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.     

肾移植2,3,5年患者的免疫抑制用药分析

目的：分析肾移植后免疫抑制剂对长期存活的影响，寻找移植后不同时间合适的免疫抑制用药方案及其用药剂量。 方法：对肾移植一年以上、肾功能正常的４９７例患者进行５年连续随访。根据移植后２、３、５年的不同免疫抑制用药将患者分为三联、二联、传统二联治疗三组。统计各组的排异发生率，排异和无排异患者免疫抑制用药的种类、剂量及ＣｓＡ浓度，对排异患者追踪排异发生前１２个月内的药物更动情况。 结果：肾移植后２、３、５     

Ultraviolet Laser-Induced Fluorescence Spectroscopy Diagnosis of Human Stomach Malignant Tissues

To evaluate the potential of laser-induced autofluorescence spectroscopy for the detection of premalignant lesions of human stomach, fluorescence properties of stomach tissues have been investigated in vitro and in vivo. A specially made optical fibre probe and the multichannel fluorescence collection system have been used successfully in our research. Paper received 26 June 1997; accepted in revised form 31 October 1997.     

GD3 expression by cultured human tumor cells of neuroectodermal origin

Summary Seven monoclonal antibodies (mAbs) reactive with ganglioside II³(NeuAc)₂-LacCer (GD3) were generated; four of these mAbs (DMAb-21, DMAb-22, DMAb-23, and DMAb-24) by immunizing mice with GD3 adsorbed to Salmonella minnesota and the remaining three (DMAb-7, DMAb-8, and DMAb-17) with melanoma line SK-MEL 28, which contains 1.4 nmol sialic acid of GD3 per mg protein. The specificities of the mAbs were defined by high-performance thin-layer chromatography (HPTLC) immunostain and solid-phase radioimmunoassay (SP-RIA) with a panel of purified gangliosides. DMAb-7 and DMAb-8 reacted with GD3, IV³(NeuAc)₂nLcOse₄Cer(3,8-LD1), and very weakly with IV³(NeuAc)₂II³NeuAc-GgOse₄Cer (GTla), but not with II³NeuAc-LacCer (GM3), II³NeuAcGgOse₃Cer(GM2), II³NeuAc-GgOse₄Cer(GM1), II³NeuAc, IV³NeuAcGgOse₄Cer (GD1a), II³(NeuAc)₂GgOse₃(GD2), II³(NeuAc)₂GgOse₄Cer (GD1b), IV³NeuAcII³(NeuAc)₂, GgOse₄Cer(GT1b), suggesting the binding epitope to be a terminal tetrasaccharide NeuAc2-8NeuAc2-3Gal1-4(Glc or GlcNAc). DMAb-7 and DMAb-8 were used to investigate the expression of GD3 on cultured human tumor cells of neuroectodermal origin. Thirteen of 19 gliomas, 3 of 5 medulloblastomas, 5 of 5 neuroblastomas, 2 of 2 melanomas, and 1 of 3 teratomas were shown to react with DMAb-8 and/or DMAb-7 by cell surface-RIA (CS-RIA) and immunofluorescence (IF) assays. HPTLC and densitometric analysis confirmed these results, as positive immunostains in the GD3 region were obtained with oligoganglioside fractions from 9 glioma, 1 medulloblastoma, 2 neuroblastoma, 1 melanoma, and 1 teratoma cell line. Glioma cell line U-105 MG and medulloblastoma cell line Daoy contain GD3 as shown by HPTLC immunostain analysis of extracts, although GD3 was undetectable on the cell surface as determined by CS-RIA and IF. There was no detectable GD3 found in gangliosides isolated from cell lines U-373 MG, D-54 MG, TE-671, and PA-1, which were negative for both DMAb-7 and DMAb-8 by CS-RIA and IF assay. Our results provide evidence that GD3 is expressed extensively with significant quantitative heterogeneity on cultured human neuroectodermal tumor cells including glioma, medulloblastoma, neuroblastoma, and melanoma.Supported by NIH grants R37 CA11898, NS 20023, and CA32672 and by grants from the Swedish Medical Research Council (project no. 03X-627), Swedish Cancer Society (project no. 2260-B88-01X) and the National Swedish Board for Technical Development (project no. 84-4667)     

Glial cell and fibroblast cytotoxicity study on 4026-cyclotene photosensitive benzocyclobutene (BCB) polymer films

Photosensitive benzocyclobutene (photo-BCB) is a class of polymers with the trade name Cyclotene. The photoimagable property of Cyclotene makes it suitable for the manufacture of microelectronic devices. The motivation behind this study is that we see an exciting application of photo-BCB as substrates in implantable microelectronic biomedical devices due to several desirable properties distinctive from other polymer materials. To our knowledge, however, photo-BCB has never been tested for biomedical implant applications, as evidenced by the lack reported data on its biocompatibility. This study takes the first step towards assessing photo-BCB biocompatibility by evaluating the cytotoxicity and cell adhesion behavior of Cyclotene 4026 coatings exposed to monolayers of glial and fibroblast cells in vitro. It can be concluded from these studies that photo-BCB films deposited on silicon wafers using microfabrication processes did not adversely affect 3T3 fibroblast and T98-G glial cell function in vitro. We also successfully rendered photo-BCB films non-adhesive (no significant fibroblast or glial cell adhesion) with surface immobilized dextran using methods developed for other biomaterials and applications. Future work will further develop prototype photo-BCB microelectrode devices for chronic neural implant applications.     

Dipole-tracing of 'awareness' attenuating the cortical components of somatosensory evoked potentials

Using the dipole-tracing method, the source generators of N18, P22 and P40 of the somatosensory evoked potential (SEP) were estimated as the equivalent dipole. After voluntary action of the thumb flexion, no changes were observed in N18 or P40, but the amplitude of P22 was suppressed. The after-effects of intention accompanied by a voluntary action or the subject's awareness that electrical stimulation will be given after the voluntary action were treated as 'awareness'. By subtracting the pure SEP from SEP during 'awareness', it was found that the equivalent dipole of 'awareness' of P22 was located at the same region of pure P22, but the vector was of opposite orientation. 'Awareness' attenuated the perceptive potential of SEP like P22 generated in the cortex.     

猕猴输精管内注射高分子聚合物HFMC后对睾丸组织结构的影响

本实验选用具有生育力成年雄性猕猴７只，在直视下行双侧ＨＦＭＣ输精管内注射，每侧剂量分别为３０ｍｇ１只，６０ｍｇ和１００ｍｇ各３只；于注射后２．５年和３．５年分别处死动物，取睾丸组织进行光镜和电镜观察．结果发现：猕猴注射ＨＦＭＣ２．５年后，睾丸光镜大部分曲细精管生精上皮结构完整，排列整齐。仅见局部少数管腔生精上皮层数减少，上皮细胞轻度水样变性等病理改变。电镜下曲细精管内除支持细胞内脂褐素增多，轻度基底膜增厚和精母细胞内质网扩张外，各级生精细胞，支持细胞及细胞间连接复合体等超微结构未见明显异常。注射ＨＦＭＣ３．５年后猕猴的光镜、电镜结果与注射后２．５年结果相似，但局部改变较２．５年组轻。上述结果表明：猕猴输精管内注射一定剂量ＨＦＭＣ节育不会引起睾丸组织的严重病理改变。但是，由于注射ＨＦＭＣ后，ＨＦＭＣ释放Ｈ＋及其对输精管的暂时阻塞，改变了精子生存的内环境，使睾丸出现局部轻度病理改变，随着ＨＦＭＣ逐渐溶解排出，睾丸功能相继恢复正常，配对产仔。为ＨＦＭＣ应用提供了安全性依据。     

ASK1 associates with troponin T and induces troponin T phosphorylation and contractile dysfunction in cardiomyocytes

There is increasing support for the idea that excessive production of proinflammatory mediators such as tumor necrosis factor (TNF) and reactive oxygen species (ROS) contribute to the pathogenesis of cardiac dysfunction. However, the mechanisms by which cytokine/ROS production mediates cardiac dysfunction have not been established. Given that apoptosis signal-regulating kinase 1 (ASK1) is highly expressed in cardiac muscle and that ASK1 is an important mediator in the signaling pathways induced by tumor necrosis factor, interleukin-1, and ROS, we used the yeast two-hybrid system with ASK1 as bait to identify ASK1 substrates from a human heart cDNA library. The cDNA encoding the cardiac troponin T (cTnT) was isolated. ASK1 specifically interacted with cTnT, but not cTnI, in vitro and in vivo via the C-terminal ASK1 domain. ASK1 specifically phosphorylated cTnT in vitro and in vivo. Mutations in cTnT (T194/S198) at an ASK1-phosphorylation consensus sequence significantly reduced phosphorylation by ASK1. ROS-induced ASK1 activation, cTnT phosphorylation, and contractile dysfunction in cardiomyocytes showed similar kinetics. Moreover, overexpression of constitutively active ASK1 induces cTnT phosphorylation and inhibits shortening and calcium transient in adult cardiomyocytes. We conclude that ASK1 plays an important role in regulation of cardiac contractile function by phosphorylating cTnT and may participate in cytokine/ROS-induced pathogenesis of cardiomyopathy and heart failure.     

In Situ Thermal Denaturation of Proteins in Dunning AT-1 Prostate Cancer Cells: Implication for Hyperthermic Cell Injury

The in situ thermal protein denaturation and its correlation with direct hyperthermic cell injury in Dunning AT-1 prostate tumor cells were investigated in this study. The in situ thermal protein denaturation was studied using both Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The FTIR spectra at different temperatures show changes in protein secondary structure (from alpha helix to extended beta sheet) during in situ thermal protein denaturation within AT-1 cells. Calorimetric studies using DSC show that endothermic heat release is associated with the in situ thermal protein denaturation. Furthermore, both the secondary structure changes detected by FTIR and the calorimetric changes detected by DSC were quantified and the kinetics of the overall in situ thermal protein denaturation was derived under different heating conditions. The onset temperature where the overall in situ thermal protein denaturation is first detectable was found to be scanning rate dependent (approximately 41 degrees C at 2 degrees C min(-1) and approximately 44 degrees C at 5 degrees C min(-1)). The kinetics of the overall in situ thermal protein denaturation was derived from both DSC and FTIR measurements and was fit using kinetic and statistical models. The kinetic data determined by FTIR and DSC under the same heating conditions match well with each other. The activation energy of the overall in situ thermal protein denaturation is found to be strongly dependent on the temperature range considered (the activation energy ranges from approximately 110 kJ mol(-1) between 44 and 90 degrees C to approximately 750 kJ mol(-1) between 44 and 50 degrees C). However, its dependence on heating rate is negligible. Several denaturation peaks, including a dominant one between approximately 62 and 65 degrees C, are identifiable from both the DSC and the FTIR results. To investigate directly the relationship between thermally induced cell injury and the in situ thermal protein denaturation, both acute (propidium iodide dye exclusion, assessed 3-h postthermal treatment) and chronic (clonogenics, assessed 7-day postthermal treatment) cell injury were quantified using AT-1 cells prepared under the same conditions as for the DSC protein studies. Comparisons of the results from the cell injury studies and the DSC protein denaturation studies show that the overall in situ thermal protein denaturation correlates well with both the acute and the chronic cell injury, which suggests that overall in situ thermal protein denaturation is an important mechanism of direct hyperthermic cell injury in AT-1 cells at the macromolecular level.