CD4＋CD25＋调节性T细胞与Foxp3表达在白癜风发病中的作用

目的：研究CD4＋CD25＋调节性T细胞（CD4＋CD25＋Treg细胞）与Foxp3基因表达与白癜风发病的关系及可能机制。方法：以确诊为进展期白癜风且治疗显效的24例患者为研究对象,患者确诊符合白癜风诊断及疗效标准。以16例健康志愿者作为对照。应用流式细胞术检测正常对照、白癜风患者治疗前后各自外周血中CD4＋CD25＋调节性T细胞的比例。采用密度梯度离心法从治疗前后的外周血中提取单个核细胞,以逆转录聚合酶链扩增方法检测其中Foxp3 mRNA的表达水平。结果：进展期且治疗显效的24例白癜风患者外周血中CD4＋CD25＋调节性T细胞的比例以及CD4＋CD25＋调节性T细胞在总CD4＋T细胞中所占比例明显均低于正常对照组（P〈0．05）。治疗后显效的进展期白癜风患者CD4＋CD25＋调节性T细胞的比例与治疗前相比明显升高（P〈0．05）,CD4＋CD25＋调节性T细胞中Foxp3 mRNA的表达水平比治疗也前明显增加（P〈0．05）。结论：进展期白癜风患者体内存在CD4＋CD25＋调节性T细胞的异常,CD4＋CD25＋Treg细胞的数量的减少和Foxp3表达的降低所造成的CD4＋CD25＋Treg细胞免疫抑制功能爱损可能是白癜风发病的一个因素。     

来氟米特对狼疮鼠体内CD4^＋CD25^＋Treg细胞的影响

目的：探讨来氟米特（leflunomide）对狼疮小鼠体内CD4^＋CD25^＋调节性T细胞（Treg）及其特异性转录因子Foxp3基因表达水平的影响,分析其发挥免疫调节作用的可能机制。方法：取MRL/Lpr系狼疮小鼠18只随机分成对照组、来氟米特组、醋酸泼尼松组,对其进行药物干预,8周后流式细胞仪检测脾组织CD4^＋CD25^＋Treg数量变化,荧光定量RT-PCR检测各组脾组织Foxp3 mRNA基因表达变化。结果：来氟米特组、醋酸泼尼松组与对照组相比其体内CD4^＋CD25^＋Treg在CD4＋T细胞中所占比例及Foxp3 mRNA的表达均有所增加,差异有统计学意义（P〈0.05）;其中以来氟米特组增加较为显著。结论：来氟米特及醋酸泼尼松均能提高狼疮鼠体内CD4^＋CD25^＋Treg细胞水平及其特异性转录因子Foxp3 mRNA表达水平。推测其缓解SLE发展的机制之一可能是促进了具有抑制自身免疫反应的CD4^＋CD25^＋调节性T细胞增殖所致。     

白细胞介素-37在人外周血CD4＋CD25＋调节性T细胞中的表达

目的：观察白细胞介素-37（IL-37）在健康人外周血 CD4＋CD25＋调节性 T细胞（Treg）中的表达情况。方法：采用免疫磁性分离法分离人外周血 CD4＋CD25＋Treg,流式细胞技术分析其分离纯度,观察刺激剂组（每1×105个 CD4＋CD25＋Treg细胞加入20μl Dynabeads（R） Human Treg Expander）、非刺激剂组、空白对照组（单纯10%FCS-RPMI 1640培养）CD4＋CD25＋Treg细胞增殖活性及其差异。分别采用 Western blot和免疫荧光共聚焦技术检测 IL-37的表达水平与定位改变。结果：MACS分离人外周血 CD4＋CD25＋Treg 细胞纯度为93%,台盼蓝染色细胞活性为98%。与非刺激剂组和空白对照组相比,刺激剂组 CD4＋CD25＋Treg 增殖活性随时间延长而逐渐增加;非刺激剂组与空白对照组其增殖活性差异无显著性。Western blot 结果显示,正常CD4＋CD25＋Treg细胞表达 IL-37,在刺激剂作用下随作用时间延长 IL-37表达量增加。免疫荧光显微镜和免疫荧光共聚焦图像显示,刺激72 h后 IL-37在 CD4＋CD25＋Treg细胞内明显表达,并且在细胞质中表达丰富,近胞膜处表达密度较高。结论：IL-37在健康人外周血 CD4＋CD25＋Treg 细胞中表达,当受到有效刺激后 IL-37表达明显上升。     

精索静脉曲张不育患者CD_4~+ CD_(25)~+ Foxp3~+调节性T细胞的检测及临床意义

目的:探讨精索静脉曲张(VC)不育症患者外周血CD4+CD25+Foxp3+调节性T细胞(Treg)的变化及其临床意义。方法:72例VC不育症患者(VC组)作为研究对象,以30例非VC且正常生育者为对照(对照组)。流式细胞术检测外周血CD4+CD25+Foxp3+调节性T细胞的比例,ELISA法检测血清抗精子抗体(AsAb)水平,计算机辅助精液分析系统检测精液参数。结果:VC组的精子浓度、活率及活力均低于对照组(P0.05),精子畸形率及AsAb阳性率高于对照组(P0.05);Ⅲ度VC组CD4+CD25+Foxp3+调节性T细胞在总CD4+T细胞所占比例低于正常生育组(P0.05)。AsAb阳性VC组的精子活率、精子活力及CD4+CD25+Foxp3+调节性T细胞占总CD4+T细胞比例均低于AsAb阴性VC组(P0.05),相关分析提示AsAb的存在与Treg/CD4+T比例、精子活率及精子活力负相关(r=-0.245,P0.05;r=-0.314,P0.05;r=-0.263,P0.05)。结论:精索静脉曲张不育患者外周血CD4+CD25+Foxp3+调节性T细胞的水平降低,且AsAb阳性组患者更低,这群调节性T细胞的异常可能是导致精索静脉曲张免疫性不育的重要因素之一。     

高迁移率族蛋白B1对小鼠调节性T细胞抑制性相关分子表达的影响

目的观察高迁移率族蛋白B1（HMGB1）对调节性T细胞（Treg）T淋巴细胞毒性相关抗原4（CTLA-4）及叉头翼状螺旋转录因子（Foxp3）表达的影响，并对其机制进行初步探讨。方法免疫磁珠法分离正常BALB／C小鼠脾脏CIM^＋CD25^＋ Treg。采用固相包被抗CD3及可溶性CD28辅助活化，观察HMGB1刺激与CTLA-4及Foxp3表达的时间-效应关系及剂量-效应关系。结果HMGB1刺激Treg时，CTLA-4表达在24～72h均有所下调（P〈0．05或P〈0．01），其中以作用48、72h表达下调尤为明显（P〈0．01）；而不同剂量HMGB1（10、100、1000μg/L）刺激均可诱导CTLA-4表达下调（P〈0．05或P〈0．01），其中HMGB1浓度在1000μg／L时其表达下调最明显。Foxp3表达与CTLA-4呈现出相同的趋势。结论HMGB1能诱导Treg CTLA-4表达减弱。HMGB1可能通过下调Foxp3表达进而影响Treg免疫调节活性。     

乳腺癌患者外周血CD4+CD25+Foxp3+调节性T细胞水平的检测及意义

目的：探讨乳腺癌患者外周血CD4+CD25+Foxp3+调节性T细胞（Treg）水平检测的意义。 方法：流式细胞术检测74例乳腺癌患者与30例健康对照者外周血CD4+CD25+Foxp3+Treg占CD4+T细胞百分比,分析CD4+CD25+Foxp3+Treg细胞水平与乳腺癌患者临床病理特征及相关免疫组化指标的关系。 结果：乳腺癌患者外周血CD4+CD25+Foxp3+Treg占CD4+T细胞的百分比高于健康对照者[（9.15± 2.24）% vs.（2.29±1.36）%],差异有统计学意义（P<0.05）。统计分析显示,乳腺癌患者外周血CD4+CD25+Foxp3+Treg细胞水平与肿瘤组织学分级、淋巴结转移、pTNM分期以及HER-2、pS2、nm23的表达有关（均P<0.05）,而与肿瘤大小、病理类型以及雌激素受体（ER）、孕激素受体（PR）、p53、Ki-67表达无关（均P>0.05）。进一步相关性分析显示,CD4+CD25+Foxp3+Treg细胞水平与肿瘤组织学分级、淋巴结转移数、pTNM分期、HER-2的表达呈正相关（r=0.583,r=0.333,r=0.919,r=0.604,均P<0.05）而与pS2、nm23表达呈负相关（r=-0.229,r=-0.401,均P<0.05）。 结论：乳腺癌患者外周血CD4+CD25+Foxp3+Treg细胞水平升高,并与与乳腺癌的进展、转移密切相关,对其检测可能有助于患者预后及治疗效果的评估。

     

大鼠肝内胆管癌发生发展与调节性T细胞动态变化的相关性

目的 探讨大鼠外周血、脾脏、肝肿瘤组织中CD4~+CD25～+Foxp3～+调节性T细胞动态变化与肝内胆管癌发生发展之间的关系.方法 实验组大鼠40只,每日饮用0.03%硫代乙酰胺溶液,对照组大鼠40只,正常饮水.自诱癌第10周起至第24周,每隔2周处死实验组及对照组大鼠各5只,流式细胞仪检测外周血、脾脏CD4~+CD25~+Foxp3~+调节性T细胞数量,免疫组织化学法检测组织内Foxp3蛋白表达.结果 (1)在诱癌的过程中,大鼠肝脏依次发生以胆管腺体增生(12～14周),重度不典型增生(16周),浸润性癌(18～24周)为主的病理变化.(2)自诱癌第14周起,大鼠外周血Treg占CD4~+T细胞比例实验组和对照组分别为(4.25±0.64)%和(3.76±0.60)%,差异有统计学意义(P<0.01),实验组Treg比例之后逐渐升高,到18周时到高峰,为(6.18±0.64)%,此后开始下降.大鼠脾脏中Treg占CD4~+T细胞比例诱癌第18周实验组和对照组分别为(5.22±1.20)%和(3.30±0.24)%,差异无统计学意义(P>0.05),实验组Treg比例第20周时达到高峰(8.54±0.36)%.(3)实验组大鼠肝肿瘤组织、癌旁肝组织Treg在18周时较对照组升高(P<0.05),且随着肿瘤的进展持续升高.结论 外周血、脾脏Treg参与肝内胆管癌良性病变到恶性病变转变的这一过程,与肿瘤的发生有关,肿瘤微环境内Treg浸润水平与肿瘤进展密切相关.     

不同分期前列腺癌患者外周血CD4~+ CD25~+ Foxp3~+调节性T细胞的变化及与胰岛素抵抗的相关性

目的 :观察不同分期前列腺癌患者外周血单个核细胞CD4+CD25+Foxp3+调节性T细胞的变化及与胰岛素抵抗的关系。方法:采用流式细胞术检测62例前列腺癌患者(患者组,临床TNM分期Ⅰ期5例、Ⅱ期16例、Ⅲ期21例、Ⅳ期20例)外周血单个核细胞(PBMC)中CD4+CD25+Foxp3+调节性T细胞数目,计算CD4+CD25+Foxp3+调节性T细胞占CD4+T淋巴细胞的百分率;并检测其空腹胰岛素及空腹血糖水平,计算胰岛素抵抗指数(HOMA-IR);采用ELISA法测定外周血胰岛素样生长因子1(IGF-1)水平,分析CD4+CD25+Foxp3+调节性T细胞与胰岛素抵抗的相关性,并与42例健康体检者进行对照。结果:与健康对照组相比,前列腺癌患者HOMAIR明显升高(6.68±1.66 vs 3.68±1.42),IGF-1水平明显下降[(96.39±21.21)ng/ml vs(164.56±30.58)ng/ml],PBMC CD4+CD25+Foxp3+Treg占CD4+T淋巴细胞的百分率[(13.88±0.96)%vs(5.33±0.65)%]及CD4+CD25+Foxp3+Treg绝对值[(3.55±0.29)×107vs(1.99±0.78)×107]明显升高(P0.05,P0.01)。患者PBMC CD4+CD25+Foxp3+Treg占CD4+T淋巴细胞的百分率及CD4+CD25+Foxp3+Treg绝对数﹑HOMA-IR均随TNM分期逐渐加重而增加,IGF-1逐渐下降;相关性分析表明:CD4+CD25+Foxp3+Treg/CD4+T及CD4+CD25+Foxp3+Treg绝对数均与HOMA-IR呈明显正相关(r分别为0.689、0.722,P0.01),与IGF-1呈明显负相关(r分别为-0.896、-0.747,P0.01)。结论:前列腺癌患者存在不同程度的胰岛素抵抗,且随着疾病程度的加重,外周血CD4+CD25+Foxp3+调节性T细胞数目和比例及胰岛素抵抗逐渐加重;CD4+CD25+Foxp3+调节性T细胞可能通过调节胰岛素抵抗参与其形成和发展。     

原发性肝癌患者外周血Th17与CD4^＋CD25^＋调节性T细胞表达的相关性研究

目的探讨原发性肝癌患者外周血Th17和CD4^＋CD25^＋调节性T细胞的表达水平及其相关性。方法选取2008年6月—2009年5月浙江大学医学院附属第一医院30例原发性肝癌患者和25名健康人群,采血并分离其外周血单个核细胞。利用流式细胞仪分别测定Th17和CD4^＋CD25^＋Foxp3^＋调节性T细胞的表达,采用t检验分析两组的表达差异。同时,采用Spearman检验对原发性肝癌患者外周血中Th17和CD4^＋CD25^＋调节性T细胞的表达进行相关性分析。结果健康对照组外周血中Th17细胞为（2．10±0．87）％,CD4^＋CD25^＋调节性T细胞为（7．10±2．32）％,原发性肝癌组外周血中Th17细胞为（3．38±1．68）％,CD4^＋CD25^＋调节性T细胞为（11．78±5．62）％,两组差异具有统计学意义（t=3．640和4．162,P值均〈0．01）。原发性肝癌患者组外周血Th17细胞与CD4^＋CD25^＋Fosp3^＋调节性T细胞表达呈正相关（r=0．821,P〈0．01）。结论原发性肝癌患者外周血Th17和CD4^＋CD25^＋Fosp3^＋调节性T细胞表达水平较高,二者呈正相关。CD4^＋CD25^＋Fosp3^＋调节性T细胞可能通过促进Th17细胞分化导致肿瘤的发生与发展。     

血必净促进内毒素/脂多糖刺激调节性T淋巴细胞凋亡并介导辅助性T淋巴细胞漂移的作用

目的 了解血必净注射液促进LPS刺激CD4+CD25+调节性T淋巴细胞(Treg细胞)凋亡过程及介导辅助性T淋巴细胞(Th)漂移的调节作用.方法 免疫磁珠法分选获得大鼠脾脏CD4+CD25+Treg细胞,分为常规培养对照组、抗CD3/CD28组、抗CD3/CD28+LPS组、抗CD3/CD28+血必净组和抗CD3/CD28+LPS+血必净组,培养3 d后应用流式细胞术检测Treg细胞凋亡率及叉头翼状螺旋转录因子3(Foxp3)表达.将CD4+CD25+Treg细胞与CD4+CD25+T淋巴细胞1:1培养,伴刀豆球蛋白A刺激68 h,检测上清液中Th1分泌的γ干扰素(IFN-γ)、Th2分泌的IL-4、Th17分泌的IL-17水平.结果 抗CD3/CD28+LPS+血必净组Treg细胞凋亡率为(45.1±2.7)%,明显高于抗CD3/CD28+LPS组[(29.4±1.6)%,P＜0.01];2组Foxp3平均荧光强度分别为95±9、140±18,差异有统计学意义(P＜0.01).同时,抗CD3/CD28+LPS+血必净组IFN-γ分泌水平显著高于抗CD3/CD28+LPS组(P＜0.01),IL-4则呈相反变化(P＜0.05),抗CD3/CD28+LPS+血必净组IFN-γ/IL-4较对照组升高(P＜0.01);抗CD3/CD28+血必净组IL-17分泌水平较抗CD3/CD28组明显下降(P＜0.05).结论 CD4+CD25+Treg细胞活化介导了Th1向Th2功能性极化;血必净对LPS诱导的T淋巴细胞免疫功能有重要调节作用,可促进CD4+CD25+Treg细胞凋亡并介导Th2向Th1漂移,从而缓解细胞免疫抑制状态.     

Histogenesis of hyperosteoidosis in 1,25(OH)2D3-treated rats fed high levels of dietary calcium

The purpose of this investigation was to determine by sequential quantitative morphometry the histogenesis of metaphyseal changes induced in rats fed high levels of dietary calcium and treated with pharmacologic doses of 1,25(OH)2D3. Young adult female rats were placed on a diet containing 2.5% calcium and 0.3% phosphorus and administered either ethanol or 135 ng (5 units) 1,25(OH)2D3 in ethanol IP daily for 10 days. Rats were terminated at Days 1, 2, 3, 4, 6, 8, and 10. At Day 1 the proximal tibias from rats treated with 1,25(OH)2D3 had a dramatic increase in osteoclasts/mm total trabecular surface perimeter compared with placebo-treated rats. Osteoclast numbers decreased in 1,25(OH)2D3-treated rats to the levels in placebo-treated rats by Days 3 and 4 and decreased significantly below placebo-treated levels at Days 6, 8, and 10. Active resorbing surface was significantly increased at Days 1 and 2 and decreased at Days 8 and 10 in 1,25(OH)2D3-treated rats compared with placebo-treated rats. From Day 4 through Day 10 in 1,25(OH)2D3-treated rats, there was a progressive increase in osteoblasts/mm total trabecular surface perimeter, osteoid surface, active osteoid surface, and metaphyseal osteoid. Metaphyseal osteoid increased markedly at Days 8 and 10 in 1,25(OH)2D3-treated rats and caused a significant increase in the amount of osseous tissue in the metaphysis. Metaphyseal mineralized bone, however, was not consistently affected by 1,25(OH)2D3 treatment. Serum calcium and phosphorus were elevated in 1,25(OH)2D3-treated rats at more time periods. In rats fed high levels of dietary calcium, repeated supraphysiologic doses of 1,25(OH)2D3 result in a net increase in metaphyseal osseous tissue, predominantly osteoid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of 1,25-dihydroxy-vitamin D3 and 19-nor-1,25-dihydroxy-vitamin D2 on calcium and phosphorus resorption in bone

1,25-Dihydroxy-vitamin D3 [1,25-(OH)2D3] suppresses the secretion and synthesis of parathyroid hormone (PTH) and has been used in the treatment of secondary hyperparathyroidism. However, 1,25-(OH)2D3 can induce hypercalcemia, which often precludes its use. Therefore, an analog of 1,25-(OH)2D3 that would retain its therapeutic effects but produce minor effects on calcium and phosphorus metabolism could be an ideal tool for the treatment of secondary hyperparathyroidism. It has been shown that 19-nor-1,25-dihydroxy-vitamin D2 [19-nor-1,25-(OH)2D2], an analog of 1,25-(OH)2D3, can suppress PTH levels in uremic rats at doses that do not affect plasma ionized calcium levels. The experiments presented here, using parathyroidectomized rats fed diets deficient in either calcium (0.02%) or phosphorus (0.02%), were performed to compare the effects of 1,25-(OH)2D3 and 19-nor-1,25-(OH)2D2 on calcium and phosphorus resorption in bone. Parathyroidectomized rats received daily intraperitoneal injections of vehicle, 1,25-(OH)2D3 (100 ng), or 19-nor-1,25-(OH)2D2 (100 or 1000 ng) for 9 d. Plasma calcium and phosphorus levels were monitored during the study, and ionized calcium levels were determined at the end of the study. By 9 d, 1,25-(OH)2D3 (100 ng/d) increased total calcium levels to 12.4+/-0.26 mg/dl, compared with 6.32+/-0.25 mg/dl (P<0.001) in control animals. The same dose of 19-nor-1,25-(OH)2D2 (100 ng/d) was much less potent (9.45+/-0.28 mg/dl, P<0.001). Similar results were seen with ionized calcium levels [19-nor-1,25-(OH)2D2, 3.61+/-0.12 mg/dl; 1,25-(OH)2D3, 5.03+/-0.16 mg/dl; P<0.001]. Ionized calcium levels were also lower in rats receiving the higher dose (1000 ng) of 19-nor-1,25-(OH)2D2 (4.59+/-0.09 mg/dl, P<0.05). Similar results were seen in rats fed the phosphorus-deficient diet. 1,25-(OH)2D3 (100 ng) increased plasma phosphorus levels from 4.30+/-0.39 mg/dl in vehicle-treated rats to 7.43+/-0.26 mg/dl (P<0.001). The same dose of 19-nor-1,25-(OH)2D2 had no effect (5.19+/-0.32 mg/dl), whereas the high dose (1000 ng) increased plasma phosphorus levels (7.31+/-0.24 mg/dl) in a manner similar to that of 1,25-(OH)2D3 (100 ng). Therefore, 19-nor-1,25-(OH)2D2 is approximately 10 times less effective in mobilizing calcium and phosphorus from the skeleton, compared with 1,25-(OH)2D3. With its ability to suppress PTH at noncalcemic doses, 19-nor-1,25-(OH)2D2 is a potential therapeutic tool for the treatment of secondary hyperparathyroidism in chronic renal failure.     

Acute effect of 1,25-dihydroxyvitamin D3, prednisone, and 1,25-dihydroxyvitamin D3 plus prednisone on serum osteocalcin in normal individuals

Suppression of osteoblastic function plays an important pathogenic role for the development of glucocorticosteroid-induced osteoporosis. Serum osteocalcin (OC) is a sensitive marker of bone formation. The diurnal rhythm in serum OC can be changed by administration of single doses of either 1,25-(OH)2D3 or prednisone. However, the two steroids have opposing effects: 1,25-(OH)2D3 increases and prednisone decreases serum OC. The aim of the present study was to examine whether 1,25-(OH)2D3 can oppose the acute suppressive effect of prednisone on serum OC in normal subjects. We compared the effect of a combined dose of 2 micrograms 1,25-(OH)2D3 and 10 mg prednisone on the diurnal rhythm of serum OC with the effect of 2 micrograms 1,25-(OH)2D3 + placebo in a crossover study. Seven normal subjects aged 23-36 years were investigated twice at an interval of 1 week. Blood samples were collected every 60 minutes from 1900 until 1100 h the following day. Study drugs were given at 2000 h. The data from the present investigation were compared with data obtained from a similar study with placebo and prednisone in the same subjects. After administration of 1,25-(OH)2D3 serum OC followed the placebo curve during the first 8 h, but in contrast to the placebo curve it then continued to increase and remained elevated throughout the observation period (p less than 0.05). Prednisone inhibited and reversed the nocturnal rise in serum OC levels (p less than 0.01). The course of serum OC after administration of 1,25-(OH)2D3 + prednisone almost paralleled the course after placebo. We conclude that 1,25-(OH)2D3 and prednisone have opposing effects on serum OC.     

Ultrastructural development of hyperosteoidosis in 1,25(OH)2D3-treated rats fed high levels of dietary calcium

To evaluate the sequential ultrastructural pathogenesis of the increase in osseous tissue and hyperosteoidosis previously demonstrated in rats administered supraphysiologic doses of 1,25(OH)2D3 and fed high levels of dietary calcium, young adult female rats were placed on a 2.5% calcium and 0.3% phosphorus diet, administered ethanol or 135 ng (5 units) 1,25(OH)2D3 IP daily, and killed after 2, 4, 6, 8, and 10 days. Metaphyseal trabeculae from 1,25(OH)2D3 and placebo-treated rats were examined. Osteoblast hypertrophy characterized by increased cytoplasmic area, abundant rough endoplasmic reticulum, and prominent Golgi apparatus was evident in 1,25(OH)2D3-treated rats at Day 4. These osteoblasts were interpreted to be active in matrix synthesis. Widened osteoid seams were present at Day 6. Osteoblast hypertrophy and widened osteoid seams persisted through Day 10 in 1,25(OH)2D3-treated rats. The unmineralized bone matrix in 1,25(OH)2D3-treated rats contained more numerous cytoplasmic processes from adjacent osteoblasts than did control animals and loosely arranged collagen fibrils, which failed to aggregate in regions adjacent to the osteoid-mineralized bone interface as in placebo-treated rats. Osteoid seams in 1,25(OH)2D3-treated rats contained irregular electron-dense foci, which were often concentrated around embedded cytoplasmic processes. Osteocytic hypertrophy characterized by increased cytoplasmic area, developed rough endoplasmic reticulum, and increased numbers of mitochondria was evident at Day 2 and was sustained through Day 10 in 1,25(OH)2D3-treated rats. Variable-sized aggregates of electron-dense deposits similar to those concentrated around osteoblast cytoplasmic processes were observed in the pericellular space and on and immediately adjacent to the plasma membranes of osteocytes and embedding osteoblasts in 1,25(OH)2D3-treated rats as early as Day 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,25(OH)2 vitamin D3 inhibits parathyroid cell proliferation in experimental uremia

Parathyroid cell proliferation and parathyroid hyperplasia are features of renal secondary hyperparathyroidism. Since parathyroids have recently been recognized as an important target for 1,25(OH)2D3, the effects of administration of variable doses of 1,25(OH)2D3 on ex vivo radiothymidine incorporation in the parathyroid glands, on parathyroid cell mitoses, on parathyroid weight, morphometric indices and on parathyroid protein/DNA ratio were examined in rats with uremia (subtotal nephrectomy; NX) or with calcium deficiency. 3H-thymidine incorporation (3 hr; 37 degrees C; PBS with 10 mmol glucose) was elevated in NX animals, that is, 204 +/- 51 dpm/micrograms DNA versus 96 +/- 28 in controls. In vivo pretreatment with 1,25(OH)2D3, either by intermittent i.p. injection or by osmotic minipump, dose-dependently decreased 3H-thymidine incorporation and parathyroid cell mitoses without affecting morphometric indices of parathyroid cells. Prophylactic administration (i.p.) of 1,25(OH)2D3, starting on the day of nephrectomy, prevented parathyroid hyperplasia (NX + 1,25(OH)2D3 0.84 micrograms tissue/g body wt vs. 1.25 micrograms in untreated NX and 0.54 in ad libitum fed controls), but 10 days of treatment beginning on the 21st day of uremia did not reverse existing hyperplasia (NX + 1,25(OH)2D3 1.5 micrograms/g body wt vs. 1.37 micrograms in untreated NX and 0.56 micrograms in ad libitum fed controls). The inhibitory effect was specific for 1,25(OH)2D3 and not imitated by Dexamethason. However, the effect was not specific for parathyroid hyperplasia of uremia, since similar inhibition of 3H-thymidine incorporation by 1,25(OH)2D3 was also observed in rats on low calcium diet.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of 2-methylene-19-nor-(20S)-1 alpha-hydroxy-bishomopregnacalciferol (2MbisP), an analog of vitamin D, on secondary hyperparathyroidism.

Vitamin D analogs are being developed that retain therapeutic effects but are less calcemic and phosphatemic, a concern in CKD patients who are prone to vascular calcification. We tested a new analog of vitamin D, 2MbisP, and found that it suppresses PTH at doses that do not affect serum Ca or P. INTRODUCTION: Calcitriol is used for the treatment of secondary hyperparathyroidism. However, its use is often limited by the development of hypercalcemia and hyperphosphatemia, an important consideration in patients with chronic kidney disease (CKD) because they are prone to vascular calcification. To minimize this toxicity, structural modifications in the vitamin D molecule have led to the development of calcitriol analogs with selective actions. MATERIALS AND METHODS: In this study, we compared the effects of 1,25(OH)(2)D(3) and a new analog, 2-methylene-19-nor-(20S)-1 alpha-hydroxy-bishomopregnacalciferol (2MbisP), on the development of secondary hyperparathyroidism and established secondary hyperparathyroidism in uremic rats and on mobilization of calcium and phosphorus from bone in parathyroidectomized rats. The clearance from circulation, half-life, and binding affinities to the vitamin D-binding protein and vitamin D receptor of this compound were also evaluated. RESULTS: Uremia produced a marked rise in plasma PTH, but treatment every other day for 2 wk with either 1,25(OH)(2)D(3) (4 ng) or 2MbisP (250, 750, 1500, or 3000 ng) suppressed this increase by >50%. The suppression by 1,25(OH)(2)D(3), however, was accompanied by increases in ionized calcium, phosphorus, and the calcium x phosphorus product, whereas these three parameters were unchanged by 2MbisP. The binding affinity of 2MbisP was 10-20 times less for the vitamin D receptor and 1000 times less for the serum vitamin D-binding protein compared with 1,25(OH)(2)D(3). Also, 2MbisP was cleared more rapidly from the circulation (t1/2 = 10 min) than 1,25-(OH)(2)D(3) (t1/2=7-9 h). In parathyroidectomized rats fed calcium-or phosphorus-deficient diets, daily injections of 2MbisP (1500 or 3000 ng), unlike 1,25(OH)(2)D(3) (50 ng), had no effect on calcium or phosphorus mobilization from bone. CONCLUSIONS: In uremic rats, 2MbisP can suppress PTH at doses that do not affect plasma calcium, phosphorus, and calcium x phosphorus product. This new vitamin D analog may represent an important tool in the treatment of secondary hyperparathyroidism in patients with CKD.     

Effect of intravenous 1-alpha-hydroxyvitamin D3 on secondary hyperparathyroidism in chronic uremic patients on maintenance hemodialysis

The effect of intravenous 1 alpha(OH)D3 on circulating intact parathyroid hormone (PTH) and COOH-terminal immunoreactive PTH was examined in 21 patients on chronic hemodialysis. The patients were treated for 3 months with increasing doses of 1 alpha(OH)D3 under careful control of serum Ca2+. 1 alpha(OH)D3 was given intravenously at doses of up to 4 micrograms three times a week, and blood samples were obtained every week, including 1 week before treatment (basal control). No patients were treated with oral vitamin D metabolites. At the end of the study intact PTH levels were reduced by an average of 67 +/- 6%, and COOH-terminal immunoreactive PTH levels were reduced by 35 +/- 6%. Serum Ca2+ was kept within normal levels, but showed a slight increase from 1.17 to 1.30 mmol/l. An effect of calcium on PTH secretion could not be excluded, but an effect of 1 alpha(OH)D3, independent of serum Ca2+ was also found. This effect may be mediated by 1,25(OH)2D3, assuming a large capacity of the 25-hydroxylase in the liver to convert 1 alpha(OH)D3 to 1,25(OH)2D3. Also, the parathyroid glands may possess receptors for 1 alpha(OH)D3 with an effect similar to that established for the 1,25(OH)2D3 receptors. Thus, although the exact mechanisms of the action of 1 alpha(OH)D3 have not yet been completely clarified, it is concluded that intravenous administration of 1 alpha(OH)D3 may be of benefit in the treatment of secondary hyperparathyroidism of uremia.     

Extrarenal metabolism of 25-hydroxycholecalciferol in the rat: regulation by 1,25-dihydroxycholecalciferol

To determine the role of the kidney in regulation of 25-hydroxycholecalciferol (25OHD3, metabolism, the effects of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] on 3H-25OHD3 were compared in intact and nephrectomized vitamin D-deficient rats. Sixteen hours after the intravenous administration of 3H-25OHD3, extracts of serum and pooled small intestinal mucosa were fractionated by Sephadex LH-20 column chromatography followed by high performance liquid chromatography. In intact rats, 1,25(OH)2D3 (50 ng/day i.p. for 7 days) increased mean serum 3H-24,25-dihydroxycholecalciferol [3H-24,25(OH)2D3] from 2 +/- 2-210 +/- 80 fmol/ml (mean +/- 1 SD), increased mean serum 3H-25,26-dihydroxycholecalciferol [3H-25,26(OH)2D3] from 2 +/- 2-12 +/- 6 fmol/ml and lowered mean serum 3H-1,25(OH)2D3 from 210 +/- 40-4 +/- 4 fmol/ml. Similarly, in nephrectomized animals, 1,25(OH)2D3 increased mean serum 3H-24,25-(OH)2D3 from 6 +/- 11-115 +/- 30 fmol/ml and increased mean serum 3H-25,26(OH)2D3 from 3 +/- 3-26 +/- 10 fmol/ml. Nephrectomy increased serum 3H-25(OH)D3 in untreated (from 1450 +/- 225-2675 +/- 225 fmol/ml serum) and 1,25(OH)2D3 treated rats (from 1600 +/- 175-3075 +/- 100 fmol/ml). 3H-1,25(OH)2D3 averaged 74 +/- 16% of total radioactivity in intestinal mucosa of untreated intact rats and was not detected in either the serum or intestinal mucosa of nephrectomized animals. The results suggest that in intact animals, extrarenal synthesis can account for substantial 24,25(OH)2D3 production and for most 25,26(OH)2D3 production.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of vitamin D metabolites on the calcification of cartilage matrix and the C-propeptide of type II collagen (chondrocalcin)

The influence of vitamin D metabolites (at 1 X 10(-10) M) on the calcification of cartilage matrix (measured by 45Ca2+ uptake) and the C-propeptide of type II collagen (measured by radioimmunoassay) has been studied using organ cultures and chondrocytes isolated from growth plates of vitamin D-deficient and -sufficient 11-day-old rats. Vitamin D-deficient rats had reduced amounts of C-propeptide in their serum and freshly isolated growth plate chondrocytes. In all chondrocytes cultured from vitamin D-deficient animals, the C-propeptide content was maximal at 24 hr whereas calcification continued to increase for up to 72 hr. In organ and chondrocyte cultures of tissue from vitamin D-sufficient rats, both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) were required for maximal stimulation of calcification and maximal increases in C-propeptide content. In these D-replete tissues, 24,25-(OH)2D3 had a less stimulatory effect on both calcification (organ and cell cultures) and C-propeptide (organ cultures only), while 1,25(OH)2D3 alone had no effect in cell cultures but an inhibitory effect in organ cultures. Studies of cells or tissue from growth plates of vitamin D-deficient rats demonstrated that 24,25(OH)2D3 alone produced maximal calcification and maximal increases in the C-propeptide content. 1,25(OH)2D3 generally had an inhibitory effect on both calcification and C-propeptide when used alone. In the presence of 1,25(OH)2D3, the stimulatory effect of 24,25(OH)2D3 was partly abrogated. Maximal stimulation of calcification and increases in C-propeptide by 24,25(OH)2D3 were observed at 1 X 10(-9) M and 1 X 10(-10) M. In none of these studies was there any effect on proteoglycan content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics of doxercalciferol, a new vitamin D analogue that lowers parathyroid hormone.

BACKGROUND: This is the first detailed pharmacokinetic report published on the administration of doxercalciferol [1alpha(OH)D(2)] recently introduced to treat secondary hyperparathyroidism. METHODS: 1alpha(OH)D(2) was administered in a range of single and multiple doses to volunteers with and without normal renal and/or hepatic function. Subsequent serial blood samples were assayed by HPLC/radioimmunoassay for the metabolite 1,25-dihydroxyvitamin D(2) [1,25(OH)(2)D(2)], the major active species. RESULTS: Bioavailability of 1,25(OH)(2)D(2) from a single 5 micro g 1alpha(OH)D(2) oral-capsule dose was estimated to be normally approximately 42% of that from a 5 micro g intravenous injection. Steady-state serum concentrations of 1,25(OH)(2)D(2) were attainable within 8 day, and fluctuated approximately 2.5-fold from peak to trough when oral 1alpha(OH)D(2) doses were taken every second day, and the terminal half-life was 34+/-14 h. Mean steady-state serum concentrations rose less than proportionally (from 20 to 45 pg/ml) on increasing oral 1alpha(OH)D(2) doses from 5 to 15 micro g every 48 h. Renal patients showed 39+/-37% increase in serum 1,25(OH)(2)D(2) concentration during 3-4 h haemodialysis sessions, but no other difference in steady-state pharmacokinetics was found between these or hepatically impaired patients and normal subjects. CONCLUSIONS: Given the sensitivity limits of current assays, the pharmacokinetics of this and other vitamin-D compounds is best elucidated from steady-state studies. The pharmacokinetics of 1,25(OH)(2)D(2) from 1alpha(OH)D(2) doses appears to be similar to that of 1,25(OH)(2)D(3) from 1alpha(OH)D(3) doses, albeit D(3) data have to date largely derived from single-dose studies. Deviation of 1,25(OH)(2)D(2) pharmacokinetics from linearity appears to be marginal enough to be clinically manageable with adequate precaution.