Immortalized chromaffin cells disimmortalized with Cre/lox site-directed recombination for use in cell therapy for pain after partial nerve injury

To prepare immortalized adrenal chromaffin cells for eventual clinical use, the immortalizing oncogene must be removed. We have utilized a Cre-mediated excision of a loxP-flanked Tag sequence to test whether immortalized chromaffin cells could be disimmortalized by this method. Cultures of embryonic rat adrenal cells were immortalized with the tsA-TN retroviral vector encoding the loxP-flanked temperature-sensitive allele of SV40 large T antigen (tsA-TN) and a positive/negative neo/HSV-TK sequence for selection with either G418 or gancyclovir, respectively. These cells were then infected with the 1710-CrePR1 bicistronic retroviral vector coding for a form of Cre modulatable by the synthetic steroid RU486. These immortalized loxTsTag/CrePR1/RAD cells expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). After initial incubation at 37 degrees C with RU486 for 3 days, followed by the addition of gancyclovir for 7 days, Tag-ir was not detectable in most of the surviving chromaffin cells, compared to 100% expression in immortalized loxTsTag/CreR1/RAD cells not treated with RU486 and gancyclovir. The expression of TH, DbetaH, and PNMT was increased after disimmortalization and the ability of disimmortalized cells to synthesize norepinephrine was also significantly increased compared to immortalized cells. When both types of chromaffin cells were transplanted in a model of neuropathic pain and partial nerve injury, both cell grafts were equally able to reverse the behavioral hypersensitivity induced by the injury. The use of Cre/lox site-directed disimmortalization of chromaffin cells that are able to deliver neuroactive molecules offers a novel approach to cell therapy.     

Induction of labor with RU 486 (mifepristone) in relaxin-deficient rats: antepartum administration of relaxin facilitates delivery and increases pup survival

OBJECTIVE: This study was conducted to determine whether antepartum administration of relaxin improves RU 486-induced delivery at term in rats that lack circulating endogenous relaxin. STUDY DESIGN: Pregnant rats were modified two ways to obtain circulating levels of relaxin and progesterone that resemble those of pregnant humans: relaxin was immunoneutralized throughout the second half of the 23-day pregnancy and high progesterone levels were sustained until term by inserting progesterone implants on day 20. Porcine relaxin was administered subcutaneously from 8 AM on day 20 until delivery. Labor was induced by administering RU 486 subcutaneously at 4 AM on day 22. RESULTS: After induction of labor with RU 486, labor and delivery were faster, and the incidence of live births was higher when rats were also administered relaxin during the antepartum period. CONCLUSION: Antepartum administration of relaxin in combination with RU 486 has beneficial effects on delivery in relaxin-deficient rats.     

Dexamethasone regulates endothelin-1 and endothelin receptors in human non-pigmented ciliary epithelial (HNPE) cells

Endothelin-1 (ET-1) lowers intraocular pressure (IOP) in animal models by regulating aqueous humour dynamics through both inflow and outflow mechanisms. Moreover, ET's concentration is elevated in glaucoma patients and in animal models of glaucoma. Glucocorticoid therapy often can lead to increase IOP in susceptible individuals including patients with primary open angle glaucoma (POAG). In this study, we examined the effects of dexamethasone (Dex), a frequently used anti-inflammatory glucocorticoid, on the synthesis and release of endothelin-1 and on the expression of endothelin receptors in human non-pigmented ciliary epithelial (HNPE) cells, an established source for ET-1 in the anterior chamber. As measured by ET-1 immunoreactivity, ET-1 was concentration-dependently increased following 24hr Dex treatment, with a maximum concentration (100 nM) causing a threefold increase of ET-1 release. Western blot analysis of HNPE cells showed the expression of endothelin receptor A (ET(A)) and endothelin receptor B (ET(B)) with approximate molecular weights of 40 kDa. Dex treatment decreased ET(A) receptor expression at all Dex doses, but up-regulated ET(B) receptors with 10nM Dex having the greatest effect. Quantitative PCR demonstrated that Dex also increased the mRNA of pre-pro-ET-1 (ppET-1) and ET(B) but decreased the mRNA of ET(A). RU486, a glucocorticoid receptor antagonist, was able to block Dex's actions on ET release and ET(B) receptor expression, but did not block its action on ET(A) receptor expression. Endothelin receptors were minimally expressed in HNPE cells as determined in binding experiments (B(max): ET(A) 17, ET(B) 25 fmolmg(-1) membrane protein). However Dex treatment stimulated a dramatic increase in ET(B) receptor density while decreasing ET(A) receptors (B(max): ET(A) 11, ET(B) 116 fmolmg(-1) membrane protein). The regulation of endothelin and its receptors could be a novel mechanism associated with glucocorticoid's effects on intraocular pressure. The increase in ET-1 and disproportionate regulation in ET receptor expression by Dex could promote dysregulation in ET's mechanism on both inflow and outflow, thus affecting aqueous humour dynamics in the anterior chamber of the eye.     

Involvement of hepatic glucocorticoid receptor-mediated functions in steroid-induced cataract formation

Determination of whether the steroid-induced cataract formation is caused through glucocorticoid (GC) receptor-mediated process was conducted by using GC antagonist (RU486) and anti-GC receptor antibody, and by sucrose density gradient ultracentrifugation analysis. (1) When 15 day-old chick embryos were treated with dexamethasone (DEX, 0.025 micromol per egg), their lenses started to form an opaque ring around the peri-nuclear region (stage II-III) after 12 hr and developed into nuclear-like cataract (stage IV-V) after 44 hr. The cataract formation examined at the 44 hr could be effectively prevented by administration of RU486 (0.2 micromol per egg) ranging from 2 hr before to 12 hr after the DEX administration. (2) GC receptor was present in liver, but could not be determined in lens by western blot analysis using monoclonal anti-GC receptor antibody. (3) Sucrose gradient ultracentrifugation analysis indicated that the receptor (9S) in the liver could be transformed to the 4S form after 0.4M NaCl treatment. Combined with our previous data, this suggests that changes in hepatic functions mediated by the GC receptor after the GC administration may be involved in the process of the cataract formation.     

Interleukin 1 beta enhances conditioned fear memory in rats: possible involvement of glucocorticoids

Central administration of 15 ng interleukin (IL)-1beta in the rat significantly enhanced conditioned fear memory assessed by a passive avoidance task, when retested at 24 and 48 h post-training. Pain threshold was unaffected by 15 ng IL-1beta administration. IL-1beta treatment also increased serum corticosterone. This increase in serum corticosterone was further enhanced in rats given both IL-1beta and footshock. Furthermore, the glucocorticoid receptor antagonist mifepristone blocked IL-1beta-induced elevation in corticosterone and also attenuated the enhanced conditioned fear memory. Central administration of IL-1beta significantly increased prostaglandin E2 and decreased the anti-inflammatory cytokine IL-10 release from whole blood cultures; therefore this treatment appears to be effective in inducing an inflammatory response in both the periphery and the brain. The present study confirms that IL-1beta can enhance conditioned fear memory, an effect which is correlated with changes in glucocorticoid function. This facilitation of defensive behaviour could reflect adaptive responses which may enhance survival during sickness.     

抗早孕新药RU486的药理研究

RU 486经口服、皮下、肌肉和阴道四种途径给药,对大鼠有显著的抗早孕作用;幼兔子宫内膜增生试验证明其在大剂量时有明显的抗孕酮活性;大鼠子宫体外孕激素胞浆受体结合力试验证明与孕酮受体有很强的结合能力。此外还进行了血浆药物半衰期及急性毒性测定。     

孕酮拮抗物RU486对妊娠小鼠子宫巨噬细胞的影响及与妊娠相关性的研究

目的　研究孕酮拮抗物RU4 86对妊娠小鼠子宫巨噬细胞的影响及与妊娠的相关性。　方法　取孕4、10d的小鼠 ,皮下注射RU4 86 (每只 15 0mg L) ,对照组以等量生理盐水代替。在注射后 12、2 4、36h密切观察妊娠结果 ,并用免疫组织化学方法检测子宫内的巨噬细胞。　结果　孕 4d ,注射RU4 86可以完全阻止胚泡的着床 ,且注射后 12～ 36h有大量的巨噬细胞涌入子宫内膜、肌层和肌间血管层 ,细胞数量极显著地高于对照组 (P <0 0 0 1)。孕 10d ,RU4 86处理可以导致大多数胚胎吸收 ,并在处理后 4～ 36h子宫巨噬细胞的数量和分布发生急剧的变化 ,尤其是 12～ 36h细胞数量极显著地高于对照组 (P <0 0 0 1)。　结论　RU4 86诱导小鼠妊娠早期的着床失败和妊娠中期的胚胎吸收与大量巨噬细胞侵入子宫有密切的关系 ,拮抗孕酮的免疫抑制功能可能是RU4 86的抗孕机制之一     

米索前列醇在中期妊娠引产中的作用

米索前列醇(misoprostol)用于终止妊娠及促宫颈扩张已有广泛研究。近年来，misoprostol单独或与米非司酮(RU486)联合用于终止中期妊娠也成为重点研究方向，以获得一种更安全有效的引产方法。本文综述misoprostol不同用法对中期妊娠的作用，以探讨其最佳用药方案。     

Plasma levels of SP1-like immunoreactive material (SP1) and progesterone throughout pregnancy and following RU 486-induced early abortion in the cynomolgus monkey (Macaca fascicularis)

A human-SP1 immunoassay was used to detect SP1-like material (SP1) in the plasma of cynomolgus monkeys. In 27 females remaining non-pregnant during a mating period of 4 months, SP1 was occasionally detected at a mean concentration of 2.5 ng/ml. In 14 non-pregnant females of subsequent proven fertility, SP1 was detected 20 days following unfertile mating at a mean concentration of 4.8 ng/ml in 86% of cycles. At day 20 of proven pregnancies, SP1 was at 12 ng/ml in 93% of these animals. SP1 levels during pregnancy increased in two steps, a slow rise between days 20 and 50, followed by an abrupt rise between days 50 and 60 and afterwards a plateau at 600 ng/ml. Seven other pregnant monkeys received 10 mg/kg of the antiprogestin RU 486 at 28 days. Four aborted and the others continued their gestation. SP1 was always dramatically depressed by this treatment; in animals with abortion failure, it remained at a low concentration for 3 months, then the normal concentration range was recovered. The assay of SP1-like material does not allow early diagnosis of pregnancy, however, it remains interesting as a follow-up parameter after 60 days. Also, antiprogestins appear to be useful tools to analyse the metabolism of SP1.