Optical Pretargeting of Tumor with Fluorescent MORF Oligomers

Objective Pretargeting with radioactivity has significantly improved tumor to normal tissue radioactivity ratios over conventional antibody imaging in both animal studies and clinical trials. This laboratory is investigating DNA analogues such as phosphorodiamidate morpholinos (MORFs) for pretargeting using technetium-99m (^99mTc) for detection. However, the unique properties of fluorescence activation and quenching combined with oligomers with their unique properties of hybridization may be particularly useful when used together for pretargeting with optical detection. The use of linear fluorophore-conjugated oligomer duplexes have been little used in animals, and to our knowledge, have not previously been considered for pretargeting applications. Methods A MORF/cDNA pair was selected such that when hybridized, the fluorescence of the Cy5.5-conjugated 25 mer MORF (Cy5.5–MORF25) is inhibited with a BHQ3-conjugated 18 mer complementary DNA (BHQ3–cDNA18). The short BHQ3–cDNA18 was selected to dissociate in the presence of a long cMORF25 in the pretargeted tumor, thus releasing the inhibitor from the Cy5.5 emitter. In this manner, the Cy5.5 fluorescence will be inhibited everywhere but in the target. The dissociation was first examined in vitro by adding the duplex to the cMORF25 both in solution and immobilized on polystyrene microspheres and by surface plasmon resonance (SPR). Thereafter, biotinylated cMORF25 immobilized on streptavidin polystyrene microspheres were administered intramuscularly in one thigh of hairless SKH-1 mice as target while an identical weight of the identical microspheres but without the cMORF25 was administered in the contralateral thigh as control. The animals then received IV the Cy5.5–MORF25/BHQ3–cDNA18 duplex or equal molar dosage of single-chain Cy5.5–MORF25 and were imaged. Results The SPR studies showed that the immobilized cDNA18 rapidly captured the flowing MORF25 to provide a duplex with a slow dissociation rate constant. Furthermore, when cMORF25 was next allowed to flow over the now immobilized duplex, the cDNA18 was unable to prevent dissociation of the heteroduplex and the formation and release of the cMORF25-MORF25 homoduplex. Images of animals obtained soon after receiving the Cy5.5–MORF25 singlet showed intense whole body fluorescence obscuring the target thigh. However, only 5 minutes after receiving the Cy5.5–MORF25/BHQ3–cDNA18 duplex, the target thigh was clearly visible along with only the kidneys. Conclusions This first study of optical pretargeting provides a proof of concept that oligomer pretargeting found to be useful with radioactivity detection is applicable with fluorescent detection as well. In addition, our results demonstrate that by using linear oligomers for optical pretargeting, chain lengths (and base sequences) may be manipulated to provide duplexes with stabilities and fluorescence inhibition optimized for pretargeting and other in vivo applications of optical imaging.     

综合医院精神科会诊患者失眠的临床研究

目的：探讨综合医院精神科会诊患者失眠的临床特征及与其它精神障碍的关系。方法：对168例精神科会诊患者进行分析。结果：128例(76、2％)会诊患者有失眠．其中61．7％(79／128)为慢性失眠。原发性失眠仅占5．5％(7／128)，85．1％(109／128)是与其它精神障碍有关的失眠。常见的精神障碍诊断为焦虑障碍、心境障碍和谵妄。60．9％的失眠患者在会诊前得到了处理。结论：应对临床医师进行精神卫生教育，提高他们对失眠等常见精神障碍的识别和处理能力。     

A model of sterile vesicoureteric reflux in the sheep

Eighteen Coopworth ewe lambs were divided into three groups based on the initial cystourethrogram and cystometry findings at 5 – 7 weeks of age: group 1, 6 lambs with spontaneous low-pressure bilateral vesicoureteric reflux (VUR) on bladder filling were used to study the natural history of reflux; group 2, 5 lambs with no VUR detected were used to establish an experimental model of bilateral VUR using an unroofing surgical procedure; group 3, 7 lambs with spontaneous VUR detected during micturition had the same surgical procedure to increase the degree of VUR. All three animal groups were followed for 4 – 10 months. Spontaneous VUR was demonstrated in 13 of 18 lambs (25/36 ureters). The presence and severity of spontaneously occurring reflux in group 1 lambs diminished with increasing age. VUR was created successfully in group 2 and increased in degree in group 3 animals. The only significant histological finding in all three animal groups with grades II and III VUR was distal renal tubular dilatation. The sheep is a useful and readily available animal for studying VUR. During 4 – 10 months of follow-up, sterile reflux without bladder outflow obstruction resulted in distal renal tubular dilatation, but no renal parenchymal damage. Received April 17, 1997; received in revised form August 5, 1997; accepted August 21, 1997     

Effect of Renal Impairment on the Pharmacokinetics and Tolerability of Tiagabine

Summary: Purpose: We wished to determine the effect of renal impairment on the pharmacokinetics and tolerability of the new antiepileptic drug tiagabine (TGB).
Methods: We assessed TGB pharmacokinetics and tolerability in 25 subjects with various degrees of renal function (based on creatinine clearance, n = 4–6 per group) from healthy (group I) to requiring hemodialysis (group V) in a single and multiple dose (every 12h), one-period (groups I-IV) or a single dose, two-period (group V) study (4-mg oral doses of TGB · HCl). Blood samples were collected after the first dose (both periods for group V) and after the last dose on day 5 (groups I-IV). TGB plasma concentrations and plasma protein binding were determined by high-performance liquid chromatography (HPLC) and ultrafiltration, respectively.
Results: TGB was well tolerated by all study subjects. The pharmacokinetics of TGB were similar in all subjects; no pharmacokinetic parameter (based on either total or unbound concentrations) was statistically correlated with creatinine clearance. For total TGB in plasma, single-dose mean values of the maximum plasma concentration, clearance, and half-life (t1/2) ranged from 52 to 108 ng/ml, from 7.14 to 11.02 I/h, and from 6.4 to 8.4 h, respectively.
Conclusions: TGB pharmacokinetics and tolerability were independent of renal function; therefore, dosage adjustment is unnecessary for epilepsy patients with renal impairment.     

金属离子及氨基糖对大鼠肾脏N－乙酰氨基葡萄糖转移酶Ⅲ活性测定的影响

采用反相ＨＰＬＣ荧光测定法对大鼠肾脏Ｎ－乙酰氨基葡萄糖转移酶（ＧｎＴ）Ⅲ活性测定时二价金属离子及氨基糖的影响进行了研究。结果表明：ＧｎＴⅢ必须有二价金属离子激活，最佳激活离子为Ｍｎ￣（２＋）离子，其次为Ｍｇ￣（２＋）离子；二协金属离子对ＧｎＴⅢ的激活作用依次为Ｍｎ￣（２＋）＞Ｍｇ（２＋）＞Ｃｏ（２＋）＞Ｃａ￣（２＋）＞Ｎｉ￣（２＋）＞Ｂａ￣（２＋）＞Ｚｎ￣（２＋）。四种氨基糖（Ｎ－乙酰氨基葡萄糖ＧｌｃＮＡｃ，Ｎ－乙酰氨基半轧糖ＧａＩＮＡｃ，氨基葡萄糖ＧｌｃＮ，氨基半乳糖ＧａｌＮ），除ＧｌｃＮ使ＧｎＴ活性增加外，其余三种对ＧｎＴⅢ均有不同程度的抑制作用。     

Development and clinical evaluation of immunoluminometric assays for lactoferrin and elastase-alpha 1-proteinase inhibitor complexes in body fluids with special references to bronchoalveolar lavage and neonatal sepsis

Immunoluminometric assays for lactoferrin and elastase-alpha 1-proteinase inhibitor complexes were developed using solid-phase methodology, which has already been published from this laboratory. The aim of the study was to develop a rapid method to see whether elevated granulocyte activity was present in the lung, as for example in neonatal sepsis. The lactoferrin assay gave reliable results within 30 minutes, the elastase-alpha 1-proteinase inhibitor complexes, within 5 hours. The correlation between both analytes was good, so that the lactoferrin assay could replace the elastase-alpha 1-proteinase inhibitor assay in emergency cases. The lactoferrin assay was used for rapid answer, the elastase-alpha 1-proteinase inhibitor complex assay for "fine" monitoring of the progress of the disease. Both assays could be used to measure concentrations in plasma or bronchoalveolar lavage using a 10 microliters sample. Plasma for the elastase-alpha 1-proteinase inhibitor complex determination had to be diluted 1:50 before being assayed. Only EDTA plasma was used in the assay, as either heparin plasma or serum resulted in granulocyte destruction, thus giving rise to elevated, and non-reproducible results. The results from bronchoalveolar lavage show an excellent correlation between elastase-alpha 1-proteinase inhibitor complexes and lactoferrin. No interference was seen from lipaemic or icteric plasma samples. Results from haemolytic samples i.e. where lysis of erythrocytes and leukocytes had occurred, had to be treated with care if no clinical indication of intravascular haemorrhage was present. The assays lend themselves to perinatal diagnosis, as the total volume of plasma or lavage needed is theoretically under 50 microliters, i.e. ethically acceptable for regular monitoring of neonates.(ABSTRACT TRUNCATED AT 250 WORDS)     

肉桂挥发油对“虚证”模型环核苷酸系统反应性的影响

本实验用纯系CFW小鼠与Wistar大鼠建立“虚证”模型。以环核苷酸系统反应性为指标观察了肉桂挥发油的作用。通过气相色谱及气质联用等方法研究表明,本实验所提取的挥发油中桂皮醛含量约为92％。肉桂挥发油可显著降低“阳虚”——甲状腺素缺乏(甲减)模型升高了的cGMP系统反应性而使其降低了的耗O_2率升高;这是一种治疗作用。肉桂挥发油可显著升高“阴虚”——甲状腺素过多(甲亢)模型已升高了的cAMP系统反应性并使其升高了的耗O_2率进一步升高,这是一种恶化作用。这两种作用与中医对症治疗的原则是一致的。     

猪卵透明带单克隆抗体的研制

用热溶猪卵透明带抗原免疫BALB/C 小鼠,取其脾细胞和SP2/O 细胞融合产生的杂交瘤。用间接免疫荧光和ELISA 方法筛选,经四次克隆化后,获得三株分泌猪卵透明带单克隆抗体杂交瘤细胞株(LPD_8,LPC_4,LPD_2)。通过间接免疫荧光试验,其中二株单克隆抗体(LPD_8,LPC_4)与人卵透明带有交叉反应。但它们在猪与人卵透明带上呈现的荧光部位有明显的不同,LPD_8位于整个透明带上,而LPC_4只见于透明带外层。LPD_2荧光位于透明带内层且与人卵无交叉反应。我们认为这种差别是由于抗原决定簇的不同所致。