).

bathed the dogs in not a specific but a certain
manner. Furthermore, worsening condition of
the ventral abdominal lesions and increased
CADESI-03 score were observed in case 3 at week
4. The ventral abdominal skin is comparatively
thin in the breed phenotype of case 3. Therefore,
it may be preferable to tailor the frequency ofbathing in each dog based on breed characteristics
to avoid excessive washing.
A limitation of the present trial was treatment
necessity, and it was not possible to obtain
permission from the owners for their animals to
be bathed with only tap water as a control group
participating in this trial. Further clinical trials
with this control group are needed based on thispilot study. In conclusion, we propose weekly
bathing as useful in controlling canine allergic
dermatitis by preventing recurrent MRS skininfections.
This study was partially supported by theYoung Scientist Research Training Award (2012)

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Yasuda, N., Ohmori, K., Sakaguchi, M.,
Asami, Y. and Tsujimoto, H. 2010. Clinicalefficacy of a novel elimination diet composedof a mixture of amino acids and potatoes indogs with non-seasonal pruritic dermatitis.
J. Vet. Med. Sci., 72: 1413‑1421.
6) Lloyd, D. 2013. Chapter 4, Bacterial skindiseases. In: Muller and Kirk’s small animaldermatology, 7 th ed., pp. 184‑186, Miller, W.
H., Jr, Griffin, C. E. and Campbell, K. L.,
eds., Elsevier Mosby, St. Louis, Missouri.
7) McEwan, N. A., Mellor, D. and Kalna, G.
2006. Adherence by Staphylococcus intermedius
to canine corneocytes: a preliminary study
comparing noninflamed and inflamed atopic
canine skin. Vet. Dermatol., 17: 151‑154.
8) Olivry, T., Marsella, R., Iwasaki, T. and
Mueller, R. 2007. International task force
on canine atopic dermatitis. Validation of592

CASE REPORTS

Canine Pemphigus Foliaceus with Concurrent
Immune-Mediated Thrombocytopenia
Shinpei Kawarai, PhD, DVM, Masaharu Hisasue, PhD, DVM, Shinobu Matsuura, PhD, DVM, Tetsuro Ito, DVM,
Yukari Inoue, DVM, Sakurako Neo, PhD, DVM, DACVP, Yoko Fujii, PhD, DVM, DACVIM (Cardiology),
Hiroo Madarame, PhD, DVM, DJCVP, Kinji Shirota, PhD, DVM, DJCVP, Ryo Tsuchiya, PhD, DVM

ABSTRACTA 3 yr old wirehaired fox terrier was presented to his primary care veterinarian with fever, thrombocytopenia, andgeneralized crusting dermatitis. The skin lesion had progressed for at least 18 days, and thrombocytopenia had
developed 3 days before presentation. Histopathology and direct immunofluorescence studies of the skin wereconsistent with pemphigus foliaceus (PF). Immunofluorescence revealed immunoglobulin G deposition around the
keratinocytes in the stratum spinosum. A diagnosis of immune-mediated thrombocytopenia (IMT) was confirmed by thepresence of platelet surface-associated immunoglobulin using flow cytometry. Systemic immunosuppressive therapy
with cyclosporine and azathioprine was effective, and the dog survived for .2 years from the initial presentation. IMT israrely associated with PF. This appears to be the first detailed report of a definitive diagnosis of concurrent PF and IMTin a dog. The authors’ findings indicate that canine PF could be complicated by hematologic immune-mediateddiseases such as IMT. (J Am Anim Hosp Assoc 2015; 51:56–63. DOI 10.5326/JAAHA-MS-6044)

Introduction

different epidermal staining patterns were identified by indirect IF
using healthy canine footpad epithelium as a substrate.11 Compar-

Canine pemphigus foliaceus (PF) is the most common autoimmune

isons of IF staining patterns of desmosomal and nondesmosomal

pustular skin disease in dogs characterized by pustules, crusting,adhesion molecules of keratinocytes with those of canine PF sera

erosions, and alopecia of the head, face, nasal planum, pinnae, andshowed that desmocolin-1 was the major autoantigen of canine

footpads.1–3 When generalized, skin lesions spread over the entire

PF.11,12 Although circulating antikeratinocyte autoantibodies arebody, and symptoms include fever, lethargy, and limb edema.

considered to cause acantholysis only on the epidermis, canine PFHistopathologically, acantholytic keratinocytes are found within

reportedly occurs either concurrently with or following hypothy-

intraepidermal-to-subcorneal neutrophilic and eosinophilic pus-roidism, leishmaniasis, and systemic lupus erythematosus

tules. Direct immunofluorescence (IF) has shown that antikerati-(SLE).3,13–15 Dysregulated antibody production based on polyclonal

nocyte autoantibodies are deposited in vivo in the subcorneal layers,

B cell activation, cross-reacting antigens, and the innocent

especially the stratum spinosum and granulosum.1–9 Indirect IF

bystander reaction due to adsorption of circulating immune

using canine PF sera revealed the presence of circulatingcomplexes onto keratinocytes are thought to cause these autoim-

antikeratinocyte immunoglobulin (Ig) G in vitro.10 Recently, several

mune disorders.3,13–15

From Laboratory of Small Animal Clinics, Veterinary Teaching Hospital

CRP, C-reactive protein; IF, immunofluorescence; Ig, immunoglob-(S.K., T.I., H.M.) and Laboratory of Veterinary Internal Medicine II

ulin; IMT, immune-mediated thrombocytopenia; IV, intravenous(M.H., Y.I., S.N., R.T.), Laboratory of Veterinary Surgery I (Y.F.),

injection; PF, pemphigus foliaceus; PO, per os; PSA, platelet

and Laboratory of Veterinary Pathology (K.S.), Department of

surface-associated; RF, rheumatoid factor; SLE, systemic lupus

Veterinary Medicine, Azabu University, Fuchinobe, Chuo-ku,

erythematosus

Sagamihara, Kanagawa, Japan; and Center for Advanced Biomedical Research, Boston University School of Medicine, Boston, MA(S.M.).
Correspondence: hisasue@azabu-u.ac.jp (M.H.)

56

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FIGURE 1

Appearance of the wirehaired fox terrier at the time of presentation. A, B: Note the generalized dermatitis with pustules, crusts,

erosions, and alopecia, on the face (A), ear (B), and trunk (C) at the primary care hospital. The dog’s hair was clipped by the primary careveterinarian.
Thrombocytopenia in dogs is often immune mediated.16 The

before presentation. At the time of examination, the dog was

pathogenesis of canine immune-mediated thrombocytopenia

pyrexic (39.78C) and the skin lesions were bilateral, symmetrical,(IMT) involves platelet surface-associated (PSA) Ig binding, which

and manifested as pustules, crusting, and erosions. The pustules

leads to the increased removal of antibody-coated platelets byinitially appeared on the ventral trunk, in the ear canal, and on themacrophages through phagocytosis in the reticuloendothelial

pinnae. Multifocal crusting and erosion then progressed over the16

Canine IMT is difficult to diagnose because it occurs

entire body to cover the nasal planum, top of the head, muzzle,

not only primarily without obvious underlying causes, but also

dorsal and ventral trunk, feet, and paw pads over the next 18 days

secondarily to various systemic diseases including infectious,(Figures 1A, B, and C). Prior to referral, commercially-available

neoplasias, adverse reactions to drugs (e.g., sulfonamide, cephalo-

rapid drug sensitivity testing (Monoris Inc., Tokyo, Japan) was

sporin, cyclophosphamide, etc.), and systemic autoimmune

performed without bacterial identification from swabs collected

system.

16–20

Typically, IMT is diagnosed by excludingfrom underneath a crust on the ventral trunk. Although theother causes of thrombocytopenia, such as disseminated intravas-

crusting dermatitis did not respond to a systemic antibiotic

cular coagulation and deficient platelet production by bone

(cephalexina, 25 mg/kg per os [PO] q 12 h) selected by the drugdiseases such as SLE.

16–20

Kristensen et al. (1994) established a direct assay

sensitivity test, the fever was reduced with a single dose offor measuring PSA-Ig using flow cytometry, which can be used to

diclofenac sodiumb (1.25 mg PO). Because leukocytosis, anemia,

marrow.

16,17,19

Tsuchiya et al. (2010) recently

and thrombocytopenia were diagnosed after the administration of

improved the accuracy of the assay using established artificial

cephalexin and diclofenac sodium, the dog was referred to thediagnose canine IMT.

positive-control platelets.21 The authors of this study routinely use

study authors’ hospital for further examination, diagnosis, andthis assay when canine IMT is suspected in their hospital.

treatment.

The purpose of this article is to describe a rare association of

Twelve days after the initial presentation at the primary care

PF with concurrent IMT. Diagnosis was made based on

hospital (day 1), the dog had lost 10% of its body weight (new body

histopathological findings, immunohistochemical detection ofweight was 9 kg). Body temperature, pulse, and respiratory rates

IgG deposition around keratinocytes, and flow cytometric

were 37.98C, 120 beats/min, and 25 breaths/min, respectively. A

detection of PSA-Ig.

physical examination revealed lameness; swelling of all limbs in the
synovial joints of the carpus, stifle, and hock; conjunctivitis; andCase Report

peripheral lymphadenopathies on the superficial cervical and

A 3 yr, 10 mo old male wirehaired fox terrier was presented to his

popliteal lymph nodes. A grade 2/6 systolic heart murmur, loudest

primary care veterinarian with pruritus and generalized dermatitis.

over the left thorax, was audible during cardiac auscultation. The

The owner noticed that the dog’s skin lesions had worsen 6 days

skin showed generalized erythema, pustules, crusting, and erosions.

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Those lesions were distributed symmetrically on the dog’s face(including the muzzle, nasal planum from the top of the muzzle to

TABLE 1the front of the glabella, the periocular region, ear canal andpinnae), ventral and dorsal trunk, feet, and paw pads. The oral

Complete Blood Cell Count and Serum Biochemical Profile
Results at the Time of Initial Presentation

mucosa and mucocutaneous junctions, such as the eyelids andVariableanus, were free of lesions. Impression smears were obtained from9

WBC (3 10 /L)

Value

Reference range

1.76(0.6–1.7)

the erosive skin lesions under the crusting. Cytology findings

Red blood cell count (3 10 /L)

4.3(5.5–8.5)

showed eosinophilic, pyogranulomatous inflammation comprising

Hemoglobin (g/L)

96(120–180)

numerous degenerate and nondegenerate neutrophils, many

Packed cell volume

0.29(0.37–0.55)

eosinophils and macrophages, and a few small lymphocytes. A

Mean corpuscular volume (fL)67

(66–77)few oval and intensely basophilic acantholytic keratinocytes with a

Mean corpuscular hemoglobin (pg/cell)

22.3(19.9–24.5)

centrally placed nucleus were individually surrounded by abundant

Mean corpuscular hemoglobin concentration (g/L)

333(320–360)

neutrophils. Infectious organisms such as bacteria and Malassezia

Platelet count (3 109/L)

70(200–500)

spp. were unremarkable in smears. Swabs collected from pustules

Total protein (g/L)

47(51–77)in the axillary area were examined for bacterial and fungal cultures

Albumin (g/L)

18(25–40)at Hoken Kagaku Laboratory (Kanagawa, Japan) using sheep blood

Blood urea nitrogen (mmol/L)

7.43(3.28–10.42)

agar and Sabouraud agar, respectively. Sarcoptic mange andCreatinine (lmol/L)

35.36(44.2–132.6)

Demodex canis were not detected in skin scrapings or plucked hairs.

Phosphorus (mmol/L)

1.39(0.71–1.78)

Calcium, total (mmol/L)

2.2(2.15–2.85)

primary care veterinarian and based on the physical exam findings

Alanine aminotransferase (lkat/L)

0.25(0.3–1.19)

conducted at the authors’ hospital, immunologic tests, diagnostic

Alkaline phosphatase (lkat/L)

68.17(0.6–4.33)

imaging, arthrocentesis, fine-needle aspiration of lymph nodes,

Total cholesterol (mmol/L)

5.26(2.77–8.13)

skin biopsy, and a histopathological examination were performed.

Triglycerides (mmol/L)

1.34(0.19–1.15)

Table 1 shows the hematologic and serum biochemical

Total bilirubin (lmol/L)

16.93(0.0–5.13)

12

Considering the diagnosis of hematologic abnormalities by thefindings. Complete blood cell counts showed a slightly elevatedGlucose (mmol/L)

4.11(3.83–6.77)

white blood cell count (1.76 3 109/L; reference range, 0.6–1.7 109/

Sodium (mmol/L)

152.4(142–152)L), a low red blood cell count (4.3 3 1012/L; reference range, 5.5–

Potassium (mmol/L)

8.5 1012/L), a packed cell volume of 0.29 (reference range, 0.37–

Chloride (mmol/L)

4.48(3.7–5.3)

118.6(105–117)0.55), and a remarkably low platelet count (PLT; 70 3 109/L;
reference range, 200–500 109/L). The ratio and cell counts ofphosphatase (68.17 lkat/L; reference range, 0.6–4.33 lkat/L), andreticulocytes were 0.0032 proportion of red blood cells and 13.76 3
10 /L, respectively (the range of reticulocyte counts in regenerative

bilirubin (16.93 lmol/L; reference range, ,5.13 lmol/L). C-

response to mild anemia (a packed cell volume of 0.26–0.38) arereactive protein (CRP) was measured using a laser nephelometric100–150 3 109/L). A slightly low serum iron concentration (16.11

immunoassayc to evaluate the degree of systemic inflammation.

lmol/L; reference range, 16.83–21.84 lmol/L) and low unsaturated

Examination of globulin protein fraction by serum protein

iron binding capacity (6.80 lmol/L, reference range; 22.73–60.86

electrophoresis was performed because hypoalbuminemia waslmol/L) indicated that chronic inflammation caused the mild

observed. CRP was obviously elevated at 114.29 nmol/L (reference,

nonregenerative anemia. Blood coagulation tests showed an,9.52 nmol/L) and serum protein electrophoresis revealed elevatedelevated plasma fibrinogen (13.35 lmol/L; reference range, 2.59–

a2-globulin level.2299.88 lmol/L), a normal prothrombin time (8.1 s; reference range,

To determine the cause of the hypoalbuminemia, diagnostic

6.8–8.6 s), a slightly prolonged activated partial prothrombin time

imaging was performed, focusing on possible renal and hepatic(28.1 s; reference range, 13.1–26.9 s), and a normal fibrinogen-abnormalities. Thoracic and abdominal radiography revealed nofibrin degradation product (,2.5 mg/L; reference range, 0–2.5 mg/

apparent abnormalities. An abdominal ultrasound showed hepaticL). The blood coagulation test results excluded disseminatedvenous congestion and mild hyperechogenicity of the liver

intravascular coagulation. The serum biochemical analysis showedparenchyma and renal cortex. The dog underwent a cardiac

moderately low total protein (47 g/L; reference range, 51–77 g/L)

work-up to determine the cause of its heart murmur and hepaticand albumin (18 g/L; reference range, 25–40 g/L) with high alkaline

venous congestion. Echocardiography revealed mild pulmonary

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Concurrent Pemphigus Foliaceus and IMT in a Dog

FIGURE 2

Flow cytometric analysis of platelet surface-associated (PSA) immunoglobulin (Ig) M, -IgG, and complement 3 (-C3). Platelets

from sick (filled curve) and healthy control (open curve) dogs stained with anti-canine IgM, IgG, and C3 antibodies conjugated with fluorescein
isothiocyanate. Numbers in the graphs indicate binding percentage (%) of PSA-IgM, -IgG, and -C3 on platelets obtained from sick dog. Referencelines for PSA-IgM, -IgG, and -C3 positive platelets from healthy control are set at 5%.
hypertension on the basis of increased tricuspid regurgitation

ma multiforme, cutaneous vasculitis, IMT, and SLE. An additional

velocity (3.41 m/s, reference range ,3.0 m/s; estimated pressure

skin biopsy and immunologic tests were conducted to reach a

gradient across the tricuspid valve, 46 mm Hg). The dog’s systolicdefinitive diagnosis.

blood pressure was 120 mmHg, which was considered normal for

A commercial laboratory (Monoris Inc, Tokyo, Japan)

dogs. Urinalysis revealed a specific gravity of 1.045, a protein

performed a direct Coombs’ test and measured serum antinuclear

content of 1.8 g/L, and a protein/creatinine ratio of 1.5, indicatedantibodies and rheumatoid factor (RF) to determine the presence

mild proteinurea (reference ranges, 1.020–1.050, ,0.5 g/L, andof SLE. The direct Coombs’ test (378C and 48C) and antinuclear,0.5, respectively). The findings of proteinurea and a hyperechoic

antibodies were negative, but RF was positive (titer was 320;

renal cortex indicated protein-losing nephropathy. The leakage ofreference value; 0). Nine days after the initial presentation, flow

antithrombin III from the kidneys is considered a possible cause ofcytometry for PSA-IgM, -IgG, and -C3 was performed as describedpulmonary thromboembolism, which leads to pulmonary hyper-by Tsuchiya et al (2010) for the diagnosis of canine IMT.21 The

tension; however, further work-up was not performed.

gates for positive events were set based on healthy control samples

To exclude lymphoma, a fine-needle aspirate of the lymph

at 0–5%. Values .10% were judged positive. The results showed

nodes was obtained, which showed mild-to-moderate reactive

apparently positive anti-canine IgM (89.3%) and anti-canine IgGlymphoid hyperplasia with mild neutrophilic infiltration. the(47.1%) antibodies, but negative anti-canine C3 (7%) antibody

authors performed arthrocentesis because idiopathic polyarthritis

compared with the healthy control (Figure 2).

was suspected on the basis of the history of fever, physical findings

Skin biopsies obtained using a 4 mm biopsy punch from theof lameness, and increased serum CRP level. A gross examination

crusting skin lesions on the dog’s head, neck, and dorsal trunk wereof joint fluid showed turbidity and decreased viscosity. A cytologic

examined by routine histopathology. Those findings revealed severe

examination identified low-to-moderate cellularity and mild
inflammation consisting of large mononuclear cells (94%), small

crusting of the superficial epidermis and large intraepidermal tomononuclear cells (5%), and neutrophils (1%). Those findings

subcorneal pustules (Figure 3A) that mainly comprised neutrophils

were consistent with polyarthropathy associated with chronicand eosinophils, and some included acantholytic keratinocytes

inflammation.

(Figure 3B). The pustules involved the hair follicles and extended

At that point, a mild anemia, polyarthropathy (possibly due tointo the follicular infundibula. Neutrophilic and eosinophilicchronic inflammation), thrombocytopenia, hypoalbuminemia

inflammation was seen in the pustules. Primarily neutrophils and(probably due to protein-losing nephropathy), and pulmonary

then mononuclear cells infiltrated the superficial-to-middle dermis.

hypertension were identified. Based on those findings, the authors’

Direct IF using anti-canine IgG antibodiesd (1:800) identified IgG

differential diagnoses included PF, pemphigus erythematous,

deposition throughout the superficial-to-middle epidermis layer,

panepidermal pustular pemphigus, cutaneous lymphoma, erythe-

particularly in the stratum spinosum (Figure 3C). Canine PF and

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FIGURE 3

A: Histopathological features of the superficial pustules. B: High-power magnification of the area circled in A. The pustules arecomprised of neutrophils with some acantholytic keratinocytes (arrows). C: Direct immunofluorescence shows immunoglobulin G deposition fromthe superficial to middle epidermal layers, especially the stratum spinosum. Hematoxylin and eosin stainining (A and B) and Immunofluorescencestaining with anti-dog IgG (green) and antinuclear stain, DAPI (blue) (C). Bar ¼ 400 lm (A), 100 lm (B), and 100 lm (C).
Follow-up examination on day 22 after initial therapy revealed

IMT were diagnosed based on those histopathological anda 50% improvement in the skin lesions. The physical findings, PLT

immunologic findings.
The dog was hospitalized for 7 days and received fluid therapy(359 3 109/L), and CRP (46.67 nmol/L) were also improved.

with acetate Ringer’s solution (3 mL/kg/hr) supplemented with low

Because of persistent erythema and pruritus on the skin lesion andmolecular weight heparin (dalteparin sodiume, 75 U/kg/day) andhyperthermia (39.88C), azathioprineo (2 mg/kg PO q 24 hr) was

monoammonium glycyrrhizinatef (1 mg/kg intravenous injection

coadministered with cyclosporine. On day 36, the red blood cell[IV] q 12 hr). Cefazolin sodium hydrateg (25 mg/kg IV q 8 hr) andcount and packed cell volume had recovered (5.14 3 1012/L andenrofloxacinh (5 mg/kg BW q 24 hr) were administered to prevent

0.37, respectively) and CRP had decreased to 11.43 nmol/L.

bacterial growth, and ursodeoxycholic acidi (10 mg/kg PO q 12 hr)

Cyclosporine was discontinued immediately. On day 64, 90% of

was administered as liver function support. Hand-mixed topical

the skin lesions were improved and were completely resolved byj

day 120 (Figure 4). The dog’s physical condition and complete

corticosteroid (0.12% betamethasone valerate and 0.3% heparinkloid ) and 0.5% vitamin A oil was applied to reduce the inflam-

blood cell count had also improved. The azathioprine dosage wasmation and as a moisturizer. After cutaneous lymphoma had been

gradually tapered to 0.8 mg/kg PO q 48 hr. On day 309 from theexcluded by frozen-section histopathology by day 3 from the firsttime of referral, PSA-IgM, -IgG, and -C3 were re-evaluated by flowmpresentation at the authors’ institution, cyclosporine (5.5 mg/kg

cytometry. The results were negative for IgM (7%), IgG (5.1%),

PO q 24 hr) was started. A bacterial culture and sensitivity tests were

and C3 (3.6%). The skin symptoms were controlled by azathio-

performed on day 7. Escherichia coli and Staphylococcus spp. withprine monotherapy until day 785.

methicillin resistance (i.e., penicillin, oxacillin, cephalexin, cefazolin, imipenem, lincomycin, enrofloxacin, minocycline, gentamicin,

Discussion

and sulfamethoxazole-trimethoprim) were isolated on the basis of

To the author’s knowledge, this is the first report to describe a dog

23

the Clinical Laboratory Standard Institute guidelines. Based on thedefinitively diagnosed with concurrent PF and IMT. Some forms ofculture, the previously prescribed antibiotics were substituted withimmune-mediated skin disease have been considered as possible

fosfomycin calcium hydraten (25 mg/kg PO q 12 hr). The results ofcauses of IMT and an epidemiological survey by Grindem et al.

fungal cultures were negative on day 17.

(1991) found that IMT in pemphigus complex is very rare.16,18 The

60

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Concurrent Pemphigus Foliaceus and IMT in a Dog

A diagnosis of canine IMT is difficult because it is usually only
concluded by excluding other potential causes of thrombocytopenia.16–20 Dogs with IMT develop an increased risk of spontaneous
hemorrhage when PLT fall below 10–30 3 109/L, whereas O’Marra
et al. (2011) reported that signs of bleeding were absent in 19% of
73 dogs showing platelet counts ,50 3 109/L.16–20,24 Those asymptomatic dogs with moderate thrombocytopenia are not thoroughly
evaluated in routine clinical practice because an invasive bonemarrow examination is needed to exclude nonimmune-mediatedthrombocytopenia. The authors of the current study diagnosed
IMT in a dog by measuring PSA-Ig using flow cytometry at thetime of the initial presentation at their hospital. Furthermore, thepositive PSA-IgM and -IgG findings became negative when the
FIGURE 4

Appearance of the wirehaired terrier following

thrombocytopenia was improved by immunosuppressive therapy

treatment with azathioprine 120 days after initially being presented

with azathioprine. The study authors thus considered that the PSA-

to the authors’ hospital.

Ig level correlated with disease status and was useful for the
diagnosis and evaluation of treatment in dogs with IMT.

following forms of histological types of pemphigus are recognized

Vaughan et al. (2010) reported that complicated cases of canine

in dogs and cats: PF, pemphigus erythematosus, panepidermal

PF concurrent with allergic skin disease and other systemic diseases

pustular pemphigus, pemphigus vulgaris, and paraneoplastic

are significantly associated with the presence of eosinophil

pemphigus.1–9 The most common form of pemphigus complex isinfiltration in intraepidermal pustules.8 In the present case,PF, of which symptoms are commonly less severe than in othereosinophil infiltration was detected in the pustules, and the dog

Although Grindem et al.

presented with pruritus at onset and a history of recurrent pyoderma.

(1991) did not specify the forms of pemphigus, recent retrospective

Concurrent IMT was also diagnosed. Those finding are consistentstudies have not reported those diseases to occur simultaneous-with those of Vaughan et al. (2010).8 One case, positive forly.7,8,19,20 Therefore, the presence of PF and IMT seems to be rare.

eosinophil infiltration, was provisionally diagnosed with drug

The present findings of PSA-Ig analysis reflect immunologicalhypersensitivity against ampicillin, amoxicillin, enrofloxacin, and

1–4

forms, such as pemphigus vulgaris.

metronidazole.8 Because PF and IMT, as observed in the present case,signs of canine PF in addition to known physiological andare clinical symptoms in drug hypersensitivity, it was possible that

hematological abnormalities. In canine PF, symptoms are generally

those symptoms persisted at the initial presentation to the authors’

localized to the skin. Although systemic symptoms, including

hospital.2,6,8,16 If canine PF is accompanied with eosinophilicanorexia, depression, fever, and weight loss are rare, they become

infiltration, it may be necessary to examine underlying diseases.

obvious when erosive skin lesions progress over the entire body.3

Skin lesions had been evident in the case described herein

Various hematologic abnormalities, such as moderate to severe

since the first examination at the primary care veterinary hospital.

leukocytosis and neutrophilia, mild nonregenerative anemia,

However, the PLT (194 3 109/L; reference range, 200–500 3 109/L)

thrombocytopenia, mildly to moderately elevated a2-, b2-, and c-at that time were essentially normal and gradually decreased

globulins, and mild hypoalbuminemia have also been report-

thereafter. The possible causes of secondary IMT include infectious

ed.7,8,14,15 Those features are usually associated with the severity of

diseases, neoplasia, adverse reactions to drugs, and systemicskin symptoms; however, the results of routine blood tests,

autoimmune diseases such as SLE. In the current case, PF anddiagnostic imaging, and urinalysis have received relatively little

adverse drug reaction were considered as the possible etiologies.

focus because they are not considered to support a specific

IMT may also arise in association with circulating antikeratinocyte

diagnosis.3 Focusing on thrombocytopenia among systemic

autoantibodies.3 Secondary IMT develops following SLE in some

symptoms, IMT was diagnosed on the basis of PSA-Ig levels

dogs, suggesting that immune complexes bound to platelets bymeasured using flow cytometry. Therefore, if dogs with PF havecomplement-mediated immune adherence or nonspecific interac-obvious systemic signs, systemic work-ups and immunological tests

tions cause IMT concurrent with SLE.16 The current case had IgGshould be conducted to identify complicating factors andantibodies on the epidermis, had IgM and IgG antibodies on theassociated diseases.

platelet-cell surface, and was RF positive. Those findings indicate

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that in this case, PF may have been the cause of hematologicg

Cefamezin a; Astellas, Tokyo, Japanimmune-mediated diseases such as IMT. The other possible causehBaytril; Bayer Health Care, Tokyo, Japanof IMT was an adverse drug reaction. Prior to referral, IMTi

URSO; Mitsubishi Tanabe Pharma Co., Osaka, Japanoccurred after administration of cephalexin, which is a knownjRinderon-V Ointment; Shionogi Co. Ltd., Osaka, Japancause of secondary IMT. In the present case, cefazolin sodiumk

Hirudoid Soft Ointment; Maruho, Osaka, Japanhydrate, which belongs to the same drug class as cephalexin, hadlSahne Oint; Eisai, Tokyo, Japan

been administered IV for 3 days before administration ofm

Atopica; Novartis Animal Health, Tokyo, Japan

cyclosporine. Because the symptoms did not worsen after cefazolinn

FOSMICIN; Meiji Seika Pharma Co. Ltd., Tokyo, Japan

administration without any immunosuppressant drug, the possi-oImuran, GlaxoSmithKline, Tokyo, Japanbility of an adverse drug reaction was considered low. Epidemiological studies are warranted to determine the prevalence and

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10. Olivry T, Dunston SM, Walker RH, et al. Investigations on the natureand pathogenicity of circulating antikeratinocyte antibodies in dogs
with pemphigus foliaceus. Vet Dermatol 2009;20(1):42–50.
11. Bizikova P, Linder KE, Olivry T. Immunomapping of desmosomal andnondesmosomal adhesion molecules in healthy canine footpad, haired
skin and buccal mucosal epithelia: comparison with canine pemphigus
foliaceus serum immunoglobulin G staining patterns. Vet Dermatol
2011;22(2):132–42.
12. Bizikova P, Dean GA, Hashimoto T, Olivry T. Cloning andestablishment of canine desmocollin-1 as a major autoantigen incanine pemphigus foliaceus. Vet Immunol Immunopathol 2012. In press.
13. Ginel PJ, Mozos E, Fernandez A, et al. Canine pemphigus foliaceus
associated with leishmaniasis. Vet Rec 1993;133(21):526–7.
14. Foster AP, Sturgess CP, Gould DJ, et al. Pemphigus foliaceus inassociation with systemic lupus erythematosus, and subsequent
lymphoma in a cocker spaniel. J Small Anim Pract 2000;41(6):266–70.
15. Werner LL, Bloomberg MS, Calderwood et al. Progressive polysystemic
immune-mediated disease in a dog. Vet Immunol Immunopathol
1985;8(1–2):183–92.
16. Scott MA, Jutkowitz LA. Immune-Mediated Thrombocytopenia. In:
Veterinary Hematology. 6th ed. Philadelphia (PA): WB Saunders;
2011:586–604.

Conclusion
Canine PF is autoimmune pustular disease with symptoms that are
generally localized to the skin. Although a low incidence ofthrombocytopenia associated with pemphigus complex is reported,
the study authors confirmed the diagnosis of concurrent PF and
IMT based on histology findings, direct IF, and the presence of
PSA-Ig. The dermatologic and hematologic signs were treated withimmunosuppressive therapy using cyclosporine and azathioprine,
and the dog has survived for .2 yr since the day of firstpresentation at the authors’ animal hospital. Positive findings for
PSA-IgM and IgG became negative when thrombocytopenia was
improved after immunosuppressive therapy. Those findings
indicate that IMT can develop with canine PF; thus, systemic
evaluation should include precise hematologic assessment.
The authors would like to thank Dr. Takao Otsuki (Heiwa Animal
Hospital, Japan) for referring patients, Dr. Yoko Kakinuma for thetechnical assistance with the histological sections, Dr. Ryoji
Takahashi and Dr. Shinsuke Fujimoto for supporting patient careat the clinic, and Jonathan Bloch for proofreading the manuscript.
The authors declare that there are no conflicts of interest associatedwith the present report. This study was supported by the Grant-inAid for Matching Fund Subsidy for Private University and by the
Science Research Promotion Fund of The Mutual Aid Corporation
for Private Schools of Japan.
FOOTNOTESa

Larixin; Toyama chemical, Tokyo, Japanb

Voltaren; Novartis Pharma, Tokyo, Japanc

Canine CRP measurement system CRP-2; Arrows Co. Ltd, Osaka, Japand

Fluorescein-conjugated goat affinity purified antibody to dog IgG(whole molecule); MP bio Japan K.K., Tokyo, Japane

Hepagumin a; Sawai Pharma, Osaka, Japanf

Stronger neo-minophagen C, Eisai, Tokyo, Japan

62

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Concurrent Pemphigus Foliaceus and IMT in a Dog

17. Kristensen AT, Weiss DJ, Klausner JS, Laber J, Christie DJ. Comparisonof microscopic and flow cytometric detection of platelet antibody indogs suspected of having immune-mediated thrombocytopenia. Am J
Vet Res 1994;55(8):1111–4.
18. Grindem CB, Breitschwerdt EB, Corbett WT, Jans HE. Epidemiologic
survey of thrombocytopenia in dogs: a report on 987 cases. Vet Clin
Pathol 1991;20(2):38–43.
19. Dircks BH, Schuberth HJ, Mischke R. Underlying diseases andclinicopathologic variables of thrombocytopenic dogs with and without
platelet-bound antibodies detected by use of a flow cytometric assay: 83
cases (2004–2006). J Am Vet Med Assoc 2009;235(8):960–6.
20. Botsch V, Küchenhoff H, Hartmann K, Hirschberger J. Retrospective
study of 871 dogs with thrombocytopenia. Vet Rec 2009;164(21):647–51.

21. Tsuchiya R, Komatsu T, Ishikawa T, et al. Preparation of positive control
platelets for detection of canine platelet surface-associated IgG, IgM andcomplement (C3) by flow cytometry. J Vet Med Sci 2010;72(8):1063–6.
22. Onishi T, Inokuma H, Ohno K, et al. C-reactive protein concentrationin normal and diseased dogs measured by laser nephelometricimmunoassay. J Jpn Vet Med Assoc 2000;53:595–601 [in Japanese].
23. Clinical Laboratory Standards Institute. Performance standards for
antimicrobial susceptibility testing; seventeenth informational supplement. In: CLSI document M100-S17. Pennsylvania (PA): Clinical andLaboratory Standards Institute; 2007.
24. O’Marra SK, Delaforcade AM, Shaw SP. Treatment and predictors ofoutcome in dogs with immune-mediated thrombocytopenia. J Am Vet
Med Assoc 2011;238(3):346–52.

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CASE REPORTS

A Case of Cutaneous Sterile Pyogranuloma/
Granuloma Syndrome in a Maltese
Shinpei Kawarai, PhD, DVM, Shinobu Matsuura, PhD, DVM, Saburo Yamamoto, PhD, Akio Kiuchi, PhD, DVM,
Nobuyuki Kanemaki, PhD, DVM, DAiCVO, Hiroo Madarame, PhD, DVM, DJCVP, Kinji Shirota, PhD, DVM, DJCVP

ABSTRACT
Cutaneous sterile pyogranuloma/granuloma syndrome (SPGS) is a locally restricted multinodular dermatitis. Affected dogs are
typically healthy, but a few show systemic signs. Herein, a case of a dog presenting with generalized ulcerative dermatitis withsystemic signs of mild anemia and an increased C-reactive protein level is described. Cutaneous SPGS was diagnosed byhistopathology, negative staining causative organisms, and polymerase chain reaction for Mycobacterium spp. Successfultreatment was achieved by immunosuppressive drugs, including prednisolone and azathioprine, administered for at least 20
mo. Recurrences of skin lesions were observed when prednisolone and/or azathioprine were discontinued. Long-term management with immunosuppressive agents may be required if the affected dog exhibits severe symptoms of cutaneous SPGS.
(J Am Anim Hosp Assoc 2014; 50:278–283. DOI 10.5326/JAAHA-MS-6009)

Introduction

The diagnosis of cutaneous SPGS is challenging and can be

Cutaneous sterile pyogranuloma/granuloma syndrome (SPGS) is

made only after ruling out other granulomatous and pyogranu-

a skin disease that is uncommon in dogs and extremely rare inlomatous skin diseases.1–4 According to Santoro et al. (2008), the

cats.

1–4

In the literature, SPGS is also termed idiopathic peri-

etiology of granulomatous and pyogranulomatous skin diseases

adnexal multinodular granulomatous dermatitis.2 No age or gendercan be divided into two major groups. The ﬁrst group is infectious

predisposition is reported; however, boxers, golden retrievers, col-diseases caused by protozoans, mycobacteria, pathologic dimor-

lies, Great Danes, and Weimaraners may be predisposed, and malephic fungi, etc. and noninfectious diseases with a known etiology

1–5

Characteristic

such as a foreign body, hair, sebum, etc. The second groupskin lesions are usually observed on the bridge of the nose, muzzle,

includes unknown etiologies, such as idiopathic “sterile” derma-dogs seem to be affected at a higher frequency.
1–5

These lesions comprisetitis.4 Histopathologically, those criteria were also based on visible

dermal papules and nodules, which are multiple, well demarcated,

infectious and known etiological agents and etiologic agents notﬁrm, painless, and nonpruritic.

periocular region, pinnae, and paws.
1–5

They may become alopecic anddetectable by analytical methods and possible immune-mediatedulcerated and may get secondarily infected, particularly on the(idiopathic) mechanisms. Some organisms such as Mycobacterium

paws.1–5 Although systemic signs such as lymphadenopathy andspp. and Leishmania spp. are not easily detected by histopa-

hypercalcemia have been reported in dogs and cats, the affectedthology; therefore, polymerase chain reaction (PCR) analysis is

animals are typically healthy.1–7

required.4,7,8 A diagnosis of cutaneous SPGS can be made after

From the Veterinary Teaching Hospital Laboratory of Small Animal
Clinics (S.K., N.K., H.M.), Department of Veterinary Medicine Laboratory of Veterinary Pathology (K.S.), and Laboratory of Microbiology I (A.K.), Azabu University, Sagamihara, Kanagawa, Japan;
Whitaker Cardiovascular Institute, Boston University School of
Medicine, Boston, MA (S.M.); and Central Laboratory, Japan BCG
Laboratory, Kiyose, Tokyo, Japan (S.Y.).

CRP, C-reactive protein; Ig, immunoglobulin; PCR, polymerase chain reaction;
PO, per os; SPGS, sterile pyogranuloma/granuloma syndrome

Correspondence: kawarai@azabu-u.ac.jp (S.K.)

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Cutaneous SPGS in a Maltese

ruling out noninfectious granulomas of unknown etiologies,

Hematologic analysis revealed results within reference ranges,

such as reactive histiocytosis, juvenile sterile granulomatous

except for a slight decrease in the red blood cell count (3.92 3 1012/L;

dermatitis and lymphadenitis, cutaneous xanthoma, and caninereference range, 5.5–8.5 3 1012/L) and packed cell volume (0.31%;

sarcoidosis.1–4

reference range,) and a remarkable increase in CRP level (142.86

Herein, the successful long-term treatment of a dog pre-nmol/L; reference range 0.37–0.55, , 9.52 nmol/L) as described insenting with cutaneous SPGS characterized by generalized ulcer-

Table 1. After severe inﬂammation was suspected in the dog, CRP

ative dermatitis, systemic signs of mild anemia, and an increased

level was monitored using a laser nephelometric immunoassay

C-reactive protein (CRP) level is described. Cutaneous SPGS

systema for dogs.9 Serum protein electrophoresis revealed an in-

was diagnosed by histopathology, negative staining, and PCR for

creased a2-globulin fraction level, but did not indicate gammopathy.
Differential diagnoses from the ﬁndings at the ﬁrst visit were

Mycobacterium spp.

both aerobic and anaerobic bacteria, including Nocardia andCase Report

Actinomyces spp.; deep and intermediate fungi; dermatophytosis;

An indoor 3 yr old castrated male Maltese weighing 4.2 kg was

pemphigus foliaceus; pemphigus vulgaris; erythematosus para-

referred to the Veterinary Teaching Hospital, Azabu University,

neoplastic pemphigus; discoid lupus erythematosus; systemic lu-

with generalized ulcerative dermatitis. Initially, the skin lesions

pus erythematosus; cutaneous lymphoma; erythema multiforme;

occurred on the dog’s head, identiﬁed by mild scales. The lesions

cutaneous vasculitis; toxic epidermal necrolysis; hepatocutaneous

progressed to crusting and multifocal ulcers, which spread to the

syndrome; and idiopathic ulcerative dermatosis.

head, dorsal and ventral trunk, and limbs over a period of 8 mo.

Antimicrobial susceptibility testing was conducted at Hoken

Prior to being referred, the dog was administered systemic anti-

Kagaku Laboratory (Kanagawa, Japan) using the Clinical Labo-bacterial and antifungal drugs [30 mg/kg cephalexin per os (PO) q

ratory Standards Institute method. Aerobic bacterial culture from

12 hr and 6 mg/kg ketoconazole PO q 12 hr, respectively] fora swab of a dorsal ulcerative skin lesion using sheep blood agar was

4 wk; however, the dog was unresponsive. Physical and derma-

negative. Although the association of anaerobic bacteria could not

tological examinations, bacterial culture with antimicrobial sus-

be ruled out, anaerobic culture was not performed because theceptibility testing, fungal culture, blood tests, diagnostic imaging,ulcerative lesion was aerobically exposed on the superﬁcial epi-

urinalysis, arthrocentesis, skin biopsy, and histopathological exami-

dermis. Fungal culture performed and analyzed at Hoken Kagaku

nations were subsequently performed at the time of referral.

Laboratory from the same skin lesion was negative. Sabouraud

On physical examination, the dog presented with severe pain,

agar, which is used for detection of dermatophytes, yeast-like

mild pruritus of the skin, and a body temperature of 398C. Bilateralorganisms, and Cryptococcus spp., was used for the culture. Be-

lymphadenopathy of the superﬁcial cervical and popliteal lymph

cause speciﬁc bacterial and fungal features were not identiﬁed bynodes was observed. Skin lesions comprised alopecia and ery-the impression smears, macerated tissue culture and specialized

thema with epidermal detachment and multifocal ulcers with

culture were not performed.

crusting. Although haired skin on the top of the muzzle remained,Thoracic and abdominal radiographs and ultrasonography

demarcated alopecic skin lesions were observed on the dog’s face,

did not reveal any apparent abnormalities. Dermatoses associatedincluding the periocular, periauricular regions, and the planumwith systemic diseases such as paraneoplastic pemphigus andnasale from the top of the muzzle to the head. Other affected areas

hepatocutaneous syndrome was ruled out because none of ﬁndings

were the dorsal trunk, extending from the neck to the front of theindicated either a tumor mass or a remarkable abnormality of thetail; the ventral trunk extending from the anterior chest; and theliver parenchyma. To examine the possibility of systemic lupus

axilla to the inguinal region, limbs, paws, and perianal region

erythematosus, urinalysis (Table 1), and arthrocentesis of synovial(Figure 1). Impression smears were obtained from the ulcerative

ﬂuid from the left knee joint were performed. Urine test strips

skin lesions of the dorsal and ventral regions and stained by thewere positive for protein (21) and negative to occasionally pos-

Wright’s-Giemsa method. Cytology revealed a pyogranulomatous

itive leukocytes, nitrate, urobilinogen, blood, ketones, bilirubin,inﬂammation comprising macrophages and nondegenerate neu-

and glucose. Zero to occasional red blood cells, white blood cells,

trophils; however, acantholytic keratinocytes, bacteria such as

and transitional cells were seen, but no crystals or casts were

cocci and bacilli, and Malassezia spp. were not apparent. Infection

detected by urinary sediment examination. The synovial ﬂuid waswith Demodex canis was ruled out by skin scraping and hairclear, colorless, and extremely viscous. Cytology revealed a few

plucking, and sarcoptic mange was ruled out by only mild pru-large and small mononuclear inﬂammatory cells, which was notritus of the skin.

suggestive of arthropathy.

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FIGURE 1

Photographs of the dog’s head (A), right paw (B), dorsal region (C), and ventral region (D) on day 1 showing alopecia anderythematous dermatitis with ulcerative plaques.
59-CATGTCTTGTGGTGGAAAGCG-39; RI16S, 59-TACCGTCSpecimens were collected using a 4 mm punch biopsy fromskin lesions on the dog’s head and dorsal trunk. Histopathology

AATCCGAGAGAACCC-39) were used to amplify a 288-base pairrevealed a nodular to diffuse dermatitis characterized by intensefragment. The ﬁrst and second PCR ampliﬁcation consisted oflymphohistiocytic inﬁltration and ﬁbrosis involving the superﬁcial

one cycle at 988C for 3 min, followed by 35 cycles of denatur-dermis and subcutis (Figure 2). The adnexa, such as the hairation (988C for 30 sec), annealing (65.78C for 30 sec), and ex-follicles and sebaceous glands, were obscured by ﬁbrosis. Depo-

tension (728C for 30 sec). The PCR products were separated bysition of autoantibodies in the tissue was not detected by immu-

2% agarose gel electrophoresis, stained with ethidium bromide,

noﬂuorescence using ﬂuorescein isothiocyanate-labeled anti-dog

and visualized using an UV transilluminatorf. DNA was extractedbimmunoglobulin (Ig) G antibodies (diluted 1:800). Based on thefrom a BCG vaccine straing and used as a positive control. The

histopathological evaluation, pemphigus foliaceus, pemphigus

PCR results were negative, and mycobacteriosis was ruled out invulgaris, and systemic and discoid lupus erythematosus, cutaneous

the present case.

lymphoma, erythema multiforme, cutaneous vasculitis, toxic epi-

Clinical ﬁndings (age of onset and breed), histopathologicaldermal necrolysis, and idiopathic ulcerative dermatosis were ruledﬁndings, and diagnostic imaging results ruled out the otherout. Histopathological analysis revealed the presence of nodular

noninfectious granulomas of unknown etiologies; therefore, a di-

granuloma, which suggested a deep pyoderma and fungal infection

agnosis of cutaneous SPGS was considered most suitable for theby an antibiotic-resistant microorganism, atypical mycobacteriosis

present case. Consequently, prednisoloneh (1 mg/kg PO q 12 hr)(canine leproid granuloma syndrome), and cutaneous SPGS. Cu-

and fosfomycin Ca hydratei (25 mg/kg PO q 12 hr) therapy

taneous leishmaniasis was ruled out because it is not endogenous

was initiated. In addition, as a supportive therapy, famotidine j

to Japan and the dog has never been outside Japan. Periodic acid-(1 mg/kg PO q 12 hr) and ursodeoxycholic acidk (10 mg/kg PO q

Schiff, Ziehl-Neelsen, Grocott’s methenamine silver, and gram

12 hr) was administered for protection against the adverse effects

staining were used, but failed to detect any etiologic agent in theof prednisolone. The treatment of skin barrier restoration in-tissue specimens.

cluded 0.5% vitamin A oill and a topical corticosteroid prepara-

Using the primers described by Cornegliani et al. (2005), PCR

tion comprising 0.12% betamethasone valeratem and 0.3%

was performed to detect Mycobacterium spp.8 DNA extraction

heparinoidn. One wk after the initiation of therapy (day 7), thefrom parafﬁn-embedded tissues was conducted using a commer-

ulcerative dermatitis improved and CRP level decreased (4.76ccial kit . Five microliters of the extracted DNA solution were used

nmol/L). After 1 mo (28 days), the dog was free of grossly visible

for the nested PCR ampliﬁcation of a 16S rRNA fragment withulcerative skin lesions (Figures 3A–C) and the packed cell volume

a DNA polymerased using a gradient thermal cyclere. The external

increased to 43%. Subsequently, the dose of prednisolone wasprimers (FO16S, 59-GATAAGCCTGGGAAACTGGGTC-39; RO16S,

tapered by 0.5 mg/kg BW at 2 wk intervals and administration of

59-TTCTCCACCTACCGTCAATCCG-39) were used to amplify

azathioprineo (1 mg/kg PO q 24 hr) was initiated. After decreasinga 344-base pair fragment and the internal primers (F116S,

the dose of prednisolone to 0.5 mg/kg q 48 hr (day 82), it was

280

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Cutaneous SPGS in a Maltese

TABLE 1
Complete Blood Cell Count, Serum Biochemical Proﬁle, andUrinalysis Results on the Day of Referral
AnalyteValue
9

Reference range

WBC (3 10 /L)

1.66(0.6–1.7)

Red blood cell count (3 1012/L)

3.92(5.5–8.5)

Hemoglobin (g/L)

99(120–180)

Packed cell volume

0.31(0.37–0.55)

Mean corpuscular volume (ﬂ)77.8(66–77)

Mean corpuscular hemoglobin (pg/cell)

25.3(19.9–24.5)

Mean corpuscular hemoglobin
concentration (g/L)

325(320–360)

Platelet count (310 /L)

388(200–500)

intense lymphohistiocytic inﬁltration and ﬁbrosis throughout the

Total protein (g/L)

61(51–77)superﬁcial dermis and subcutis. B: A nodular lesion mainly com-

Albumin (g/L)

27(25–40)Blood urea nitrogen (mmol/L)

18.6(6.57–20.9)

prised of macrophages and neutrophils in the subcutis. Hematoxylin

Creatinine (mmol/L)
Phosphorus (mmol/L)

26.5
1.2

(44.2–132.6)(0.7–1.8)

Ca, total (mmol/L)

2.4(2.2–2.9)

reported an apparent recurrence of pruritus and crusting on the9

FIGURE 2

A: Nodular to diffuse dermatitis characterized byand eosin stain, bar represents 500 mm (A) and 100 mm (B).

Alanine aminotransferase (mkat/L)

0.2(0.3–1.2)

dog’s face and back within 2 wk after discontinuing prednisolone

Alkaline phosphatase (mkat/L)

4.3(0.6–4.3)

and azathioprine. The recurrent lesions were treated with pred-Total cholesterol (mmol/L)

5.1(2.8–8.1)

nisolone and azathioprine that were prescribed by the referring

Triglycerides (mmol/L)

0.6(0.2–1.2)

Total bilirubin (mmol/L)

0(0.0–5.1)

5.7
142(3.8–6.8)(142–152)

Glucose (mmol/L)
Na (mmol/L)
Potassium (mmol/L)
Chloride (mmol/L)veterinarian. No hair regrowth was observed during the course of
the treatment (day 624).

4.49(3.7–5.3)

Discussion

106.8(105–117)

At the time of referral, the study authors considered a diagnosis of(1.020–1.050)

pustular skin disease; however, based on the histopathologicalﬁndings of granulomatous dermatitis, that diagnosis was changed

Urine speciﬁc gravity

1.074

Urine pH

7.0

(5.5–7.5)

Urine protein/creatinine ratio

0.22(, 0.5)to granulomatous and pyogranulomatous skin disease. Based on
the criteria described by Santoro et al. (2008), a diagnosis of
cutaneous SPGS was made.4

further decreased to q 72 hr for 2 mo (day 110). Following the

The etiology and pathogenesis of cutaneous SPGS is un-

cessation of prednisolone on day 138, mild skin lesions withknown.1–8 The absence of infectious agents and foreign materials

crusting and erosion were observed on the dorsum of the neck on

together with a good response to systemic glucocorticoids sug-day 166 (Figures 3D, E). Those relapses were treated with pred-gested the involvement of immune-mediated mechanisms.1–7 In

nisolone, and the dosage of azathioprine was decreased to 1 mg/kg

the present case, neither causative organisms nor foreign bodies

q 48 hr. Maintenance doses of prednisolone (0.5 mg/kg q 48 hr)

were detected by culture, impression smears, special histopatho-

and azathioprine (1 mg/kg q 48 hr) were selected on day 197;

logical staining, or PCR for Mycobacterium spp. Because Leish-

however, the owner had administered a single dose of predniso-

mania spp. is not endogenous to Japan, diagnoses were only madelone (0.5 mg/kg) 1–2 times/wk according to the owner’s judg-

for Mycobacterium spp.4,8 Although potentially infectious bacteria,

ment, although the dog received azathioprine as scheduled. At a 3such as antimicrobial-resistant bacteria, anaerobic bacteria, andmo routine follow-up examination (day 278), the dog exhibitedbacteria requiring special culture methods, were not ruled out, themild skin lesions of erythema and crusting on the limbs andauthors attempted to limit the possibility of infection using fos-dorsal and ventral trunk. A fungal culture using a dermatophyte

fomycin. Fosfomycin is a broad-spectrum antibiotic for gram-test agar medium was negative. The owner was reminded topositive and gram-negative bacteria and is well distributed incontinue prednisolone. The skin symptoms did not become ag-

tissues with clinically relevant concentrations in the serum, kid-

gravated and remained unchanged for at least 9 mo. However, on

neys, bladder wall, prostate, lungs, inﬂamed tissues, bone, cere-day 537 during a routine follow-up examination, the ownerbrospinal ﬂuid, abscess ﬂuid, and heart valves.10 The efﬁcacy of

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FIGURE 3 Photographs of the head (A), right paw (B), dorsal region (C), ventral neck (D), and ventral abdomen (E) indicating that theerythematous dermatitis and ulcerative plaques improved by day 28; however, no hair regrowth was observed (A, B, and C). A recurrence ofskin lesions was observed on day 166 after discontinuation of prednisolone (D, E).

fosfomycin against cephalexin-resistant Staphylococcus spp. iso-in the present case. The examination of surface cluster of differ-lated from a patient with pyoderma and otitis externa has been

entiation antigens and major histocompatibility complex mole-

reported in Japan.

11

cules on inﬁltrated cells could provide clues to elucidate theIn the present case, systemic inﬂammation

immune mechanisms involved in cutaneous SPGS.

was indicated by fever, an increased CRP level, and lymphadenopathy. The dog was also treated with immunosuppressive
drugs, including prednisolone and azathioprine, although recur-

Conclusion

rences of skin lesions were observed when both drugs were dis-Cutaneous SPGS is an uncommon skin disease in dogs.1–4 The

continued. Those ﬁndings suggested that systemic inﬂammation,affected animals are typically healthy, but a few exhibit systemic

which was treated with prednisolone and azathioprine, was as-

signs.1–7 This report documented a case of SPGS diagnosed byhistopathological ﬁndings of a nodular to diffuse granulomatous

sociated with the pathogenesis in the present case.
Hair regrowth was not observed even after successful treat-dermatitis with intense lymphohistiocytic inﬁltration, negative

ment with prednisolone and azathioprine, possibly due to thestaining for causative organisms, and PCR for Mycobacterium spp.

presence of extensive ﬁbrosis involving the superﬁcial dermis and

The dog presented with generalized ulcerative dermatitis andsubcutis at the site of adnexa. The dermal papilla and the bulge

systemic signs of mild anemia and an increased CRP level. Suc-

region, where arrector pili muscles insert into hair follicles, arecessful treatment was achieved by administration of immuno-

considered to contain stem cells capable of hair and skin regen-suppressive drugs (prednisolone and azathioprine) for at least

The presence of ﬁbrosis suggested severe inﬂamma-

20 mo. Recurrence of skin lesions after cessation of prednisolone

tion that possibly destroyed the hair follicles and sebaceous glands

and/or azathioprine was observed, suggesting that long-termat the affected sites.

management using immunosuppressive drugs may be required

eration.

12,13

if the dog exhibits severe systemic signs of cutaneous SPGS.

In the present case, lymphohistiocytic inﬁltrations were observed by histopathology; however, deposition of the IgG antibody
was not detected using immunoﬂuorescence. It has been proposed

The authors would like to thank Tadayoshi Yamaguchi (Ann Animal

that cutaneous SPGS is associated with immune dysfunction in

Hospital) for providing the initial information, Yoko Kakinuma (Re-response to chronic antigenic stimulation.1 The absence of IgG

search Institute of Biosciences, Azabu University) for technical as-

autoantibodies suggested that the pathogenesis of the diseases was

sistance with the histologic sections, Takahiro Hamada (Kurume

not associated with humoral-mediated immunity (as observed in

University) for his advice regarding the present case, Dr. Ryoji

pemphigus complex) but with cell-mediated immunity, in which

Takahashi and Dr. Yusuke Matsuura (Veterinary Teaching Hospital,

effecter cells comprise macrophages and lymphocytes, as observed

Azabu University) for supporting patient care, and Jonathan Bloch

282

JAAHA |

50:4 Jul/Aug 2014605

Cutaneous SPGS in a Maltese(Service Employees International Union Training and Education

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diagnosis. 2nd ed. Oxford, UK: Blackwell Publishing; 2005:320–3.
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pyogranulomas, leishmaniasis and mycobacterial infections. J Small
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12. Mercati F, Pascucci L, Ceccarelli P, et al. Expression of mesenchymal
stem cell marker CD90 on dermal sheath cells of the anagen hairfollicle in canine species. Eur J Histochem 2009;53(3):159–66.
13. Mercati F, Pascucci L, Gargiulo AM, et al. Immunohistochemicalevaluation of intermediate ﬁlament nestin in dog hair follicles.
Histol Histopathol 2008;23(9):1035–41.

Funds, Renton, WA) for proofreading the manuscript. This report
was partially supported by a project grant (Young Scientist ResearchTraining Award, 2010 and 2011) funded by the Azabu University
Research Service Division and Grant-in-Aid for Young Scientists(B) by the Japan Society for the Promotion of Science (23780330).
The authors declare that there are no conﬂicts of interest associatedwith the present report.
FOOTNOTESaCanine CRP measurement system CRP-2; Arrows Co. Ltd., Osaka,Japanb
Fluorescein-conjugated goat afﬁnity puriﬁed antibody to dog IgG(whole molecule); MP Bio Japan K.K., Tokyo, Japanc
DEXPAT; Takara Bio Inc., Siga, Japand
MightyAmp DNA Polymerase Ver. 2; Takara Bio Inc., Siga, Japane
MJ Mini; Bio-Rad Laboratories, Hercules, CA
f
DigDoc-It Imaging System; UVP, CAg
BCG vaccine strain; Japan BCG Laboratory, Tokyo, Japanh
Prednisolone; Sanwa Kagaku Kenkyusho Co. Ltd., Aichi, Japani
FOSMICIN; Meiji Seika Pharma Co. Ltd., Tokyo, Japanj
Gaster; Astellas, Tokyo, Japank
URSO; Mitsubishi Tanabe Pharma Co., Osaka, Japanl
Sahne Oint; Eisai, Tokyo, Japanm
Rinderon-V Ointment; Shionogi Co. Ltd., Osaka, Japann
Hirudoid Soft Ointment; Maruho, Osaka, JapanoImuran; Glaxo Smith Kline, Tokyo, Japan
REFERENCES
1. Scott DW, Miller WH, Grifﬁn CE, eds. Miscellaneous skin diseases.
In: Muller and Kirk’s Small Animal Dermatology. 6th ed. Philadelphia(PA): WB Saunders; 2001:1136–40.

JAAHA.ORG

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Ꮫ఍Ⓨ⾲せ᪨₇⪅ྡS. Kawarai, A. Fujimoto, G. Nozawa, N. Kanemaki,
H. Madarame, T. Shida, A. Kiuchi

₇㢟ྡDecreasing methicillin-resistant Staphylococcal colonization inallergic dog skin by shampoo

Ꮫ఍ྡ

The Asian Meeting of Animal Medicine Specialtiesሙᡤࠊ㛤ദ᪥

Bogor,Indonesia, Dec, 2013

We evaluated the effects of shampoo against methicillin-resistant Staphylococcus(MRS) colonization in dogs with allergic dermatitis in a pilot clinical trial. Six allergic
dogs were referred to our hospital for weekly shampoo for 1 month. Diagnosis of canine
atopic dermatitis (CAD) and adverse food reactions were made based on Favrot’s
criteria for CAD, serum allergen-specific immunoglobulin E (IgE) tests and foodelimination and/or provocation trials. The Canine Atopic Dermatitis Extent and Severity
Index (CADESI-03) by veterinarians, pruritus score by owners, and isolation
frequencies of mecA-positive Staphylococcus spp. from skin surface of the three bodysite between weeks 0 and 4 were compared. Polymerase chain reaction was used forevaluation of mecA gene. There was no significant difference in CADESI-03; however,the pruritus scores showed the significance (p < 0.05, Wilcoxon signed-rank test). The
frequencies of MRS isolated from the skin surface also decreased in 4 of the 6 dogs (p <0.05, Mann-Whitney U test). MRS is usually colonized and associated with recurrent
pyoderma in allergic dog skin. Decrease of MRS by the shampoo presents useful forcontrol of allergic dog skin.

615

Ꮫ఍Ⓨ⾲せ᪨₇⪅ྡᮧୖᘯṇࠊ⣽ᕝ⪷▮ࠊᕝཎ஭᫴ᖹࠊᩬ┠ᗈ㑻₇㢟ྡῶឤస⒪ἲࢆᐇ᪋ࡋࡓ≟࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࡢ 2 ౛Ꮫ఍ྡ᪥ᮏᑠື≀⋇་Ꮫ఍ᮾ໭ᆅ༊኱఍

ሙᡤࠊ㛤ദ᪥

㒆ᒣᕷࠊ2013 ᖺ 10 ᭶࠙┠ⓗࠚῶឤస⒪ἲࡣ≟࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡟ᑐࡍࡿ᰿ᮏⓗ἞⒪ἲࡢࡦ࡜ࡘ࡛࠶ࡿࠋẁ㝵ⓗ࡟⃰ᗘࢆ⃰ࡃㄪᩚࡋࡓᢠཎᾮࢆᐃᮇⓗ࡟⓶ୗᢞ୚ࡍࡿࡇ࡜࡛ࠊᢠཎ࡟ᑐࡍࡿ㐣ᩄ཯ᛂࢆῶ
ᑡࡉࡏࡿࠋ୍⯡ⓗ࡟ึᅇᢠཎᾮࡢ⃰ᗘࡣࠊឤసᢠཎẖ࡟ Protein Nitrogen Unit㸦PNU㸧ࢆ⏝࠸ࡓ୍ᐃࡢᕼ㔘ಸ⋡࡟ࡼࡾᐃࡵࡽࢀ࡚࠸ࡿࡀࠊᢠཎ࡟ᑐࡍࡿឤసࡣಶࠎࡢ⑕౛࡟࠾࠸࡚
␗࡞ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋ௒ᅇࠊึᅇ࡟ᢞ୚ࡍࡿᢠཎ⃰ᗘࢆ⓶ෆ཯ᛂ࡟ࡼࡗ࡚Ỵᐃࡋࠊ㔜⠜࡞๪స⏝ࡀ࡞ࡃῶឤస⒪ἲࢆᐇ᪋࡛ࡁࡓ≟ࡢ 2 ౛࡟ࡘ࠸࡚ሗ࿌ࡍࡿࠋ
࠙ᮦᩱ࡜᪉ἲࠚ⑕౛ 1 ࡣ 10 ṓ 1 ࢝᭶㱋ࡢ㞤ࡢᰘ≟࡛ࠊ⒍⑛࡜㢦㠃㸦ཱྀ၁࣭║࿘ᅖ㸧ࠊୗ⭡㒊ࠊᅄ⫥➃ࠊ㝜㒊࠿ࡽ⫠㛛ࠊᑿ᰿㒊࡟࠿ࡅ࡚⣚ᩬࠊⰍ⣲ỿ╔ࠊⱏⓄ໬ࢆ࿊ࡋ࡚࠸ࡓࠋFavrot
㸦2010㸧ࡢデ᩿ᇶ‽ࢆࡶ࡜࡟≟࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡜デ᩿ࡋࡓࠋ⓶ෆ཯ᛂ᳨ᰝ࡟ࡼࡾࢥࢼࣄ
ࣙ࢘ࣄࢲࢽ࡟ᑐࡋ࡚㝧ᛶ཯ᛂࢆ♧ࡋࡓࡇ࡜࠿ࡽࠊ᳨ᰝ㝜ᛶ࡜࡞ࡿᢠཎᾮࡢ⃰ᗘ࠿ࡽῶឤస⒪ἲࢆᐇ᪋ࡋࡓࠋ⑕౛ 2 ࡣ 3 ṓ 9 ࢝᭶㱋ࡢཤໃ㞝ࡢ࣌࢟ࢽ࣮ࢬ࡛ࠊయᖿ࡟⒍⑛ࢆㄆࡵࠊ㢕㒊ࠊ⭆ୗࠊ⭡㒊ࠊ⫼㒊యᖿࠊᅄ⫥➃࡟⬡₃࡜ୣ⑈ࢆㄆࡵ࡚࠸ࡓࠋ㝖ཤ㣗ヨ㦂࡟཯ᛂࡋ࡚⮫ᗋ⑕≧ࡢᨵၿࡀㄆࡵࡽࢀࠊFavrot㸦2010㸧ࡢデ᩿ᇶ‽ࢆࡶ࡜࡟≟࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡜㣗≀᭷ᐖ཯ᛂࡢేⓎ࡜デ᩿ࡋࡓࠋ⓶ෆ཯ᛂ᳨ᰝࢆᐇ᪋ࡋ࡚ࢥࢼࣄࣙ࢘ࣄࢲࢽ࡟ᑐࡍࡿ≟࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡜デ᩿ࡋࠊ᳨ᰝ㝜ᛶ࡜࡞ࡿᢠཎᾮࡢ⃰ᗘ࠿ࡽῶឤస⒪ἲࢆᐇ᪋ࡋࡓࠋ
࠙⤖ᯝ࠾ࡼࡧ⪃ᐹࠚ⑕౛㸯ࡣᐇ᪋ 2 ࢝᭶ᚋ࡟ࠊ2 ᪥࡟ 1 ᅇฎ᪉ࢆ⾜ࡗ࡚࠸ࡓࣉࣞࢻࢽࢰࣟ
ࣥ㸦0.5 ਺/਻㸧ࡢఇ⸆ࡀྍ⬟࡜࡞ࡗࡓࠋᐇ᪋ 4 ࢝᭶༙ᚋ࡟ᢠཎᾮࡢ⥔ᣢ㔞࡟㐩ࡋࠊ௨㝆 1
ᖺ㛫ࠊ1 ࢝᭶࡟ 1 ᅇࡢᢞ⸆࡜ࡋࡓࠋ⌧ᅾࠊእ⏝๪⭈⓶㉁࣍ࣝࣔࣥ๣㸦ࢥࣝࢱࣂࣥࢫࠊVirbacࠊ
኱㜰㸧ࢆ⑕≧ᝏ໬᫬ࡢࡳ࡟ᑡ㔞౑⏝ࡍࡿࡔࡅ࡛Ⰻዲ࡟⑕≧ࡀ⥔ᣢ࡛ࡁ࡚࠸ࡿࠋ⑕౛ 2 ࡣࠊῶឤస⒪ἲࢆࣇ࣐ࣝ㓟ࢡ࣐ࣞࢫࢳࣥ㸦ࢱ࣋ࢪ࣮ࣝ㘄ࠊࣀࣂࣝࢸ࢕ࢫࣇ࢓࣮࣐ࠊᮾி㸧࡜ే⏝ࡋ࡚ᐇ᪋ࡋࡓࠋᐇ᪋ 6 ࢝᭶ᚋ࡟ᢠཎᾮࡀ⥔ᣢ㔞࡟㐩ࡋࠊ௨㝆 8 ࢝᭶ࠊ1 ࢝᭶࡟ 1 ᅇࡢᢞ⸆ࢆ⾜ࡗࡓࠋ⌧ᅾࠊ㣗≀᭷ᐖ཯ᛂࡢⓎ⑕࡟ὀពࡋ࡞ࡀࡽࠊᢠࣄࢫࢱ࣑ࣥ๣ࡢෆ᭹࠾ࡼࡧእ
⏝๪⭈⓶㉁࣍ࣝࣔࣥ๣㸦ࢥࣝࢱࣂࣥࢫ㸧ࢆᑡ㔞౑⏝ࡍࡿࡔࡅ࡛⑕≧ࡀⰋዲ࡟⥔ᣢ࡛ࡁ࡚࠸
ࡿࠋ⓶ෆ᳨ᰝࢆᣦᶆ࡟ࠊึᅇᢠཎ⃰ᗘࢆ⑕౛࡟ྜࢃࡏ࡚Ỵᐃࡋࠊῶឤస⒪ἲࢆᐇ᪋࡛ࡁࡓࠋ
௒ᚋࠊ⑕౛ᩘࢆቑࡸࡋ࡚ࠊ๪స⏝ࡢᑡ࡞࠸ຠ⋡ⓗ࡞἞⒪᪉ἲࢆ᳨ウࡋ࡚࠸ࡃᚲせࡀ࠶ࡿࠋ

616

Ꮫ఍Ⓨ⾲せ᪨₇⪅ྡ㣤㔝⍞㈗ࠊすᮏඃᏊࠊ⸨ᮏ࠶ࡺࡳࠊ㔝⃝※ኴࠊᕝཎ஭᫴ᖹࠊಙ⏣༟⏨ࠊ
ᮌෆ᫂⏨

₇㢟ྡ

≟ࡢ࢔ࣞࣝࢠ࣮ᛶ⓶⭵⅖ࡢ⑕≧⥔ᣢ࡟࠾ࡅࡿ࣐࢖ࢡࣟࣂࣈࣝࢆ⏝࠸ࡓࢩࣕࣥࣉ࣮⒪ἲࡢ᭷ຠᛶࡢ᳨ウᏛ఍ྡ᪥ᮏື≀┳ㆤᏛ఍㛵すᆅ༊➨ 5 ᅇ౛఍ሙᡤࠊ㛤ദ᪥

኱㜰ࠊ2012 ᖺ 3 ᭶࠙┠ⓗࠚ≟ࡢ࢔ࣞࣝࢠ࣮ᛶ⓶⭵⅖ࡣࠊ᰿἞ᅔ㞴࡞⑌ᝈ࡛࠶ࡾ᪥ᖖⓗ࡞⑕≧ࡢ⥔ᣢࡀ㔜せ࡛࠶ࡿࠋࡑࡢቑᝏᅉᏊ࡜ࡋ࡚⸆๣ឤཷᛶࣈࢻ࢘⌫⳦ࡢᏑᅾࡀ▱ࡽࢀ࡚࠸ࡿࠋࢩࣕࣥࣉ࣮⒪ἲࡣ⿕ẟ࡟௜╔ࡋࡓ࢔ࣞࣝࢤࣥࡢ㝖ཤ࠾ࡼࡧឤᰁ⑕ࡢண㜵࡟᭷ຠ࡛࠶ࡿࠋ࣐࢖ࢡࣟࣂࣈࣝࢆ
⏝࠸ࡓࢩࣕࣥࣉ࣮⒪ἲ࡟ࡼࡗ࡚⮫ᗋ⑕≧ࡢ⥔ᣢࡀྍ⬟࡛࠶ࡿ࠿ࠊ᭷ຠᛶࢆ᳨ウࡋࡓࠋ࠙ᮦᩱ࡜᪉ἲࠚ㯞ᕸ኱Ꮫ㝃ᒓື≀⑓㝔࡟᮶㝔ࡋࡓ࢔ࣞࣝࢠ࣮ᛶ⓶⭵⅖࡜デ᩿ࡉࢀࡓ≟ 3 㢌ࠋ
㐌 1 ᅇࠊࢩ࣮ࣕ࣡࣊ࢵࢻᆺ࣐࢖ࢡࣟࣂࣈࣝࢆ⏝࠸࡚ࢩࣕࣥࣉ࣮⒪ἲࢆᐇ᪋ࡋࡓࠋヨ㦂๓࡜
ヨ㦂 4 㐌ᚋ࡟⋇་ᖌ࡟ࡼࡿ⓶⭵㔜⑕ᗘࢫࢥ࢔ࠊ㣫࠸୺࡟ࡼࡿ⑛ࡳࡢࢫࢥ࢔ࠊ⓶⭵࠿ࡽᇵ㣴ࡋࡓ⳦✀ࡢྠᐃ࠾ࡼࡧ⸆๣ឤཷᛶࠊ┳ㆤᖌ࡟ࡼࡿ⿕ẟࡢほᐹࢆ⾜ࡗࡓࠋ࠙⤖ᯝ࠾ࡼࡧ⪃ᐹࠚ⋇་ᖌ࡟ࡼࡿ⓶⭵㔜⑕ᗘࢫࢥ࢔࡟ኚ໬ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࡀࠊ㣫࠸୺࡟ࡼࡿ⑛ࡳࡢࢫࢥ࢔ࡣ 3 ౛୰ 2 ౛࡟࠾࠸࡚ᨵၿࡋࡓࠋ⣽⳦ᇵ㣴᳨ᰝ࡛ࡣࠊ3 ⑕౛୰ 2 ౛࡟࠾࠸࡚ヨ㦂 4 㐌ᚋࡢ᳨ᰝ⤖ᯝࡀ㝜ᛶ࡜࡞ࡗࡓࠋ┳ㆤᖌࡀࡳࡓ⓶⭵࡜⿕ẟࡢ≧ែ࡟ࡘ࠸࡚ࡣࠊ
ヨ㦂๓࡜ẚ࡭࡚ࠊ඲⑕౛࡛⓶⭵ࡢⓎ㉥ࡀῶᑡࡋࠊⓎẟࡀㄆࡵࡽࢀࡓࠋ௨ୖ࠿ࡽ≟ࡢ࢔ࣞࣝࢠ࣮ᛶ⓶⭵⅖࡟࠾ࡅࡿ⮫ᗋ⑕≧ࡢ⥔ᣢ࡟࣐࢖ࢡࣟࣂࣈࣝࢆ⏝࠸ࡓࢩࣕࣥࣉ࣮⒪ἲࡣ᭷ຠ࡛
࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ௒ᅇࡣᑐ㇟⑕౛ࡀ 3 㢌ࡔࡗࡓࡓࡵࠊ௒ᚋᑐ㇟⑕౛ࢆቑࡸࡋ᳨࡚ウࡋ࡚࠸ࡃࡇ࡜ࡀᚲせ࡛࠶ࡿࠋ

617

Ꮫ఍Ⓨ⾲せ᪨₇⪅ྡ⣽ᕝ⪷▮ࠊᕝཎ஭᫴ᖹࠊὠஂ஭฼ᗈࠊஂᮎṇᬕࠊ༳∾ಙ⾜ࠊᩬ┠ᗈ㑻ࠊᅵᒇுࠊᑠ᪉᐀ḟ₇㢟ྡᅜ⏘ࢥࢼࣄࣙ࢘ࣄࢲࢽᢠཎᾮࡢ᭷⏝ᛶ࡟㛵ࡍࡿ෌⪃.

Ꮫ఍ྡ➨ 154 ᅇ᪥ᮏ⋇་Ꮫ఍Ꮫ⾡㞟఍.

ሙᡤࠊ㛤ദ᪥

ᒾᡭࠊ2012 ᖺ 9 ᭶࠙┠ⓗࠚ≟ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࡢཎᅉ࢔ࣞࣝࢤࣥࢆྠᐃࡍࡿࡓࡵࡢ࢔ࣞࣝࢠ᳨࣮ᰝࡣ୺࡟
⓶ෆ཯ᛂ᳨ᰝ࡜⾑Ύᢠཎ≉␗ⓗ IgE ᳨ᰝࡀᐇ᪋ࡉࢀ࡚࠸ࡿࠋ⾑ᾮ᳨ᰝࢆእὀࡍࡿ⾑Ύᢠཎ≉␗ⓗ IgE ᳨ᰝࡣ⡆౽࡛࠶ࡿࡀࠊཷクᶵ㛵࡟࠾࠸࡚ከᵝ࡞᪉ἲ࡛ᐇ᪋ࡉࢀ࡚࠾ࡾࠊ᳨ᰝᶵ
㛵࡟࠾࠸࡚⤖ᯝࡀ␗࡞ࡿࡇ࡜ࡀㄢ㢟࡜࡞ࡗ࡚࠸ࡿࠋࡑࡇ࡛ࠊ㉁ࡢ㧗࠸≟ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖⨯ᝈ⑕౛ࡢࢤࣀ࣒ DNA ࢆ᥇ྲྀࡍࡿࡓࡵࠊ⓶ෆ཯ᛂ᳨ᰝࢆ㯞ᕸ኱Ꮫ࡟࡚❧ࡕୖࡆࢆ⾜ࡗࡓࠋ࠙ᮦᩱ࡜᪉ἲࠚ 㯞ᕸ኱Ꮫ㝃ᒓື≀⑓㝔࡟᮶㝔ࡋࡓ CAD ࡀ␲ࢃࢀࡿ⑕౛ 29 㢌ࢆᑐ㇟࡜ࡋࠊ
IDT ࡜ IgE test ࢆᐇ᪋ࡋࡓࠋIDT ࡣᅜ⏘ᢠཎᾮ࡜ࡋ࡚㫽ᒃ⸆ရ㸦ᰴ㸧〇ࢆ 1:100㹼
1:10000(w/v)ࡢ⃰ᗘ࡜ࠊ㍺ධᢠཎᾮ࡜ࡋ࡚ Greer ♫〇ࢆ 1:50000㸦w/v㸧ࡢ⃰ᗘ࡛⏝࠸ࠊ⣚ᩬࡢ࠶ࡿ⭾⑈ᙧᡂࢆ㝧ᛶ࡜ࡋࡓࠋIgE test ࡣࣄࢺ㧗ぶ࿴ᛶ IgE ࣞࢭࣉࢱ࣮Ș㙐ࢆ⏝࠸ࡓ
ELISA ἲࢆ⏝࠸࡚ 150LU ௨ୖࢆ㝧ᛶ࡜ࡋ࡚እ㒊ᶵ㛵࡛ᐃࡋࡓࠋࡑࢀࡒࢀࡢ㛵㐃ᛶࢆ

Fisher's exact test ࢆ⏝࠸࡚⤫ィゎᯒࡋࡓࠋ࠙⤖ᯝ࠾ࡼࡧ⪃ᐹࠚ IgE test ࡜㍺ධᢠཎᾮࢆ⏝࠸ࡓ IDT ࡢ㛫࡟᭷ពᕪࢆㄆࡵࡓ㸦P<0.01㸧
ࠋ
1:100㸦w/v㸧࡜ 1:10000㸦w/v㸧ࡢᅜ⏘ᢠཎᾮࡣࠊIgE test ࠾ࡼࡧ㍺ධᢠཎᾮ࡜ࡢ㛫࡟᭷ពࠋ௨ୖࡼࡾࠊᅜ⏘ᢠཎᾮࡢ≟࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࡢデ᩿࡟࠾ࡅࡿ᭷
ᕪࡀㄆࡵࡽࢀࡓ㸦P<0.05㸧
⏝ᛶࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ

618

Ꮫ఍Ⓨ⾲せ᪨
₇⪅ྡKawarai S, Takahashi R, Madarame H, Shirota K.

₇㢟ྡA case of granulomatous dermatitis in a dog.

Ꮫ఍ྡ

The Asian Meeting of Animal Medicine Specialties.

ሙᡤࠊ㛤ദ᪥

Thailand , May, 2011.

Cutaneous sterile pyogranuloma /granuloma syndrome (SPGS) is a locally restricted
multinodular dermatitis. Affected dogs are typically healthy, but a few show systemic
signs. Herein, a case of a dog presenting with generalized ulcerative dermatitis withsystemic signs of mild anemia and an increased C-reactive protein level is described.
Cutaneous SPGS was diagnosed by histopathology, negative staining causative
organisms, and polymerase chain reaction for Mycobacterium spp. Successful treatment
was achieved by immunosuppressive drugs, including prednisolone and azathioprine,
administered for at least 20 mo. Recurrences of skin lesions were observed whenprednisolone and/or azathioprine were discontinued. Long-term management withimmunosuppressive agents may be required if the affected dog exhibits severe
symptoms of cutaneous SPGS.

619

≟ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࡢ㑇ఏᏊゎᯒᮧୖ⿱ಙ (㯞ᕸ኱Ꮫ⾨⏕Ꮫ➨ 2)ࠊబࠎᮌៅ஧(♫ᅋἲே␆⏘ᢏ⾡༠఍㝃ᒓື≀㑇ఏ◊✲ᡤ)ࠊᒸᮏ᠇᫂ࠊᓥ಴⚽຾ࠊ㜰ཱྀ㞞ᘯ (㯞ᕸ኱Ꮫᚤ⏕≀Ꮫ➨ 1)◊✲せ᪨≟ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖㻔㼏㻭㻰㻕ࡣ⎔ቃࡸ㣗≀୰࡟ྵࡲࢀࡿᢠཎ࡬ࡢ㐣ᩄ཯ᛂ࡛࠶ࡾࠊே
ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡜⑓ែࡀ㢮ఝࡋ࡚࠸ࡿࡔࡅ࡛࡞ࡃࠊ⑌⑓࡜㑇ఏⓗ⫼ᬒࡀᐦ᥋࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ␲ࢃࢀ࡚࠸ࡿࠋࡑࡇ࡛ࠊ㼏㻭㻰 ࡟㛵㐃ࡍࡿ㑇ఏᏊࢆ⥙⨶ⓗ࡟᥈⣴ࡍࡿࡓࡵ࡟ࠊ㼏㻭㻰 ࢆⓎ⑕ࡋࡓ≟ࡢ⾑ᾮࢧࣥࣉࣝࢆ཰㞟ࡋࠊࡉࡽ࡟཰㞟ࡋࡓࢧࣥࣉࣝࡢ⾑Ύ࠿ࡽࢲࢽ࡟ᑐࡍࡿ≉␗ⓗ 㻵㼓㻱 ࢆ᳨ฟࡋࡓࠋࡑࡢ⤖ᯝࠊ㼏㻭㻰 ࡛࠶ࡾࠊ㻵㼓㻱 㝧ᛶࡢᰘ≟ 㻞㻥 㢌࡜ࠊ㼏㻭㻰
ᮍⓎ⑕࡛ 㻵㼓㻱 ࡀ᳨ฟࡉࢀ࡞࠿ࡗࡓᰘ≟ 㻝㻥 㢌ࢆ཰㞟࡛ࡁࡓࡓࡵࠊࡇࢀࡽࡢ 㻰㻺㻭 ࢧࣥࣉࣝࢆ⏝࠸࡚ 㻰㻺㻭 ࢔ࣞ࢖ࢆ⾜࠸ࠊ㼏㻭㻰 ࡟㛵㐃ࡍࡿ㑇ఏᏊࡀ࠶ࡿࡢ࠿ࢆゎᯒࡋࡓࠋ㼏㻭㻰 Ⓨ⑕≟࡛ࡣ➨ 㻞㻥 ␒࠾ࡼࡧ 㻤 ␒ᰁⰍయࡢ㝈ᒁࡋࡓ㡿ᇦ࡟୍ሷᇶከᆺ㻔㻿㻺㻼㻕ࡀㄆࡵࡽࢀࠊࡑࡢ࠺
ࡕࡦ࡜ࡘࡣ➨ 㻞㻥 ␒ᰁⰍయ࡟ࢥ࣮ࢻࡉࢀ࡚࠸ࡿᶵ⬟୙᫂࡞ 㻾㻮㻹㻝㻞㻮 㑇ఏᏊࡢ࢚ࢡࢯࣥୖࡢ 㻿㻺㻼 ࡛࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋᮏ◊✲࡛ࡣ࢔ࣞࣝࢠ࣮࡟㛵㐃ࡍࡿ㑇ఏᏊୖࡲࡓ
ࡣ㏆ഐࡢ 㻿㻺㻼 ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࡀࠊ➨ 㻞㻥 ␒ᰁⰍయୖ࡟ 㼏㻭㻰 ࡟㛵㐃ࡍࡿ㑇ఏᏊ㡿ᇦࡀ᭷ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ㻌㻌┠ⓗ㻌≟ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖㻔㼏㻭㻰㻕ࡣ⎔ቃࡸ㣗≀୰࡟ྵࡲࢀࡿᢠཎ࡬ࡢ㐣ᩄ཯ᛂ࡛࠶ࡾࠊே
ࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡜⑓ែࡀ㢮ఝࡋ࡚࠸ࡿࠋࡲࡓࠊ㼏㻭㻰 ࡣᐙ᪘ᛶ⣲ᅉࡔࡅ࡛࡞ࡃ≟✀࡟
ࡼࡾⓎ⏕⋡ࡀ␗࡞ࡿࡇ࡜࠿ࡽࠊே࡜ྠᵝࠊ⑌⑓࡜㑇ఏⓗ⫼ᬒࡀᐦ᥋࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜
ࡀ␲ࢃࢀࡿࠋࡇࡢࡇ࡜࠿ࡽࠊ㼏㻭㻰 Ⓨ⑕࡟ᐤ୚ࡍࡿ㑇ఏᏊࢆྠᐃࡍࡿࡇ࡜࡟ࡼࡾࠊ㼏㻭㻰 ࡢ
἞⒪࡟ᐤ୚ࡍࡿࡔࡅ࡛࡞ࡃேࡢ࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡟㛵㐃ࡍࡿ㑇ఏᏊ࡛࠶ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࠊ἞⒪ࡢᇶ♏ⓗ࡞▱ぢ࡜࡞ࡿࡓࡵࠊ㼏㻭㻰 ࡟㛵㐃ࡍࡿ㑇ఏᏊ㡿ᇦࢆ⥙⨶ⓗ࡟ゎᯒࡋࡓࠋᮦᩱ࡜᪉ἲᰘ≟ࠊࣇࣞࣥࢳࣈࣝࢻࢵࢡࠊ࣑ࢽࢳࣗ࢔ࢲࢵࢡࢫࣇࣥࢺࠊࢺ࢖ࣉ࣮ࢻࣝࡢ࠸ࡎࢀ࠿ࡢ
≟✀࡛࠶ࡾࠊྠࡌᆅᇦ࡛ᐊෆ㣫⫱ࡉࢀ࡚࠸ࡿ≟࡟㝈ᐃࡋ࡚⾑ᾮࢧࣥࣉࣝࡢ཰㞟ࢆ⾜ࡗࡓࠋ㼏㻭㻰 ࡢデ᩿࡟ࡘ࠸࡚ࡣࠊ㻲㼍㼢㼞㼛㼠 ࡢデ᩿ᇶ‽㻔㼂㼑㼠㼑㼞㼕㼚㼍㼞㼥㻌㻰㼑㼞㼙㼍㼠㼛㼘㼛㼓㼥㻘㻌㻞㻜㻜㻥㻕࡟‽ࡌࡓ 㻣 㡯┠㻔㻟 ṓ௨ୗ࡛Ⓨ⑕ࠊࢫࢸࣟ࢖ࢻᢞ⸆࡛⑛ࡳࡀ἞ࡲࡿࠊᦆയࡢకࢃ࡞࠸⑛ࡳࠊ๓⫥࡟⑓ኚࡀ࠶ࡿࠊ⪥௓࡟⑓ኚࡀ࠶ࡿࠊ⪥ࡢ㎶⦕࡟ࡣ⑓ኚࡀ࡞࠸ࠊ⭜⫼㒊࡟⑓ኚࡀ࡞࠸㻕ࡢ࠺ࡕ 㻡 㡯┠ࡀᙜ࡚ࡣࡲࡗࡓሙྜ࡟ 㼏㻭㻰 ࡜デ᩿ࡋࡓࠋࡲࡓࠊ㼏㻭㻰 ࢆⓎ⑕ࡋ࡚࠸࡞࠸೺ᖖ≟ࡣ 㻣 ṓ௨ୖ࡛ 㼏㻭㻰 ࢆ␲࠺⑕≧ࡀ࡞ࡃࠊ㐣ཤ࡟࠾࠸࡚ࡶ⑕≧ࡀ࡞࠸ಶయ࡜ࡋࡓࠋࡇࢀࡽࡢデ᩿ࡣྠࡌ⋇་ᖌ࡟ࡼࡗ࡚⾜࠺ࡇ࡜࡟ࡼࡾࠊデ᩿ᇶ‽ࢆ⤫୍ࡋࡓࠋࡇࢀࡽࡢ 㼏㻭㻰 Ⓨ⑕≟࠾620

ࡼࡧᮍⓎ⑕≟࠿ࡽ᥇⾑ࢆ⾜࠸ࠊ⾑ᾮࢧࣥࣉࣝ࠿ࡽ⾑Ύศ㞳࠾ࡼࡧ 㻰㻺㻭 ᢳฟࢆ⾜ࡗࡓࠋࡇࢀࡽࡢࢧࣥࣉࣝࡣࠊ
⾑Ύࢆ⏝࠸ࡓ 㻱㻸㻵㻿㻭 ࡟ࡼࡾࢲࢽ࢔ࣞࣝࢤࣥ≉␗ⓗ 㻵㼓㻱 ࡢ᳨ฟࠊ㻰㻺㻭ࡣ 㻰㻺㻭 ࢔ࣞ࢖࡟ࡼࡾࠊ㻿㻺㻼 ゎᯒࢆ⾜ࡗࡓࠋ㻌⤖ᯝ࠾ࡼࡧ⪃ᐹcAD ዲⓎ≟✀࡛࠶ࡿᰘ≟ࠊࣇࣞࣥࢳࣈࣝࢻࢵࢡࠊ࣑ࢽࢳࣗ࢔ࢲࢵࢡࢫࣇࣥࢺࠊࢺ࢖
ࣉ࣮ࢻࣝࡢ⾑ᾮࢧࣥࣉࣝࡢ཰㞟ࢆ⾜࠸ࠊ㼏㻭㻰 Ⓨ⑕≟ࡣࠊᰘ≟ 㻠㻡 㢌ࠊ࣑ࢽࢳࣗ࢔ࢲࢵࢡ
ࢫࣇࣥࢺ 㻟㻟 㢌ࠊࢺ࢖ࣉ࣮ࢻࣝ 㻟㻜 㢌ࠊࣇࣞࣥࢳࣈࣝࢻࢵࢡ 㻞㻜 㢌ࡢィ 㻝㻞㻤 㢌࠿ࡽ 㻰㻺㻭 ࢧࣥࣉࣝࢆᚓࡽࢀࡓࠋࡲࡓࠊ೺ᖖ≟ࡣᰘ≟ 㻞㻜 㢌ࠊ࣑ࢽࢳࣗ࢔ࢲࢵࢡࢫࣇࣥࢺ 㻠㻝 㢌ࠊࢺ࢖ࣉ࣮ࢻࣝ 㻝㻢 㢌ࠊࣇࣞࣥࢳࣈࣝࢻࢵࢡ 㻞 㢌ࡢィ 㻣㻥 㢌࠿ࡽ 㻰㻺㻭 ࢧࣥࣉࣝࢆᚓࡿࡇ࡜ࡀ࡛ࡁࡓࠋࡉࡽ࡟ࠊ㼏㻭㻰 Ⓨ⑕≟࡟࠾࠸࡚ࢲࢽ࢔ࣞࣝࢤࣥ≉␗ⓗ 㻵㼓㻱 ᢠయ᳨ᰝࢆ⾜ࡗࡓ࡜ࡇࢁࠊ
ᰘ≟࡛ࡣ 㻣㻞㻚㻡㻑࡜௚ࡢ≟✀ࡼࡾ㧗࠸ᢠయಖ᭷⋡㻔ࣇࣞࣥࢳࣈࣝࢻࢵࢡ㻦㻡㻡㻚㻢㻑㻘㻌 ࣑ࢽࢳࣗ࢔
ࢲࢵࢡࢫࣇࣥࢺ㻦㻡㻜㻚㻜㻑㻘㻌 ࢺ࢖ࣉ࣮ࢻࣝ㻦㻞㻜㻑㻕࡛࠶ࡗࡓࠋࡇࢀࡽࡢࢧࣥࣉࣝࢆ⏝࠸࡚ࠊ㼏㻭㻰ࡢཎᅉ㑇ఏᏊࢆ⥙⨶ⓗ࡟᥈⣴ࡍࡿࡓࡵࠊᰘ≟ࡢࢧࣥࣉࣝࢆ⏝࠸࡚ࢲࢽ࢔ࣞࣝࢤࣥ≉␗ⓗ㻵㼓㻱 㝧ᛶࡢ 㼏㻭㻰 Ⓨ⑕≟ 㻞㻥 㢌࡜ 㻵㼓㻱 㝜ᛶ࡛ 㼏㻭㻰 ᮍⓎ⑕≟ 㻝㻥 㢌࡟࠾࠸࡚ 㻰㻺㻭 ࢔ࣞ࢖ࢆ⏝࠸ࡓ 㻿㻺㻼 ࡢ⥙⨶ⓗ㑇ఏᏊゎᯒࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊ㼏㻭㻰 㝧ᛶ⩌࡛ㄆࡵࡽࢀࡓ 㻿㻺㻼 ࡢୖ఩ 㻡 ࡘ㻔ᅗ 㻝㻕ࡀ➨ 㻞㻥 ␒ᰁⰍయ࡟Ꮡᅾࡋࠊୖ఩ 㻢ࠥ㻞㻜 ࡀ➨ 㻤 ␒ᰁⰍయ࡟Ꮡᅾࡋ࡚࠸ࡓ㻔⾲㻝㻕ࠋࡇࡢ➨ 㻞㻥 ␒ᰁⰍయࡢ 㻡 ࡘࡢ 㻿㻺㻼 ࡢ࠺ࡕ 㻞 ࡘࡢ 㻿㻺㻼 ࡣ㑇ఏᏊࡀࢥ࣮ࢻࡉࢀ࡚࠸ࡿ㡿ᇦࡢࡶࡢ࡛࠶ࡾࠊ㻌 ➨ 㻞㻥 ␒ᰁⰍయ 㻠㻝㻘㻢㻝㻤㻘㻡㻝㻠 ␒ሷᇶࡢ 㻿㻺㻼 ࡣヲ⣽࡞ᶵ⬟ࡀ୙᫂࡞㻾㻮㻹㻝㻞㻮 㑇ఏᏊࡢ࢚ࢡࢯࣥ࡟ࠊ㻠㻝㻘㻢㻠㻜㻘㻡㻠㻣 ␒ሷᇶࡢ 㻿㻺㻼 ࡣ⥺ẟᵓ㐀࡟㛵୚ࡍࡿ 㼀㻹㻱㻹㻢㻣ࡢ࢖ࣥࢺࣟࣥ࡟఩⨨ࡋ࡚࠸ࡓࠋࡑࡢ௚ࡢ 㻟 ࡘࡢ 㻿㻺㻼 ࡣ㑇ఏᏊࡀࢥ࣮ࢻࡉࢀ࡚࠸࡞࠸㡿ᇦ࡟఩⨨ࡋ࡚࠸ࡓࡀࠊࡇࢀࡽ 㻡 ࡘࡢ 㻿㻺㻼 ࡣ⣙ 㻢㻜㻜㼗㼎㼜 ෆ࡟㞟୰ࡋ࡚Ꮡᅾࡋ࡚࠸ࡓࠋࡲࡓࠊ➨ 㻤 ␒ᰁⰍయࡢ 㻝㻝 ࡢ 㻿㻺㻼㻔ሷᇶ␒ྕ㻦㻌 㻠㻘㻥㻣㻢㻘㻜㻜㻢ࠊ㻌 㻠㻘㻥㻤㻥㻘㻡㻟㻥ࠊ㻌 㻠㻘㻥㻥㻜㻘㻞㻣㻣ࠊ㻌 㻠㻘㻥㻥㻞㻘㻢㻡㻝ࠊ㻌㻡㻘㻜㻜㻣㻘㻝㻤㻠ࠊ㻌 㻡㻘㻜㻞㻝㻘㻥㻞㻞ࠊ㻌 㻡㻘㻜㻠㻝㻘㻢㻥㻞ࠊ㻌 㻡㻘㻜㻢㻢㻘㻞㻢㻥ࠊ㻌 㻡㻘㻜㻣㻞㻘㻤㻞㻡ࠊ㻌 㻡㻘㻜㻤㻟㻘㻞㻞㻥ࠊ㻌 㻡㻘㻜㻥㻥㻘㻢㻥㻥㻕ࡣ⣽⬊Ṛ࡟㛵୚ࡍࡿ 㻵㻼㼀㻷㻝 㑇ఏᏊୖࡢ࢖ࣥࢺࣟࣥ㡿ᇦ࡟఩⨨ࡋ࡚࠾ࡾࠊ௚ࡢ 㻠 ࡘࡢ 㻿㻺㻼㻔ሷᇶ␒ྕ㻦㻌㻠㻘㻥㻞㻢㻘㻥㻥㻤ࠊ㻌 㻠㻘㻥㻟㻥㻘㻟㻢㻟ࠊ㻌 㻠㻘㻥㻠㻜㻘㻝㻞㻥ࠊ㻌 㻠㻘㻥㻡㻠㻘㻣㻡㻝㻕ࡣ㑇ఏᏊࡀࢥ࣮ࢻࡉࢀ࡚࠸࡞࠸㡿ᇦ࡟఩⨨ࡋ࡚࠸ࡓࠋࡇࢀࡽࡢ➨ 㻤 ␒ᰁⰍయୖࡢ 㻝㻡 ࡢ 㻿㻺㻼 ࡣ⣙ 㻥㻜㼗㼎㼜 ෆ࡟ᐦ㞟ࡋ࡚఩⨨ࡋ࡚࠸ࡓࠋࡇࢀࡽࡢ 㻿㻺㻼 ࡢ㏆ഐ࡟ࡣ࢔ࣞࣝࢠ࣮࡟㛵୚ࡍࡿ㑇ఏᏊࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࡀࠊ➨ 㻞㻥 ␒ᰁⰍయࡢ 㻿㻺㻼 ࡢ㏆ഐ࡟ࡣ 㻾㻮㻹㻝㻞㻮 ྠᵝ࡟ᶵ⬟ࡀ୙᫂࡞㑇ఏᏊ࡛࠶ࡿ
㻸㻻㻯㻝㻜㻜㻢㻤㻡㻢㻜㻡 ࡞࡝ࡀ఩⨨ࡋ࡚࠸ࡓࠋ௨ୖࡢࡇ࡜࠿ࡽ➨ 㻤 ࠾ࡼࡧ 㻞㻥 ␒ᰁⰍయୖ࡟ 㼏㻭㻰࡟㛵㐃ࡍࡿ 㻿㻺㻼 ࡀㄆࡵࡽࢀࠊ≉࡟➨ 㻞㻥 ␒ᰁⰍయ࡟ࡣ 㼏㻭㻰 Ⓨ⑕≟࡛㧗㢖ᗘ࡟ 㻡 ࡘࡢ 㻿㻺㻼ࡀ㝈ᒁࡋ࡚ㄆࡵࡽࢀࡓࡇ࡜࠿ࡽࠊ㼏㻭㻰 ࡟㛵㐃ࡍࡿ㑇ఏᏊࡀ➨ 㻞㻥 ␒ᰁⰍయ࡟Ꮡᅾࡍࡿࡇ
࡜ࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊࡇࡢ➨ 㻞㻥 ␒ᰁⰍయࡢ 㻿㻺㻼 ࡣ 㼏㻭㻰 Ⓨ⑕≟࡛㧗㢖ᗘ࡟ㄆࡵࡽࢀࡿ
ࡇ࡜࠿ࡽࠊ㼏㻭㻰 Ⓨ⑕ண ࡟᭷⏝࡛࠶ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࠋ㻌621

ᅗ 1 cAD Ⓨ⑕≟ࡢࢤࣀ࣒࣡࢖ࢻ㛵㐃ゎᯒ
A. Q-Q ࣉࣟࢵࢺB. ࣐ࣥࣁࢵࢱࣥࣉࣟࢵࢺ

622

⾲ 1 cAD Ⓨ⑕≟࡛㧗㢖ᗘ࡟ㄆࡵࡽࢀࡿ SNP㡰఩ ᰁⰍయ

఩⨨

P್

㢖ᗘ
Case

Control

12941126533 1.45282E-06

0.79310.29412

2941618514 3.78361E-06

0.50000.0294132941300904 3.80005E-06

0.79310.323542941640547 4.03532E-06

0.74140.264752941703308 1.39326E-05

0.65520.1765684926998

6.58203E-05

0.13790.47067849393636.58203E-05

0.13790.4706884940129

6.58203E-05

0.13790.4706984954751

6.58203E-05

0.13790.47061084976006

6.58203E-05

0.13790.470611

849895396.58203E-05

0.13790.47061284990277

6.58203E-05

0.13790.470613

84992651

6.58203E-05

0.13790.470614

85007184

6.58203E-05

0.13790.470615

850219226.58203E-05

0.13790.470616

85041692

6.58203E-05

0.13790.470617

85066269

6.58203E-05

0.13790.470618

85072825

6.58203E-05

0.13790.470619

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Yuan Y, Kitamura-Muramatsu Y, Saito S, Ishizaki H, Nakano M, Haga S, Matoba K, OhnoA, Murakami H, Takeshima SN, Aida Y. Detection of the BLV provirus from nasal
secretion and saliva samples using BLV-CoCoMo-qPCR-2: Comparison with blood
samples from the same cattle. Virus Res 210:248-54,2015.

2)ᮧୖ⿱ಙࠊ㛫㝧Ꮚ: HIV-1 ࢔ࢡࢭࢧ࣮ࣜ⺮ⓑ㉁ Vpr ࡜ᐟ୺ᅉᏊ. ⮫ᗋච␿࣭࢔ࣞࣝ
ࢠ࣮⛉. 63:484-488,2015.

3)

Aida Y, Murakami H, Takahashi M, Takeshima S.: Mechanisms of pathogenesis inducedby bovine leukemia virus (BLV) as a model for human T-cell leukemia virus (HTLV)”
Front. Microbiol 4:328, 2013.

2.Ꮫ఍Ⓨ⾲
1) ᮧୖ⿱ಙࠊ஧㝵ᇽ⣪ᜨࠊబ⸨♩୍㑻ࠊሯᮏ೺ྖ㸸 ∵ⓑ⾑⑓࢘࢖ࣝࢫ G4 㑇ఏᏊḞᦆᰴࡢ࢘࢖ࣝࢫ⏘⏕㔞. ➨ 63 ᅇ᪥ᮏ࢘࢖ࣝࢫᏛ⾡㞟఍ࠊ⚟ᒸࠊ11 ᭶ 2015 ᖺ2) 㕥ᮌ༓⿱ࠊᡞ㧘ᬕ⳯ࠊ஧㝵ᇽ⣪ᜨࠊబ⸨♩୍㑻ࠊሯᮏ೺ྖࠊᮧୖ⿱ಙ: BLV ឤᰁᛶศᏊࢡ࣮ࣟࣥࢆ⏝࠸ࡓ࢘࢖ࣝࢫ⏘⏕㔞࡜ࢤࣀ࣒ኚ␗ࡢゎᯒ. ➨ 63 ᅇ᪥ᮏ࢘࢖ࣝࢫ
Ꮫ⾡㞟఍ࠊ⚟ᒸࠊ11 ᭶ 2015 ᖺ3) ᮧୖ⿱ಙࠊⴗཎᜤ஧ࠊ㕥ᮌ೺⿱ࠊᇽ๓┤ࠊ㛫㝧Ꮚ: ࣉࣟࢸ࣑࢜ࢡࢫἲ࡟ࡼࡾྠᐃࡋ
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REVIEW ARTICLE
published: 08 November 2013
doi: 10.3389/fmicb.2013.00328

Mechanisms of pathogenesis induced by bovine leukemia
virus as a model for humanT-cell leukemia virus
Yoko Aida1 *, Hironobu Murakami 1,2 , Masahiko Takahashi 3 and Shin-Nosuke Takeshima1
1

Viral Infectious Diseases Unit, RIKEN, Wako, Saitama, Japan
Laboratory of Animal Health II, Azabu University, Sagamihara, Kanagawa, Japan
3
Division of Virology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
2

Edited by:
Akio Adachi, The University ofTokushima Graduate School, Japan
Reviewed by:
Toshiki Watanabe, The University ofTokyo, Japan
Takeo Ohsugi, Kumamoto University,
Japan*Correspondence:
Yoko Aida, Viral Infectious Diseases
Unit, RIKEN, 2-1 Hirosawa, Wako,
Saitama 351-0198, Japane-mail: aida@riken.jp

Bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) make up aunique retrovirus family. Both viruses induce chronic lymphoproliferative diseases with
BLV affecting the B-cell lineage and HTLV-1 affecting the T-cell lineage. The pathologies of
BLV- and HTLV-induced infections are notably similar, with an absence of chronic viraemia
and a long latency period. These viruses encode at least two regulatory proteins, namely,
Tax and Rex, in the pX region located between the env gene and the 3 long terminal
repeat. The Tax protein is a key contributor to the oncogenic potential of the virus, and isalso the key protein involved in viral replication. However, BLV infection is not sufﬁcient forleukemogenesis, and additional events such as gene mutations must take place. In thisreview, we ﬁrst summarize the similarities between the two viruses in terms of genomic
organization, virology, and pathology. We then describe the current knowledge of the BLVmodel, which may also be relevant for the understanding of leukemogenesis caused by
HTLV-1. In addition, we address our improved understanding of Tax functions through the
newly identiﬁed BLV Tax mutants, which have a substitution between amino acids 240 and
265.
Keywords: BLV, HTLV-1, EBL, B-cell lymphoma, Tax, leukemogensis, transactivation, apoptosis

INTRODUCTION
Bovine leukosis was ﬁrst reported in 1871 as the presenceof slightly yellow nodules in the enlarged spleen of cattle(Leisering, 1871). Spleen disruption consecutive to tumor formation is one of the most important clinical manifestations ofbovine leukemia. Bovine leukosis is classiﬁed into two types,
sporadic bovine leukosis (SBL) and enzootic bovine leukosis (EBL), which are characterized by T- and B-cell leukosis,
respectively (Gillet et al., 2007). The occurrence of EBL in cattle is much higher than that of SBL (Theilen and Dungworth,
1965; Onuma et al., 1979). Bovine leukemia virus (BLV), which
belongs to the Retroviridae family and Deltaretrovirus genus,
is the etiologic agent of EBL, although it remains unknown
what causes SBL (Gillet et al., 2007). The natural hosts of
BLV are domestic cattle and water buffaloes; however, experimental infection with BLV in sheep can lead to the development of lymphoma (Djilali and Parodi, 1989). Interestingly,
BLV is consistently associated with leukemia only in cattle andsheep, even though it can infect many cell lines (Graves andFerrer, 1976) and can be experimentally transmitted to rabbits (Wyatt et al., 1989; Onuma et al., 1990), rats (Altanerova
et al., 1989), chickens (Altanerova et al., 1990), pigs, goats, andsheep (Mammerickx et al., 1981). Most BLV-infected cattle are
asymptomatic, but approximately one-third of them suffer frompersistent lymphocytosis (PL) characterized by non-malignant
polyclonal B-cell expansion and 1–5% of them develop B-cellleukemia/lymphoma after a long latency period (Gillet et al., 2007).
On the other hand, sheep that are experimentally inoculatedwith BLV develop B-cell tumors at a higher frequency andwith a shorter latency period than those observed in naturally
infected cattle (Ferrer et al., 1978; Burny et al., 1979; Kenyon
et al., 1981; Aida et al., 1989). Interestingly, the transformed
B-lymphocytes in cattle are CD5+ IgM+ B-cells (Aida et al.,1993), whereas in sheep they are CD5− IgM+ B-cells (Murakami
et al., 1994a,b), suggesting that the mechanisms of leukemogenesis induced by BLV may differ (Graves and Ferrer, 1976;
Djilali and Parodi, 1989).
BLV is closely related to human T-cell leukemia virus type 1(HTLV-1), which is the causative agent of adult T-cell leukemia(ATL) and a chronic neurological disorder known as tropicalspastic paraparesis or HTLV-1-associated myelopathy HAM/TSP(Gessain et al., 1985; Osame et al., 1986; Gillet et al., 2007). Therefore, studies on BLV may facilitate our understanding of themechanism of leukemogenesis induced by HTLV-1.

BLV AND HTLV-1
All retroviruses are encoded by gag, pro, pol, and env essential
genes, which are necessary for the production of infectious virions, and are ﬂanked by two identical long terminal repeats (LTRs;
Figure 1). The gag, pro, pol, and env genes encode the internal
structural proteins of the virion, the viral protease, the reverse
transcriptase, and the envelope glycoproteins of the virion, respectively. The genome sequences of BLV and HTLV-1 are different,
but have a unique sequence called the pX situated between the env
gene and the 3 LTR and encoded by the regulatory gene (Figure 1).
The pX sequence is not of host cell origin; that is, it is not an
oncogene. It has been reported that both viruses have an ability toimmortalize primary cells in vitro (Grassmann et al., 1989; Willems

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FIGURE 1 | Schematic representation of genome organization of retroviruses. HTLV-1 and BLV encode unique regulatory and accessory proteins in the pX
region. RSV, Rous sarcoma virus; MuLV, murine leukemia virus.

et al., 1990). Because their structure and properties differ from anyother class of retroviruses, BLV and HTLV-1 viruses were classiﬁed into a new group of retroviruses (Gillet et al., 2007). In bothviruses the regulatory proteins Tax and Rex are encoded in the pX
region. The R3 and G4 proteins are encoded in the BLV pX region,
while p12I , p13II , and p30II are encoded in the HTLV-1 pX region(Sagata et al., 1984b; Franchini et al., 2003; Figure 1). Interestingly,
the HTLV-1 genome codes for HBZ, a unique gene encoded by theminus strand chain (Gaudray et al., 2002; Figure 1). The major
functions of the viral proteins encoded in the BLV and HTLV-1
pX regions are summarized in Table 1. The Tax protein has been
extensively studied, and it is believed to play a critical role in leukemogenesis induced by BLV and HTLV-1 (Katoh et al., 1989; Tanaka
et al., 1990; Willems et al., 1990). The Rex protein is responsible
for nuclear export of viral RNA and promotes cytoplasmic accumulation and translation of viral messenger mRNA in BLV- and
HTLV-1-infected cells (Felber et al., 1989). BLV R3 and G4 proteins contribute to the maintenance of high viral load (Willems
et al., 1994; Florins et al., 2007). The G4 protein is particularly
relevant to leukemogenesis, since it can immortalize primary rat
embryo ﬁbroblasts (REFs; Lefebvre et al., 2002). HTLV-1 p12I issimilar to the R3 protein, in that it contributes to the maintenanceof infectivity (Collins et al., 1998), and both proteins are locatedin the nucleus and cellular membranes (Gillet et al., 2007). On theother hand, HTLV-1 p13II protein resembles the G4 protein, sinceboth proteins bind to farnesyl pyrophosphate synthetase, which

farnesylates Ras (Lefebvre et al., 2002), and the p13II protein promotes Ras-dependent apoptosis (Hiraragi et al., 2005). HTLV-1
p30II protein regulates gene transcription through its interaction with the cAMP responsive element (CRE) binding protein(CREB)/p300 (Zhang et al., 2001). The HBZ protein plays a critical role in the leukemogenesis of HTLV-1, and HBZ knockdown
inhibits the proliferation of ATL cells (Satou et al., 2006). However, since the BLV genome does not code for HBZ, it is assumed
that the Tax protein plays a central role in the leukemogenesis
of BLV.
The infection route of BLV and HTLV is by horizontal andvertical transmission. BLV is transmitted via direct contact (Konoet al., 1983), milk, and insect bites (Ferrer and Piper, 1978), while
HTLV-1 is transmitted via milk and sexual intercourse (Bangham, 2003). Moreover, the artiﬁcial transmission of BLV is caused
by iatrogenic procedures such as dehorning, ear tattooing, andreuse of needles (Hopkins and DiGiacomo, 1997), whereas theartiﬁcial transmission of HTLV-1 is caused by blood transfusion
and needle sharing among drug abusers (Robert-Guroff et al.,1986). Since cell contact is required for the efﬁcient transmissionof both BLV and HTLV-1, cell-free infection by these viruses isbelieved to be very inefﬁcient, most probably due to virion instability (Voneche et al., 1992; Johnston et al., 1996; Igakura et al.,2003).
As shown in Figure 2, an infection with BLV is characterized
by three progressive stages of disease, including an asymptomatic

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Table 1 | Viral proteins are encoded in BLV and HTLV-1 pX regions.
Virus

Viral protein

Major reported functions

Reference

BLV

Tax

Transcriptional activator of viral expression

Derse (1987), Willems et al. (1987), Katoh et al. (1989)

Oncogenic potential

Willems et al. (1990)

Activation of NF-kappa B (NF-κB) pathway

Szynal et al. (2003), Klener et al. (2006)

HTLV-1

Rex

Nuclear export of viral mRNAs

Felber et al. (1989)

G4

The maintenance of high viral load

Willems et al. (1994), Florins et al. (2007)

Oncogenic potential

Kerkhofs et al. (1998), Lefebvre et al. (2002)

R3

The maintenance of high viral load

Willems et al. (1994), Florins et al. (2007)

Tax

Transcriptional activator of viral expression

Kashanchi and Brady (2005)

Oncogenic potential

Matsuoka and Jeang (2011)

Induction of DNA damage, cellular senescence and apoptosis

Chlichlia and Khazaie (2010)

Functional regulation of many cellular proteins by direct binding

Boxus et al. (2008)

Inhibition of HTLV-1 transcription

Lemasson et al. (2007)

Suppression of the classical pathway of NF-κB

Zhao et al. (2009)

Enhancement of TGF-β signaling

Zhao et al. (2011)

HBZ

Oncogenic potential

Satou et al. (2006, 2011)

Rex

Nuclear export of viral mRNAs

Felber et al. (1989)

p12I

Maintenance of viral infectivity

Collins et al. (1998)

p13II

p30II

Activation of nuclear factor of activated T-cells (NFAT) pathway

Ding et al. (2002)

Suppression of viral replication

Andresen et al. (2011)

Interaction with farnesyl pyrophosphate synthetase

Lefebvre et al. (2002)

Activation of Ras-mediated apoptosis

Hiraragi et al. (2005)

Suppression of viral replication

Nicot et al. (2004)

Regulation of gene transcription by binding with p300

Zhang et al. (2001)

Enhancement of Myc transforming potential

Zhang et al. (2001)

stage, PL, and lymphoma. Most BLV-infected cattle are asymptomatic, but approximately one-third of them suffer from PL
characterized by a permanent and relatively stable increase in thenumber of B-lymphocytes in the peripheral blood. PL is considered to be a benign form of the disease resulting from theaccumulation of untransformed B-lymphocytes. Finally, 1–5% of
BLV-infected cattle develop B-lymphoma in various lymph nodes
after a long latency period (Schwartz and Levy, 1994; Florins et al.,2008). Although BLV can also infect CD4+ T-cells, CD8+ T-cells,γ/δ T-cells, monocytes, and granulocytes in cattle (Williams et al.,
1988; Stott et al., 1991; Schwartz et al., 1994; Mirsky et al., 1996; Wu
et al., 1996; Panei et al., 2013), a large number of the tumor cells arederived from CD5+ IgM+ B-cell subpopulations (Schwartz andLevy, 1994). Interestingly, the full-length BLV proviral genome
is maintained in each animal throughout the course of the disease (Tajima et al., 1998a). In addition, previous studies haveshown that both large and small deletions of proviral genomes
may be very rare events in BLV-infected cattle. Thus, the proviralloads were signiﬁcantly increased at the PL stage compared withthe aleukemic stage and were further increased at the lymphoma
stage (Jimba et al., 2010, 2012; Figure 2B). These ﬁndings clearly
demonstrated that the BLV proviral copy number increases withincreasing severity of the disease. On the other hand, unlike BLV,HTLV-1 is associated with ATL and with the chronic neurologicaldisorder, HAM/TSP, and induces not only a malignant tumor but
also an inﬂammatory disease (Gessain et al., 1985; Osame et al.,1986). Although the pathogenesis of HTLV-1 is slightly different
from BLV, HTLV-1, like BLV, can infect many cells in addition to
T-cells, including B-cells and monocytes (Koyanagi et al., 1993;
Schwartz and Levy, 1994). In contrast to BLV, defective HTLV-1
proviral genomes have been found in more than half of all examined patients with ATL (Konishi et al., 1984; Korber et al., 1991;
Ohshima et al., 1991; Tsukasaki et al., 1997).

MECHANISM OF LEUKEMOGENESIS BY BLV
Animal retroviruses, which belong to the Alpharetrovirus andGammaretrovirus genera, induce tumors by one of two mechanisms: either by activation of the “viral oncogene” or by “insertional activation” of a cellular gene such as a proto-oncogene(Weiss et al., 1985; Figure 1). By contrast, BLV lacks a known oncogene (Sagata et al., 1984a,b) and does not integrate into preferredsites in their host cell genomes, which related to the disruption of
the host gene but not to the suppression of viral gene expression(Murakami et al., 2011b).
Most studies of BLV-induced leukemogenesis have focused on
the Tax protein because it is believed to be a potent transcriptional

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FIGURE 2 | BLV-induced leukemogenesis is a multistep process. (A) An
infection with BLV is characterized by three progressive stages of disease:
asymptomatic stage, persistent lymphocytosis (PL), and lymphoma. BLV
infects to cells non-speciﬁcally. Among them, BLV Tax protein immortalizes apart of BLV-infected cells, probably only CD5+ IgM+ B-cells, and induces

polyclonal proliferation of the cells. However, the Tax protein does not havethe ability to transform the cells. For lymphoma to develop, a malignant
transformation needs to occur with the help of host factors, such as p53
mutation, TNF-α activities or bovine leukocyte antigen (BoLA) class II
phosphorylation. (B) The provirus load increases with disease progression.

activator of viral gene expression. In addition to its function as atranscriptional activator, Tax induces immortalization of primary
REFs (Willems et al., 1990, 1998). Furthermore, Tax cooperates
with the Harvey rat sarcoma viral oncoprotein (Ha-ras) for the
induction of full transformation of primary REF (Willems et al.,1990). Importantly, the Tax transformed cells induce tumors innude mice. The ability of the Tax protein to induce immortalization may be the ﬁrst step in the BLV-mediated transformation
process. Moreover, after the infection of cattle and during thelatency period, the expression of BLV becomes blocked at thetranscriptional level (Kettmann et al., 1982; Lagarias and Radke,1989). Such repression appears to be very important for the escapeof BLV from the host’s immunosurveillance system, and later onlya certain small proportion of infected animals rapidly develop aterminal disease (Gillet et al., 2007). Indeed, transcription of theBLV genome in fresh tumor cells or in fresh peripheral bloodmononuclear cells (PBMCs) from infected individuals is almost
undetectable by conventional techniques (Kettmann et al., 1982;
Tajima et al., 2003b; Tajima and Aida, 2005). In situ hybridization
has revealed the expression of viral RNA at low levels in manycells, and at a high level in only a few cells within PBMCs freshly
isolated from BLV-infected asymptomatic animals (Lagarias andRadke, 1989). Thus, BLV infection is probably not sufﬁcient forleukemogenesis and some additional events such as gene mutations might be involved in the leukemogenic process (Figure 2A).
Taken together, Tax may induce immortalization of only CD5+
IgM+ B-cells among BLV-infected B-cells, CD4+ T-cells, CD8+
T-cells, γ/δ T-cells, monocytes, and granulocytes in cattle, thereby
conferring a selective transformation advantage to the infected
CD5+ IgM+ B-cells by a second event.

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A mutation in the p53 tumor suppressor gene is one of several genetic changes known to be involved in the developmentof lymphoma (Figure 2A). The protein encoded by the p53
tumor suppressor gene plays a critical role in transducing a signal from the damaged DNA to genes that control cell cycle andapoptosis. Approximately half of the solid tumors induced by
BLV in cattle (Dequiedt et al., 1995; Ishiguro et al., 1997; Zhuang
et al., 1997; Tajima et al., 1998b) and three of four bovine B-cell
lymphoma lines (Komori et al., 1996) were shown to harbor missense mutations in p53. By contrast, very few mutations were
found in B-cells from cows with PL and none of the uninfected
cattle harbored a mutated p53 gene. These observations indicate that p53 mutations frequently occur at the ﬁnal stage oflymphoma in cattle. A previous study of the molecular mechanism of mutations at codons 206, 207, 241, and 242, whichwere identiﬁed in lymphoma, showed that these mutations may
potentially alter the wild-type function of the bovine p53 protein, including the conformation and transactivator and growth
suppressor activities, and then cause lymphoma (Tajima et al.,1998b). These four mutations were clearly divided into two functionally distinct groups: (i) the mutant forms with substitutions
at codons 241 and 242, which were mapped within an evolutionarily conserved region and corresponded to the human“hot-spot” mutations, and had completely lost the capacity fortransactivation and growth suppression while gaining transdominant repression activity in p53-null SAOS-2 cells; and (ii) the
mutations at codons 206 and 207, which were located outside
the evolutionarily conserved regions and partially retained thecapacity for transactivation and growth suppression. Collectively,
these naturally occurring mutations may potentially alter thewild-type function, and in addition, out of the four missense
mutations, at least two mutations may be sufﬁcient to cause
lymphoma. However, since the other two mutations may beinsufﬁcient to induce lymphoma, it is possible that other cancerrelated genes may contribute to lymphoma in concert with the p53
mutations.
A major factor involved in the clinical progression of
BLV-infected animals is the bovine leukocyte antigen (BoLA;
Figure 2A), which plays a crucial role in determining immune
responsiveness (Lewin and Bernoco, 1986; Lewin et al., 1988;
Zanotti et al., 1996; Takeshima and Aida, 2006). Several studies have shown that genetic variations in BoLA-DRB3, which isa functionally important and the most polymorphic BoLA class
II locus in cattle, inﬂuence resistance and susceptibility to a wide
variety of infectious diseases, including lymphoma (Aida, 2001)
and PL (Xu et al., 1993; Sulimova et al., 1995; Starkenburg et al.,
1997; Juliarena et al., 2008), and affect BLV proviral load (Miyasaka
et al., 2013). For example, the presence of the amino acids Glu–
Arg (ER) at positions 70–71 of the BoLA-DRβ chain was associatedwith resistance to PL in BLV-infected cattle (Xu et al., 1993). Furthermore, the BoLA-DRB3 alleles encoding Glu, Arg, and Val at
positions 74, 77, and 78, respectively, of the BoLA-DRβ chain
might be associated with resistance to tumor development (Aida,
2001). In a related study, Nagaoka et al. (1999) and Konnai et al.
(2003) found that the ovine leukocyte antigen (OLA)-DRB1 alleles
encoding the Arg–Lys (RK) and the Ser–Arg (SR) motifs at positions 70–71 of the OLA-DRβ chain are associated with resistance(RK motif) and susceptibility (SR motif) to the development oflymphoma after experimental infection of sheep with BLV. The
sheep with alleles encoding the RK motif produced neutralizing
antibodies against BLV and interferon-γ, eliminated BLV completely, and did not develop lymphoma (Konnai et al., 2003).
The susceptibility to the monoclonal expansion of BLV-infected
B-lymphocytes is thus associated with speciﬁc alleles of BoLA
system.
A polymorphism in the promoter region of the tumor necrosis factor (TNF)-α gene is one of several genetic changes involvedin the development of lymphoma (Figure 2A). A previous study
found that, in sheep experimentally infected with BLV, the frequency of the TNF-α-824G allele, which has been associated with
low transcription activity of the promoter/predicted enhancer
region of the bovine TNF-α gene, was higher in animals withlymphoma than in asymptomatic carrier animals. In addition, atendency was observed for increased BLV-provirus load in cattle homozygous for the TNF-α-824G/G allele compared to cattle
homozygous for the TNF-α-824A/A or TNF-α-824A/G alleles.
These data suggest that the observed polymorphism in the promoter region of the TNF-α gene could at least in part contributeto the progression of lymphoma in BLV infection (Konnai et al.,2006).
The BLV studies have also focused on understanding theprocess of signal transduction such as B-cell receptor (BCR)
signaling (Alber et al., 1993), since many signal transduction
factors have been implicated in leukemogenesis of B-cells inhumans (Murakami et al., 2011a). For example, the immunoreceptor tyrosine-based activation (ITAM) motifs present in thetransmembrane gp30 proteins of the BLV envelope are important for the incorporation of envelope proteins into the virion(Inabe et al., 1999) and are required for infectivity in vivo
(Willems et al., 1995). In addition to the viral signaling motif,
the spleen tyrosine kinase (Syk) mRNA expression was significantly increased in PL samples, whereas it was decreased intumor samples, suggesting that Syk mRNA expression dynamics is closely related to the progression of BLV-induced disease(Murakami et al., 2011a).

BLV Tax FUNCTION
As mentioned above, the Tax gene is a key contributor to the
oncogenic potential, as well as a key protein involved in the replication of the virus. Table 1 summarizes the functions of the Tax
protein. The Tax open reading frame is mainly encoded in thepX region, and its translation occurs upstream of the pol stopcodon. The Tax protein is modiﬁed by phosphorylation of two
serine residues and is detected as a 34–38 kDa product (Chenet al., 1989; Willems et al., 1998). In addition, the Tax protein has
T- and B-cell epitopes corresponding to regions 110–130/131–
150 and 261–280, respectively (Sakakibara et al., 1998). One of
the best characterized functions of Tax is the activation of viraltranscription. The Tax protein acts on a triplicate 21 bp enhancer
motif known as the Tax-responsive element (TxRE) in the U3
region of the 5 LTR, and it stimulates transactivation of the viralgenome (Derse, 1987; Willems et al., 1987; Katoh et al., 1989).
The TxRE consists of a cyclic AMP-response element (CRE)-likesequence, and it has been suggested that Tax binds to this element

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indirectly through cellular factors, such as the members of the
CREB/activating transcription factor (ATF) family of basic leucine
zipper proteins that have been shown to bind to the CRE-likesequence (Adam et al., 1994, 1996; Boros et al., 1995). Furthermore, the Tax protein modulates the expression of cellular genesthat are involved in the regulation of cell growth (Tajima and Aida,
2002). In addition to its function in the regulation of cellularand viral transcription, the Tax protein can induce immortalization of primary REF and cooperates with Ha-Ras oncogene
to fully transform the primary cells (Willems et al., 1990). On
the other hand, the transactivation and transformation of Tax
may be independently induced by each mechanism, since phosphorylation of Tax is required for its transformation but notfor its activation (Willems et al., 1998). Moreover, the expression of Tax in primary ovine B-cells, which depends on CD154
and interleukin-4, affects B-cell proliferation, cell cycle phase distribution, and survival, leading to cytokine-independent growth(Szynal et al., 2003). This immortalization process is also associated with increased B cell leukemia/lymphoma 2 (Bcl-2) protein
levels, nuclear factor kappa B (NF-κB) accumulation, and a series
of intracellular pathways that remain to be characterized (Kleneret al., 2006). In addition, Tax inhibits base-excision DNA repair ofoxidative damage, thereby potentially increasing the accumulationof ambient mutations in cellular DNA (Philpott and Buehring,1999).

FIGURE 3 | Schematic representations of BLV Tax protein mutants andfunction. (A) Missense mutations between amino acids 240 and 265
containing D247G and S240P inﬂuence the transactivation activity of the BLVNEGATIVE REGULATION OF BLV Tax BY THE REGIONBETWEEN RESIDUES 240–265
Our studies (Tajima and Aida, 2000) demonstrated new functionsof the region between amino acids 240 and 265 of BLV Tax. As
shown in Figure 3, a series of mutants with at least one amino acidsubstitution between amino acids 240 and 265 of BLV Tax wereidentiﬁed, including TaxD247G and TaxS240P, which exhibit anenhanced ability to stimulate and reduce viral LTR-directed transcription respectively, compared to the wild-type protein (Tajima
and Aida, 2000). Transient expression analysis revealed that the
TaxD247G mutant increased the production of viral protein andparticles from a defective recombinant proviral BLV clone to agreater extent than the wild-type Tax (TaxWT). Conversely, theTaxS240P mutant was unable to induce the release of viral particles. The microarray data in human HeLa cells and its validationof differentially expressed genes at the RNA and protein levels inbovine 23CLN cells revealed several alterations in genes involved inmany cellular functions such as transcription, signal transduction,
cell growth, apoptosis, and the immune response (Arainga et al.,2012). In both of human HeLa cells and bovine 23CLN cells, theTaxD247G mutant induced higher gene expression compared with
TaxWT and TaxS240P and many of these genes were expressed at
the lowest level in the TaxS240P-transfected cells. In particular, ourresults showed that Tax activates the proteins which are involvedin activator protein 1 (AP-1) signaling pathway [FBJ osteosarcoma

Tax protein. A putative zinc ﬁnger structure (amino acids 30–53) and aleucine-rich activation domain (amino acids 157–197) are also indicated.
(B) Multiple functions of the region between amino acids 240 and 265.

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BLV-induced leukemogenesis

oncogene (FOS), jun proto-oncogene (JUN), etc.] via interactions
with other transcriptional pathways (G-protein, GTP-binding
proteins, etc.). Likewise, the TaxD247G mutant induced apoptosisin transfected cells more effectively than the TaxWT (Takahashi
et al., 2005). These results suggest that the region between aminoacids 240 and 265 of the Tax protein might act as a negative regulatory domain, and missense mutations in this region might leadto enhanced transactivation activity of Tax, expression of manycellular genes and induction of apoptosis. Our results raise the possibility that the target sequence speciﬁcity of retroviral enhancersof Tax might be limited by this region because TaxD247G, but not
TaxS240P, was found to activate other retroviral enhancers suchas HTLV-1, HIV-1, and mouse mammary tumor virus (MMTV)
and Moloney murine leukemia virus (M-MuLV), and c-fos, which
are not activated by TaxWT (Tajima and Aida, 2000; Figure 3B).
The microarray data also raised the possibility that BLV Tax regulates the innate immune response (Figure 3B): the largest groupof downregulated genes was related to the immune response,
and the majority of these genes belonged to the interferon family of antiviral factors, such as interferon-induced protein withtetratricopeptide repeats 1 (IFIT1; Arainga et al., 2012). Interferons are major components of the innate immune system, andare recognized for their antiviral function in addition to theirantiproliferative and immunomodulatory effects on cells (Huet al., 1993). It is likely that BLV Tax downregulates the innate
immune response, thereby increasing the production of viralprotein.
An infectious molecular clone of BLV encoding the TaxD247G
was examined for the viral expression and propagation, as well
as for the induction of apoptosis in a sheep model (Tajima et al.,2003a; Takahashi et al., 2004, 2005). Interestingly, the infectious
molecular clone of BLV encoding the TaxD247G produced moreviral particles and was transmitted at an elevated rate in vitro,
but with no signiﬁcant differences in the proviral load and theexpression of viral RNA between sheep experimentally injectedwith BLVs encoding the TaxWT or the mutant TaxD247G proteins (Tajima et al., 2003a). These ﬁndings suggest the presence ofa dominant host defense mechanism regulating BLV–LTR-directedtranscription by Tax that may play an important role in viralsilencing in vivo (Figure 4). Likewise, although the transient

FIGURE 4 | Proposed mechanism for the regulation of BLV expression
and apoptosis induction in TaxD247G-encoded BLV-infected sheep.

expression of TaxD247G induced apoptosis in transfected cells
in vitro more effectively than TaxWT, higher level of protection against apoptosis was observed in PBMCs isolated fromsheep infected with TaxD247G-encoded BLV compared to TaxWTencoded BLV (Takahashi et al., 2005; Figure 4). These ﬁndings
demonstrate that TaxD247G has an increased potential to induce
apoptosis, which could be beneﬁcial for BLV propagation like otherviruses (Wurzer et al., 2003; Richard and Tulasne, 2012). One possible explanation for our results might be that TaxD247G-induced
apoptosis is modulated by a dominant mechanism ex vivo, so thefunction might be suppressed.

CONCLUSION
BLV is the etiologic agent of EBL, which is the most common
neoplastic disease in cattle. It infects cattle worldwide, thereby
imposing a severe economic burden on the dairy cattle industry.
In this review, we evaluated existing information on the mechanism of BLV-induced leukemogenesis. We propose that, since
BLV Tax induces immortalization of only CD5+ IgM+ B-cells
within BLV-infected B-cells, CD4+ T-cells, CD8+ T-cells, γ/δ
T-cells, monocytes, and granulocytes in cattle, it may confer aselective transformation advantage to the infected CD5+ IgM+
B-cells by a second event, such as p53 mutation, polymorphismsof BoLA, or the promoter region of the TNF-α gene. We also propose new functions of the region between amino acids 240 and
265 of BLV Tax (Figure 3). Namely, the transactivation activity
and target sequence speciﬁcity of BLV Tax might be limited or
negatively regulated by this region. The most interesting point
regarding the ability of TaxD247G to enhance BLV expression andapoptotic induction in vitro is that it might be suppressed in vivo
or ex vivo. Thus, we hypothesize that there could be dominantmechanisms controlling the functions of TaxD247G ex vivo and invivo, as shown in Figure 4. For HTLV-1, it has been reported
that CD8+ cell-mediated cytotoxic T-lymphocytes (CTLs) target Tax-expressing cells, thereby reducing the number of infected
cells (Hanon et al., 2000). Likewise, BLV-infected cells expressing
Tax may be exposed to the host defense system, and BLV may
evolve in a manner that promotes the shielding of their potential
abilities. Therefore, a strong transactivation activity of BLV Tax
might not be advantageous for the propagation of BLV in vivo.
Taken together, the ﬁndings discussed in this review suggest that
there might be a dominant mechanism involved in the inductionof apoptosis and expression of HTLV-1 in vivo. To address our
hypothesis, it seems necessary to evaluate whether possible host
responses against BLV infection, such as the induction of CTLs,
genetic, and epigenetic alterations in apoptosis-regulatory genes,
and DNA and chromatin modiﬁcations of BLV promoter for the
suppression of viral expression, could be enhanced in animals
infected with TaxD247G-encoded BLV. Thus, future investigationsof the relationship between apoptosis and viral expression using
BLV containing the mutant D247G Tax as a model will broaden
our understanding of the replication and propagation of HTLV-1,
and leukemia progression.
ACKNOWLEDGMENTS
The studies on BLV were supported by Grants-in-Aid for ScientiﬁcResearch (A, B, and C) from the Japan Society for the Promotion ofNovember 2013 | Volume 4 | Article 328 | 7

www.frontiersin.org

638

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BLV-induced leukemogenesis

Science (JSPS), and by a grant from the Program for the Promotionof Basic and Applied Research for Innovations in Bio-oriented
Industry.

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Conflict of Interest Statement: The authors declare that the research was conductedin the absence of any commercial or ﬁnancial relationships that could be construed
as a potential conﬂict of interest.

Received: 30 July 2013; accepted: 17 October 2013; published online: 08 November
2013.
Citation: Aida Y, Murakami H, Takahashi M and Takeshima S-N (2013)
Mechanisms of pathogenesis induced by bovine leukemia virus as a model forhuman T-cell leukemia virus. Front. Microbiol. 4:328. doi: 10.3389/fmicb.2013.
00328
This article was submitted to Virology, a section of the journal Frontiers in Microbiology.
Copyright © 2013 Aida, Murakami, Takahashi and Takeshima. This is an openaccess article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with theseterms.

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ࢡࢫࣇࣥࢺ㸦 ಶయ㸧ࠊࣅ࣮ࢢࣝ㸦 ಶయ㸧ࠊ࣏࣓ࣛࢽ࢔ࣥ  ಶయ ࠊࢩ࣮ࢬ࣮㸦 ಶయ㸧ࠊࢩ࣋ࣜ࢔ࣥࣁࢫ࣮࢟㸦 ಶయ㸧ࠊᰘ≟㸦 ಶయ㸧ࠊ⛅⏣≟㸦 ಶయ㸧ࠊ⏥ᩫ≟㸦 ಶయ㸧ࡢ ≟✀  ಶయࢆ⏝࠸ࡓࠋ࢖ࢾ  ␒ᰁⰍయࡢࣜࣇ࢓ࣞࣥࢫࢤࣀ࣒ 1&B ࡟࠾࠸
࡚ࠊJ ࠿ࡽ J ࡲ࡛ࡢ⣙ NE ࡢ㓄ิࢆᑐ㇟࡟ࠊ⾲㸯࡟♧ࡋࡓ  ⤌ࡢࣉࣛ࢖࣐࣮ࢭࢵࢺࢆ⏝࠸࡚ ,/ 㑇ఏᏊࡢ '1$ ᩿∦ࢆቑᖜࡋࡓࠋ
ࡇࡢ༊㛫ࡣࠊ
࢖ࢾ ,/
㑇ఏᏊࡢ඲࡚ࡢ࢚ࢡࢯࣥ࡜࢖ࣥࢺࣟࣥ࠾ࡼࡧࠊ᥎ᐃ㌿෗㛤ጞⅬ࠿ࡽ vഃ࡟ NE ୖὶ࡜೵Ṇࢥࢻࣥ࠿ࡽ vഃୗὶ 㹩㹠ࢆྵࢇ࡛࠸ࡿࠋࡇࢀࡽࡢ '1$ ᩿∦ࢆ⢭〇ࡋࡓᚋࠊ%LJ'\H7HUPLQDWRUY&\FOH6HTXHQFLQJ.LW ࠾ࡼࡧ $%,ࢩ࣮ࢡ࢚ࣥࢧ࣮ 7KHUPR)LVKHU6FLHQWLILF:DOWKDP0DVVDFKXVHWWV86$ ࢆ⏝࠸࡚ሷᇶ㓄ิࢆỴᐃࡋࡓࠋᚓ
ࡽࢀࡓ㓄ิࡣࠊࣜࣇ࢓ࣞࣥࢫࢤࣀ࣒㓄ิ㸦≟✀ࡣ࣎ࢡࢧ࣮㸧࡜┦஫࡟ẚ㍑ࡋከᆺࢆ᳨ฟ
ࡋࡓࠋ⤖ᯝ࠾ࡼࡧ⪃ᐹ ಶయࡢ㓄ิ࠾ࡼࡧࣜࣇ࢓ࣞࣥࢫࢤࣀ࣒㓄ิࢆẚ㍑ࡋࡓ⤖ᯝࠊ⾲㸰࡟♧ࡋࡓ  ࠿ᡤࡢ ሷᇶከᆺ㸦613㸧࡜ࠊ$**7***&$ ࡢ  ሷᇶࢆ༢఩࡜ࡍࡿ⦪ิ཯᚟ከᆺࡀ  ࠿ᡤ࡛ぢࡘ࠿ࡗࡓࠋࡇࡢ࠺ࡕ  ࡘࡢከᆺࡣࠊGE613 ࡟Ⓩ㘓ࡉࢀ࡚࠸࡞࠸᪂つࡢࡶࡢ࡛࠶ࡗࡓࠋࡇࢀࡽ
ࡢከᆺࡢ୰࡛ࠊUV ࡣࠊ,/ ๓㥑యࢱࣥࣃࢡ㉁ࡢ  ␒┠ࡢ࢔࣑ࣀ㓟ࢆࢺࣞ࢜ࢽࣥ࠿ࡽ࢔ࣛࢽࣥ࡟ኚ᭦ࡍࡿ㠀ྠ⩏⨨᥮࡛࠶ࡗࡓࠋࡇࡢ఩⨨ࡣࠊ ಶࡢ࢔࣑ࣀ㓟࠿ࡽ࡞ࡿࢩࢢࢼࣝ࣌ࣉࢳࢻࢆ㝖ཤࡋࡓ ,/ ࡛ࡣࠊ ␒┠ࡢṧᇶ࡟఩⨨࡙ࡅࡽࢀࡿࠋࡇࡢ
࢔࣑ࣀ㓟⨨᥮㸦௨ᚋ ,/ S7KU!$OD ࡜⾲グ㸧ࡢ㐍໬Ꮫⓗ఩⨨࡙ࡅࢆ⾜࠺ࡓࡵ࡟ࠊ
࣐࢘ࠊࣄࢺࠊࢳࣥࣃࣥࢪ࣮ࠊ࢝ࢽࢡ࢖ࢨࣝࠊ࢘ࢩࠊࣈࢱࠊࣛࢵࢺ࠾ࡼࡧ࣐࢘ࢫࡢ ,/ࡢ࢔࣑ࣀ㓟㓄ิ࡜ẚ㍑ࡋࡓ㸦ᅗ㸯㸧
ࠋS7KU!$OD ከᆺࡣࠊ,/ ࡢᵓ㐀ୖ㔜せ࡞Ș࡬ࣜࢵࢡࢫ & ࡢ୰ኸ࡟఩⨨ࡍࡿ஦ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡲࡓࠊẚ㍑࡟⏝࠸ࡓ඲࡚ࡢ✀࡟࠾࠸࡚ࢺࣞ࢜ࢽࣥ࡟ᅛᐃࡉࢀ࡚࠸ࡓࠋࡺ࠼࡟ࠊ,/ ␒┠ࡢ࢔࣑ࣀ㓟ṧᇶࡀࢺࣞ࢜ࢽ࡛ࣥ࠶
ࡿ஦ࡀ ,/ ࡢᶵ⬟࡟㔜せ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋࡉࡽ࡟ࠊ3RO\SKHQ
㸦KWWSJHQHWLFVEZKKDUYDUGHGXSSK㸧ࢆ⏝࠸ࡓࢩ࣑࣮ࣗࣞࢩࣙࣥ࠿ࡽ S
7KU!$OD ከᆺࡣࠊࢱࣥࣃࢡ㉁ࡢ㧗ḟᵓ㐀࡟኱ࡁ࡞ࢲ࣓࣮ࢪࢆ୚࠼ࡿࡇ࡜ࡀண

644

ࡉࢀࡓࠋ

௨ୖࡢ஦࠿ࡽࠊUV ከᆺࡣࠊ,/ ࡢᶵ⬟࡟኱ࡁ࡞ኚ᭦ࢆຍ࠼ࡿ஦ࡀᙉࡃ♧၀ࡉࢀࡓࠋࡺ࠼࡟ࡇࡢከᆺࡣࠊ࢖ࢾ࡟࠾࠸࡚ ,/ ࢩࢢࢼࣝࡢ␗ᖖࡀ㛵୚ࡍࡿ⑌ᝈ࡟ᑐࡍࡿ㔜せ࡞ೃ⿵ኚ␗࡛࠶ࡿ஦ࡀ♧ࡉࢀࡓࠋࡲࡓࠊᮏ◊✲࡛Ⓨぢࡋࡓ ,/ 㑇ఏᏊࡢ v㞄᥋㡿ᇦ࡟࠾ࡅࡿ⦪ิ཯᚟ከᆺࡢ୰࡟ࡣࠊ1)$7 ࡸ 63 ࢆྵࡴ」ᩘࡢ㌿෗ᅉᏊ࡜₯ᅾⓗ࡟⤖ྜࡍࡿࣔࢳ࣮ࣇࡀྵࡲࢀ࡚࠸ࡓࠋࣉ࣮ࣟࣔࢱෆ࡛ࡢ཯᚟㓄ิከᆺࡀࠊ㑇ఏᏊࡢ㌿෗άᛶ࡟ᙳ㡪ࢆཬࡰࡍ౛ࡀከࡃ▱ࡽࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊv㞄᥋㡿ᇦෆࡢከᆺࡶྠ᫬࡟ㄪᰝࡍࡿᚲせࡀ࠶ࡿࠋ

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645

ᅗ㸯,/ ࡢ࢔࣑ࣀ㓟㓄ิࡢẚ㍑㸬࢔࣑ࣀ㓟ṧᇶࡢ࣏ࢪࢪࣙࣥࡣࢩࢢࢼࣝ࣌ࣉࢳࢻࡀ㝖ཤࡉࢀ࡚࠸ࡿࠋ
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㸰㸬≟ ,/ 㑇ఏᏊୖࡢ㑇ఏᏊከᆺࡢ㢖ᗘㄪᰝ◊✲せ᪨
࢖ࢾ ,/ 㑇ఏᏊ࡟࠾࠸࡚ࠊ㌿෗ㄪᩚ࡟ᙳ㡪ࢆ୚࠼ࡿྍ⬟ᛶࡢ࠶ࡿ v㞄᥋㡿ᇦ࡟Ꮡᅾࡍࡿ㹥$**7***&$㸸㹙㹛!㹙㹛
ࠊJ$!&ࠊUV ࠾ࡼࡧࠊUVࡢ  ࢝ᡤ࡜ ,/ ࡢ㠀ྠ⩏⨨᥮ UV ࡢᑐ❧㑇ఏ㢖ᗘࢆࠊ ≟✀ࢆᑐ㇟࡟ㄪᰝࡋ

ࡓࠋㄪᰝ⤖ᯝࡣࠊ⾲㸰࡟♧ࡋࡓࠋ㸳ࡘࡢᗙ఩ࡣࠊ ≟✀࠿ࡽ  ≟✀࡟࠾࠸࡚ᑐ❧㑇ఏᏊࡢከᆺࡀㄆࡵࡽࢀࡓࠋ୍㒊ࡢရ✀࡛࢔ࣞࣝࡢᅛᐃࡀㄆࡵࡽࢀࡓࡶࡢࡢࠊᮏ◊✲ࡢᑐ㇟࡜ࡋࡓ㸳ࡘࡢᗙ఩ࡣࠊ࠸ࡎࢀࡶᗈ⠊ᅖࡢ≟✀࡛ᑐ❧㑇ఏᏊࡢከᵝᛶࡀᏑᅾࡋࡓࡇ࡜࠿ࡽࠊᐙ
␆≟࡟ᗈࡃศᕸࡍࡿࢥࣔࣥከᆺ࡛࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ
5 ࡘࡢᗙ఩ࡢ࠺ࡕ J
$!& ࢆ㝖ࡃ㸲ࡘࡢከᆺ㒊఩࡛ࡣࠊရ✀㛫࡛ᑐ❧㑇ఏᏊ㢖ᗘ࡟኱ࡁ࡞ᕪࡀㄆࡵࡽࢀࠊ࣓ࢪ࣮ࣕ
࢔ࣞࣝ࡜࣐࢖ࢼ࣮࢔ࣞࣝࡢ㛵ಀࡀ㏫㌿ࡍࡿ⌧㇟ࡶㄆࡵࡽࢀࡓࠋ20 ≟✀࡟࠾ࡅࡿᑐ❧㑇ఏ㢖ᗘࡢഴྥࢆ᫂ࡽ࠿࡟ࡋࡓࡇ࡜࡛ࠊ࢖ࢾ IL-13 㑇ఏᏊከᆺࢆᑐ㇟࡜ࡍࡿ┦㛵ゎᯒࢆᐇ᪋ࡍࡿ
ࡓࡵࡢᇶ♏᝟ሗࢆᚓࡿࡇ࡜ࡀ࡛ࡁࡓࠋ◊✲┠ⓗ๓㡯ࡢ◊✲࡛࢖ࢾ ,/ 㑇ఏᏊ࡟࠾࠸࡚  ࢝ᡤࡢከᆺࢆ᳨ฟࡋࡓࠋࡇࡢ୰࡛ࠊv㞄᥋㡿ᇦ࡟Ꮡᅾࡍࡿ㹥$**7***&$㸸㹙㹛!㹙㹛ࠊJ$!&ࠊUV ࠾ࡼࡧࠊUV ࡢ  ࢝ᡤࡢከᆺࡣࠊ,/ ࡢ㌿෗ㄪ⠇࡟㛵୚ࡍࡿྍ⬟ᛶࡢ࠶ࡿ」ᩘࡢ㌿෗ᅉᏊࡢ⤖ྜࣔࢳ࣮ࣇࢆྵࢇ࡛࠸ࡓࠋࡲࡓࠊUV ࡣࠊ,/ ࡢᵓ㐀ୖ㔜せ࡜⪃࠼ࡽࢀࡿ఩

⨨࡛ࡢ࢔࣑ࣀ㓟㓄ิࢆ⏕ࡌࡉࡏࡿࠋࡺ࠼࡟ࠊ,/ ࡀᐤ୚ࡍࡿ⑌ᝈ࡜ࡢ┦㛵ࡍࡿྍ⬟ᛶࡀ࠶ࡿࠋࡋ࠿ࡋࠊࡇࢀࡽࡢከᆺ࡟࠾ࡅࡿᑐ❧㑇ఏᏊ㢖ᗘ࡟㛵ࡍࡿ᝟ሗࡣࠊࡇࢀࡲ࡛඲ࡃ
▱ࡽࢀ࡚࠸࡞࠸ࠋ୍⯡࡟ࠊ࢖ࢾࡢ⦾Ṫࡣရ✀ࡈ࡜࡟⊂❧ࡋ࡚⾜ࢃࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊ㑇ఏᏊᾋືࡢᙳ㡪ࢆཷࡅࡸࡍࡃࠊရ✀ࡈ࡜࡟ᑐ❧㑇ఏᏊ㢖ᗘࡀ኱ࡁࡃ␗࡞ࡿ஦ࡀண᝿ࡉࢀ

646

ࡿࠋᮏ◊✲࡛ࡣࠊ,/ 㑇ఏከᆺ࡜⑌ᝈ࡜ࡢ㛵㐃ࢆㄪᰝࡍࡿ๓ẁ㝵࡜ࡋ࡚ࠊ≟✀ูࡢᑐ❧㑇ఏᏊ㢖ᗘࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ
ᮦᩱ࡜᪉ἲࢦ࣮ࣝࢹࣥࣞࢺ࣮ࣜࣂ࣮ࠊࣛࣈࣛࢻ࣮ࣝࣞࢺ࣮ࣜࣂ࣮ࠊࣅ࣮ࢢࣝࠊࢩ࢙ࢵࢺࣛࣥࢻࢩ࣮ࣉ
ࢻࢵࢢࠊ࣐ࣝࢳ࣮ࢬࠊ࣭ࢥ࣮ࢠ࣮࣮ࣚࢡࢩ࣮ࣕࢸࣜ࢔ࠊࢺ࢖ࣉ࣮ࢻࣝࠊ࣑ࢽࢳࣗ࢔ࢲࢵࢡࢫ
ࣇࣥࢺࠊࢳ࣡࣡ࠊࢪ࣮࣐ࣕࣥࢩ࢙ࣃ࣮ࢻࠊࢩ࣮ࢬ࣮ࠊ࣏࣓ࣛࣜ࢔ࣥࠊࣃࣆࣚࣥࠊ࢙࢘ࣝࢵ
ࢩ࣭ࣗࢥ࣮ࢠ࣮ࠊࣃࢢࠊ࣑ࢽࢳࣗ࢔ࣆࣥࢩ࣮ࣕࠊࢩ࣋ࣜ࢔ࣥࣁࢫ࣮࢟ࠊᰘ≟ࠊ⏥ᩫ≟࠾ࡼࡧ⛅⏣≟ࡢ 20 ≟✀࡟࠾࠸࡚ࠊぢ࠿ࡅୖ೺ᗣ࡞ᡂ≟ࢆ㑅ᢥࡋࠊ1 ≟✀࠶ࡓࡾ 8 ࠿ࡽ 35 ಶయ
ࡢ DNA ヨᩱࢆゎᯒࡋࡓࠋ㹥20957075 AGGTGGGCA㸸
㹙1㹛>㹙2㹛ࠊg.20957831 A>Cࠊ
rs8973298ࠊrs8973300 ࠾ࡼࡧࠊrs22147008 ࡢከᆺ㒊఩ࢆྵࡴ DNA ᩿∦ࢆ⾲㸯࡟♧ࡋࡓ
5 ⤌ࡢࣉࣛ࢖࣐࣮ࢭࢵࢺࢆ⏝࠸࡚ PCR ቑᖜࡋࡓࠋ
ࡇࡢ࠺ࡕ㹥20957075 AGGTGGGCA㸸
㹙1㹛>㹙2㹛ከᆺࡣࠊPCR ⏘≀ࢆ࢔࣮࢞ࣟࢫࢤࣝ㟁ẼὋືࡍࡿ஦࡛ࠊቑᖜ᩿∦㛗ከᆺ࡜ࡋ࡚㑇ఏᏊᆺࢆุᐃࡋࡓࠋṧࡾࡢ 4 ࢝ᡤ࡟ SNP ࡟ᑐࡋ࡚ࡣࠊ⾲㸯࡟♧ࡋࡓไ㝈㓝⣲ࢆ⏝࠸࡚ DNA᩿∦ࢆฎ⌮ࡋࡓᚋ࡟ࠊ࢔࣮࢞ࣟࢫࢤࣝ㟁ẼὋືࢆ⾜࠸ࠊไ㝈㓝⣲ษ᩿᩿∦㛗ከᆺ࡜ࡋ࡚㑇ఏᏊᆺࢆุᐃࡋࡓࠋ
⤖ᯝ࠾ࡼࡧ⪃ᐹ࢖ࢾ IL-13 㑇ఏᏊ࡟Ꮡᅾࡍࡿ 5 ࡘࡢከᆺᗙ఩࡟ᑐࡍࡿᑐ❧㑇ఏᏊ㢖ᗘࢆ⾲ 2 ࡟♧ࡋࡓࠋ
rs8973300 ࡛ࡣࠊㄪᰝࡋࡓ 20 ≟✀඲࡚࠿ࡽከᆺࡀ᳨ฟࡉࢀࡓࠋࡲࡓࠊ㹥20957075
AGGTGGGCA㸸
㹙1㹛>㹙2㹛࡜ rs8973298 ࡛ࡶࠊࢩ࣋ࣜ࢔ࣥࣁࢫ࣮࢟ࢆ㝖ࡃ 19 ≟✀࡛ከᆺࡀㄆࡵࡽࢀࡓࠋࡇࢀ࡟ᑐࡋ࡚ࠊrs22147008 ࡜ g.20957831 A>C ࡛ࡣࠊከᆺㄆࡵࡽࢀࡓ≟✀ࡣࠊࡑࢀࡒࢀ 14 ≟✀࡜ 9 ≟✀࡛࠶ࡾࠊṧࡾࡢ≟✀࡛ࡣ࣐࢖ࢼ࣮࢔ࣞࣝࡀ඲ࡃ᳨ฟࡉࢀ࡞࠿
ࡗࡓࠋg.20957831 A>C ࡛ࡣࠊㄪᰝࡋࡓ 20 ≟✀࡟࠾࠸࡚ᖖ࡟ A ᆺࡀ࣓ࢪ࣮ࣕ࢔࡛ࣞࣝ࠶ࡗ
ࡓࠋࡇࢀ࡟ᑐࡋ࡚ࠊṧࡾࡢ㸲ࡘࡢከᆺ࡛ࡣࠊ≟✀࡟ࡼࡗ࡚࣓ࢪ࣮ࣕ࢔ࣞࣝ࡜࣐࢖ࢼ࣮࢔ࣞ
ࣝࡢ㛵ಀࡀ㏫㌿ࡍࡿࡇ࡜ࡀ࠶ࡗࡓࠋ
୍㒊ࡢရ✀࡛࢔ࣞࣝࡢᅛᐃࡀㄆࡵࡽࢀࡓࡶࡢࡢᮏ◊✲ࡢㄪᰝᑐ㇟࡜ࡋࡓ 5 ࡘࡢከᆺࡣࠊ
࠸ࡎࢀࡶᗈ⠊ᅖࡢ≟✀࡟ศᕸࡋ࡚࠸ࡿࡇ࡜࠿ࡽࠊಶࠎࡢရ✀ࡀ☜❧ࡋࡓᚋ࡟᪂ࡋࡃ⏕ࡌࡓኚ␗࡛ࡣ࡞ࡃࠊᐙ␆≟࡟ᗈࡃศᕸࡍࡿࢥࣔࣥከᆺ࡛࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡲࡓࠊ࢔
ࣞࣝࡀᅛᐃࡉࢀ࡚࠸ࡓရ✀㛫࡟᫂☜࡞㛵㐃ᛶࡣㄆࡵࡽࢀ࡞࠸ࡇ࡜࠿ࡽ㑇ఏᏊᾋື࡟ࡼࡗ࡚
⏕ࡌࡓ⌧㇟࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ๓ 㡯 ࡢ ◊ ✲ ࡛ ࠊ IL-13 ࢱ ࣥ ࣃ ࢡ ㉁ ࡢ ᵓ 㐀 ࡟ ኱ ࡁ ࡞ ᙳ 㡪 ࢆ ࠾ ࡼ ࡰ ࡍ ࡜ ⪃ ࠼ ࡽ ࢀ ࡓ
rs22147008 ࡢ G ᆺ࡟ࡘ࠸࡚ࠊ㢖ᗘ 10㸣ᮍ‶ࡢရ✀ࡀ 20 ≟✀୰ 10 ≟✀࡜᭱ࡶከࡃࠊ⤖ᯝ࡜ࡋ࡚࣍ࣔ᥋ྜయࡣᴟࡵ࡚ᑡᩘ࡛࠶ࡗࡓࠋࡇࡢࡓࡵࠊ⑌⑓࡜ࡢ┦㛵ゎᯒࢆ㐍ࡵࡿ࡟࠶ࡓࡾࠊ
rs22147008 ࡢ G ᆺ࣍ࣔ᥋ྜࡢಶయࢆぢࡘࡅࡿࡇ࡜ࡀᅔ㞴࡞ရ✀ࡀከࡃᏑᅾࡍࡿ஦ࡀ♧ࡉࢀࡓࠋࡋ࠿ࡋࠊ࣑ࢽࣗࢳࣗ࢔ࢲࢵࢡࢫࣇࣥࢺࠊࢳ࣡࣡ࠊ࣏࣓ࣛࢽ࢔ࣥ࠾ࡼࡧࠊࣃࢢ࡛ࡣࠊG

647

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648

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IL-13 㑇ఏᏊከᆺࡢᐤ୚ࢆゎᯒࡋࡓࠋᮦᩱ࡜᪉ἲᮏࣉࣟࢪ࢙ࢡࢺ◊✲࡛཰㞟ࡉࢀࡓ࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࢆⓎ⑕ࡋࡓᰘ≟ 10 ಶయ࡜ࠊ࢔ࢺࣆ࣮ᛶ
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1. ㄽᩥⓎ⾲
1) Okubo T, Hayashi D, Yaguchi T, Fujita Y, Sakaue M, Suzuki T, Tsukamoto A,
Murayama O, Lynch J, Miyazaki Y, Tanaka K, Takizawa T.: Differentiation of rat
adipose tissue-derived stem cells into neuron-like cells by valproic acid, a histone
deacetylase inhibitor. Exp Anim [Epub ahead of print] , PMID: 26411320,2015.
2) Kurihara Y, Suzuki T, Sakaue M, Murayama O, Miyazaki Y, Onuki A, Aoki T, Saito M,
Fujii Y, Hisasue M, Tanaka K, Takizawa T. Valproic acid, a histone deacetylase
inhibitor, decreases proliferation of and induces specific neurogenic differentiation ofcanine adipose tissue-derived stem cells. J Vet Med Sci 76:15-23, 2014.
3) Watanabe M, Tanaka K, Takizawa T, Segawa K, Neo S, Tsuchiya R, Murata M,

650Murakami M, Hisasue M.:Characterization of a canine tetranucleotide microsatellite
marker located in the first intron of the tumor necrosis factor alpha gene. J Vet Med

Sci 76,119-22. 2014.
4) Ishii Y, Takizawa T, Iwasaki H, Fujita Y, Murakami M, Groppe JC, Tanaka K.:
Nucleotide polymorphisms in the canine Noggin gene and their distribution amongdog (Canis lupus familiaris) breeds. Biochem Genet 50, 12-8,2012.
2. Ꮫ఍Ⓨ⾲1) ⏣୰࿴᫂ࠊ ⁪ἑ㐩ஓࠊ ᒣᮏᮍဏࠊ ᒸᮏ᠇᫂ࠊᓥ಴⚽຾ࠊ 㜰ཱྀ㞞ᘯ㸸࢖ࢾ IL-13 㑇ఏࡢከᆺ࡜ᰘ≟࡟࠾ࡅࡿ࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡜ࡢ㛵㐃ㄪᰝ. ᪥ᮏ␆⏘Ꮫ఍➨ 121 ᅇ኱఍, ᮾி㒔Ṋⶶ㔝ᕷ 3 ᭶ 20162) బ⸨⨾⣖ࠊᐑᓮ㝧Ꮚࠊ㟷ᮌ༟☻ࠊ⸨⏣ᖾᘯࠊᅭᑿᣅஓࠊ㰺⸨ᘺ௦Ꮚࠊ⸨஭ὒᏊࠊஂᮎṇᬕࠊ⏣୰࿴᫂ࠊ⁪ἑ㐩ஓ:࢖ࢾ⬡⫫⤌⧊ᖿ⣽⬊(ASC)ࡢ⣽⬊ቑṪ࡜⚄⤒ศ໬࡟ཬࡰࡍࣂࣝ
ࣉࣟ㓟࡜ప㓟⣲ࡢᙳ㡪. ➨ 87 ᅇ᪥ᮏ⏕໬Ꮫ఍ࠊி㒔 10 ᭶ 20143) Ώ㎶ᚁ, ⏣୰࿴᫂, ⁪ἑ㐩ஓ, ℩ᕝ࿴ோ, ᰿ᑿḈᏊ, ᅵᒇு, ᮧ⏣೔Ꮚ,ᮧୖ㈼, ஂᮎṇᬕ㸸࢖ࢾ⭘⒆ቯṚᅉᏊ(TNFA)㑇ఏᏊࡢ➨㸯࢖ࣥࢺࣟࣥ࡟Ꮡᅾࡍࡿ 4 ሷᇶ཯᚟࣐࢖ࢡࣟࢧ
ࢸࣛ࢖ࢺࡢ≉ᚩ. ➨ 156 ᅇ᪥ᮏ⋇་Ꮫ఍ࠊᒱ㜧ࠊ9 ᭶ 20134) ᰩཎᗣᘯࠊ୰ᒸඃᕼࠊ⸨஭♸㍤ࠊ⸨⏣㞝኱ࠊᒣᮏᮍဏࠊᐑᓮ㝧Ꮚࠊ⏣୰࿴᫂ࠊ⁪ἑ㐩ஓ㸸࢖ࢾ⬡⫫⤌⧊⏤᮶㛫ⴥ⣔ᖿ⣽⬊ࡢ⚄⤒ศ໬࡟ཬࡰࡍࣄࢫࢺࣥ⬺࢔ࢭࢳࣝ໬㓝⣲㜼ᐖ๣ࡢᙳ㡪㸬➨ 154 ᅇ᪥ᮏ⋇་Ꮫ఍Ꮫ⾡㞟఍ࠊᒾᡭࠊ9 ᭶ 2012 ᖺ5) ᰩཎᗣᘯࠊ୰ᒸඃᕼࠊ⸨஭♸㍤ࠊ⸨⏣㞝኱ࠊᐑᓮ㝧Ꮚࠊ⏣୰࿴᫂ࠊ⁪ἑ㐩ஓ㸸࢖ࢾ⬡⫫⤌⧊⏤᮶㛫ⴥ⣔ᖿ⣽⬊ࡢ⣽⬊ቑṪ࠾ࡼࡧከศ໬⬟࡟ཬࡰࡍࣄࢫࢺࣥ⬺࢔ࢭࢳࣝ໬㓝⣲㜼ᐖ๣ࡢᙳ㡪㸬 ➨ 152 ᅇ᪥ᮏ⋇་Ꮫ఍Ꮫ⾡㞟఍ࠊሜࠊ9 ᭶ 2011 ᖺ

651

Exp. Anim. 65(1), 45–51, 2016

—Original—

Differentiation of rat adipose tissue-derived stem
cells into neuron-like cells by valproic acid, a histone
deacetylase inhibitor
Takumi OKUBO1), Daiki HAYASHI1), Takayuki YAGUCHI1), Yudai FUJITA1),
Motoharu SAKAUE1), Takehito SUZUKI1), Atsushi TSUKAMOTO1), Ohoshi MURAYAMA2),
Jonathan LYNCH2), Yoko MIYAZAKI1), Kazuaki TANAKA1), and Tatsuya TAKIZAWA1)
1)Graduate School of Veterinary Medicine, Azabu University, Fuchinobe, Chuo-ku, Sagamihara, Kanagawa

252–5201, Japan
2)School of Life and Environmental Science, Azabu University, Fuchinobe, Chuo-ku, Sagamihara, Kanagawa

252–5201, Japan

Abstract: Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported
to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans anddogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiationof ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat
ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced
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associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction.
Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by
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histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. TheseUHVXOWVLQGLFDWHWKDW93$VLJQL¿FDQWO\SURPRWHVWKHGLIIHUHQWLDWLRQRIUDW$6&VLQWRQHXURQOLNHFHOOV
through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing
transplantable cells for future applications in clinical treatments.
Key words: adipose tissue-derived stem cells, histone deacetylase inhibitor, neuronal differentiation,neuron marker, rats

Introductionrogenic lineage cells [16, 18]. However, unlike BMSCs,
ASCs can easily be obtained in large quantities and withminimum risk from invasive surgery [5]. Therefore, it isreasonable to conclude that ASCs will be the preferredadult stem cells for future clinical applications [14].
Valproic acid (VPA), well known as an antiepileptic
and anticonvulsant drug, is an inhibitor of class I histone
deacetylase (HDACs) [2, 15]. The acetylation of histone

Adipose tissue-derived stem cells (ASCs) are mesenchymal stem cells that are isolated from the stromal
vascular fraction of adipose tissues [3, 14, 19]. In asimilar way to bone marrow-derived mesenchymal stem
cells (BMSCs), ASCs can differentiate into not only
mesenchymal lineage cells [17, 18], but also into neu-

(Received 16 April 2015 / Accepted 16 August 2015 / Published online in J-STAGE 25 September 2015)
Address corresponding: T. Takizawa, Graduate School of Veterinary Medicine, Azabu University, Fuchinobe, Chuo-ku, Sagamihara, Kanagawa
252–5201, Japan©2016 Japanese Association for Laboratory Animal Science652

T. OKUBO, ET AL.

46

N-terminal tails is thought not only to alter the interaction between histone and DNA, but also to bind acetylated histone to bromodomain proteins and transcription
activators [8], thus inducing the expression of genes on
the loci [4, 6, 7, 10]. We have previously shown that VPA
induces neuronal differentiation of canine ASCs [11], a
result that suggests a safer and more direct method for
preparing the cell source for future applications. However, Lee et al. [12] showed that the pretreatment of VPA
GLPLQLVKHGHI¿FLHQF\RIQHXURQDOGLIIHUHQWLDWLRQRIKXman ASCs. Thus there is debate over the role of VPA as
an inducer of neuronal differentiation of ASCs. To address this, we examined the effect of VPA on neuronaldifferentiation of ASCs isolated from adipose tissue inrats in the present study. The results indicate that VPA
induced differentiation of rat ASCs into neuron-like cells
through acetylation of histone H3.
Materials and Methods
Isolation and culture of rat ASCs
ASCs were isolated as previously described [11].
%ULHÀ\  WR ZHHNROG PDOH:LVWDU UDWV &KDUOHVRiver Japan, Yokohama, Japan) were euthanatized bydecapitation under light anesthesia. Subcutaneous adipose tissue was excised from the inguinal regions andextensively washed with a washing buffer of sterile
phosphate-buffered saline (PBS) containing penicillin(100 U/ml) and streptomycin (100 ȝg/ml) to remove
contaminating blood cells and local anesthetics. The tissue was minced into small pieces and then incubated in
washing buffer with added 0.05% collagenase type 1A(Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 1 h
with vigorous shaking. The top lipid layer was removed,
and the remaining liquid portion was centrifuged at
200 × g for 10 min. The pellet was resuspended in a
JURZWKPHGLXPRI'XOEHFFR¶VPRGL¿HG(DJOH¶VPHGLXP(DMEM, Nissui, Tokyo, Japan) supplemented with 10%
newborn bovine serum (NBS, Invitrogen, Carlsbad, CA,
USA) and antibiotics above described, and then spreadin 100-mm collagen type 1-coated dishes (Iwaki, Tokyo,
Japan) at a density of 1 × 106 cells per dish. Cells weremaintained in growth medium at 37°C and 5% CO2.
After 24 h, the unattached cells were removed by rinsing
ZLWKZDVKLQJEXIIHU$6&VZHUHLGHQWL¿HGE\FRQ¿UPLQJ
multilineage potentials to adipogenic, osteogenic andneurogenic cells (data not shown). Twice-passaged ASCs
were used in the present study. All animal experiments653

in the present study were carried out according to theguidelines of the Committee for Animal Experimentation
at Azabu University.
In vitro neuronal differentiation assay
In vitro assay of neuronal differentiation was carried
RXWDVSUHYLRXVO\GHVFULEHG>@ZLWKDPLQRUPRGL¿FDWLRQ %ULHÀ\$6&V ZHUH VHHGHG RQ QRQFRDWLQJ JODVVcoverslips in 35-mm dishes at a density of 1 × 104 cells
per dish. ASCs were incubated for one or three days ingrowth medium containing 2 mM of VPA. After pretreatment with and without VPA, vehicle dimethylsulfoxide(DMSO), the medium was changed to a neuronal induction medium (NIM) of DMEM supplemented with100 ȝM dibutyryl cyclic adenosine monophosphate(dbcAMP, Wako Pure Chemical Ind., Osaka, Japan) and125 ȝM isobutylmethylxanthine (IBMX, Wako Pure
Chemical Ind.) for 2 h [1, 11]. Control for NIM was NIM
minus dbcAMP and IBMX. After pretreatment of VPA
IROORZHGE\1,0WKHFHOOVZHUH¿[HGLQ3%6FRQWDLQLQJ
3.7% formaldehyde for 15 min at room temperature.
Similarly incubated ASCs on non-coating 35-mm dishes were removed by a cell-scraper and the removed-cells
were immersed in ISOGEN (Nippon Gene, Tokyo, Japan) and stored at –80°C until further analysis. Neuronal
GLIIHUHQWLDWLRQZDVGHWHUPLQHGE\LPPXQRÀXRUHVFHQFH
VWDLQLQJIRUȕ,,,WXEXOLQDQGQHXUR¿ODPHQWPHGLXPSRO\peptide (NEFM) and by RT-PCR analysis for expression
RI JHQHV ȕ,,,WXEXOLQ TUBB3), NEFM, microtubuleassociated protein 2 (MAP2 JOLDO¿EULOODU\DFLGLFSURtein (GFAP) and housekeeping gene hypoxanthineguanine phosphoribosyl-transferase (HPRT).
,PPXQRÀXRUHVFHQFHVWDLQLQJ,PPXQRF\WRFKHPLFDODQDO\VHVIRUȕ,,,WXEXOLQ1()0and acetylation of histone H3 were performed. FormalGHK\GH¿[HG$6&VZHUHULQVHGZLWK3%6WKUHHWLPHVThe cells were then permeated with 0.2% Triton X-100
in PBS for 10 min at room temperature, and then incuEDWHGZLWKDQDQWLȕ,,,WXEXOLQDQWLERG\ $EFDP
ab74978, Cambridge, UK), an anti-NEFM antibody
 3URWHXV %LR6FLHQFHV ,QF  5DPRQD
CA, USA) and an anti-acetylated histone H3 (K9) antiERG\ 1RYXV%LRORJLFDOV1%66/LWWOHton, CO, USA) for 1 h at room temperature. After being
washed with PBS, the cells were incubated with secondary antibody (Cy3-conjugated goat anti-rabbit IgG,RU),7&FRQMXJDWHGJRDWDQWLPRXVH,J*NEURONAL DIFFERENTIATION AND ACETYLATED H3

Jackson ImmunoResearch, West Grove, PA) for 30 min
at room temperature. The cells were then rinsed with
PBS and counterstained with 4’, 6-diamidino-2-phenylLQGROH '$3, IRUQXFOHDUVWDLQLQJEHIRUHÀXRUHVFHQFH
PLFURVFRSLFREVHUYDWLRQ7KHSHUFHQWDJHRIȕ,,,WXEXOLQ
positive cells was determined based on the total numberof counting cells. At least 300 cells were evaluated per
culture.
Reverse transcription-PCR
Total RNA was extracted using ISOGEN and reversetranscribed for single-strand cDNA, using oligo (dT)primer and Superscript III reverse transcriptase (Invitrogen) according to the manufacture’s instruction. PCR
was performed using Taq DNA polymerase (KAPA BioV\VWHPV:REXUQ0$86$ DQGVSHFL¿FSULPHUVDQG
HDFKF\FOHFRQVLVWHGRIWKHIROORZLQJVWHSVGHQDWXUDWLRQat 98°C for 10 s, annealing at 57°C to 65°C for 30 s, andelongation at 72°C for 30 s (Table 1). Reaction products
were electrophoresed on a 2.0% agarose gel and visualized with ethidium bromide.
Effect of VPA on the acetylation of histone H3
7R FRQ¿UP WKH HIIHFW RI93$ RQ WKH DFHW\ODWLRQ RI
histone H3, ASCs were incubated for three days using asimilar method to that described above, in growth medium containing 2 mM VPA or valpromide (VPM), an
analogue of VPA without HDAC inhibitory activity.
$IWHUWUHDWPHQWZLWKWKHVHUHDJHQWVWKHFHOOVZHUH¿[HG
with formaldehyde in a similar method to that describedabove. The effect of VPA on the acetylation of histone+ZDVH[DPLQHGE\LPPXQRÀXRUHVFHQFHVWDLQLQJIRU
acetylated-histone H3.
Chromatin Immunoprecipitation assay
Tissue samples were treated with chromatin immuno-

47

precipitation (ChIP) reagents (Nippon Gene), following
WKH PDQXIDFWXUHU¶V LQVWUXFWLRQV ZLWK PLQRU PRGL¿FDtions. Cells were harvested and mixed with formaldehyde
DWD¿QDOFRQFHQWUDWLRQRIIRUPLQDWURRPWHPperature to cross-link protein to DNA. Cells were thensuspended in lysis buffer (Protease Inhibitors, Pierce,
Rockford, IL, USA) and incubated on ice for 10 min.
DNA cross-linked with protein was sonicated into fragments of 200–1,000 bp by an Ultrasonic Homogenizer(Taitec, Koshigaya, Japan). Samples were subsequently
incubated at 4°C with Dynabeads (Invitrogen) to obtainthe soluble chromatin fraction. Dynabeads were previously incubated with 4 ȝg rabbit anti-histone H3 (acetyl
K9) antibody (Novus Biologicals, Littleton) or ChIP
rabbit IgG control antibody (Santa Cruz Biotechnology,
Santa Cruz, CA, USA) for 16 h overnight at 4°C. The
chromatin-antibody complexes were eluted with ChIP
direct elution buffer. Protein-DNA cross-links were reversed with 5 M NaCl for 12 h incubation at 65°C. Proteinase K treatment and phenol-chloroform extraction
were carried out, and the DNA was then precipitated in
HWKDQRODQGXVHGDVDWHPSODWHIRU3&53&5DPSOL¿FDWLRQZDVFDUULHGRXWZLWKSULPHUVVSHFL¿FIRUSURPRWHU
region of TUBB3. Primer sequences were shown as folORZVIRUZDUG¶$&7&&$7$&&&&&7&777*&¶UHYHUVH¶$*7&&$7**&7&&$&$$$$*¶,QSXWDNA and DNA immunoprecipitated by anti-IgG served
as positive and negative control, respectively.
Statistical analysis
Results are expressed as means ± standard error. Multiple comparisons were performed with the TurkeyKramer test after one-way analysis of variance (ANOVA).
A P value of less than 0.05 was considered statistically
VLJQL¿FDQWTable 1. Primer sequences used in RT-PCR
Gene
TUBB3
NEFM
MAP2
GFAP
HPRT

Primer sequence (5’-3’)
Forward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
Reverse5’-GGCCTCCTCTCACAAGTATGT-3’
5’-CGCCCTCTGTATAGTGC-3’
5’-AGGCTGAGTCCCCAGTGAAA-3’
5’-TCCACCTCCCCATTGATAGC-3’
5’-ACCTTCCTCCATCCTCCCTC-3’
5’-AGTAGGTGTTGAGGTGCCGC-3’
5’-ACATCGAGATCGCCACCTAC-3’
5’-GCACACCTCACATCACATCC-3’
5’-AATGTCTGTTGCTGCGTC-3’
5’-TGTCTGTCTACAAGGGAAG-3’

654

Anealing temperature Product length (bp)
58°C

167

58°C

220

57°C

151

58°C

228

55°C

92

T. OKUBO, ET AL.

48

Results
VPA promotes neuronal differentiation in a timedependent manner
Using immunocytochemistry and RT-PCR analysis,
neuronal differentiation was determined in ASCs withand without VPA pretreatment for 1 or 3 days, followed
by treatment with and without NIM for 2 h. Regardlessof the duration of VPA treatment, differentiated ASCs
showed neuron-like morphology with several branchingneurite-like cell processes, in which most of the cells
KDGSRVLWLYHVLJQDOVRIDQLPPDWXUHQHXURQPDUNHUȕ,,,
tubulin and a mature neuron marker NEFM. Three-days
WUHDWPHQWZLWK93$VLJQL¿FDQWO\HQKDQFHGWKHSRVLWLYH
VLJQDORIȕ,,,WXEXOLQDQG1()0ZLWKDQGHYHQZLWKRXW
1,0 WUHDWPHQW7KH PDMRULW\ RI ȕ,,,WXEXOLQ SRVLWLYHFHOOVDOVRH[SUHVVHG1()0DQGERWKLPPXQRÀXRUHVcence intensities were reduced without VPA pretreatment(Fig. 1A 1HXURQDOLQGXFWLRQVLJQL¿FDQWO\LQFUHDVHGWKH
UDWLR RI ȕ,,,WXEXOLQSRVLWLYH FHOOV IURP   
without VPA to 85.6 ± 1.9% with three-days treatment
of VPA (Fig. 1B). Three-days treatment of VPA also
VLJQL¿FDQWO\LQFUHDVHGWKHUDWLRRIȕ,,,WXEXOLQSRVLWLYH
cells from 35.4 ± 1.2% without NIM to 85.6 ± 1.9% with
1,0:LWKRXW1,0JURXSV93$VLJQL¿FDQWO\LQFUHDVHGWKHUDWLRRIȕ,,,WXEXOLQSRVLWLYHFHOOVLQDWLPHGHSHQdent manner (Fig. 1B).
mRNA expressions of immature neuron marker
TUBB3 were observed in all groups with and without
pretreatment of VPA, but mRNA expression of the glial
cell marker GFAP was not detected in any groups(Fig. 1C). Furthermore, three-days treatment of VPA
markedly elevated not only the mRNA expression levelsof immature neuron marker TUBB3, but also matureneuron markers MAP2 and NEFM in ASCs with andwithout NIM. One-day treatment of VPA also induced
the expression of these mature neuron markers (data notshown). In addition, NIM treatment without VPA did not
VLJQL¿FDQWO\ HQKDQFH WKH H[SUHVVLRQV RI WKHVH QHXURQmarkers when compared to those observed in the groups
with VPA.
VPA induces acetylation of histone H3
7RFRQ¿UPWKHHIIHFWRI93$RQDFHW\ODWLRQRIKLVWRQH
H3, we examined the acetylated histone H3 by immuQRÀXRUHVFHQFHVWDLQLQJ0LQLPDODFHW\ODWLRQRIKLVWRQH
H3 were observed in the control ASCs. Three-days pretreatment of VPA gave morphological changes of the655$6&VVXFKDVÀDWWHQHGDQGH[SDQGHGVKDSHVDQGLQcreased their acetylation of histone H3 (Fig. 2A). In
contrast, VPM, an analogue of VPA without HDAC inhibitory activity, did not cause any morphological
FKDQJHV RU VLJQL¿FDQW FKDQJHV LQ DFHW\ODWLRQSRVLWLYH
signal of histone H3 in ASCs.
VPA increases H3K9 acetylation levels at the proximal
promoter region of TUBB3
The state of the histone tails of ASCs pretreated with
VPA for three days was analyzed by ChIP assay using
antibody to acetylated lysine 9 on histone H3 (antiAcH3K9). The acetylated state of histone H3 tails associated with the proximal region of TUBB3 increased
in ASCs pretreated by VPA compared to those in theASCs without VPA (Fig. 2B), indicating that the increaseof histone acetylation of H3K9 correlated with the expression of the TUBB3 gene of ASCs pretreated with
VPA.
Discussion
In this study we demonstrated that VPA promoted the
GLIIHUHQWLDWLRQRIDSSUR[LPDWHO\RI$6&VLQWRȕ,,,
tubulin-positive neuron-like cells after three-days treatment followed by neuronal induction. The differentiatedcells showed neuron-like morphology with branching
QHXULWHOLNHFHOOSURWUXVLRQVDQGZHUHVLJQL¿FDQWO\LPPXQRVWDLQHGWRDQLPPDWXUHQHXURQPDUNHUȕ,,,WXEXOLQ
and a mature neuron marker NEFM. Pretreatment with
93$IROORZHGE\QHXURQDOLQGXFWLRQDOVRVLJQL¿FDQWO\enhanced mRNA expressions of TUBB3, MAP2 andNEFM, compared to the groups without VPA. We also
demonstrated that VPA induced differentiation of ASCs
into neuron-like cells in a time-dependent manner. The
present study is in agreement with our previous study
demonstrating that VPA promotes neuronal differentiation of canine ASCs [11], and further indicates that VPA
not only promotes neuronal differentiation but can also
promote more mature neuron-like cells. The present dataalso show that VPA enhances expression of neuron markers, whereas no effect was observed for glial marker.
This suggests that VPA induces the differentiation ofASCs into neuron-like cells by neuronal induction, alWKRXJKIXUWKHUFRQ¿UPDWLRQE\PHDQVRIDGHWDLOHGVWXG\
is required.
In the present study, we used a well-known neuronaldifferentiation medium including IBMX and dbcAMP.

NEURONAL DIFFERENTIATION AND ACETYLATED H3

49

Fig. 1. Valproic acid promotes neuronal differentiation. Adipose tissue-derived stem cells (ASCs) were pretreatedwith valproic acid (VPA) for 1 or 3 days followed by neuronal induction medium (NIM) for 2 h. (A) NeuroQDOGLIIHUHQWLDWLRQZDVDVVHVVHGE\LPPXQRF\WRFKHPLVWU\XVLQJDQWLȕ,,,WXEXOLQDQWLERG\ JUHHQ DQGDQWL
1()0DQWLERG\ 5HG DIWHU¿[LQJDWWKHHQGRILQFXEDWLRQLQGD\V93$WUHDWPHQWJURXS1XFOHLZHUH
counterstained with DAPI (blue). Scale bar, 100 ȝP % 7KHSHUFHQWDJHRIȕ,,,WXEXOLQSRVLWLYHFHOOVZDVdetermined based on the total number of counting cells in 1 or 3 days VPA treatment. At least 300 cells were
evaluated per culture. Data are the means ± S.E. of 4 independent experiments. Open and solid bars represent
RUGD\V93$WUHDWPHQWUHVSHFWLYHO\DEFGEDUVZLWKGLIIHUHQWOHWWHUVDWWKHWRSGLIIHUVLJQL¿FDQWO\DPRQJVDPHGXUDWLRQJURXSDVWHULVNLQGLFDWHVVLJQL¿FDQWO\GLIIHUHQWFRPSDUHGWRRQHGD\WUHDWPHQWJURXS
P<0.05. (C) RT-PCR analysis of neuronal markers TUBB3, MAP2 and NEFM, the glial marker GFAP andhouse keeping gene HPRT was performed using total RNA extracted from ASCs after 3-days VPA treatment
followed by 2 h of neuronal induction.

occurs through inducing the acetylation of histone H3.
7KHLQYHVWLJDWLRQGHPRQVWUDWHGWKDW93$ÀDWWHQHGDQG
expanded the shape of rat ASCs and markedly induced
their acetylation of histone H3. These observations are
LQDFFRUGDQFHZLWKWKH¿QGLQJVRI/HHet al. [12] andKurihara et al. [11]. Lee et al. [12], however, showed
WKDW93$GLPLQLVKHGWKHHI¿FLHQF\RIQHXURQDOGLIIHUentiation of human ASCs. The study showed that one-day
pretreatment of VPA (1, 10 mM) followed by neuronal

We showed that VPA increased the neuronal morphological changes and neuron markers expression even
without neuronal induction treatment, thus indicatingthat VPA can induce neuronal differentiation in the absence of neuronal induction. We also demonstrated that
VPA increased the acetylation of histone H3K9 on theproximal region of TUBB3 in rat ASCs, leading to theneuronal differentiation. Present results suggest that thepromotional effect of VPA on neuronal differentiation

656

50

T. OKUBO, ET AL.

Fig. 2. Effects of valproic acid on histone H3 acetylation. (A)Adipose tissue-derived stem cells (ASCs) from rats wereincubated with valproic acid (VPA) or valpromide (VPM),
an analogue of VPA without histone deacetylase inhibitory activity, for 3 days. Control group was treated withsolvent, DMSO. ASCs were immunostained with an antiacetylated histone H3 (K9) antibody (Red). Acetylated
histone H3 (K9) antibody was visualized with Cy3-conMXJDWHGJRDWDQWLUDEELW,J*6FDOHEDUȝP % 93$
increased acetylation levels at the proximal region of
TUBB3. The state of the histone tails of ASCs pretreatedwith VPA for 3 days was analyzed by ChIP assay using
antibody to acetylated lysine 9 on histone H3 (antiAcH3K9). Typical electrophoresis of PCR analysis of
TUBB3 was shown.

induction decreased neurite formation and expression of
ȕ,,,WXEXOLQLQKXPDQ$6&V>@2QWKHRWKHUKDQGWKH
study of Kurihara et al. [11] showed that VPA induced
neuronal differentiation of canine ASCs with three-days
pretreatment of VPA (4 mM) followed by neuronal induction [11]. Differing treatment regimens of VPA were
implemented in these studies and this can partially ex-

657

plain the different effect on neuronal differentiation ofASCs. In the present study, we examined the time-course
effects of VPA on the neuronal differentiation of ASCs
by immunocytochemistry and RT-PCR analysis. This
investigation clearly demonstrated that VPA induced
differentiation of ASCs into neuron-like cells, not only
as a result of three-days treatment, but also from a oneday course of treatment. Together with the previous study[11], the present results demonstrate that VPA is highly
effective in neuronal differentiation of ASCs at least for
rats and dogs, when VPA treatment is applied over one
and three days. Taken together with the present study,
an appropriate treatment of VPA promotes the differentiation of rat ASCs into neuron-like cells, although species difference should be examined in future.
It is well known that the differential potential of neural stem cells (NSCs) into neurons depends on the
VWDJHVRFFXUULQJGXULQJEUDLQGHYHORSPHQWHDUO\VWDJH
NSCs prefer to differentiate into neurons, whereas latestage NSCs into astrocytes [13]. VPA has also beenshown to promote the differentiation of adult hippocampal neural progenitors into neurons, but inhibited theirglial differentiation in adult neural progenitor cells [6, 9].
These reports offer another explanation that the different
effect of VPA on neuronal differentiation depends on thestages of ASCs similarly observed in NSCs during brain
development.
In conclusion, the results have shown that, through
WKH DFHW\ODWLRQ RI KLVWRQH +93$ VLJQL¿FDQWO\ SURmotes the differentiation of rat ASCs into neuron-likecells, with expression of mature neuron markers. The¿QGLQJVVXJJHVWWKDW93$PD\SURYHWREHDXVHIXOWRRO
for producing transplantable cells for future applicationsin clinical treatments.
Acknowledgment
This work was supported in part by the Science Research Promotion Fund of The Promotion and Mutual
Aid Corporation for Private Schools of Japan. This work
was also supported in part by the MEXT Program for
the Strategic Research Foundation at Private Universities, 2011–2015.
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M.A., and Klein, P.S. 2001. Histone deacetylase is a directtarget of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J. Biol. Chem.  ±
[Medline] [CrossRef]
16. Safford, K.M., Hicok, K.C., Safford, S.D., Halvorsen, Y.D.,
Wilkison, W.O., Gimble, J.M., and Rice, H.E. 2002. Neurogenic differentiation of murine and human adipose-derived
stromal cells. Biochem. Biophys. Res. Commun.  ±379. [Medline] [CrossRef]
17. Wu, P., Sato, K., Yukawa, S., Hikasa, Y., and Kagota, K.
2001. Differentiation of stromal-vascular cells isolated fromcanine adipose tissues in primary culture. J. Vet. Med. Sci.
±[Medline] [CrossRef]
18. Zuk, P.A., Zhu, M., Ashjian, P., De Ugarte, D.A., Huang,J.I., Mizuno, H., Alfonso, Z.C., Fraser, J.K., Benhaim, P.,
and Hedrick, M.H. 2002. Human adipose tissue is a sourceof multipotent stem cells. Mol. Biol. Cell  ±
[Medline] [CrossRef]
19. Zuk, P.A., Zhu, M., Mizuno, H., Huang, J., Futrell, J.W.,
Katz, A.J., Benhaim, P., Lorenz, H.P., and Hedrick, M.H.
 0XOWLOLQHDJH FHOOV IURP KXPDQ DGLSRVH WLVVXH LPplications for cell-based therapies. Tissue Eng.±
[Medline] [CrossRef]

FULL PAPER

Surgery

Valproic Acid, a Histone Deacetylase Inhibitor, Decreases Proliferation of and Induces

Yasuhiro KURIHARA1), Takehito SUZUKI1), Motoharu SAKAUE1), Ohoshi MURAYAMA2), Yoko MIYAZAKI1),
Atsushi ONUKI1), Takuma AOKI1), Miyoko SAITO1), Yoko FUJII1), Masaharu HISASUE1), Kazuaki TANAKA1) and
Tatsuya TAKIZAWA1)*
1)Graduate School of Veterinary Medicine, Azabu University, Fuchinobe, Chuo-ku, Sagamihara 252–5201, Japan
2)School of Life and Environmental Science, Azabu University, Fuchinobe, Chuo-ku, Sagamihara 252–5201, Japan(Received 30 April 2013/Accepted 7 August 2013/Published online in J-STAGE 27 August 2013)
ABSTRACT. Adipose tissue-derived stem cells (ADSCs) isolated from adult tissue have pluripotent differentiation and self-renewal capability.

The tissue source of ADSCs can be obtained in large quantities and with low risks, thus highlighting the advantages of ADSCs in clinicalapplications. Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to affect ADSC differentiation inmice and rats; however, few studies have been performed on dogs. We aimed to examine the in vitro effect of VPA on canine ADSCs.
Three days of pretreatment with VPA decreased the proliferation of ADSCs in a dose-dependent manner; VPA concentrations of 4 mM andabove inhibited the proliferation of ADSCs. In parallel, VPA increased p16 and p21 mRNA expression, suggesting that VPA attenuatedthe proliferative activity of ADSCs by activating p16 and p21. Furthermore, the effects of VPA on adipogenic, osteogenic or neurogenic
differentiation were investigated morphologically. VPA pretreatment markedly promoted neurogenic differentiation, but suppressed theNSE, TUBB3 and MAP2, a decrease in the adipogenic marker, LPL, but noof neurogenic differentiation of canine ADSCs and is a useful tool for studying the interaction between chromatin structure and cell fate
determination.
KEY WORDS: adipose tissue-derived stem cell, cell proliferation, histone deacetylase inhibitor, pluripotency, valproic acid.
doi: 10.1292/jvms.13-0219; J. Vet. Med. Sci. 76(1): 15–23, 2014

Spinal cord injury (SCI) often occurs in dogs, due to motor vehicle accidents or intervertebral disease (IVDD). Most
canine patients suffer from sustained incontinence and lossof walking ability, and the prognosis of severe SCI cases ispoor. Unfortunately, no effective drug or surgical therapy has
been established for severe SCI cases, and there is a needfor new therapeutic approaches. One possibility is stem celltransplantation therapy, which is used as a radical cure treatment for refractory SCI [2, 14, 25, 26].
Adipose tissue-derived stem cells (ADSCs) are isolated
from the stromal vascular fraction of adipose tissues [8, 24,39]. ADSCs, similar to pluripotent adult mesenchymal stem
cells, can differentiate into mesenchymal lineage cells, suchas adipocytes, osteocytes, chondrocytes and myocytes [36,38]. ADSCs have characteristics similar to those of bonemarrow-derived mesenchymal stem cells (BMSCs), including gene expression and differentiation potential [1, 3, 5, 17,
19, 22, 33, 35, 37, 39]. Unlike BMSCs, however, the tissuesource of ADSCs can be obtained in large quantities and withlow risks [11]. Therefore, it is reasonable that ADSCs will be
the preferred adult stem cells for future clinical applications[24]. Moreover, the dog has been found to be a good animalmodel of human disease [32], and thus, the study of canine
ADSCs is particularly useful. Stem cells derived from bonemarrow and from olfactory ensheathing glia (OEG) havebeen studied for spinal cord regenerative therapy in dogs [6,14, 16, 25, 26, 31]. However, only a few studies have been
performed on the differentiation of canine ADSCs, except
for a comparative study showing that BMSCs and ADSCs
could be differentiated into neurospheres and neuron-likecells in dogs [2].
Valproic acid (VPA), a widely used antiepileptic and anticonvulsant drug, is an inhibitor of class 1 histone deacetylases (HDACs) [7, 27]. Histone acetylation correlates withterminal tails through acetylation or deacetylation can alter
the interaction between histones and DNA, affecting theregulation of gene expression [9, 12, 13, 18, 34]. Therefore,
HDAC inhibitors have been a useful tool for studying the*C ORRESPONDENCE TO: TAKIZAWA, T., Graduate School of Veterinary
Medicine, Azabu University, Fuchinobe, Chuo-ku, Sagamihara
252–5201, Japan.
e-mail: takizawa@azabu-u.ac.jp©2014 The Japanese Society of Veterinary Science
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-ncnd) License <http://creativecommons.org/licenses/by-nc-nd/3.0/>.

of hippocampal neural progenitors into neurons, but inhibit
their glial differentiation in adult rats [12].
In the present study, we have investigated the effects
of VPA on the proliferation and differentiation of canine
ADSCs isolated from subcutaneous adipose tissue in the

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Y. KURIHAR A ET AL.
Table 1.

Primers used in RT-PCR and real-time PCR
Product
length (bp)

Gene
p16
p21

FABP4
LPL
BMP2
Dlx5
COL1A1
NSE
TUBB3
MAP2
NEFH
GFAP
GAPDH

ForwardCGATCCAGGTCATGATGATGG

Reverse

ACCACCAGCGTGTCCAGGAA

ForwardCATCCCTCATGGCAGCAAG

ReverseAGGCAGGGAGACCTTGGACA

ForwardACACGATGCTGGCGTCCTTGATG

ReverseTGGCTCCATGAAGTCACCAAAGG

Forward

ATCAGTGTAAACGGGGATGTG

ReverseGACTTTTCTGTCATCCGCAGTA

ForwardACACATTCACAAGAGGGTCACC

ReverseCTCTGCAATCACACGGATGGC

ForwardCACTAACCACGCCATTGTTCA

ReverseACAACCCTCCACAACCATGTC

ForwardTGCTCTCCTACCTCGGCTTC

ReverseTTGCCATTCACCATCCTCAC

ForwardGTAGACACCACCCTCAAGAGC

ReverseTTCCAGTCGGAGTGGCACATC

ForwardGACCAACCCAAAGCGTATTGA

ReverseGCAATGAACGTGTCCTCAGTC

ForwardAGCCAAGTTCTGGGAAGTCA

Reverse

CCCACTCTGACCAAAGATGAA

ForwardAGAGGAGGTGTCTGCAAGGA

ReverseGTGATGGAGGTGGAGAAGGA

ForwardCTCAAAGGCACCAAGGACTC

ReverseCAAAGCCAATCCGACATTCT

ForwardAGATCCACGATGAGGAGGTG

ReverseTCTTAGGGCTGCTGTGAGGT

ForwardGCTGAACGGGAAGCTCACTG

ReverseCGTCGAAGGTGGAAGAGTGG

145
208
119
117
134
163
224
119
180
238
161
244
104
221

contaminating blood cells and local anesthetics. The tissue
was minced into small pieces and then incubated in a solution containing 0.05% collagenase type IA (Sigma-Aldrich,
St. Louis, MO, U.S.A.) at 37°C for 1 hr with vigorous
shaking. The top lipid layer was removed, and the remaining liquid portion was centrifuged at 200 × g for 10 min.

inguinal region.
MATERIALS AND METHODS
Animals: Subcutaneous adipose tissue was obtained fromthe inguinal region of 8 healthy laboratory beagles (age, 1–2
years) (Kitayama Labes, Ina, Japan). All animals were anesthetized with propofol, before tissue samples were obtained.
rane (2.0%) in oxygen. At the end of each experiment, theanimals were euthanized by additional doses of anesthesia(pentobarbital, 100 mg/kg). The protocol of this study wasapproved by the Committee for Animal Experimentation at
Azabu University.
Isolation and culture of canine ADSCs: Adipose tissue
samples were processed for ADSC isolation as described

medium (DMEM, Nissui, Tokyo, Japan) supplemented with
10% newborn bovine serum (NBS, Invitrogen, Carlsbad,
CA, U.S.A.) and spread in 100-mm collagen type I-coateddishes (Iwaki, Tokyo, Japan) at a density of 1 × 106 cells
per dish. Cells were maintained in growth medium (DMEM
supplemented with 10% NBS, penicillin [100 U/ml] andstreptomycin [0.25 μg/ml]) at 37°C and 5% CO2. After 24
hr, the unattached cells were removed by washing with PBS.
Canine ADSCs from passages 1–3 were used, and no difference was observed in ADSCs between these passages.
Measurement of proliferation potential: The effects of
VPA or valpromide (VPM; Wako Pure Chemical Ind.,
Osaka, Japan), an analogue of VPA with no HDAC inhibi-

adipose tissue removed was extensively washed with sterile
phosphate-buffered saline (PBS) containing penicillin (100U/ml) and streptomycin (0.25 μg/ml) in order to remove

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PLURIPOTENCY OF ADIPOSE STEM CELLS FROM DOGS

tory activity, on ADSC proliferation were measured usinga 3-(4, 5-dimethyl-thiazol-2-yl)-2, 3-diphenyltetrazolium
bromide (MTT) assay kit (Roche Applied Science, Basel,
Switzerland) according to the manufacturer’s instructions.

17

Reverse transcription-PCR and real-time PCR: Total
RNA was extracted using ISOGEN (NIPPON GENE, Tokyo,
Japan) and reverse-transcribed to single-strand cDNA using
oligo-dT primer and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. PCR
was performed using Taq DNA polymerase (KAPA Biosys-

density of 1 × 104 per well and cultured in growth medium
for 48 hr. VPA or VPM (0–8 mM) was then added to themedium, and cultures were incubated for 3 days. Subsequently, 10 μl of MTT stock solution was added, and theplates were further incubated for 4 hr at 37°C. Diluted HCl(100 μl) was then added to solubilize the formazan crystals,
and the absorbance of each well at 570 nm was measured
with a microplate reader LS-PLATE manager 2004 (Wako
Pure Chemical Ind.); the average of measurements of 6 wells
per sample has been presented.
In vitro differentiation assay: In vitro assay of cell differentiation into adipogenic, osteogenic and neurogenic
lineages was performed as described previously [4, 29, 39]each cycle consisted of the following steps: denaturation for
10 sec at 98°C, annealing for 30 sec at 53–65°C and a 30sec elongation at 72°C (Table 1). Reaction products were
electrophoresed on a 2.0% agarose gel and visualized withethidium bromide. Real-time PCR of the mRNAs for p16,
p21 and GAPDH was performed using an ABI PRISM 7500
Sequence Detection System (Applied Biosystems Japan,
Tokyo, Japan) according to the manufacturer’s instructions.
Analysis of the results was carried out using ABI PRISM
7500 Dissociation Curve Software v 1.0 (Applied Biosystems Japan). The relative amount of mRNA was normalized
to that of GAPDH.
Statistical analysis: Results are expressed as the mean
± standard error. Multiple comparisons were done with theTurkey–Kramer test after one-way analysis of variance. A
p35-mm dishes at a density of 1 × 105 cells per dish. The
cells were incubated on glass coverslips in growth medium
containing 4 mM VPA for 3 days and then transferred toadipogenic induction medium (DMEM supplemented with
10% FBS, 1 μM dexamethasone, 10 μM insulin and 0.5 mM
isobutylmethylxanthine) or to osteogenic induction medium(DMEM supplemented with 10% FBS, 0.1 μM dexamethasone, 50 μM L-ascorbate-2-phosphte and 10 mM glycerophosphate) for 14 days [39] and then to neurogenic induction
medium (DMEM supplemented with100 μM dibutyryl cyclic
adenosine monophosphate and 125 μM isobutylmethylxanthine) for 2 hr [23, 24]. Intracellular lipid accumulation, as
an indicator of adipogenic differentiation, was visualized by

RESULTS
VPA induces acetylation of histone H3 and decreased cellproliferation
acetylation of histone H3, we examined the expression ofcence staining. Minimal HDAC1 and acetylation of histone

calcium deposition. Neurogenic differentiation was assessed

the morphology of ADSCs and increased the expression of
HDAC1 and the acetylation of histone H3 in ADSCs after 3
days of treatment (Fig. 1A and 1B). In contrast, VPM did not

were purchased from Wako Pure Chemical Ind.
: Immunocytochemical
analyses of HDAC1, acetylation of histone H3 (ace H3),
cubated in growth medium for 3 days as described above.
Some cultures were processed for neurogenic induction. At

HDAC1 and histone H3 staining in ADSCs.
liferation of ADSCs in a dose-dependent manner: about 20%(2 mM VPA), 40% (4 mM VPA) and 80% (8 mM VPA) of
the control group (Fig. 1C). However, VPM treatment did
not substantially affect ADSC proliferation. No dead cells
were observed in any VPA-treated groups by phase-contrast
microscopy.
VPA induces upregulation of cyclin-dependent kinase
inhibitors: To assess the effect of VPA on the expression of
cyclin-dependent kinase (CDK) inhibitors, we examined theexpression of these genes by real time-PCR. The expression
levels of p16
treated with 4 mM VPA (4.6 fold vs. control); however, p16
mRNA expression levels did not change in the cells treatedwith 8 mM VPA (2.1 fold vs. control; Fig. 2A). p21 mRNA

3.7% formaldehyde for 15 min at 4°C. After PBS washes,
the cells were permeabilized with 0.2% Triton X-100 for 10
min at room temperature. The cells were then incubated withan anti-HDAC1 antibody (sc-7872, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), an anti-histone H3 (acetyl
K9) antibody (Novus Biologicals, Littleton, CO, U.S.A.),
U.K.) or an anti-NSE antibody (PA1-46203, Thermo ScienAfter further PBS washes, cells were incubated with secondary antibody (Cy3-conjugated goat anti-rabbit IgG or FITCconjugated goat anti-mouse IgG, Jackson ImmunoResearch,West Grove, PA, U.S.A.) for 30 min at room temperature.
The cells were then washed with PBS and counterstainedwith 8 mM VPA (Fig. 2B). The expression levels of p21
mRNA were approximately 2.6 fold (4 mM VPA) and 3.0
fold (8 mM VPA) of that of the control group.

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Y. KURIHAR A ET AL.

Fig. 1. Effects of valproic acid on histone deacetylase 1 expression, histone H3 acetylation and cell proliferation. Canine adipose tissue-derived stem cells (ADSCs) were treated with valproic acid (VPA) or valpromide (VPM). (A) ADSCs were then

HDAC1 (A) and acetylation of histone H3 (B). Scale bar, 50 μm. (C) Cell proliferation was measured by MTT assay andexpressed as percentage of the negative control (DMSO). Data represent the means ± S.E. (% of control) of 4 independent
experiments; each measurement was the average for 6 wells. *P

VPA suppresses adipogenic and osteogenic differentiation: To assess the effect of VPA on the pluripotency of
ADSCs, we investigated whether VPA treatment alters thedifferentiation of ADSCs into adipogenic and osteogenic
lineages using an in vitro differentiation assay. Oil red O
staining revealed that ADSCs that differentiated into theadipogenic lineage accumulated lipid droplets in the cytosol, as compared to undifferentiated cells, which did notaccumulate lipid droplets (Fig. 3A). VPA pretreatment fol-

affected by VPA pretreatment (Fig. 4B).
VPA promotes neurogenic differentiation: We further
examined the effect of VPA on the neurogenic lineage ining revealed that ADSCs that differentiated into neurogenic
cells had typical neuron-like cell protrusions and higher

accumulation of lipid droplets. RT-PCR analysis showed thatthe mRNA expression levels of adipogenic markers, peroxi), fatty acidbinding protein 4 (FABP4) and lipoprotein lipase (LPL) were
elevated by adipogenic induction (Fig. 3B). On the other
hand, VPA pretreatment followed by adipogenic induction
LPL mRNA expression level, inparallel with the decreased accumulation of lipid droplets.
Alizarin red S staining revealed that ADSCs differentiatedinto osteogenic lineage cells with accumulated calcium deposition, as compared with the undifferentiated cells, which
demonstrated no calcium deposition (Fig. 4). VPA pretreat-).
mRNA levels of neurogenic markers, viz., NSE, TUBB3
and microtubule-associated protein 2 (MAP2), were also
elevated by neurogenic induction, but mRNA expression of
the glial cell marker, GFAP, was not observed in any groups(Fig. 5B). Pretreatment with VPA followed by neurogenic
induction increased the expression of NSE, TUBB3 andNEFH), as
MAP2compared to that in the neurogenic induction group. VPA

positive cells also stained for NSE. VPA pretreatment fol-cells, even without neurogenic induction. Furthermore, VPA
elevated the mRNA expression levels of neurogenic markers
in ADSCs with and without neurogenic induction.

calcium deposition (Fig. 4A). mRNA expression levels of
osteogenic markers, viz., bone morphogenetic protein 2(BMP2) and distal-less homeobox 5 (DLX5), were also el-

DISCUSSION-

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PLURIPOTENCY OF ADIPOSE STEM CELLS FROM DOGS

19

ogy of canine ADSCs and markedly induced their expressionof HDAC1 and acetylation of histone H3. In contrast, VPM,
an analogue of VPA with no HDAC inhibitory activity, dideffects on HDAC1 and histone H3, indicating that the H3
acetylation was increased by VPA. These observations supet al. [20]. Thus, our results clearly
indicated that VPA induced H3 acetylation by reducing
HDAC1 activity in canine ADSCs.
pendent decrease in the proliferation of ADSCs, suggestingthat VPA suppresses ADSC proliferation through acetylationof histone H3. p21 and p16, well-known CDK inhibitors,
regulate cell cycle arrest. VPA induces expression of these
CDK inhibitors in human ADSCs and mesenchymal stem
mRNA expression levels of p16 at 4 mM and of p21 at 8mM without inducing cell death. p21 is also a well-known
HDAC-inhibitor responsive gene that is upregulated byhyperacetylation of histones H3 and H4 [10, 20, 28]. In
acetylation was markedly increased by VPA and that p21
viability was further reduced by 8 mM VPA treatment, again
et al. [20] who reported that
VPA causes cell cycle arrest through increased p21 expression in the absence of p16 mRNA expression in human ADSCs. Therefore, the inhibitory effect of VPA on proliferationof canine ADSCs was due to cell cycle arrest, although theunderlying mechanism needs to be further examined.
Furthermore, VPA promoted differentiation of approximately 90% of ADSCs into a neuronal cell lineage after 3
days of treatment. The differentiated cells have neuron-like
Pretreatment with VPA followed by neurogenic induction
also promoted mRNA expression of the neuronal markers
NSE, TUBB3, MAP2 and NEFH as compared to the pretreatment without VPA, suggesting that promotional effects of
VPA on neuronal differentiation of ADSCs were induced byupregulation of these genes. Previous reports have shownthat VPA promotes differentiation of neural stem cells into
neurons in adult rats [12, 15]. To our knowledge, the present
tiation of ADSCs.
Interestingly, VPA pretreatment in the absence of neurogenic induction caused moderate differentiation into neuronlike cells and also increased mRNA expression levels ofneurogenic markers in ADSCs. Thus, our data indicated that
VPA could induce neurogenic differentiation in the absenceof neurogenic induction. We also demonstrated that VPA
increased the acetylation of histone H3 that has been correlated with gene activation [12, 13]; thus, VPA caused gene
expression in part through H3 acetylation. In addition, we
showed that the neuron-like differentiated cells all stained
et al.
[21] recently reported that neuronal differentiation could
modulate gene transcription, translation and post-translational modulation of Ca2+ channels to change the Ca2+ ion

Fig. 2. Effects of valproic acid on cyclin-dependent kinase inhibitor expression. Adipose tissue-derived stem cells (ADSCs) weretreated with valproic acid (VPA) or valpromide (VPM). Total
RNA was extracted from ADSCs after 3 days of treatment with
VPA (4 or 8 mM) or VPM (8 mM). The relative expression of thecyclin-dependent kinase (CDK) inhibitors p16(A) and p21(B)dehydrogenase (GAPDH) was used as an internal standard.
Data are the means ± S.E. of 4–7 independent experiments. a, b:
P<0.05.

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Y. KURIHAR A ET AL.

Fig. 3. Valproic acid suppresses accumulation of lipid droplets. Adipose tissue-derived stem cells (ADSCs) werepretreated with valproic acid (VPA) for 3 days followed by adipogenic induction for 14 days. (A) Adipogenic differentiation was visualized by oil red O staining after 14 days of induction with adipogenic medium. Arrowheads
show cells that accumulated lipid droplets. Scale bar, 50 μm. (B) RT-PCR analysis of adipogenic markers,,
FABP4 and LPL, was performed using total RNA extracted from ADSCs after 14 days of adipogenic induction. AIM,
adipogenic induction medium.

Fig. 4. Valproic acid suppresses calcium deposition. Adipose tissue-derived stem cells (ADSCs) were pretreated withvalproic acid (VPA) for 3 days followed by osteogenic induction for 14 days. (A) Osteogenic differentiation was
evaluated by alizarin red S staining after 14 days of induction with osteogenic medium. Arrowheads show cells that
accumulated calcium in the cytosol. Scale bar, 200 μm. (B) RT-PCR analysis of osteogenic markers, BMP2, Dlx5
and COL1A1, was performed using total RNA extracted from ADSCs after 14 days of osteogenic induction. OIM,
osteogenic induction medium.

currents; our results suggested that neuronal differentiation
could modulate TUBB3 transcription and translation. In thepresent study, VPA promoted ADSCs differentiation into
neuronal cells in the absence of neurogenic induction factors
and induced acetylation of histone H3, indicating that VPA
is a useful tool for studying the interaction between chromatin structure and cell fate determination. Further studies
are needed to examine the molecular mechanism underlying
the neurogenic differentiation induced by VPA using canine

664

ADSCs.
In contrast, VPA suppressed the late stage of differentiation into adipogenic and osteogenic lineage cells. Two of the
3 adipogenic marker genes examined showed no reduction
after VPA pretreatment; however, lipid accumulation appeared to be suppressed, suggesting that the VPA inhibits
accumulation of lipid droplets rather than an inhibiting thewhole adipogenic differentiation process.
Similarly, osteogenic marker genes showed no changes

PLURIPOTENCY OF ADIPOSE STEM CELLS FROM DOGS

21

Fig. 5. Valproic acid promotes neurogenic differentiation. Adipose tissue-derived stem cells (ADSCs) were pretreatedwith valproic acid (VPA) for 3 days followed by neurogenic induction. (A) Neurogenic differentiation was assessed

Scale bar, 50 μm. (B) RT-PCR analysis of the neurogenic markers, NSE, TUBB3, MAP2 and NEFH and the glial
marker, GFAP, was performed using total RNA extracted from ADSCs after 2 hr of neurogenic induction. NIM, neurogenic induction medium.

after VPA treatment, but accumulation of calcium deposition
ing that VPA inhibits calcium deposition rather than inhibiting the osteogenic differentiation process as a whole. The
mechanism underlying the differential effects of VPA on thepluripotent capacity of ADSCs remains unclear. A previous
report has shown that VPA decreases adipogenic and neurogenic differentiation, but increases osteogenic differentiation in human ADSCs [20]. The reason for VPA acting as a
stimulator for differentiation of canine ADSCs and as a suppressor for that of human ADSCs is not immediately clear.
Therefore, chromatin structure and cell fate determinationneed to be further examined in relation to the pluripotency of
ADSCs, including this difference between humans and dogs.
In conclusion, pretreatment with VPA dose-dependently
decreased proliferation of canine ADSCs. In parallel with
its inhibitory effects, VPA increased p16 and p21 mRNA
expression, implying induction of cell cycle arrest throughactivation of p16 and p21. In addition, pretreatment with
VPA followed by adipogenic, osteogenic or neurogenic
induction markedly promoted in vitro neurogenic differentiation, but suppressed accumulation of lipid droplets andcalcium deposition. These in vitro
by pretreatment with VPA were associated with changes in
expression of relevant markers. These results suggested thatcanine ADSCs and is a useful tool for studying the interaction between chromatin structure and cell fate determination.
ACKNOWLEDGMENTS. This work was supported in partby the Science Research Promotion Fund of The Promotionand Mutual Aid Corporation for Private Schools of Japan.
This work was also supported in part by the MEXT Program

665

for the Strategic Research Foundation at Private Universities, 2011–2015.
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Ꮫ఍Ⓨ⾲せ᪨₇⪅ྡ⏣୰࿴᫂, ⁪ἑ㐩ஓ, ᒣᮏᮍဏ, ᒸᮏ᠇᫂, ᓥ಴⚽຾, 㜰ཱྀ㞞ᘯ₇㢟ྡ

࢖ࢾ IL-13 㑇ఏࡢከᆺ࡜ᰘ≟࡟࠾ࡅࡿ࢔ࢺࣆ࣮ᛶ⓶⭵⅖࡜ࡢ㛵㐃ㄪᰝ.

Ꮫ఍ྡ᪥ᮏ␆⏘Ꮫ఍➨ 121 ᅇ኱఍ሙᡤࠊ㛤ദ᪥

ᮾி㒔Ṋⶶ㔝ᕷ 3 ᭶ 2016 ᖺ࠙┠ⓗࠚIL13 ࡣࠊIgE ⏕⏘ㄪ⠇࡟㔜せ࡞ࢧ࢖ࢺ࢝࢖࡛ࣥ࠶ࡿ㸬ᮏ◊✲࡛ࡣࠊ࢖ࢾ IL13 㑇ఏᏊࡢከᆺ᳨⣴࡜⑌⑓࡜ࡢ┦㛵ゎᯒࢆ⾜ࡗࡓ㸬࠙ᐇ㦂᪉ἲࠚ
15 ≟ ✀ 34 ಶ య ࡢ IL13 㑇 ఏ Ꮚ ࡢ ሷ ᇶ 㓄 ิ ࢆ Ỵ ᐃ ࡋ ࡓ 㸬 ࡲ ࡓ IL13 㑇 ఏ Ꮚ ୖ ࡢ
rs22147008(p.63 Thr>Ala)࡜ rs8973298(5v㞄᥋㡿ᇦ)࡟ࡘ࠸࡚ࠊ࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࢆⓎ⑕ࡋࡓᰘ≟ 10 ಶయ࡜ࠊ⓶⭵⅖ࡀㄆࡵࡽࢀ࡞࠸ᰘ≟ 33 ಶయࢆ⏝࠸ࡓ⑕౛ᑐ↷ゎᯒࢆ⾜ࡗࡓ㸬࠙⤖ᯝࠚ
NC_006593 ࢆᇶ‽࡜ࡋ࡚ࠊg.20957075 AGGTGGGCA:[1]>[2]⦪ิ཯᚟ከᆺ࡜ 15 ࢝ᡤࡢ
SNP ࢆ᳨ฟࡋࡓ㸬ᰘ≟ࢆ⏝࠸ࡓ⑕౛ᑐ↷ゎᯒ࡛ࡣࠊrs22147008 ࡛ࡣ G ᆺᑐ❧㑇ఏᏊ㢖ᗘࡣࠊ⑕౛⩌࡛ 0.05ࠊᑐ↷⩌࡛ 0.11 ࡛࠶ࡾ᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ㸬ࡲࡓ G ᆺᑐ❧㑇ఏᏊࢆᣢࡘಶయࡣ඲࡚࣊ࢸࣟ᥋ྜయ࡛࠶ࡗࡓ㸬rs8973298 ࡢ㑇ఏᏊᆺࡣࠊᝈ␆⩌࡛ࡣ CC ᆺ 4
ಶయࠊCT ᆺ 3 ಶయࠊTT ᆺ 3 ಶయ࡛࠶ࡗࡓࡢ࡟ᑐࡋ࡚ࠊ㠀ᝈ␆⩌࡛ࡣ CC ᆺ 4 ಶయࠊCT
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ࡼࡿ࢝࢖஧஌್ࡀ 3.939㸦p=0.047㸧࡛᭷ព࡟␗࡞ࡗ࡚࠸ࡓ㸬ࡇࡢ᫬ࠊC ᆺᑐ❧㑇ఏᏊࡢ࣍ࣔ᥋ྜ㸦CC ᆺ㸧ࢆࠊ࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࡢࣜࢫࢡᆺ࡜᝿ᐃࡍࡿ࡜࢜ࢵࢬẚࡀ 4.833㸦95㸣ಙ㢗༊㛫 1.018㹼23.455㸧࡛࠶ࡗࡓ㸬ࡇࢀ࡟ࡼࡾࠊᰘ≟࡛ࡣ rs8973298 ࡢ C ࡢ࣍ࣔ᥋ྜᆺࡣࠊ
࢔ࢺࣆ࣮ᛶ⓶⭵⅖ࡢⓎ⑕ࣜࢫࢡࢆ㧗ࡵࡿࡇ࡜ࡀ♧၀ࡉࢀࡓ㸬675

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୰㛫ሗ࿌఍࣭㐍ᤖ≧ἣホ౯᭩እ㒊ホ౯ጤဨ

ᮾி኱Ꮫ኱Ꮫ㝔㎰Ꮫ⏕࿨⛉Ꮫ◊✲⛉ᩍᤵ㎷ᮏ ඖඛ⏕໭ᾏ㐨኱Ꮫ኱Ꮫ㝔⋇་Ꮫ◊✲⛉ᩍᤵ✄ⴥ ╬ඛ⏕㓗㎰Ꮫᅬ኱Ꮫᩍᤵ໭ᮧ ᾈඛ⏕㛤ദ᪥ ᖹᡂ㸰㸴ᖺ㸴᭶㸰㸶᪥㸦ᅵ㸧676

㯞ᕸ኱Ꮫ ◊✲䝥䝻䝆䜵䜽䝖㐍ᤖ≧ἣホ౯᭩ᮾி኱Ꮫ኱Ꮫ㝔㎰Ꮫ⏕࿨⛉Ꮫ◊✲⛉㎷ᮏඖ⚾❧኱Ꮫᡓ␎ⓗ◊✲ᇶ┙ᙧᡂᨭ᥼஦ᴗ࡟ࡼࡿ◊✲ࣉࣟࢪ࢙ࢡࢺࠕ≟ࡢ㑇ఏᛶ⑌ᝈ࡟࠾
ࡅࡿཎᅉ㑇ఏᏊゎᯒࡢࡓࡵࡢ㑇ఏᏊ(DNA)ࣂࣥࢡᣐⅬᙧᡂࠖࡢ㐍ᤖ≧ἣ࡟㛵ࡋ࡚ࠊ◊✲㐍ᤖ≧ἣሗ࿌᭩࠾ࡼࡧ 2014 ᖺ 6 ᭶ 28 ᪥࡟㛤ദࡉࢀࡓ◊✲⪅࡟ࡼࡿࣉࣞࢮࣥࢸ࣮ࢩࣙ
ࣥࢆ㏻ࡋ࡚ࠊࡑࡢෆᐜࢆホ౯ࡍࡿᶵ఍ࢆᚓࡓࡢ࡛ሗ࿌ࡉࡏ࡚࠸ࡓࡔࡃࠋ㻔㻝㻕 ◊✲䝥䝻䝆䜵䜽䝖䛾ᇶᮏⓗ⌮ᛕ䛻䛴䛔䛶⮫ᗋ⋇་Ꮫ࡟࠾࠸࡚ࡣࠊ2000 ᖺ௨㝆࡟࡞ࡗ࡚ᛴ㏿࡟㐍Ṍࡋࡓศ㔝ࡢ୍ࡘ࡜ࡋ࡚ࠊ⑓
⌮Ⓨ⏕࡟㛵ࡍࡿศᏊ⏕≀Ꮫⓗ◊✲ࡀ࠶ࡿࠋ2003 ᖺ࡟࢖ࢾࡢ඲ࢤࣀ࣒ࡀⓎ⾲ࡉࢀࡓࡇ࡜
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ࢾࡢ DNA ࣂࣥࢡసࡾࡀጞࡵࡽࢀ࡚࠸ࡿࡼ࠺࡟ࠊࣄࢺ࡜ྠᵝࡢ࡛ࣞ࣋ࣝࢤࣀ࣒ゎᯒࡀྍ⬟࡜࡞ࡗࡓ࢖ࢾࡢ DNA ࢧࣥࣉࣝࡣࠊ⑓Ẽࡢデ᩿ࠊ἞⒪ࠊ࠾ࡼࡧண㜵࡟㛵ࡍࡿ◊✲࡟࡜ࡗ࡚ࡁࢃࡵ࡚㈗㔜࡞◊✲㈨※࡜࡞ࡿࠋ
࢖ࢾ DNA ࣂࣥࢡసࡾ࡟㛵ࡋ࡚ࡣࠊ᪥ᮏࡢᅜᐙⓗࣉࣟࢪ࢙ࢡࢺ࡜ࡋ࡚㐍ࡵࡿ࡭ࡁࡶࡢ
࡜ᛮࢃࢀࡿࡀࠊ⌧᫬Ⅼ࡟࠾࠸࡚ࡣᮏ◊✲ࣉࣟࢪ࢙ࢡࢺࡀࡑࡢᣐⅬ࡜࡞ࡗ࡚ᑗ᮶ࡢࡼࡾ኱
ᆺࡢࣂࣥࢡࡢ᰾࡟࡞ࡿࡇ࡜ࢆᮇᚅࡍࡿࠋࡇࡢࡼ࠺࡞ほⅬ࠿ࡽࠊᮏ◊✲ࣉࣟࢪ࢙ࢡࢺࡢᖺ
㝈㸦ᖹᡂ 23~27 ᖺᗘ㸧ࡢᚋࡢᒎ㛤࡟ࡘ࠸࡚ࠊ࠶ࡽ࠿ࡌࡵࣉࣛࢽࣥࢢࡋ࡚࠾ࡃᚲせࡀ࠶ࡿࠋ㻔㻞㻕 ◊✲䝥䝻䝆䜵䜽䝖䛾యไᵓ⠏䛻䛴䛔䛶ࢧࣥࣉࣝ཰㞟ࠊ⑕౛ಶయ᝟ሗ཰㞟ࠊ࠾ࡼࡧࡑࡢ⟶⌮࡟ࡘ࠸࡚ࠊ⡆᫆ᛶ࠾ࡼࡧỗ⏝ᛶࢆවࡡഛ࠼ࡓࢩࢫࢸ࣒ࢆᵓ⠏ࡋ࡚࠾ࡽࢀࠊࡇࢀࡲ࡛ࡢດຊࢆ㧗ࡃホ౯ࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋ
ࣄࢺࡢ㑇ఏᏊ(DNA)ࣂࣥࢡᙧᡂࡢࣀ࢘ࣁ࢘ࢆ✚ᴟⓗ࡟ᑟධࡋ࡚࠸ࡿࡇ࡜ࡶࠊከᩘࢧࣥࣉ
ࣝࡢಖᏑ⟶⌮ࡢຠ⋡໬࡟ᙺ❧ࡗ࡚࠸ࡿࡶࡢ࡜⪃࠼ࡽࢀࡿࠋ
ᖹᡂ 23 ᖺᗘ࠿ࡽ⌧ᅾࡲ࡛ࡢయไᵓ⠏࡟ࡼࡾࠊ࢖ࢾ㑇ఏᏊ(DNA)ࣂࣥࢡࡢᇶᮏⓗ࡞యไࡣࡍ࡛࡟ᩚࡗ࡚࠸ࡿࠋ௒ᚋࡢࢧࣥࣉࣝᩘቑຍࢆ⪃៖ࡋࠊ᪋タࠊタഛࠊேဨࠊ࠾ࡼࡧಖ
Ꮡ⟶⌮ࢩࢫࢸ࣒ࡢ඘ᐇࢆᅗࡿ࡭ࡁ࡛࠶ࡿࠋ㻔㻟㻕 㑇ఏᏊ㻔㻰㻺㻭㻕䝞䞁䜽䜈䛾䝃䞁䝥䝹཰㞟䛻䛴䛔䛶ࢧࣥࣉࣝ཰㞟࡟㛵ࡋ࡚ࡣࠊ㯞ᕸ኱Ꮫ㝃ᒓື≀⑓㝔࡟ཷデࡋࡓ⑕౛࠿ࡽᚓࡽࢀࡓࡶࡢࡀ

677

୰ᚰ࡜࡞ࡗ࡚࠸ࡿࡀࠊ㐃ᦠࡢ௚኱Ꮫ࡜ࡢ༠ຊࢆࡉࡽ࡟㐍ࡵࡿࡇ࡜ࡀᮏࣉࣟࢪ࢙ࢡࢺᡂຌ
ࡢ㘽࡜࡞ࡿࠋࡲࡓࠊ᭱㏆࡛ࡣ኱Ꮫ㝃ᒓື≀⑓㝔࡜ྠ➼࠶ࡿ࠸ࡣࡑࢀࡼࡾࡶ⑕౛ᩘࡢከ࠸஧ḟ⋇་⒪ᶵ㛵ࡸ⥲ྜⓗ㛤ᴗື≀⑓㝔ࡀቑ࠼࡚࠾ࡾࠊࡑࢀࡽデ⒪ᶵ㛵࠿ࡽࡢࢧࣥࣉࣝ཰
㞟࡟㛵ࡍࡿ᳨ウࡶᮃࡲࢀࡿࠋ㻔㻠㻕 㑇ఏᏊ㻔㻰㻺㻭㻕䝞䞁䜽䛻཰㞟䛥䜜䛯䝃䞁䝥䝹䜢฼⏝䛧䛯◊✲䛻䛴䛔䛶㻢 ᭶ 㻞㻤 ᪥㛤ദ䛾◊✲⪅䛻䜘䜛䝥䝺䝊䞁䝔䞊䝅䝵䞁䛷䛿䚸ྛศ㔝䛻䜟䛯䛳䛶 㻰㻺㻭 䝞䞁䜽䛻཰㞟䛥䜜䛯䝃䞁䝥䝹䜢฼⏝䛧䛯◊✲ᡂᯝ䛜Ⓨ⾲䛥䜜䛯䚹ㄢ㢟䛻䜘䛳䛶䛭䛾㐍ᤖ≧ἣ䛿䛥䜎䛦䜎䛷䛒䛳䛯䛜䚸䛂≟䛾♫఍ᛶ䜢ྖ䜛㑇ఏᏊ䛾᥈⣴䛃䠄⳥Ỉ೺ྐඛ⏕䠅䛻㛵䛩䜛◊✲Ⓨ⾲䛷䛿䚸䛸䛟䛻ඃ䜜䛯ᡂᯝ䛜ሗ࿌䛥䜜䛶䛔䛯䚹ᮏ㑇ఏᏊ㻔㻰㻺㻭㻕䝞䞁䜽䜈䛾䝃䞁䝥䝹཰㞟䜢ಁ㐍䛩䜛䛯䜑䛻䛿䚸䛭䜜䛻䜘䛳䛶ᚓ䜙䜜䛯◊✲ᡂᯝ䛸ᮏ䝞䞁䜽䛸䛾䛴䛺䛜䜚䜢᫂☜䛻䛧䚸䝩䞊䝮䝨䞊䝆➼䛷䜰䝘䜴䞁䝇䛩䜛䛣䛸䛜ᮃ䜎䜜䜛䚹ᮏ◊✲ࣉࣟࢪ࢙ࢡࢺ࡛ࡣࠊ◊✲௦⾲⪅ࡢ㜰ཱྀ㞞ᘯඛ⏕ࡢ࣮ࣜࢲ࣮ࢩࢵࣉࡢࡶ࡜࡟⢭ຊⓗ࡟◊✲ࡀ㐍ࡵࡽࢀ࡚࠾ࡾࠊᙜึィ⏬ࡢࣉࣟࢪ࢙ࢡࢺ࡟ࡼࡿᡂᯝࡢከࡃࡀࡍ࡛࡟ሗ࿌ࡉ
ࢀ࡚࠸ࡿࠋ௒ᚋࠊᑗ᮶࡟ྥࡅࡓ㛗ᮇⓗ࡞ࣅࢪࣙࣥࢆᣢࡗ࡚ᮏࣉࣟࢪ࢙ࢡࢺࡀ㐙⾜ࡉࢀࡿࡇ࡜ࢆᙉࡃᮃࡴࠋ

678

㯞ᕸ኱Ꮫ

◊✲ࣉࣟࢪ࢙ࢡࢺ㐍ᤖ≧ἣホ౯᭩໭ᾏ㐨኱Ꮫ኱Ꮫ㝔⋇་Ꮫ◊✲⛉✄ ⴥ 
╬㸯㸬ࡣࡌࡵ࡟
ࠕ≟ࡢ㑇ఏᛶ⑌ᝈ࡟࠾ࡅࡿཎᅉ㑇ఏᏊゎᯒࡢࡓࡵࡢ㑇ఏᏊ '1$ ࣂࣥࢡᣐⅬᙧᡂࠖ࡟ࡘ࠸࡚ࠊࠕ◊✲㐍ᤖ≧ἣሗ࿌᭩ࠖ࡞ࡽࡧ࡟◊✲ᡂᯝⓎ⾲఍㸦㸴᭶㸰㸶᪥㛤ദ㸧࡟࠾ࡅࡿཱྀ㢌Ⓨ⾲࣭㉁␲ᛂ⟅ࢆࡶ࡜࡟ࠊ㸯㸧㐍ᤖ≧ἣ࣭◊✲ᡂᯝ➼ࠊ㸰㸧◊✲㈝ࡢຠ⋡ᛶ࣭ጇᙜᛶࠊ㸱㸧┠ⓗ㐩ᡂࡢྍ⬟ᛶࠊ㸲㸧◊✲ࡢⓎᒎᛶࡢほⅬ࠿ࡽホ౯ࢆ⾜࠸ࠊ௨ୗࡢ࡜࠾ࡾሗ
࿌ࡍࡿࠋ㸰㸬㐍ᤖ≧ἣ࣭◊✲ᡂᯝ➼
㸯㸧'1$ ヨᩱࡢ㞟✚㸸࡯ࡰィ⏬㏻ࡾ࡟ヨᩱࡢ཰㞟࣭ಖᏑࡀ㐍ࢇ࡛࠸ࡿࠋࡓࡔࡋࠊ≟ࡢ⑌ᝈ㸦≉ᐃ㑇ఏᛶ⑌ᝈࢆྵࡵ࡚㸧ࡢ㑇ఏⓗ⫼ᬒࢆ௒ᚋ⥙⨶ⓗ࡟ゎᯒࡍࡿሙྜ࡜ࠊ≉ᐃ⑌ᝈࡢゎᯒ࡟㔜Ⅼࢆ⨨ࡃሙྜ࡜࡛ࡣ㸦࠶ࡿ࠸ࡣ୧⪅㸧ࠊヨᩱࡢ཰㞟ᑐ㇟㸭᪉ἲࡀ⮬ࡎ࡜␗࡞ࡿ࠿ࡽࠊࡇࡢⅬࢆ᫂☜࡟ࡋࡓᕤኵࡀᚲせ࠿ࡶࡋࢀ࡞࠸ࠋィ⏬᫬ࡢ⑌ᝈヨᩱࡀ୙㊊ࡋ࡚㐍⾜ࡀ㐜ࢀ࡚࠸ࡿࡶࡢ࡟ࡘ࠸࡚ࡣ≉࡟ఱࡽ࠿ࡢᑐฎࡀᚲせ࡛࠶ࢁ࠺ࠋ
㸰㸧ᑐ㇟⑌ᝈ㸸⣙㸯㸮ࡢ≉ᐃ⑌ᝈࢆᑐ㇟࡟ࠊࡑࢀࡒࢀ࡯ࡰᕤ⛬⾲࡟ἢࡗࡓ '1$ ࡢゎᯒࡀ㐍ࡵࡽࢀ࡚࠸ࡿࠋ'1$ ཰㞟࡟࠶ࡓࡗ࡚ࡣࠊ㐺ษ࣭඘ศ࡛ಙ㢗ᛶࡢ㧗࠸⮫ᗋࢹ࣮ࢱ㸭᳨ᰝࢹ࣮ࢱࡢ㞟✚࣭↷ྜࡀ୙ྍḞ࡛࠶ࡿࡀࠊࡇࡢⅬ࡟ࡘ࠸࡚ࡸࡸ୙㩭࡛᫂࠶ࡗࡓࠋ඘ศ
࡟⪃៖ࡢࡇ࡜࡜ࡣᛮ࠺ࡀࠊᮏࣉࣟࢪ࢙ࢡࢺ࡟࠾ࡅࡿ᭱ࡶ㔜せ࡞せ⣲࡜ゝࡗ࡚㐣ゝ࡛ࡣ࡞ࡃࠊྍ⬟࡞㝈ࡾ୍ᐃෆᐜ㸭ᇶ‽࡛ࡢࢹ࣮ࢱ࣮࣋ࢫࡢᵓ⠏࡜฼⏝ࡀᮃࡲࢀࡿࠋ ࡲࡓࠊࡇࢀࡽᑐ㇟⑌ᝈࡢ㑇ఏᵝᘧࡸ༢୍㑇ఏᏊ⑌ᝈ㸭ከᅉᏊ⑌ᝈࡢูࡀᚲࡎࡋࡶ᫂
☜࡛࡞࠸⑌ᝈࡀ࠶ࡗࡓࡼ࠺࡟ᛮࢃࢀࡿࠋࡘࡲࡾࢱ࣮ࢤࢵࢺࡀ⑌ᝈࡢࠕཎᅉ㑇ఏᏊࠖ࡞ࡢ࠿ࠊࠕࣜࢫࢡࣇ࢓ࢡࢱ࣮㸦ឤཷᛶ㑇ఏᏊ㸧ࠖ࡞ࡢ࠿ࠊ࠶ࡿ࠸ࡣ୙᫂࡞ࡢ࠿ࠊࡇࢀࢆࢡ
ࣜ࢔࡟ᢕᥱࡋ࡚◊✲࡟࠶ࡓࡿ࡭ࡁ࡛࠶ࢁ࠺ࠋ
㸱㸧ཎᅉ㑇ఏᏊ␗ᖖࡢゎᯒ㸸⥳ෆ㞀ࠊⓑෆ㞀ࠊ࢔ࣟ࣌ࢩ࢔ ; ➼ࠊ࠸ࡃࡘ࠿ࡢ⑌ᝈ࡟ࡘ࠸
࡚ཎᅉ㑇ఏᏊ␗ᖖࡢೃ⿵࡜࡞ࡿኚ␗ࡀぢฟࡉࢀࠊᡂᯝࡣㄽᩥ㸰⦅࡜㸴௳ࡢᏛ఍Ⓨ⾲➼࡟࡚බ⾲ࡉࢀ࡚࠾ࡾࠊ⌧ẁ㝵࡛ࡣ࡯ࡰ㡰ㄪ࡟⤒㐣ࡋ࡚࠸ࡿ࡜࠸࠼ࡿࠋࡓࡔࡋࠊ⌧≧࡛ࡣఱࢀࡢሙྜࡶ㑇ఏᏊኚ␗࡜Ⓨ⑕࣭⑓ែࡢᅉᯝ㛵ಀࡣᚲࡎࡋࡶ᫂☜࡛ࡣ࡞࠸ࠋ௒ᚋࠊ
ࠕ㑇ఏᏊᆺп⑓ែ⾲⌧ᆺ㛵㐃ࡢ᫂☜໬ࠖ࡜ࠕ㑇ఏᏊ⏘≀ᶵ⬟ࡢゎ᫂ЍⓎ⑕ᶵᵓゎ᫂ࠖࡀ㐍ᒎࡍࡿࡇ࡜ࢆᮇᚅࡋࡓ࠸ࠋ౛࠼ࡤᨷᧁᛶ㸭♫఍ㄆ▱࡞࡝࡟ࡘ࠸࡚ࡣࠊ㞧✀⣔⤫ࡢゎᯒࡀ◊✲ࡢຠ⋡໬࡟᭷⏝࡜ᛮࢃࢀࠊࣂࣥࢡᵓ⠏ࡢ඲యィ⏬࡟ࡶ୍⪃ࢆせࡍ࠿ࡶࡋࢀ
࡞࠸ࠋ௒ᚋࠊཧຍ◊✲⪅㛫ࠊ≉࡟ᇶ♏⣔࡜⮫ᗋ⣔◊✲⪅㛫ࡢ㆟ㄽࠊ㐃ᦠࢆ୍ᒙᙉ໬ࡋ
࡚௒ᚋࡢゎᯒ࡟࠶ࡓࡿࡇ࡜ࡀᮃࡲࢀࡿࠋ㸱㸬◊✲㈝ࡢຠ⋡ᛶ࣭ጇᙜᛶ
࣭ࡇࡢ✀ࡢ◊✲࡛ࡣࠊヨᩱ࡜ࢹ࣮ࢱ࣮࣋ࢫࡢ཰㞟㸭⥔ᣢ⟶⌮ࠊ'1$ ゎᯒ࡟┦ᙜ࡞⤒㈝ࢆ
せࡍࡿࠋᖺ㛫  ༓෇ࡢ⤒㈝࡜ෆヂࡣጇᙜ࡛࠶ࡾࠊ⣙㸯㸮⑌ᝈࢆᑐ㇟࡜ࡋࡓゎᯒࢆᐇ᪋ࡋ࡚࠸ࡿࡇ࡜࡟㚷ࡳࢀࡤຠ⋡ᛶࡣⰋ࠸࡜ホ౯࡛ࡁࡼ࠺ࠋࡴࡋࢁࠊ௒ᚋࠊᶵ⬟ゎ

679

ᯒࢆ୪⾜ࡋ࡚඘ศ࡟ᐇ᪋ࡍࡿࡓࡵࡢ㈨㔠⋓ᚓࡀせồࡉࢀࡿ࡛࠶ࢁ࠺ࠋ
㸲㸬◊✲ࡢⓎᒎᛶ࣭ᮏࣉࣟࢪ࢙ࢡࢺࡣࠊ⮫ᗋ⋇་Ꮫ࡟Ṇࡲࡽࡎࠊᇶ♏⋇་Ꮫ࣭ື≀་⛉Ꮫࠊ⏕࿨⛉Ꮫࠊ་Ꮫࠊ♫఍⛉Ꮫ➼ࠊከᵝ࡞ศ㔝࡬ࡢᙳ㡪ࡀ኱࡛࠶ࡿࠋࡍ࡞ࢃࡕࠊ≟⑌ᝈⓎ⑕ࡢデ᩿ἲ㛤Ⓨࡸண㜵࣭ไᚚࡣࡶࡕࢁࢇࠊཎᅉ㑇ఏᏊ㸭㛵㐃㑇ఏᏊࡢᶵ⬟ゎ᫂ࢆ㏻ࡋ࡚ࡢᇶ♏⏕࿨⛉Ꮫ࡬ࡢ㈉⊩ࠊࣄࢺ⑌ᝈࣔࢹࣝື≀ࡢ㛤Ⓨ㸭἞⒪ἲࡢ㛤Ⓨ࡞࡝ࠊከᵝ࡞Ⓨᒎᛶ࡜⛉Ꮫ࡜ࡋ࡚ࡢ኱ࡁ࡞౯್ࢆ᭷ࡍࡿࠋࡑࢀࡔࡅ࡟ࠊࣂࣥࢡࡢᵓ⠏࠿ࡽᙜ㠃ࡢࢱ࣮ࢤࢵࢺ⑌ᝈ
ཎᅉ㑇ఏᏊࡢ᫂☜࡞ྠᐃ࡟⮳ࡿィ⏬ࡀ☜ᐇ࡞ಙ㢗ᛶࢆࡶࡗ࡚㐩ᡂࡉࢀࡿࡇ࡜ࢆᮇᚅ
ࡍࡿࡶࡢ࡛࠶ࡿࠋ㸳㸬⥲ホ࣭ᮏࣉࣟࢪ࢙ࢡࢺࡣࠊ᪥ᮏ࡛ࡣ᭱ึࡢẚ㍑ⓗ኱つᶍ࡞≟ࡢࣂ࢖࢜ࣂࣥࢡィ⏬࡛࠶ࡾࠊࡑ
ࡢព⩏ࡣ኱ࡁ࠸ࠋ⌧᫬Ⅼࡢ㐍ᤖ≧ἣࡣࠊ'1$ ヨᩱࡢ཰㞟࣭ಖᏑࠊࡑࢀࢆ⏝࠸࡚ࡢ≉ᐃ⑌ᝈཎᅉ㑇ఏᏊࡢゎᯒ࡜ࡶ࡟ࠊ࡯ࡰᖺᗘィ⏬ᕤ⛬⾲࡟‽ࡌ࡚࠸ࡿࠋୖグᣦ᦬ࢆྵࡵࠊᚲせ࡟ᛂࡌ࡚᪂ࡓ࡞ཧຍ࣭㐃ᦠ◊✲⪅ࢆືဨࡍࡿ࡞࡝࡟ࡼࡾᡤᐃࡢ┠ⓗࢆ㐩ᡂ࡛ࡁࠊࡲࡓ⋇་Ꮫࠊ་Ꮫࠊ⏕࿨⛉Ꮫ➼࡟኱ࡁ࡞Ἴཬຠᯝࢆࡶࡓࡽࡍࡇ࡜ࡀᮇᚅ࡛ࡁࡿࠋ
࣭ᮏࣉࣟࢪ࢙ࢡࢺࡢ⥅⥆࡟࠶ࡓࡗ࡚ࡣࠊ㑇ఏᏊゎᯒ◊✲ࡢ☜ᐇ࡞㐍ᒎࡣࡶ࡜ࡼࡾࠊ'1$ࣂࣥࢡࡢຠᯝⓗ࣭㐺ṇ࡞฼⏝➼࡟ࡘ࠸࡚ࡶ඘ศ࡞⪃៖ࡀ⾜ࢃࢀࡿࡇ࡜ࡀᮃࡲࡋ࠸ࠋ

680

㯞ᕸ኱Ꮫ

◊✲ࣉࣟࢪ࢙ࢡࢺ㐍ᤖ≧ἣホ౯᭩                                     㓗㎰Ꮫᅬ኱Ꮫᩍᤵ                   ໭ᮧ ᾈ⚾❧኱Ꮫᡓ␎ⓗ◊✲ᇶ┙ᙧᡂᨭ᥼஦ᴗࠕ≟ࡢ㑇ఏᛶ⑌ᝈ࡟࠾ࡅࡿཎᅉ㑇ఏᏊゎᯒࡢࡓࡵࡢ㑇ఏᏊ㸦DNA㸧ࣂࣥࢡࡢᣐⅬᙧᡂࠖ◊✲ࣉࣟࢪ࢙ࢡࢺࡢ㐍ᤖ≧ἣࢆ௨ୗࡢࡼ࠺࡟ホ౯ࡍࡿࠋ㸯㸧࢖ࢾ DNA ࣂࣥࢡ࡟㛵ࡋ࡚ᮏࣉࣟࢪ࢙ࢡࢺࡢ᭱኱ࡢ┠ᶆࡣ᭷⏝࡞࢖ࢾ DNA ࣂࣥࢡࡢ๰ᡂ࡛࠶ࡿࠋᮏࣉࣟࢪ࢙ࢡ
ࢺ࡛ࡣࡇࢀࡲ࡛࡟ 9,000 ᳨య⛬ᗘࡢ DNA ࡀ᥇ྲྀࡉࢀࡓࠋᙜึணᐃࡉࢀࡓ᳨యࡢ⣙༙ᩘ࡟Ṇࡲࡗࡓࡀࠊᅜෆ࡛ึࡵ࡚࢖ࢾ DNA ⮫ᗋࢧࣥࣉࣝࡢ DNA ࣂࣥࢡ໬࡟╔ᡭࡋࠊ1 ୓
᳨య㏆ࡃࡢࢧࣥࣉࣝ࡜᝟ሗࢆ⵳✚࡛ࡁࡓࡇ࡜ࡣホ౯࡟್ࡍࡿࠋࡲࡓࠊࣉࣟࢪ࢙ࢡࢺ㐙⾜࡟ᙜࡓࡾࠊ௚኱Ꮫ࡜ࢧࣥࣉࣝࡸ᝟ሗࢆඹ᭷ࡍࡿィ⏬ࡀ㐍ࢇ࡛࠸ࡿࡇ࡜ࡶホ౯࡛ࡁࡿࠋ
୍᪉࡛ࠊᮏࣉࣟࢪ࢙ࢡࢺᡂᯝࡢ౯್ࡣ㞟✚࡛ࡁࡓ᳨యᩘ࡟኱ࡁࡃ౫Ꮡࡋ࡚࠸ࡿࡇ࡜࠿
ࡽࠊࡼࡾ୍ᒙࠊ཰㞟ࢧࣥࣉࣝᩘࢆቑࡸࡍດຊࡀᚲせ࡛࠶ࡿࠋ௒ᅇࠊࢧࣥࣉࣝࡢෆヂࢆࡳࡿ࡜ࠊᅽಽⓗ࡟║⑌ᝈ࡟೫ࡗ࡚࠸ࡓࠋࡇࡢࡇ࡜ࡣཧ⏬ࡋࡓ◊✲⪅㛫ࡢᗘᕪࢆ཯ᫎࡋ࡚

࠸ࡿࠋ║⑌ᝈࢧࣥࣉࣝࡢ඘ᐇࢆࣂࣥࢡࡢ≉Ⰽ࡜ࡋ࡚⏕࠿ࡍ࠿ࠊࡼࡾᗈ⠊ᅖ࡞᝟ሗࢆ㞟✚ࡍࡿ࠿ࡣุ᩿ࡢศ࠿ࢀࡿ࡜ࡇࢁ࡛࠶ࢁ࠺ࠋࡓࡔࠊᅜᐙⓗᒎᮃ࡟❧ࡗࡓᣐⅬᙧᡂ࡜࠸࠺Ⅼࢆ㚷ࡳࡿ࡜ࠊ⌧ᅾ༠ຊᰯ࡟ྵࡲࢀ࡚࠸࡞࠸኱Ꮫࡶྵࡵࠊ௚኱Ꮫ࠿ࡽࡢࢧࣥࣉࣝཷࡅධࢀ
ࢆᙉ໬ࡍࡿ࡜ඹ࡟ࠊ࢖ࣥࣇ࢛࣮࣒ࢻࢥࣥࢭࣥࢺࢆὙ⦎໬ࡍࡿ࡞࡝ࢧࣥࣉࣝ཰㞟ࡢᕤኵࡀᚲせ࠿ࡶࡋࢀ࡞࠸ࠋ
DNA ࣂࣥࢡ࡟࠾࠸࡚ࠊDNA ࢧࣥࣉࣝ࡟௜㝶ࡍࡿ᝟ሗ㈨※ࡢ඘ᐇࡣࠊࢧࣥࣉࣝ⮬యࡢ
඘ᐇ࡜୧㍯ࢆ࡞ࡍࠋDNA ࣂࣥࢡࡢࢧࣥࣉࣝ᝟ሗ࡟ࡣヲ⣽࡞⮫ᗋࢹ࣮ࢱ࡟ຍ࠼ࠊᬑ㐢ᛶ࣭ᐈほᛶࡀồࡵࡽࢀࡿࠋⓏ㘓⏝ࡢ㟁Ꮚࣇ࢓࢖ࣝࢆసᡂࡋࠊࡑࢀࢆᇶ࡟ࢹ࣮ࢱ࣮࣋ࢫࡢ᝟ሗ
ࢆ᭦᪂ࡍࡿ௙⤌ࡳࡀฟ᮶ୖࡀࡗࡓࡇ࡜ࡣホ౯࡛ࡁࡿࠋ୍᪉࡛ DNA ࣂࣥࢡࡢᬑཬࢆど㔝࡟ධࢀࡿ࡜᝟ሗࢆࣈࣛ࢘ࢬࡍࡿᶵ⬟ࡶᕤኵࡍࡿᚲせࡀ࠶ࡿࠋࡲࡓᾏእࡢ᪤࡟බ㛤ࡉࢀ࡚࠸ࡿࣉࣟࢪ࢙ࢡࢺࢹ࣮ࢱ࡜ዴఱ࡟᝟ሗ஫᥮ᛶࢆᣢࡓࡏࡿ࠿ࠊࡑࡋ࡚⮫ᗋ⋇་ᖌࡀ┿࡟ồࡵࡿ᝟ሗ࡜ࡋ࡚බ㛤࡛ࡁࡿ࠿ࡀ௒ᚋࡢ኱ࡁ࡞ㄢ㢟࡜ゝ࠼ࡼ࠺ࠋ᝟ሗࢩ࣮ࢬࢆ౯್ࡢ㧗࠸㈨※࡟ᡂ㛗ࡉࡏࡿ࡟ࡣࠊ͆᪪࡞᫬ᮇ͇࡟᝟ሗࢆ୍⯡බ㛤ࡍࡿࡇ࡜ࡶ኱ษ࡜⪃࠼ࡿࠋྠᵝࡢࣉࣟࢪ࢙ࢡࢺࡀࡲࡔᑡ࡞࠸⌧ẁ㝵࡛ࠊẁ㝵ⓗ࡟࡛ࡶᡂᯝࢆ
බ㛤ࡋࠊࢹ࣮ࢱ࣮࣋ࢫ฼⏝⪅ࢆቑࡸࡍࡇ࡜ࡀࠊᮏ DNA ࣂࣥࢡࡢㄆ▱ᗘࢆ㧗ࡵࠊᡂᯝࡢ
Ἴཬᛶࢆ㧗ࡵࡿࡇ࡜࡟⧅ࡀࡿ࡜⪃࠼ࡿࠋ

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SNP
DNA
SNP
SNP
SNP

cause-and-effect
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DNA
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