5-10 GeV Neutrinos from Gamma-Ray Burst Fireballs
组蛋白脱乙酰酶抑制剂使癌细胞对表皮生长因子抑制剂敏感[发明专利]
专利名称:组蛋白脱乙酰酶抑制剂使癌细胞对表皮生长因子抑制剂敏感
专利类型:发明专利
发明人:萨米尔·E·威塔,小保罗·A·邦恩,哈里·A·德拉布金,罗伯特·M·格米尔,丹尼尔·钱
申请号:CN200680016308.9
申请日:20060313
公开号:CN101175492A
公开日:
20080507
专利内容由知识产权出版社提供
摘要:本发明公开了组蛋白脱乙酰酶抑制剂和表皮生长因子受体(EGFR)抑制剂的组合用于治疗癌症的用途。
申请人:科罗拉多大学董事会
地址:美国科罗拉多州
国籍:US
代理机构:北京市柳沈律师事务所
更多信息请下载全文后查看。
科学家发现一种有助于抑郁症治疗的小分子
科学家发现一种有助于抑郁症治疗的小分子作者:暂无来源:《乡村科技》 2014年第7期加拿大麦吉尔大学和道格拉斯研究所的科学家,日前发现了一种只有在人体和其他灵长类动物大脑中存在的小分子。
当这种分子水平变低时容易导致沮丧抑郁等情绪。
这一发现有望将抑郁症的治疗提高到一个新层次。
相关论文发表在最近出版的《自然·医学》杂志上。
现代社会生活节奏快,工作压力大,抑郁症患者有日渐增多的趋势。
目前虽然也有一些治疗药物,但由于缺乏针对性,疗效并不明显。
新研究中,加拿大麦吉尔大学医学院附属道格拉斯医院心理科主任医师,抑郁症治疗中心主任古斯塔沃·德瑞奇教授带领的研究团队发现了一种名为miR-1202 的小分子。
这种分子有可能为抑郁症的治疗提供一个新的着力点,并且通过对这种分子含量的测试还可以发现潜在的抑郁症风险。
物理学家组织网6月9日(北京时间)报道称,通过对道格拉斯贝尔加拿大大脑库中样本的分析和对比,德瑞奇的研究团队发现miR-1202 是抑郁症患者大脑样本与健康大脑样本的一项重要区别。
他说:“我们发现在人体和其他灵长类动物大脑中,miR-1202是调节神经递质谷氨酸的重要受体。
我们进行了多次实验,结果表明抗抑郁药物能够改变这种分子的水平。
在临床实验中,我们也发现,在治疗前,抑郁症患者体内miR-1202 含量水平要显著低于没有抑郁症的健康个体。
很显然这种物质能够帮助人体对抗抑郁反应。
”德瑞奇说,目前包括西酞普兰(一种常见的抑郁症治疗药物)在内的一些药物确实能够帮助一些患者减轻痛苦,但在此之前往往要经过对多种药物的尝试后才能发现真正起效的那一种。
而如果能够引入对miR-1202的测试,并根据患者体内这种分子含量的水平对症下药,情况或许就会完全不同。
这一发现可能为“新的、更有效地抑郁症疗法”提供帮助。
来源:《科技日报》。
治疗癌症的“奇兵”——微纳米马达
142Univ. Chem. 2023, 38 (11), 142–147收稿:2023-04-10;录用:2023-09-04;网络发表:2023-09-12*通讯作者,Email:********************.cn基金资助:国家自然科学基金(52073071);黑龙江省自然科学基金优秀青年基金(YQ2022E021)•科普• doi: 10.3866/PKU.DXHX202304034 治疗癌症的“奇兵”——微纳米马达姜满乐1,张荡1,张箭2,徐平1,王磊1,*1哈尔滨工业大学化工与化学学院,哈尔滨150001 2哈尔滨医科大学基础医学院,哈尔滨 150081摘要:目前癌症的发病率正在逐渐上升,为实现精准且低毒害的治疗目标,科学家们设计了智能的微纳米马达材料。
这类微小的智能装置可在外界条件控制下靶向于甚至进入癌细胞,从而靶向运输药物,最终实现癌症的治疗。
为了使这项技术得到更广泛的认识,本文采用拟人化手法,以微纳米马达与免疫细胞共同消灭癌细胞的故事为主体,简单介绍微纳米马达的制备、驱动方式以及治疗原理。
关键词:微纳米马达;癌症治疗;微纳米机器人;靶向治疗;免疫治疗中图分类号:G64;O6Micro/nanorobots: An “Ingenious Shocking Military” towards Cancer TreatmentManle Jiang 1, Dang Zhang 1, Jian Zhang 2, Ping Xu 1, Lei Wang 1,*1 School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China.2 School of Basic Medical Sciences, Harbin Medical University, Harbin 150081, China.Abstract: In recent decades, an escalating incidence of tumors has been observed, necessitating more precise and less side-effect-prone therapies. Micro/nanorobots have been successfully engineered to address this challenge. These minute intelligent machines are capable of navigating to, and even infiltrating, tumor cells or tissues under external control. This enables targeted drug delivery and precise tumor therapy. To elucidate this sophisticated technology to a general audience, this paper adopts a personification style. Utilizing an engaging “battle narrative between micro/nanorobots and tumor cells”, we briefly outline the design, fabrication, propulsion mechanisms, and therapeutic principles for a wider readership.Key Words: Micro/nanomotors; Tumor treatment; Micro/nanorobots; Targeted treatment; Immunotherapy1 癌细胞规避免疫,微纳米马达助消病魔一位患者体内的癌细胞正在疯狂增殖,而他的免疫细胞却仿佛抛弃了它们的主人似的,对着极有危害的敌人置若罔闻。
日本和匈牙利共同开发出改善记忆力药品
日本和匈牙利共同开发出改善记忆力药品
佚名
【期刊名称】《医学信息》
【年(卷),期】1996(000)012
【摘要】日本和匈牙利共同开发出改善记忆力药品据《日经产业新闻》报道,日本武田药品工业公司和匈牙利制药企业格德翁·利赫特公司,共同开发出一种新型的改善记忆力的药品。
这种药防止神经细胞死亡,并且有促进神经成长因子分泌的作用。
神经成长因子能够使受伤的神经再生。
研究...
【总页数】2页(P16-17)
【正文语种】中文
【中图分类】R977.9
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灌肠咖啡历史、谷胱甘肽GST、烘焙知识小讲解
灌肠咖啡历史、谷胱甘肽GST、烘焙知识小讲解早在两千年前,在欧洲的古书中就已有关于灌肠的记载。
而第一次世界大战时,德军的伤员在开刀的时候,因为缺乏麻醉剂及吗啡,因咖啡灌肠有止痛的效果,曾被用来减轻病患的痛苦。
德国有两位医学院的教授以老鼠试验进行咖啡灌肠,发现不仅能清肠、止痛,还有促进肝脏排毒的效果,进而奠定了咖啡灌肠的医疗背景。
后被自然养生排毒之父马克思.葛森医生学习沿用,并为人们所熟知…葛森博士Max Gerson.M.D(1881~1959)德国人,移民美国,自然疗法权威人士。
早在1907,马克斯葛森医生就利用全素食为主的无盐饮食,搭配咖啡灌肠治愈了自己很严重、在当时被认为是不可根治的偏头痛。
新谷弘实教授现任美国阿尔伯特、爱因斯坦医科大学外科教授。
美国最权威的内视镜外科医生,担任欧美各国多名政要的保健医生。
新谷弘实教授是国际上著名的肠胃内窥镜发明人、同时也是咖啡灌肠人体肠道跟踪观测第一人。
1971年于美国肠胃内视镜协会发表70个案例,获得在场1000多位的医生起立喝彩,从此奠定了他在大肠内视镜手术界的地位。
他通过肠道观测及自身35年的咖啡灌肠亲身实践论证了:1、咖啡灌肠副作用、依赖性等问题不存在。
2、通过观测咖啡灌肠及补充益生菌对肠道的改善,提出了肠相论;即“肠相”美丽,身体年龄就更年轻。
3、咖啡灌肠对人体的众多疾病有预防和治疗的作用。
新谷弘实认为:1.肠内停滞的大便会产生有害物质。
因大便停滞的缘故,肠内也开始腐烂,产生有害物质,这些有害物质被肠吸收后,将会严重地损害人体的健康。
2.积存在肠内的粪便,会使肠内的坏细菌繁殖,并且随着血液一起在体内循环,不仅会使肠内污浊,而肠内环境恶化又会促进身体的老化,如果置之不理,这种恶性循环会一直持续下去;反过来说,若能用某些方法来制止肠内环境的恶化,就可以防止身体的老化。
1928年,葛森医生意外发现灌肠水疗不单能治愈偏头痛,更能治愈皮肤结核病,糖尿病等各种医学上都认为不能根治的病。
氯仿-甲醇提取贝氏柯克斯体组分Q热疫苗的免疫保护性评价
不同分期恶性肿瘤患者外周血凝血功能指标、NLR检测及其临床意义
病变的相关性分析[J].医学临床研究,2020,37(5):680-682.[17]Medina-Leyte DJ,Zepeda-García O,Domínguez-Pérez M,et al.En-dothelial dysfunction,inflammation and coronary artery disease:po-tential biomarkers and promising therapeutical approaches [J].Int J Mol Sci,2021,22(8):3850.[18]Kwon Y ,Kim M,Kim Y ,et al.EGR3-HDAC6-IL -27axis mediatesallergic inflammation and is necessary for tumorigenic potential of cancer cells enhanced by allergic inflammation-promoted cellular in-teractions [J].Front Immunol,2021,12(1):680441.[19]Nie F,Zhang Q,Ma J,et al.Schizophrenia risk candidate EGR3is anovel transcriptional regulator of RELN and regulates neurite out-growth via the Reelin signal pathway in vitro [J].J Neurochem,2021,157(6):1745-1758.[20]Shin SH,Kim I,Lee JE,et al.Loss of EGR3is an independent riskfactor for metastatic progression in prostate cancer [J].Oncogene,2020,39(36):5839-5854.[21]Hua Y ,Wang H,Ye Z,et al.An integrated pan-cancer analysis ofidentifying biomarkers about the EGR family genes in human carci-nomas [J].Comput Biol Med,2022,148(1):105889.(收稿日期:2023-09-18)不同分期恶性肿瘤患者外周血凝血功能指标、NLR 检测及其临床意义赵娜1,赵宁2,申晓楠1,苗雨莉11.通用环球西安西航医院检验科,陕西西安710021;2.西安医学院药学院,陕西西安710021【摘要】目的探讨不同分期恶性肿瘤患者外周血凝血功能指标、中性粒细胞/淋巴细胞比值(NLR)检测及其临床意义。
哌嗪磺酸衍生物作为CCR1拮抗剂用于治疗纤维化、阿尔茨海默病和其它病症的用途[发明专利]
专利名称:哌嗪磺酸衍生物作为CCR1拮抗剂用于治疗纤维化、阿尔茨海默病和其它病症的用途
专利类型:发明专利
发明人:M·F·布朗,A·S·加韦科,R·P·格拉迪,M·M·海沃德
申请号:CN200380102266.7
申请日:20031020
公开号:CN1708304A
公开日:
20051214
专利内容由知识产权出版社提供
摘要:本发明涉及作为免疫调节剂使用CCR1拮抗剂的方法。
具体而言,该方法涉及使用式(I)化合物或其可药用盐:其中X、Y、a、b、c、d、R、R、R和R如此处所定义。
申请人:辉瑞产品公司
地址:美国康涅狄格州
国籍:US
代理机构:北京市中咨律师事务所
更多信息请下载全文后查看。
安捷伦产品目录
15
Real-Time PCR
16
Mx3000P QPCR System
17
Brilliant III Ultra-Fast SYBR Green QPCR and QRT-PCR Reagents
18
Brilliant III Ultra-Fast QPCR and QRT-PCR Reagents
Agilent / STRATAGENE
Agilent website: /genomics
Welgene | Agilent Stratagene
威健股份有限公司 | Stratagene 總代理
Table of Content
Table of Contents
/ XL1-Red Competent Cells SoloPack Gold Supercompetent Cells
/ TK Competent Cells Specialty Cells
/ Classic Cells / Fine Chemicals For Competent Cells
適用於 UNG 去汙染或 bisulphite
sequencing
適用於 TA Cloning
最高敏感性
取代傳統 Taq 的好選擇
-
2
威健股份有限公司 | Stratagene 總代理
PCR Enzyme & Instrument
Agilent SureCycler 8800
市場上領先的 cycling 速度和 sample 體積 10 ~ 100 μL 簡易快速可以選擇 96 well 和 384 well 操作盤 優秀的溫控設備讓各個 well 都能保持溫度的穩定 七吋的高解析度觸控螢幕讓操作上更為簡便 可以透過網路遠端操控儀器及監控儀器 Agilent 專業的技術支援可以幫助您應對各種 PCR 的問題
新型AChE抑制剂胡椒碱衍生物对阿尔茨海默病小鼠的治疗作用评价
网络出版时间:2023-10-3019:33:31 网络出版地址:https://link.cnki.net/urlid/34.1086.R.20231027.1531.022◇神经精神药理◇新型AChE抑制剂胡椒碱衍生物对阿尔茨海默病小鼠的治疗作用评价孙佳磊,朱仁德,吴 静,曹国敏,刘新华,李 荣,石静波(安徽医科大学药学院,安徽合肥 230032)收稿日期:2023-06-09,修回日期:2023-09-23基金项目:国家自然科学基金资助项目(No21977001);药学创新基金科研项目(NoYXCX202102)作者简介:孙佳磊(1998-),男,硕士生,研究方向:小分子药物的设计、合成及抗炎活性评价,E mail:2971799980@qq.com;石静波(1975-),男,博士,副教授,硕士生导师,研究方向:药物设计与合成/药物化学生物学,通信作者,E mail:sjbo616@126.com;李 荣(1979-),男,博士,教授,博士生导师,研究方向:类风湿关节炎发病机制及药物药理学,通信作者,E mail:lirong@ahmu.edu.cndoi:10.12360/CPB202306026文献标志码:A文章编号:1001-1978(2023)11-2064-06中国图书分类号:R 332;R282 71;R338 64;R345 61;R745 7摘要:目的 研究胡椒碱衍生物4a对乙酰胆碱酯酶的抑制活性及对阿尔茨海默病(Alzheimer′sdisease,AD)小鼠的神经保护的作用。
方法 雄性C57BL/6J小鼠30只,随机分:(i)空白对照组,(ii)模型组,(iii)多奈哌齐(10mg·kg-1,阳性对照)组,(iv)4a低浓度(20mg·kg-1)组,(v)4a高浓度(40mg·kg-1)组。
iii至v组,小鼠给药东莨菪碱(3mg·kg-1)30min后,口服多奈哌齐和4a,连续10d,Morris水迷宫实验观察由东莨菪碱诱导的认知功能障碍小鼠的记忆功能。
益生菌对阿尔茨海默病作用的研究进展
益生菌对阿尔茨海默病作用的研究进展发布时间:2021-12-14T06:08:15.523Z 来源:《中国结合医学杂志》2021年12期作者:宋鑫萍1,2,李盛钰2,金清1[导读] 阿尔茨海默病已成为威胁全球老年人生命健康的主要疾病之一,患者数量逐年攀升,其护理的经济成本高,给全球经济造成重大挑战。
近年来研究显示,益生菌在适量使用时作为有益于宿主健康的微生物,在防治阿尔茨海默病方面具有积极影响,其作用机制可能通过调节肠道菌群,影响神经免疫系统,调控神经活性物质以及代谢产物,通过肠-脑轴影响该病发生和发展。
宋鑫萍1,2,李盛钰2,金清11.延边大学农学院,吉林延吉 1330022.吉林省农业科学院农产品加工研究所,吉林长春 130033摘要:阿尔茨海默病已成为威胁全球老年人生命健康的主要疾病之一,患者数量逐年攀升,其护理的经济成本高,给全球经济造成重大挑战。
近年来研究显示,益生菌在适量使用时作为有益于宿主健康的微生物,在防治阿尔茨海默病方面具有积极影响,其作用机制可能通过调节肠道菌群,影响神经免疫系统,调控神经活性物质以及代谢产物,通过肠-脑轴影响该病发生和发展。
本文综述了近几年来国内外益生菌对阿尔茨海默病的作用进展,以及其预防和治疗阿尔茨海默病的潜在作用机制。
关键词:益生菌;阿尔茨海默病;肠道菌群;机制Recent Progress in Research on Probiotics Effect on Alzheimer’s DiseaseSONG Xinping1,2,LI Shengyu2,JI Qing1*(1.College of Agricultural, Yanbian University, Yanji 133002,China)(2.Institute of Agro-food Technology, Jilin Academy of Agricultural Sciences, Chanchun 130033, China)Abstract:Alzheimer’s disease has become one of the major diseases threatening the life and health of the global elderly. The number of patients is increasing year by year, and the economic cost of nursing is high, which poses a major challenge to the global economy. In recent years, studies have shown that probiotics, as microorganisms beneficial to the health of the host, have a positive impact on the prevention and treatment of Alzheimer’s disease. Its mechanism may be through regulating intestinal flora, affecting the nervous immune system, regulating the neuroactive substances and metabolites, and affecting the occurrence and development of the disease through thegut- brain axis. This paper reviews the progress of probiotics on Alzheimer’s disease at home and abroad in recent years, as well as its potential mechanism of prevention and treatment.Key words:probiotics; Alzheimer’s disease; gut microbiota; mechanism阿尔茨海默病(Alzheimer’s disease, AD),系中枢神经系统退行性疾病,属于老年期痴呆常见类型,临床特征主要包括:记忆力减退、认知功能障碍、行为改变、焦虑和抑郁等。
桦褐孔菌提取物介导LXRs信号通路调控肝纤维化
ventricle,andthenLPS(1g·L-1)2μLwasinjec tedintothelateralventricletopreparetheinflammatorymodel30minuteslater.Salidrosidewasadministeredataconcentrationof50mg·kg-1·d-1.Samplesweretakenonedayafteradministration.Thestainingofneutrophilswasobserved,andtheexpressionsofp Akt,AktproteinandNF κBweredetectedbyWest ernblot.TNF α,IL 1β,CD14,iNOSmRNAexpres sionsweredetectedbyRT PCR.Results ComparedwithLPSgroup,salidrosidecouldreduceneutrophilin filtration,promoteAktproteinphosphorylationandin hibitnuclearproteinNF κBexpression,inhibitTNF α,IL 1β,CD14,iNOSmRNAexpression.Afterin terventionofPI3KinhibitorLY294002,salidrosidehadnoobviouseffectontheaboveindicators,indicatingthatLY294002couldblocktheinhibitoryeffectofsali drosideonLPS inducedinflammationinratbrain.Conclusion SalidrosideglucosidecanreducetheLPSinducedneutrophilinfiltration,andinhibittheNF κBnucleoproteinandinflammationthroughthePI3K/Aktpathway,thusexertingananti inflammatoryeffect.Keywords:salidroside;anti inflammation;lipopo lysaccharide;PI3K/Akt;NF κB;neutrophils网络出版时间:2023-11-0208:37:00 网络出版地址:https://link.cnki.net/urlid/34.1086.r.20231101.1457.002◇肝脏药理学◇桦褐孔菌提取物介导LXRs信号通路调控肝纤维化刘赛虎,窦佳艺,崔振宇,廉丽花,南极星,吴艳玲(延边大学药学院,长白山天然药物研究教育部重点实验室,吉林延吉 133002)doi:10.12360/CPB202211022文献标志码:A文章编号:1001-1978(2023)11-2101-08中国图书分类号:R 332;R282 71;R322 47;R364 5;R575 2摘要:目的 探究桦褐孔菌提取物(Inonotusobliquusextract,IOE)介导肝X受体(liverXreceptors,LXRs)信号通路调控肝纤维化。
人类基因组概况ppt课件
2.91Gbp
54% 38% 9% 35% 26588 42% Titin(234) 约300万个 1/12500 bp
最长的染色体 最短的染色体 基因最多的染色体 基因最少的染色体 基因密度最大的染色体 基因密度最小的染色体 重复序列含量最高的染色体
It is essentially immoral not to get it (the human genome sequence) done as fast as possible.
James Watson
人类基因组计划的完成,使得我们今天有可能来探 讨基因组的概,但我们仍然无法来谈论细节。
重复序列含量最低的染色体
编码外显子序列的比例 基因的平均长度
2(240 Mbp) Y(19 Mbp) 1(2453) Y(104) 19(23/Mb) 13,Y(5/Mb) 19(57%)
2,8,10,13,18(36%)
1.1~1.4% 27 Kb
女 平均 男
染色体上距着丝粒越远,重组率越高
4. Francis S. Collins, Eric D. Green, Alan E. Guttmacher, Mark S. Guyer :A Vision for the Future of Genomics Research. A blueprint for the genomic era. Nature Apr 24 2003: 835.
而 Celera 的测序样本来自5个人:分别属于西班牙裔、 亚洲裔、非洲裔、美洲裔和高加索裔(2男3女),是从21个志 愿者样本中挑选的。
维贝格龙获FDA批准用于膀胱过度活动症
维贝格龙获FDA批准用于膀胱过度活动症Urovant Sciences24日宣布,美国食品和药物管理局(FDA)已经批准了β-3肾上腺素能受体(β3)激动剂GEMTESA®(vibegron,维贝格龙)的每日一次75毫克的新药申请(NDA),用于治疗成人中出现尿急尿失禁(UUI)、尿急和尿频等症状的膀胱过度活动症(OAB)。
此次获批标志着自2012年以来FDA批准的第一个新的口服品牌OAB药物,这也是Urovant Sciences的第一个产品批准。
GEMTESA是一种口服、每日一次的片剂,含有75毫克的维贝琼,这是一种小分子β3受体激动剂,有助于放松膀胱肌肉,使膀胱能够容纳更多的尿液,从而减轻OAB的症状。
波士顿圣伊丽莎白医学中心的临床试验研究者和主要泌尿科医生David Staskin博士说:"GEMTESA是第一个以每日一次药片形式提供的β3受体激动剂,它不需要剂量滴定。
值得注意的是,在关键的EMPOWUR研究中,与安慰剂相比,GEMTESA没有增加任何高血压的不良事件,并且与CYP2D6代谢的药物没有相互作用,这一点很重要,因为许多常用药物都是由CYP2D6代谢的。
"FDA的批准是基于一项广泛的开发计划的结果,该计划涉及4000多名OAB患者,包括为期12周的双盲、安慰剂对照的3期EMPOWUR研究,剂量为75mg,以及双盲EMPOWUR长期扩展研究。
这些数据显示,在EMPOWUR中,与安慰剂相比,使用GEMTESA治疗可显著减少每日UUI、口渴和紧急发作,并增加排空量,具有统计学意义。
Vibegron第52周达到反应终点的患者比例更高在双盲、安慰剂对照的EMPOWUR研究中,GEMTESA最常见的不良反应(≥2%)是头痛、鼻咽炎、腹泻、恶心和上呼吸道感染。
GEMTESA表现出与安慰剂相同的高血压和血压升高的不良事件发生率。
关于膀胱过度活动症膀胱炎是一种临床症状,当膀胱肌肉不自主地收缩时,就会出现这种症状。
干扰素信号通路
ORIGINAL ARTICLE1-Alpha,25-dihydroxy vitamin D3inhibits osteoclastogenesis through IFN-beta-dependent NFATc1suppressionSadaoki SakaiÆHironari TakaishiÆKenichiro MatsuzakiÆHironori KanekoÆMitsuru FurukawaÆYoshiteru MiyauchiÆAyako ShiraishiÆKeiji SaitoÆAkio TanakaÆTadatsugu TaniguchiÆToshio SudaÆTakeshi MiyamotoÆYoshiaki ToyamaReceived:12November2008/Accepted:25March2009/Published online:19May2009ÓThe Japanese Society for Bone and Mineral Research and Springer2009Abstract1-Alpha,25-dihydroxy vitamin D3(1a,25 (OH)2D3),an active form of vitamin D3,plays a critical role in calcium and bone metabolism.Although 1a,25(OH)2D3has been used for osteoporosis therapy,the direct role of1a,25(OH)2D3on human osteoclastogenesis has not been well characterized.Here we show that 1a,25(OH)2D3treatment significantly inhibited human osteoclast formation at the early stage of differentiation in a concentration-dependent manner.1a,25(OH)2D3inhib-ited the expression of nuclear factor of activated T cells c1 (NFATc1,also referred as NFAT2),an essential tran-scription factor for osteoclast differentiation,and upregu-lated the expression of interferon-b(IFN-b),a strong inhibitor of osteoclastogenesis in osteoclast progenitors. Inhibitory effects of1a,25(OH)2D3on osteoclastogenesis and NFATc1expression were restored by treatment with an antibody against IFN-b,suggesting that upregulation of IFN-b by1a,25(OH)2D3treatment results in inhibition of NFATc1expression,in turn interfering with osteoclast formation.Thus,our study may provide a molecular basis for the treatment of human bone diseases by1a,25(OH)2D3 through regulation of the IFN-b and NFATc1axis. Keywords Vitamin DÁOsteoclastogenesisÁNFATc1ÁIFN-bÁ1a,25(OH)2D3Introduction1-Alpha,25-dihydroxy vitamin D3(1a,25(OH)2D3)regu-lates calcium metabolism through the nuclear vitamin D receptor(VDR).1a,25(OH)2D3upregulates intestinal cal-cium absorption and downregulates parathyroid hormone (PTH)mRNA expression in parathyroid cells[1–3],S.SakaiÁH.TakaishiÁK.MatsuzakiÁH.KanekoÁM.FurukawaÁY.MiyauchiÁT.Miyamoto(&)ÁY.Toyama Department of Orthopedic Surgery,Keio University School of Medicine,35Shinanomachi,Shinjuku-ku,Tokyo160-8582,Japane-mail:miyamoto@sc.itc.keio.ac.jpS.SakaiÁA.ShiraishiÁK.SaitoProduct Research Department,Chugai Pharmaceutical Co.,Ltd.,Shizuoka,JapanT.MiyamotoDepartment of Musculoskeletal Reconstruction and Regeneration Surgery,Keio University School of Medicine, Tokyo,JapanT.SudaDepartment of Cell Differentiation,The Sakaguchi Laboratory of Developmental Biology,Keio University School of Medicine,Tokyo,Japan S.SakaiÁA.TanakaMedical Business and Science Sales Division, Chugai Pharmaceutical Co.,Ltd.,Tokyo,JapanT.TaniguchiDepartment of Immunology,Graduate School of Medicine and Faculty of Medicine, University of Tokyo,Tokyo,JapanJ Bone Miner Metab(2009)27:643–652 DOI10.1007/s00774-009-0084-4thereby preventing bone mineral density(BMD)reduction and maintaining bone architecture in circumstances of high bone turnover[4,5].Therefore,1a,25(OH)2D3and its analogues have been used for treatment of postmenopausal, secondary hyperparathyroidism or glucocorticoid-induced osteoporosis[6–8].Osteoclasts are bone-resorbing multinuclear cells derived from hematopoietic stem cells[9].Recent studies have shown that the interaction between receptor activator of nuclear factor kappa-B(RANK)and RANK-ligand (RANKL)is essential for osteoclast differentiation and activation[10–12].RANK signaling stimulates tumor necrosis factor(TNF)receptor-associated factor6 (TRAF6)and c-Fos,a component of the AP-1transcription factor complex,which in turn activates various down-stream molecules such as tartrate-resistant acid phospha-tase(TRAP)[13,14].c-Fos is an essential transcription factor for osteoclast differentiation,and mice deficient in c-Fos exhibit a complete lack of osteoclast formation and severe osteopetrosis[15].Nuclear factor of activated T cells(NFAT)2,also called NFATc1,is a downstream transcription factor of c-Fos in the RANKL/RANK axis and is also essential for osteoclastogenesis[16,17]. Although c-Fos positively regulates osteoclast differentia-tion by inducing NFATc1and Fos-related antigen(Fra)-1, both of which are essential transcription factors for osteo-clast differentiation[14,16,18],it also induces interferon-beta(IFN-b),a strong inhibitor of osteoclast differentia-tion,and it negatively regulates osteoclastogenesis through one of the IFN-a/b receptor components,IFNAR1,in a negative feedback manner[19].Patients with osteoporosis exhibit excessive bone resorption as a consequence of accelerated osteoclast dif-ferentiation.Extensive bone resorption brings immature bone formation,which results in decreased BMD and attenuated bone strength.Administration of active vitamin D3analogues to osteoporosis patients reduces bone resorption and bone fracture frequency[6,20].Ovariecto-mized rodents have been utilized as models of postmeno-pausal osteoporosis,and treatment with active vitamin D3 analogues has been demonstrated to reduce osteoclast for-mation and bone resorption,thereby increasing BMD and bone strength in vivo[4,5].Interestingly,1a,25(OH)2D3 has been used to induce osteoclast formation in a coculture system of bone marrow(BM)and osteoblastic cells.It has been shown that1a,25(OH)2D3acts on osteoblasts to upregulate the expression of RANKL,an essential trans-membrane ligand for osteoclastogenesis,while downregu-lating the expression of osteoprotegerin(OPG),a decoy receptor of RANKL that prevents osteoclastogenesis[10]. Thus,1a,25(OH)2D3has been considered to be an osteo-clast-inducing factor,although it inhibits osteoclast formation and increases BMD in osteoporosis patients. Recently,it was shown that1a,25(OH)2D3inhibited osteoclast formation by inhibiting c-Fos protein expression in mouse osteoclasts[21].However,the mechanisms of its inhibition of osteoclast formation remain largely unknown.In this study,we show that human osteoclastogenesis induced by macrophage colony-stimulating factor(M-CSF) and RANKL from bone marrow-derived osteoclast pro-genitor cells was strongly inhibited by1a,25(OH)2D3 treatment at an early stage of differentiation in a concen-tration-dependent manner.1a,25(OH)2D3treatment inhib-ited expression of NFATc1and upregulated IFN-b expression in osteoclast progenitor cells.The inhibitory effect of1a,25(OH)2D3on human osteoclastogenesis was restored by addition of an antibody against IFN-b,which also restored the expression of NFATc1downregulated by 1a,25(OH)2D3in osteoclast progenitor cells.Thus,our data suggest a molecular basis for the treatment of activated osteoclast-induced bone diseases such as osteoporosis by 1a,25(OH)2D3through the upregulation of IFN-b,which downregulates NFATc1expression in osteoclast progenitor cells.Materials and methodsIsolation of human bone marrow CFU-GM cellsThe study was approved by an Institutional Ethical Review Board(Keio Hospital#16-17-1),and informed consent was obtained from all study subjects.Human bone marrow-derived colony-forming unit granulocyte macrophage (CFU-GM)cells were generated as follows.Human bone marrow was obtained from patients undergoing routine hip replacement surgery.Cells were diluted1:1with Dul-becco’s phosphate-buffered saline(Invitrogen,Carlsbad, CA,USA)andfiltered through a70-l m nylon mesh cell strainer(BD Bioscience,Franklin Lakes,NJ,USA).The cell suspension was carefully layered on histopaque-1077 (Sigma-Aldrich,St.Louis,MO,USA)and centrifuged at 440g for30min at room temperature.The cell layer at the interface was transferred into a fresh tube as bone marrow mononuclear cells.Bone marrow mononuclear cells were cultured in methylcellulose semisolid medium(MethoCult H-4534;Stem cell Technologies,Vancouver,BC,Canada) containing1%methylcellulose,30%fetal bovine serum (FBS),1%bovine serum albumin,10–4M2-mercap-toethanol,2mM L-glutamine,50ng/ml recombinant human(rh)stem cell factor,10ng/ml rh GM-CSF,and 10ng/ml rh IL-3in35-mm Petri dishes.Cultures were maintained in a humidified atmosphere at5%CO2at37°C for2weeks to obtain CFU-GM cells.Osteoclast differentiation of human CFU-GM cells Human CFU-GM cells were seeded in 96-well plates (2.09104cells/well)and cultured for 6days in alpha-minimum essential medium (a -MEM)supplemented with 10%FBS,30ng/ml M-CSF (Wako,Osaka,Japan)and 30ng/ml RANKL (Wako)in the absence or presence of 10–10M,10–9M,10–8M 1a ,25(OH)2D 3(Wako).To examine the role for IFN-b in osteoclastogenesis of human CFU-GM cells,500unit/ml sheep polyclonal antibody against human IFN-b (PBL Biomedical Laboratories,New Brunswick,NJ,USA)was added in the culture medium.The culture medium was changed to fresh medium every other day.Osteoclastogenesis was evaluated by TRAP staining as described below or pit formation assay as described elsewhere [22,23].Tartrate-resistant acid phosphatase (TRAP)staining Cultured cells were fixed with 10%formalin in PBS for 10min at room temperature.After treatment with ethanol/acetone (50:50vol/vol)for 1min,the well surface was air dried and incubated for 30min at room temperature with TRAP-staining solution:0.1M sodium acetate (pH 5.0)containing 0.01%naphthol AS-MX phosphate (Sigma-Aldrich)as a substrate,and 0.03%red violet LB salt (Sigma-Aldrich)as a stain for the reaction product in the presence of 50mM sodium tartrate.TRAP-positivemultinuclear cells containing more than three nuclei were counted as osteoclasts.RNA extraction,RT-PCR,and quantitative real-time PCR analysisTotal RNA was isolated from osteoclast progenitors or osteoclasts using an RNeasy Mini kit (Qiagen,Valencia,CA,USA),and total RNA was reverse transcribed using Reverscript IV (Wako).reverse transcription-polymerase chain reaction (RT-PCR)analysis was performed using the following primer sets:NFATc1,50-TGT GCC GGA ATC CTG AAA CT-30–50-GGC GGG AAG GTA GGT GAA AC-30;c-Fos ,50-GGA CCT TAT CTG TGC GTGAAAC-30–30-CAC ACT ATT GCC AGGAACACAG-50;IFN-b ,50-TCA TCT AGC ACTGGCTGG AA-30–30-TTTCAAAA TCTTCTAGTGTCCTTTCA-50;b -actin ,50-TCC TGT GGC ATC CAC GAAACTA-30–50-CTC GGC CAC ATT GTGAACTTTG-30.PCR reactions were performed using TITANIUM Taq PCR Kit (Clontech,Mountain View,CA,USA).To quantify transcripts,cDNA was subjected to real-time PCR using Applied Biosystem 7500Fastin Real-Time PCR system (Applied Biosystems,Foster City,CA,USA)or a Thermal Cycler Dice Real Time System (Takara Bio,Otsu,Japan).Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was used for sample normaliza-tion.The probes and primers used werepredesignedFig.11-Alpha,25-dihydroxy vitamin D 3(1a ,25(OH)2D 3)inhibits osteoclast formation in colony-forming unit granulocyte macrophage (CFU-GM)cells.Human CFU-GM cells were cultured for 6days with M-CSF (30ng/ml)and receptor activator of nuclear factor kappa-B ligand (RANKL)(30ng/ml)in the presence or absence of the indicated concentrations of 1a ,25(OH)2D 3.Cells were subjected to TRAP staining (a ,b )and pit formation assay (c ).Multinuclear TRAP-positive cells containing more than three nuclei were scored as osteoclasts (b ).Vehicle,0.1%EtOH.Data are mean number ±SD of osteoclasts (*P \0.05,***P \0.001)transcripts(so-called inventoried assays)validated by Applied Biosystems bioinformatics design pipelines. Applied Biosystems assay IDs were Hs00542678_m1 (NFATc1),Hs01077958_s1(IFNB1),and Hs99999905_m1 (GAPDH).Each sample was analyzed in triplicate.Flow cytometryMonoclonal antibodies(mAbs)recognizing the following markers were used forflow cytometric analyses and cell sorting on human CFU-GM cells:CD45(HI30;BD Pharmingen,Franklin Lakes,NJ,USA),CD44(DF1485; DAKO,Glostrup,Denmark),and CD11b(ICRF44;BD Pharmingen).Osteoclast differentiation of mouse bone marrow cellsAll mice were maintained under pathogen-free conditions and cared for in accordance with the guidelines of Keio University School of Medicine.Bone marrow cells were isolated from8-to12-week-old IFN-a/b receptor1(IF-NAR1)-deficient or wild-type mice and cultured in a-MEM supplemented with10%FBS for5h.Nonadherent cells were harvested as osteoclast progenitor cells,seeded in96-well plates(2.09104cells/well),and cultured for6days in a-MEM supplemented with10%FBS,30ng/ml M-CSF, and30ng/ml RANKL in the presence or absence of 1a,25(OH)2D3.The culture medium was changed to fresh medium on day3.Osteoclastogenesis was evaluated by TRAP staining.Western blot analysisCell lysate was collected from the cells derived from IF-NARI-deficient or wild-type mice cultured in the presence of M-CSF and RANKL with or without1a,25(OH)2D3for the indicated culture period.Western blot analysis was performed as previously described[24]using polyclonal antibodies to detect c-Fos(K-25;Santa Cruz Biotechnology, Santa Cruz,CA,USA)and Actin(A2066;Sigma-Aldrich).StatisticsP values were calculated by unpaired Student’s t test.P values less than0.05were considered significant and are indicated by asterisks.Results1a,25(OH)2D3inhibits human osteoclastogenesisWefirst asked whether1a,25(OH)2D3directly affects human osteoclastogenesis from osteoclast progenitorcells. Fig.21a,25(OH)2D3inhibits osteoclastogenesis in the RANKL-RANK axis at an early stage of differentiation.Human CFU-GM cellswere cultured with the indicated concentrations of M-CSF andRANKL in the presence or absence of10–8M1a,25(OH)2D3for6days and stained with TRAP(a).Multinuclear TRAP-positive cellscontaining more than three nuclei were scored as osteoclasts.Data aremean number±SD osteoclasts(*P\0.05).Human CFU-GM cellswere cultured with M-CSF(30ng/ml)and RANKL(30ng/ml)for6days,and10–8M1a,25(OH)2D3was added to the culture mediumat days0–2,2–4,4–6,or0–6(b).1a,25(OH)2D3effectivelysuppressed the formation of osteoclasts at an early stage ofosteoclastogenesis.Vehicle,0.1%EtOH.Data are mean num-ber±SD osteoclasts(*P\0.05,**P\0.01,***P\0.001)Human osteoclast progenitor cells were prepared by culti-vation of human bone marrow mononuclear cells in methylcellulose semisolid culture medium.Human osteo-clastogenesis induced by M-CSF and RANKL in CFU-GM cells was significantly inhibited by 1a ,25(OH)2D 3in a concentration-dependent manner (Fig.1a,b).1a ,25(OH)2D 3also inhibited the bone-resorbing activity,which is the most important function of osteoclasts (Fig.1c).The inhibitory effect of 1a ,25(OH)2D 3on human osteo-clast differentiation was also observed for osteoclasto-genesis from mononuclear cells isolated from bone marrow (data not shown),indicating that 1a ,25(OH)2D 3inhibits osteoclast differentiation in human bone marrow-derivedcells.Fig.31a ,25(OH)2D 3inhibits NFATc1expression in human CFU-GM cells.Human CFU-GM cells were cultured with M-CSF (30ng/ml)and RANKL (30ng/ml)in the presence or absence of the indicated concentrations of 1a ,25(OH)2D 3for 1–2days (a ).Then,total RNA was isolated and reverse transcription-polymerase chain reaction (RT-PCR )analysis was undertaken to detect the expression of NFATc1.1a ,25(OH)2D 3inhibited the expression of NFATc1in aconcentration-dependent manner.b -actin expression is shown as an internal control.Vehicle,0.1%EtOH.Human CFU-GM cells were cultured with M-CSF (30ng/ml)and RANKL (30ng/ml)in the presence or absence of 10–8M 1a ,25(OH)2D 3for 2days (b ).Total RNA was then isolated and real-time PCR analysis performed to detect the expression of NFATc1relative to GAPDH (**P \0.01)Fig.4CD11b –cells in human CFU-GM cells have higher potential to differentiate into osteoclasts.a Human CFU-GM cells were stained with fluorescein isothiocyanate (FITC )-conjugated anti-CD45,CD44,or CD11b antibody and examined by FACS Calibur.b ,c CD45?CD44?CD11b –or CD45?CD44?CD11b ?cells were sorted,cultured with M-CSF (30ng/ml)and RANKL (30ng/ml)in the presence or absence of 10–8M 1a ,25(OH)2D 3for 6days,and the number ofmultinuclear TRAP-positive cells containing more than three nuclei was scored.d CD45?CD44?CD11b –or CD45?CD44?CD11b ?cells were sorted and cultured with M-CSF (30ng/ml)and RANKL (30ng/ml)in the presence or absence of 10–8M 1a ,25(OH)2D 3for 2days.Total RNA was then isolated and real-time PCR analysis was undertaken to detect the expression of NFATc1relative to GAPDH (*P \0.05,**P \0.01)Human osteoclastogenesis is inhibitedby1a,25(OH)2D3downstream of RANKLat an early stage of differentiationNext,we investigated whether increased concentrations of M-CSF,RANKL,or both could rescue the osteoclasto-genesis inhibited by1a,25(OH)2D3.High concentrations of RANKL or M-CSF plus RANKL partially but significantly rescued the osteoclast differentiation inhibited by1a,25 (OH)2D3(Fig.2a).To understand the inhibitory mechanism of1a,25 (OH)2D3on osteoclastogenesis,we subdivided the culture period into three parts(days0–2,days2–4,and days4–6) to assess which stage was critical for inhibition of osteo-clastogenesis by1a,25(OH)2D3.Osteoclastogenesis was strongly inhibited when1a,25(OH)2D3was added to the culture medium at the earlier stage of differentiation (Fig.2b),which suggests that1a,25(OH)2D3affects molecules that act at an early rather than a later period of osteoclast differentiation in the presence of M-CSF and RANKL.NFATc1expression is reducedin1a,25(OH)2D3-treated cells during human osteoclastogenesis induced by M-CSF and RANKLBecause osteoclast differentiation was inhibited by 1a,25(OH)2D3,we analyzed the expression of NFATc1,an essential molecule for osteoclast differentiation[16,17], in cells treated with various concentrations of 1a,25(OH)2D3(Fig.3a,b).NFATc1expression induced by M-CSF and RANKL in osteoclast progenitor cells was significantly downregulated by1a,25(OH)2D3,which suggests that the inhibitory effects of1a,25(OH)2D3on osteoclastogenesis are the result of downregulation of NFATc1expression.As CFU-GM cells may contain several types of cells, the phenotype of CFU-GM cells was analyzed to determine the target cells of1a,25(OH)2D3(Fig.4).Although CFU-GM cells showed single peaks in CD45and CD44,cells were subdivided into two populations by the expression of CD11b,CD45?CD44?CD11b–,or CD45?CD44?CD11b? cells(Fig.4a).Each cell population was sorted and cul-tured in the presence of M-CSF and RANKL with or without1a,25(OH)pared with CD11?cells, CD11b–cells showed significantly higher ability to dif-ferentiate into osteoclasts in the presence of M-CSF and RANKL(Fig.4b,c).The osteoclastogenesis induced by M-CSF and RANKL was severely inhibited by 1a,25(OH)2D3in both CD11b–and CD11b?cells (Fig.4b,c).The expression of NFATc1was analyzed by quantitative real-time PCR;the expression of NFATc1was significantly downregulated by1a,25(OH)2D3treat-ment in both populations,but CD45?CD44?CD11b–cells were more sensitive to1a,25(OH)2D3compared with CD45?CD44?CD11b?cells in inhibiting NFATc1 expression(Fig.4d).1a,25(OH)2D3upregulates IFN-b expressionin osteoclast progenitor cellsNext,we tried to elucidate the mechanism underlying inhibition of osteoclastogenesis by1a,25(OH)2D3.We found that the expression of IFN-b,a strong inhibitor of osteoclastogenesis[19],was significantly upregulated by treatment with1a,25(OH)2D3in osteoclast progenitor cells (Fig.5a,b).To analyze the role of IFN-b in the inhibitionof osteoclastogenesis by1a,25(OH)2D3,an antibody against IFN-b(500unit/ml)was added to cultures with1a,25(OH)2D3.Osteoclastogenesis inhibited by1a,25 (OH)2D3was significantly restored by treatment with the antibody against IFN-b(Fig.5c).Furthermore,the inhibi-tion of NFATc1expression by1a,25(OH)2D3also recov-ered after adding an antibody against IFN-b(Fig.5d), indicating that1a,25(OH)2D3negatively regulates osteo-clastogenesis through the upregulation of IFN-b,which in turn inhibits NFATc1expression in osteoclast progenitor cells.Fig.5Interferon-beta(IFN-b)induced by1a,25(OH)2D3inhibits human osteoclastogenesis.Human CFU-GM cells were cultured withM-CSF(30ng/ml)and RANKL(30ng/ml)in the presence or absenceof the indicated concentrations of1a,25(OH)2D3for1–2days.Then, total RNA was isolated and RT-PCR analysis was undertaken to detect the expression of IFN-b(a).b-actin expression is shown as an internal control.IFN-b expression was upregulated by1a,25(OH)2D3treat-ment in human osteoclast progenitor cells.V,vehicle(0.1%EtOH). Human CFU-GM cells were cultured with M-CSF(30ng/ml)and RANKL(30ng/ml)in the presence or absence of10–8M1a,25(OH)2D3for2days.Total RNA was then isolated and real-time PCR analysis performed to detect the expression of IFN-b relative to GAPDH(b)(*P\0.05).Human CFU-GM cells were cultured withM-CSF(30ng/ml)and RANKL(30ng/ml)in the presence or absenceof10–8M1a,25(OH)2D3with(black column)or without(white column)antibody against IFN-b for6days and stained with TRAP. Multinuclear TRAP-positive cells containing more than three nuclei were scored as osteoclasts(c).Left,representative data;right,data are mean number±SD osteoclasts(**P\0.01).Osteoclastogenesis inhibited by10–8M1a,25(OH)2D3was significantly rescued by the antibody against IFN-b.Vehicle,0.1%EtOH.Human CFU-GM cells were cultured with M-CSF(30ng/ml)and RANKL(30ng/ml)in the presence or absence of10–8M1a,25(OH)2D3with or without the antibody against IFN-b for2days,and RT-PCR analysis was undertaken to detect the expression of NFATc1(d).b-actin expressionis shown as an internal control.NFATc1expression induced by M-CSF and RANKL was inhibited by1a,25(OH)2D3treatment and was rescued by treatment with the antibody against IFN-b in human osteoclast progenitor cellscIFNARI-deficient cells are more resistantto1a,25(OH)2D3than wild-type cellsin osteoclastogenesisFinally,we tried to confirm the inhibitory effect of1a,25(OH)2D3on osteoclastogenesis through an IFN-b-dependent mechanism.To this end,we utilized an animal model deficient in IFNARI,one of the IFN-a/b receptor components,to inhibit the IFN-b-induced signals (Fig.6).The inhibition of osteoclast differentiation by 1a,25(OH)2D3was severe in wild-type cells compared with IFNARI-deficient cells(Fig.6a).The c-Fos protein was downregulated in wild-type but not IFNARI-deficient cells by1a,25(OH)2D3treatment(Fig.6b),suggesting that downregulation of c-Fos by1a,25(OH)2D3also,at least in part,results from the induction of IFN-b by 1a,25(OH)2D3.Taken together,our results demonstrate a novel mech-anism of the direct inhibition of osteoclast differentiation by1a,25(OH)2D3.DiscussionThe clinical effects of 1a ,25(OH)2D 3analogues have been considered to occur via correction of vitamin D deficiency in osteoporotic patients.However,a recent study reported that administration of 1a ,25(OH)2D 3analogues to osteo-porosis patients under vitamin D supplementation also increased BMD [25],suggesting that 1a ,25(OH)2D 3ana-logues have antiosteoporotic effects in addition to correc-tion of vitamin D deficiency.Here we provide a possible mechanism underlying the antiosteoporotic effect of 1a ,25(OH)2D 3in which 1a ,25(OH)2D 3inhibits osteo-clastogenesis by upregulating IFN-b expression,which in turn inhibits the expression of NFATc1,an essential tran-scription factor for osteoclast differentiation.1a ,25(OH)2D 3downregulates PTH transcription in parathyroid cells [2,3]and upregulates intestinal calcium absorption,thereby preventing BMD reduction [1].In spite of its usage for osteoporosis therapy in humans,it was reported that 1a ,25(OH)2D 3is an osteoclast-inducing factor in human cells [26].1a ,25(OH)2D 3has also been used as an osteoclastogenesis-stimulating agent in cocultures of murine osteoclast precursor cells with calvarial osteoblasts.1a ,25(OH)2D 3upregulates RANKL and downregulates the expression of OPG in osteoblasts [10],and this reciprocal regulation of RANKL and OPG is critical for osteo-clastogenesis.Thus,1a ,25(OH)2D 3has been considered to be an osteoclast-inducing factor through osteoblast-medi-ated activity.The regulation of the balance between direct inhibition of osteoclast progenitor cells by 1a ,25(OH)2D 3and indirect stimulation of osteoclastogenesis through osteoblasts is still unclear,but our results suggest that administration of 1a ,25(OH)2D 3or its analogues to osteo-porosis patients could directly inhibit osteoclastogenesis in circumstances with high bone turnover rates.Because we could not find any putative vitamin D response elements in 50-flanking region of the IFN-b gene,the regulation of IFN-b expression by 1a ,25(OH)2D 3is likely indirect.Further study is needed to elucidate the molecular mechanisms of the regulation of IFN-b expression by 1a ,25(OH)2D 3.Several differences have been reported between mouse and human osteoclastogenesis;granulocyte macrophage colony-stimulating factor (GM-CSF)accelerates osteoclast differentiation and osteolytic bone metastasis of human breast cancer [27],but completely inhibits osteoclasto-genesis in mouse bone marrow cells through the down-regulation of c-Fos [28].Therefore,analysis in human cells would be required in developing antihuman osteoclast therapies.c-Fos,a member of the AP-1transcription factor family,is one of the key osteoclastogenesis molecules induced by M-CSF and RANKL,and c-Fos-deficient mice exhibit osteopetrosis because of a complete lack of osteoclast differentiation.NFATc1is a downstream target of c-Fos and is also an essential transcription factor for osteoclast differentiation [14,16,17],and thus c-Fos and NFATc1cooperatively regulate osteoclast differentiation.Although several differences in osteoclastogenesis may occur between humans and mice,both c-Fos and NFATc1might be essential for osteoclastogenesis in both human and mouse systems.The physiological serum concentration of 1a ,25(OH)2D 3has been reported to be approximately 0.1nM;however,the administration of 1a ,25(OH)2D 3or its analogues suchasFig.6IFN-a /b receptor 1-dependent inhibition of osteoclastogenesis by 1a ,25(OH)2D 3.a Osteoclast progenitor cells isolated from wild-type (white column )or IFNAR-deficient (black column )mice were cultured with M-CSF (30ng/ml)and RANKL (30ng/ml)in the presence or absence of the indicated concentrations of 1a ,25(OH)2D 3for 5days and stained with TRAP.Multinuclear TRAP-positive cells containing more than three nuclei were scored as osteoclasts.Data aremean number ±SD of osteoclasts (**P \0.01,***P \0.001).V ,vehicle,0.1%EtOH.b Osteoclast progenitor cells isolated from wild-type (WT )or IFNAR-deficient (IFNARI KO)mice were cultured with M-CSF (30ng/ml)and RANKL (30ng/ml)in the presence or absence of 10–8M 1a ,25(OH)2D 3for the indicated period,and Western blot analysis was performed to detect c-Fos and actin protein1a(OH)D3was reported to increase the local concentration of1a,25(OH)2D3in bone[29,30].Thus our study may provide,at least in part,the mechanisms of the effects of 1a,25(OH)2D3and its analogues in treatment for preventing BMD reduction,and therefore inhibitory effects of NFATc1 expression and IFN-b induction levels in osteoclast pro-genitor cells may be a good index for development of 1a,25(OH)2D3analogues to provide therapies for osteo-clast-activating diseases such as osteoporosis. Acknowledgments We thank Y.Sato for technical support.T. Miyamoto was supported by Precursory Research for Embryonic Science and Technology,Japan Science and Technology Agency, Japan Society for the Promotion of Science Fujita Memorial Fund for Medical Research,and a grant-in-aid from the Global COE Program of the Ministry of Education,Culture,Sports,Science and Technol-ogy,Japan,to Keio University.References1.Gallagher JC,Riggs BL,Eisman J,Hamstra A,Arnaud SB,DeLuca HF(1979)Intestinal calcium absorption and serum vitamin D metabolites in normal subjects and osteoporotic patients:effect of age and dietary calcium.J Clin Invest64:729–7362.Okazaki T,Igarashi T,Kronenberg HM(1988)50-Flankingregion of the parathyroid hormone gene mediates negative reg-ulation by1,25-(OH)2vitamin D3.J Biol Chem263:2203–2208 3.Demay MB,Kiernan MS,DeLuca HF,Kronenberg HM(1992)Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3receptor and mediate transcriptional repression in response to1,25-dihydroxyvitamin D3.Proc Natl Acad Sci 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a r X i v :h e p -p h /0004019v 4 23 J u n 20005-10GeV Neutrinos from Gamma-Ray Burst FireballsJohn N.Bahcall 1&Peter M´e sz´a ros 1,21Institute for Advanced Study 2Pennsylvania State UniversityA gamma-ray burst fireball is likely to contain an admix-ture of neutrons,in addition to protons,in essentially all pro-genitor scenarios.Inelastic collisions between differentially streaming protons and neutrons in the fireball produce νµ(¯νµ)of ∼10GeV as well as νe (¯νe )of ∼5GeV,which could produce∼7events/year in km 3detectors,if the neutron abundance is comparable to that of protons.Photons of ∼10GeV from π0decay and ∼100MeV ¯νe from neutron decay are also produced,but will be difficult to detect.Photons with ener-gies ∼<1MeV from shocks following neutron decay produce a characteristic signal which may be distinguishable from the proton-related MeV photons.I.INTRODUCTIONGamma-ray burst (GRB)sources are distributed throughout the universe and their energy output is mea-sured to be a substantial fraction of a solar rest mass equivalent.1A variety of observations support the in-terpretation that these events are caused by cataclysmic stellar collapse or compact mergers,producing a fireball with bulk expansion Lorentz factor Γ∼102−103.In the standard GRB model a fireball made up of γ,e ±and magnetic fields with an admixture of baryons is producedby the release of a large amount of energy E ∼>1053ergs in a region r o ∼107r o 7cm (e.g.2).The observations indicate that typical fireballs are characterized by a lu-minosity L ∼1052L 52erg s −1and durations t w =10t w 1s in the observer frame,with a large spread in both quanti-ties.The outflow is controlled by the value of the dimen-sionless entropy η=(L/˙Mc2)injected at r o .Previous discussions of fireball models have generally focused on the charged particle components,since they determine directly the photon signal.However,consideration of a neutron component introduces qualitatively new effects.3In a p,n fireball,for values of η∼>400,the neutrons and protons acquire a relative drift velocity causing inelastic n,p collisions and creating neutrinos.We investigate here the neutrino and photon signals from n,p collisions following decoupling in GRB.The ∼10GeV neutrinos from this mechanism depend upon the presence of neutrons in the original explosion,but the neutrinos are created in simple physical processes occur-ring in the later stages of the fireball.On the other hand,the 105GeV neutrinos discussed in refs.4require the ac-celeration in shock waves of ultra-high energy protons interacting with photons.Thus the 10GeV and the 105GeV neutrinos reflect very different astrophysical pro-cesses and uncertainties.Other processes,e.g.neutrinos from p,p collisions 5also require shocks but have lower efficiencies,while 10-30MeV neutrinos from the original explosion 6are much harder to detect due to the lower cross sections.We show (§III)that the 10GeV neutrinos could be detectable by future km 3size detectors.The associated ∼10GeV γ-ray fluences are compatible with current de-tection rates,and may be detectable with future space missions.The dependence of these signals on the neu-tron/proton ratio ξprovides a new tool to investigate the nature of the GRB progenitor systems.Moreover the predicted neutrino event rate depends on the asymp-totic bulk Lorentz factor of the neutrons,which is linked to that of the protons.The latter affects all the electro-magnetic observables from the GRB fireball,including the photospheric and shock radii,as well as the particle acceleration and non-thermal photon production.II.DYNAMICS,N-P DECOUPLING AND PIONSAbove the fireball injection radius r o the outflow ve-locity increases through conversion of internal energy into kinetic energy,the bulk Lorentz factor Γvaryingas Γ∼T ′o /T ′=r/r o ,where T ′is the comoving tem-perature and T ′o =1.2L 1/452r −1/2o 7MeV is the initial tem-perature at r o (henceforth denoting with primes quan-tities measured in the comoving frame).The flow may be considered spherical,which is a valid approximation also for a collimated outflow of opening angle θj >Γ−1,for the conditions discussed here.In a pure proton out-flow the linear growth of Γsaturates when it reaches an asymptotic value Γf ≤η∼constant,the value ηbe-ing achieved when the fireball converts all its luminosity into expansion kinetic energy.For an n,p fireball,be-yond the injection radius r o the comoving temperature is low and nuclear reactions are rare,so the n/p ratio ξremains constant.Since the thermal velocities are non-relativistic,decoupling of the n and p fluids is essential for high-energy neutrino production.At the base of the outflow the n and p components are coupled by nuclear elastic scattering.In terms of the CM relative energy ǫrel and the relative velocityv rel between nucleons,σ′elv ′rel ∼σo c .The CM en-ergy dependence σel ∝ǫ′−1/2∝v ′−1rel is approximately 1valid between energies ∼MeV and the pion produc-tion threshold ∼140MeV,and σo ∼σπ∼3×10−26cm 2is the pion formation cross section above thresh-old.The p and n are cold in the comoving frame,and remain well coupled until the comoving n,p scatteringtime t ′np ∼(n ′p σo c )−1becomes longer than the comov-ing expansion time t ′exp ∼r/c Γ.Denoting the comovingneutron density n ′n =ξn ′p with ξ∼<1,mass conservationimplies n ′p =L/[(1+ξ)4πr 2m p c 3Γη].The n,p decoupling occurs in the coasting or accelerating regimes depending on whether the dimensionless entropy ηis below or above the critical valueηπ= Lσπ/4πm p c 3r o (1+ξ)1/4≃3.9×102L 1/452r −1/4o 7([1+ξ]/2)−1/4.(1)Figure 1shows the dependence of Γon radius for differentη.For low values,η∼<ηπ,the condition t ′np ∼>t ′exp isachieved at a radius r np /r o =ηπ(ηπ/η)3,which is beyond the saturation radius r s /r o ∼ηat which both n and p start to coast with Γ∼η=constant.In this case,even after decoupling both n and p continue to coast together due to inertia,and their relative velocities never reach the threshold for inelastic collisions.ηπηπ,2np r ,1np r ( )r lg Γr/r 012pn2lg lg lg FIG.1.Schematic behavior of the bulk Lorentz factor Γas a function of radius r for various values of the dimensionless entropy η,the decoupling radius r np being indicated with a diagonal slash.Curve 1is for η<ηπ,where the n and p achieve the same asymptotic Γnf ∼Γpf ∼η.Curve 2is for η>ηπ,and in this case n,p decoupling occurs before protons have reached their asymptotic Lorentz factor,which is larger than that of neutrons.This leads to inelastic n,p collisions,pion formation and neutrino emission at r np,2.For η∼>ηπ,on the other hand,the n,p decoupling con-dition t ′np ∼>t ′exp occurs while the protons (and neutrons)are still accelerating as Γp ≃(r/r o ),at a radius(r np /r o )=ηπ(η/ηπ)−1/3,for η∼>ηπ.(2)Beyond this decoupling radius the p can still continue toaccelerate with Γp ∝r (as long as they remain coupledto the photons).However the neutrons are no longer accelerated,since they only interact with the protons,and they continue to coast with the value of Γ∼Γnf ≃constant achieved up to that point,Γnf =(3/4)ηπ(η/ηπ)−1/3(forη∼>ηπ)≃3×102L 1/452r −1/4o 7([1+ξ]/2)−1/4(η/ηπ)−1/3,(3)where the (3/4)factor comes from a numerical solution 3of the coupling equations.When the n,p decoupling condition η∼>ηπis satisfied,the relative n,p drift velocity v rel →c and the inelastic pion production threshold ǫ′>140MeV is reached.Sinceσo ∼σπ,the condition t ′np ∼t ′exp implies that the opticaldepth to pion formation is of order unity.Thus,for η∼>ηπthe radius r np ≡r πis not only a decoupling radius but also an effective “pionospheric”radius.The lowest energy threshold processes at r πare p +n →p +p +π−→µ−+¯νµ→e −+¯νe +νµ+¯νµ→n +n +π+→µ++νµ→e ++νe +¯νµ+νµ→p +n +π0→γ+γ,(4)which occur in approximately equal ratios and with nearunit total probability.The corresponding p +p (n +n )→π±,π0processes do not involve a relative drift velocity (as do the p +n ),and are thus less probable.Processes leading to multiple baryons are also suppressed due to the higher threshold,and for simplicity we restrict ourselves to the above p +n processes.III.10GEV NEUTRINOS AND γ-RAYSThe total number of neutrons carried by the fireball isN n = ξηm p c 2∼0.83×1053E 53 2ξη ,(5)The comoving optical depth τ′∼n ′p σr/Γ∝σ/(r Γ)hasthe same dependence for pion formation and photon scat-tering,but σπ≪σT (Thomson cross section),so the pi-onosphere r πoccurs below the γ-photosphere r γ.The γ-rays in equation (4)can only escape from a skin depth be-low the γ-sphere in the essentially laminar flow with prob-ability P γ∼<τπ(r γ)∼r π/r γ∼(σπ/σT )(1+ξ/7)2∼1/25,for η∼>ηπ.Each n leads to ∼1photon of CM en-ergy ǫ′γ∼70MeV and observer energy centered broadly around ǫγ∼70Γnf /(1+z )MeV ∼ing a proper distance D p =2.8×1028h −165[1−1/√The neutrinos originate at the pionospheric radius rπ≪rγwhereτπ∼1.In this region the stable charged products andγ-rays from the reactions(4)remain in the fireball,and each n leads on average to oneνand one ¯ν.We list below the average neutrino energies for pions and muons decaying at rest.The neutrinos from muon decay have a continuum spectrum.Also,the energies are Doppler broadened by v rel/c∼0.5.ǫ′¯νµ≃30MeV,ǫ′νµ≃30MeV fromπ±ǫ′νe≃30MeV,ǫ′¯νµ≃50MeV fromµ+,(6)ǫ′¯νe≃30MeV,ǫ′νµ≃50MeV fromµ−The relevant cross section for detection averaged overνand¯νisσν∼0.5×10−38(ǫ/GeV)cm2at the observed en-ergyǫ.9The observer frame energy isǫ=ǫ′αΓnf/(1+z), whereα∼1near threshold.For the CMν¯νproduction energies of equation(6),the averageν+¯νCM energy per neutron isǫ′≃100MeV.Takingα≃1the observer ν+¯νenergy per neutron isǫ≃0.1Γnf/(1+z)GeV, and the effective detection cross section per neutron isσν¯νisRν¯νµ∼7E53N t39R b3 2ξη 4/3×h265 2−√1+z−√5×1051E53(2ξ/[1+ξ])(2/[1+z])(ηπ/η)4/3ergs depends on the total energy E of the GRB and on the neu-tron fractionξas well as on the dimensionless entropy η.For a km3detector,approximately5to10neutrino events above10GeV are predicted per year,for a neu-tron/proton ratioξ=1.These events will be coinci-dent with GRB electromagneticflashes in direction and in time(to an accuracy of∼10s),which can enable their separation from the atmospheric neutrino background. Underground water detectors of the type being planned by BAIKAL,13NESTOR,14ANTARES15and the Antar-tic detector ICECUBE16could potentially detect these relatively low energy neutrino events if a sufficiently high density of phototubes were used.About80%of these neutrinos areνµand¯νµ(in approximately equal num-bers)and the remainder areνe and¯νe.These5-10Gev ν¯νare followed by∼120MeV¯νe from neutron decay, but the event rate from neutron-decay neutrinos is very low.The higher energy neutrinos are produced for neu-tron/proton ratiosξ>0when the dimensionless entropyη=L/˙Mc2exceedsηπ≃4×102L1/452r−1/4o7(2/[1+ξ])1/4,and are accompanied by∼10GeV photons which may be detectable in low redshift cases with GLAST.8For a typical GRB at redshift z∼1the numberfluences in10 GeV neutrinos are N(¯νe+νe)∼0.5N(¯νµ+νµ)∼10−4 cm−2,and one order of magnitude less for GeV photons.In all bursts whereξ>0the lower energy(∼120MeV) neutrinos are produced,and neutron decay occurs on anobserver timescale t n∼3L−1/452r1/4o7[(1+ξ)/2]1/4(η/ηπ)1/3s.For outflows of duration t w,these decays will be as-sociated with MeV electromagnetic pulses of duration min[t n,t w],which are additional to the MeV pulses ex-pected from shocks in the original proton component. For short bursts with t w∼<3s,the proton electromag-netic pulse appearsfirst and is separated from a subse-quent neutron electromagnetic pulse,the latter having a slow rise-fast decay envelope and a softer spectrum, which may be detectable with the Swift satellite.12A systematic study of the time histories of GRB emission would be useful to search for evidence of delayed pulses that might be caused by neutron decay.The detection of5-10GeVν¯νin coincidence with GRB photonflashes will not be easy,but would provide unique astrophysical information.Constraints on the neutron fraction could provide information about the progenitor stellar system giving rise to GRB.For instance,core col-lapse of massive stars would lead to an outflow from an F e-rich core withξ∼2/3−1,while neutron star merg-ers would implyξ≥1.Photodissociation during collapse or merger,as well as n,p decoupling and inelastic colli-sions,would both driveξtoward unity,although this equalization process is likely to remain incomplete.For lowη∼<ηπ,inelastic collisions are not expected and the 5-10GeVν¯νare absent,producing only the harder to detect∼100MeV¯νe from neutron decay.An initially non-baryonic outflow of,e.g.e±and magneticfields, would acquire a baryonic load by entrainment from the progenitor environment,withξ≪1from massive stellar envelopes,butξ∼>1for,e.g.,compact mergers.Thus, lower values ofξ,leading to lower ratios of5-10GeVν¯νand a lower ratio of neutron decay MeV photons to to-talfluences would be expected from massive progenitors than from compact mergers.AcknowledgementsPartial support was received by JNB from NSF PHY95-13835and by PM from NASA NAG5-2857,the Guggen-heim Foundation and the Institute for Advanced Study. We acknowledge valuable conversations with G.Fishman, V.Fitch,Vl.Kocharovsky,P.Kumar,R.Nemiroff,J. Norris,M.J.Rees,P.Vogel and E.Waxman.。