Ex vivo

Compounds 111 having structural features of the dual cyclooxygenase (COX)/5-lipooxygenase (5-LO) inhibitor tepoxalin and the 5-LO inhibitor ABT-761 were prepared. Many of these hybrid compounds are potent COX and 5-LO inhibitors two compounds (111, r =McO, R = R" = R = H, R = NH2, R = Me and r = MeO, R = R = Me, R" = R = H, R = Cl) inhibited eicosanoid biosynthesis in an ex vivo assay, but neither improved on the main deficiency of tepoxalin, duration of 5-LO inhibitory activity (99BMCL979). Compounds 111 inhibit the production of arachidonic acid products associated with 5-lipoxygenase and cyclooxygenase and are useful in the treatment of inflammatory disorders (99USP5925769). 

We modified polyanionic polymers by use of a grafting reaction of hydrophobic groups onto the polymers. After an extensive evaluation for the affinity of the hy-drophobically modified (hydrophobized) polymers to cell membrane, the immuno-stimulating activity of polymers was investigated by in vitro or ex vivo experiments. Consequently, the increased biological activity was found in the hydrophobized polymer, indicating that... 

For additional evaluation of the effect of hydrophobization and the molecular weight of the polymers on the biological immuno-stimulating activity, we investigated the ex vivo cytokine (interIeukin-6 [IL-6], and tumor necrosis factor [TNFj-inducing activity from human peripheral whole blood cells of hydrophobized polymers by use of fractionated poly(M A-CDA) with narrow poly-dispersity. Since this assay uses the intact human cells, it shows more accurate results than in vitro assay using cultured cell line [25]. 

B. Evaluation of Biological Activity Ex Vivo Cytokine Induction... 

The ex vivo IL-6 and TNF-inducing activities of fractionated and modified or unmodified poly(MA-CDA) were performed according to the method reported [26] and shown in Figs. 12 and 13, respectively. A similar tendency was shown in IL-6 and TNF induction from peripheral whole blood cells by those of poIy(M A-CDA). 

In vitro and ex vivo studies have shown that FATPs transport LCFAs and very long-chain fatty acids (VLCFAs) but no medium-chain fatty acids, fatty acid esters, or lipid-soluble vitamins [4]. LCFA transport is inhibited by prior protease treatment. Synthetic substrates for FATPs include 14C-labeled fatty acids and the fluorescently labeled fatty acid analogue C1 -BODEP Y-Cl 2. Using the latter substrate, differences in fatty acid uptake kinetics between FATP expressing 3T3 LI adipocytes and 3T3 LI fibroblasts, which are devoid of FATPs, can be readily appreciated (Fig. 2). 

Phloretin inhibits FATP-mediated traversing of fatty acids across lipid bilayers. Phloretin is the aglycon of phlorizin and has been used to terminate the uptake of LCFAs and VLCFAs in timed in vitro uptake assays with cultured cells or in ex vivo uptake assays with isolated primary cells. 

A few ex vivo and in vivo studies have been published claiming an antigene (and antisense) effect of mixed purine/pyrimidine sequence PNA [48, 49, 78-80]. However, as pointed out by us in recent reviews [81, 82] these studies lack fundamental controls such as the inclusion of relevant internal standards as a control for sequence-specific non-antigene/antisense effects, thus confirmatory studies are warranted. The in vivo antigene studies from Richelsoris group [79, 83] completely lack a rational basis for the claimed effects. First of all there is no evidence that... 

Due to the predominantly hydrophilic nature of PNAs they do not readily cross lipid membranes [93] and enter living cells [94]. Therefore in order to explore the ex vivo and in vivo potential of PNA as an antisense and/or antigene reagent, a number of different transfection protocols have been devised over recent years. 

To obtain an increased intrinsic capacity to transgress biological membranes, a number of different modifications have been introduced to PNA. These modifications include conjugation of PNA to Hpophilic moieties [51, 97, 98], conjugation of PNA to certain so-caUed ceU-penetrating peptides [49, 55, 56, 66, 99-102] and conjugation to different moieties, which are supposed to be internahzed by specific cellular receptors [48, 103-105]. The work on cellular dehvery of PNA is, like the related work on ex vivo and in vivo effects of PNA, very difficult to summarize conclusively. First of all, the pronounced diversity of the reporter systems employed makes it impossible to directly compare the studies. Secondly, the widespread use of fluorescence studies in spite of the many inherent pitfalls of this technique makes it sometimes difficult to judge even qualitatively whether a presented result actually indicates cellular uptake. We have recently published a comprehensive review on cellular dehvery of PNA [82], with a more detailed assessment of the PNA dehvery hterature. 

A comparative study on the most promising dehvery techniques is presently being undertaken in our laboratory. Based on our experience and the prehminary results from this study, we recommend a protocol based on DNA co-transfection to be the first choice for a delivery protocol ex vivo. 

In 1995, HlV-1 latency was first documented in HIV-1-infected patients when ex vivo T cell cultures were found to contain a subpopulation of cells that produced infectious virions when stimulated with T cell activators (Chun et al. 1995 Finzi et al. 1997). Latently infected T cells are rare, to the order of one in a million resting... 

Pan XM, Saloner D, Reilly LM, Bowersox JC, Murray SP, Anderson CM, Gooding GA, Rapp JH. Assessment of carotid artery stenosis by ultrasonography, conventional angiography, and magnetic resonance angiography correlation with ex vivo measurement of plaque stenosis. J Vase Surg 1995 21(l) 82-88 [discussion 88-89]. 

The comparatively straightforward link between 5-HT and its primary metabolite, 5-HIAA, encouraged many researchers to use changes in the ratio of tissue concentrations of 5-HIAA and 5-HT as an index of the rate of release of 5-HT ex vivo. However, it has been clear for some time that the majority of 5-HT is metabolised in the cytoplasm by MAO before it is released from 5-HT nerve terminals. Consequently, the reliability of the 5-HIAA 5-HT ratio as an index of transmitter release is rather dubious, although it could be used as an acceptable measure of MAO activity. In any case, the development of in vivo microdialysis means that changes in the concentration of extracellular 5-HT can now be monitored directly which, under drug-free conditions, provides a far more reliable indication of any changes in the rate of release of 5-HT. 

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