R-isomers of Arg-Gly-Asp (RGD) mimics as potent alphavbeta3 inhibitors

The integrin alpha(v)beta(3), vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. We recently disclosed the discovery of a tripeptide Arg-Gly-Asp (RGD) mimic, which has been shown to be a potent inhibitor of the integrin alpha(v)beta(3) and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. In other investigations involving RGD mimics, only compounds containing the S-isomers of the beta-amino acids have been shown to be potent. We were surprised to find the potencies of analogs containing enantiomerically pure S-isomers of beta-amino acids which were only marginally better than the corresponding racemic mixtures. We therefore synthesized RGD mimics containing R-isomers of beta-amino acids and found them to be relatively potent inhibitors of alpha(v)beta(3). One of the compounds was examined in tumor models in mice and has been shown to significantly reduce the rate of growth and the size of tumors.     

Parallel solid-phase synthesis of vitronectin receptor (alphavbeta3) inhibitors

A combinatorial approach for rapid optimization of a vitronectin receptor (alphavbeta3) inhibitor lead was accomplished by solid-phase synthesis. Orthogonally bis protected 2,3-diaminopropionic acid was used to immobilize the C-terminus of the molecule. Selective deprotection and functionalization of the alpha-amino group followed by acyl resorcinol scaffold attachment and N-terminus diversification was used to explore structure activity relationship (SAR).     

Pharmacokinetics of buspirone as determined by ex vivo (3H)-DPAT binding

Ex vivo (3H)-8-hydroxy-2-(di-n-propylamino)-tetraline ((3H)-DPAT) binding to the hippocampus has been utilized to determine the pharmacokinetic parameters of buspirone after i.v. (30 mumol/kg) and oral (100 mumol/kg) administration of this drug to rats. Intravenous buspirone rapidly penetrated the brain as demonstrated by a maximum inhibition of (3H)-DPAT binding at 1 min. Elimination of drug from the brain was biphasic, with a first component half-life of 24.8 min and a second component half-life of 96 min. Oral buspirone at 3 times the i.v. dose produced less than one-third the maximum inhibition of (3H)-DPAT binding compared to that observed with i.v. buspirone. The pharmacokinetic parameters of buspirone observed in the present study are in agreement with those reported previously. Thus, the ex vivo binding assay could be utilized to determine the bioavailability of the drug to the brain, and its duration of action.     

Immunomodulatory effect of (--)-matairesinol in vivo and ex vivo

Matairesinol is one of the lignan compounds found in a variety of plant foodstuffs. We investigated the immunomodulatory effects of (-)-matairesinol in vivo and ex vivo by using mice. Although we found no significant differences in the IgG, IgA and IgM levels in the serum, the IgE level was strongly suppressed by the uptake of (-)-matairesinol in both intact and ovalbumin-immunized mice. The immunoglobulin produced by lymphocytes from the spleen was not activated by the intake of (-)-matairesinol. However, lymphocytes in such gut-associated lymphatic tissues as Peyer's patches and mesenteric lymph nodes were activated by the administration of (-)-matairesinol.     

Clearance of radiation-induced apoptotic lymphocytes: ex vivo studies and an in vitro co-culture model

Lymphocytes are very sensitive to radiation. Our aim was to test the possibility of detecting apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure. Our in vitro data confirmed the dose-time-effect relationships involved in radiation-induced apoptosis. The detection of in vivo induction of apoptosis in circulating lymphocytes after exposure of animals to radiation appears to depend critically on the technique used to measure apoptosis. Among the different techniques we investigated, mitochondrial modification was the most appropriate; they allowed establishment of dose-time-effect relationships when animals were observed for 72 h. A model of in vitro phagocytosis of apoptotic lymphocytes by macrophages was developed to mimic clearance of apoptotic cells occurring in vivo. Together, our data show that mitochondrial labeling may make it possible to detect ex vivo radiation-induced apoptosis of lymphocytes before macrophage ingestion occurs. We propose the measurement of apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure.     

Validation and application of Drosophila melanogaster as an in vivo model for the detection of double strand breaks by neutral Comet assay

Comet assay under neutral conditions allows detection of DNA double-strand breaks (DSBs), which has consequence to genome instability and carcinogenesis. The present study aims to validate the neutral Comet assay for genotoxicity assessment in Drosophila melanogaster (Oregon R(+)) with three well known DSBs inducers i.e. cyclophosphamide (CP), bleomycin (BLM), cisplatin (CPT) and subsequently its efficacy in detecting DSBs in the organism exposed to a well known environmental chemical, chromium [Cr(VI)]. Third instar larvae of D. melanogaster were fed different concentrations of BLM, CPT and CP (50.0-200.0μg/ml) or Cr(VI) (5.0-20.0μg/ml) mixed standard Drosophila food for 48h. Neutral Comet assay was performed in cells of mid gut and brain from control and treated larvae. Our results show a dose-dependent increase in the migration of DNA in cells of the exposed organisms. A comparison among DNA lesions per mole number of the test chemical in the exposed groups showed that both BLM and CPT induce more DSBs than CP. Interestingly, Cr(VI) at 20.0μg/ml was found to induce significantly increased (p<0.001) DSBs in the exposed organism as compared to the control. The study while validating neutral Comet assay in D. melanogaster suggests its use for in vivo assessment of environmental chemical induced DSBs.     

Green tea polyphenols (flavan 3-ols) prevent oxidative modification of low density lipoproteins: an ex vivo study in humans

Oxidation of low density lipoprotein (LDL) plays crucial roles in atherogenesis. We previously reported that green tea polyphenols (flavan 3-ols), especially epigallocatechingallate (EGCg) and epicatechingallate, exerted potent inhibitory effects on LDL oxidation in vitro. To examine whether intake of green tea polyphenols renders LDL resistant to ex vivo oxidation in humans, 22 male volunteers aged between 22 and 32 years were recruited and assigned the same dietary regimen for 2 weeks. After a 1-week baseline period, they were equally divided into two groups: control and tea. The tea group ingested 300 mg of green tea polyphenol extract twice daily for 1 week. Plasma EGCg concentration at the end of the experiment was 56 nmol/L on average (56% in free form) in the tea group; no EGCg was detected before the experiment. Plasma concentrations of lipids, ascorbate, alpha-tocopherol, and lipid peroxides did not change before and after the experiment in either group, but beta-carotene was higher in the tea group (P< 0.01 by paired Student'st-test). LDL (0.1 mg/mL) was incubated with 5 microM Cu(2+) and the oxidation was measured by absorbance at 234 nm. The lag time was significantly prolonged by 13.7 min in the tea group (P < 0.05 by paired Student'st-test, before versus after), whereas such a change was not observed in the control group. These results suggest that daily consumption of seven to eight cups (approximately 100 mL each cup) of green tea may increase resistance of LDL to in vivo oxidation, leading to reduction in the risk of cardiovascular diseases.     

An assay for functional dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) inhibitors of human dendritic cell adhesion

We report a new dendritic cell adhesion assay, using either immature or mature dendritic cells, for identifying functional dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) inhibitors. Because immature dendritic cells are responsible for pathogen binding and invasion, this in vitro assay provides an important link between in vitro results and pathogen-based in vivo assays. Furthermore, this assay does not require laborious expression, refolding, and purification of DC-SIGN carbohydrate recognition domain or extracellular domain as receptor-based assays. The assay power evaluated with Z and Z′ parameters enables screening of compound libraries and determination of IC₅₀ values in the first stage of DC-SIGN inhibitor development.     

SOS induction as an in vivo assay of enzyme-DNA interactions

We have constructed strains which are convenient and sensitive indicators of DNA damage and describe their use. These strains utilize an SOS::lac Z fusion constructed by Kenyon and Walker [Proc. Natl. Acad. Sci. USA 77 (1980) 2819-2823] and respond to DNA damage by producing beta-galactosidase. They can be used to characterize restriction systems and screen for restriction endonuclease mutants. Applications include the study of other enzymes involved in DNA metabolism, such as DNA methyltransferases, topoisomerases, recombinases, and DNA replication and repair enzymes.     

Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.     

Astroglial injury in an ex vivo model: contributions to its analysis in enriched cell cultures

In vitro cell culture models have been proposed to analyze some of the complex structural and functional characteristics involved in astroglial changes after neural injury in vivo. This report contributes to analyze the proposed hypothesis that an experimentally induced discontinuity of a confluent cellular culture could represent a useful model for the analysis of the processes involved in a neural lesion. For this purpose, it was decided to characterize astroglial proliferation and dye coupling state after a “scratch wound” applied to confluent, astrocyte-enriched cell cultures, obtained from several rat brain regions. Proliferation was assessed in terms of bromodeoxyuridine nuclear incorporation as a function of lesion width, serum deprivation, time after confluence, brain region origin, postlesional culture medium changes and agitation, and after application of a gap-junction uncoupling agent. The proliferative reaction after injury was neither cell type-specific nor brain region specific, nor was significantly affected by neither of the above-mentioned variables. Furthermore, injury failed to significantly affect the astroglial dye coupling state. Results suggest that the proliferative response observed under present conditions would depend on the disruption of contact inhibition rather than on astroglial mitogenic signals released from the wound and operating by either extracellular or cell coupling mechanisms. Present results question the validity of astrocyte-enriched cell cultures as an experimental model of neural tissue injury in vivo, as they do not appear to reproduce fundamental characteristics expressed in situ.     

Absence of metabolic rate allometry in an ex vivo model of mammalian skeletal muscle

Within mammalian species, standard metabolic rate (SMR) increases disproportionately with body mass (Mb), such that the mass-specific SMR correlates negatively with Mb. This phenomenon can be explained in part by reduced cellular metabolic rates in larger species. To better understand the cause(s) of this cellular metabolic rate allometry we have used an ex vivo approach to isolate and identify potential contributors. Skeletal myoblasts from mammalian species ranging inMb from 30 g to over 300,000 g were isolated and differentiated into myotubes in vitro. Oxygen consumption rates, citrate synthase (CS) activity, and lactate dehydrogenase (LDH) activity were measured in myotubes under standardized conditions. No correlation of any of these parameters was observedwith speciesMb, suggesting that there is no genetic contribution to between-species differences in cellular metabolic rates. Myotubes were incubated in serum from species ranging from 30 g to 400,000 g to determine whether between-species differences in the levels of metabolically important hormones might produce allometric trends in the cultured cells. However, there was no observed effect of serum donor Mb on any of the metabolic characteristicsmeasured. Thus, there is no evidence for a relationship between skeletal muscle oxidative metabolism and Mb in an ex vivo model.     

Design and application of an in vivo reporter assay for phenylalanine ammonia-lyase

Phenylalanine ammonia-lyase (PAL) is an important enzyme that links primary metabolism to secondary metabolism. Its efficiency is often a critical factor that affects the overall flux of a related metabolic pathway, the titer of the final products, and the efficacy of PAL-based therapies. Thus, PAL is a common target for metabolic engineering, and it is of significant interest to screen efficient PALs for industrial and medical applications. In this study, a novel and efficient visible reporter assay for screening of PAL efficiency in Escherichia coli was established based on a plant type III polyketide biosynthetic pathway. The candidate PALs were co-expressed with a 4-coumarate:CoA ligase 4CL1 from Arabidopsis thaliana and curcuminoid synthase (CUS) from Oryza sativa in E. coli BL21(DE3) to form a dicinnamoylmethane biosynthetic pathway. Taking advantage of the yellow color of the product, a microplate-based assay was designed to measure the titer of dicinnamoylmethane, which was validated by HPLC analysis. The different titers of the product reflect the overall performance (expression level and enzymatic activity) of the individual PALs in E. coli. Using this system, we have screened three PALs (PAL1, PAL3, and PAL4) from Trifolium pratense, among which PAL1 showed the best performance in E. coli. The engineered E. coli strain containing PAL1, 4CL1, and CUS led to the production of dicinnamoylmethane at a high level of 0.36 g/l. Supplement of 2-fluoro-phenylalanine yielded two fluorinated dicinnamoylmethane derivatives, 6,6′-difluoro-dicinnamoylmethane and 6-fluoro-dicinnamoylmethane, of which the latter is a new curcuminoid.     

Targeting myeloid leukemia with a DT(390)-mIL-3 fusion immunotoxin: ex vivo and in vivo studies in mice.

The IL-3 receptor was expressed on a high frequency of myeloid leukemia cells and also on hematopoietic and vascular cells. We previously showed that a recombinant IL-3 fusion immunotoxin (DT(390)IL-3) expressed by splicing the murine IL-3 gene to a truncated diphtheria toxin (DT(390)) gene selectively killed IL-3R(+) expressing cells and was not uniformly toxic to uncommitted BM progenitor cells (Chan,C.-H., Blazar,B.R., Greenfield,L., Kreitman,R.J. and Vallera,D.A., 1996, Blood, 88, 1445-1456). Thus, we explored the feasibility of using DT(390)IL-3 as an anti-leukemia agent. DT(390)IL-3 was toxic when administered to mice at doses as low as 0.1 microg/day. The dose limiting toxicity appeared to be related to platelet and bleeding effects of the fusion toxin. Because of these effects, DT(390)IL-3 was studied ex vivo as a means of purging contaminating leukemia cells from BM grafts in a murine autologous BM transplantation. In this setting, as few as 1000 IL-3R-expressing, bcr/abl transformed myeloid 32Dp210 leukemia cells were lethal. An optimal purging interval of 10 nM/l for 8 h eliminated leukemia cells from 32Dp210/BM mixtures given to lethally irradiated (8 Gy) C3H/HeJ syngeneic mice. Mice given treated grafts containing BM and a lethal dose of 32Dp210 cells survived over 100 days while mice given untreated grafts did not survive (P < 0.00001). DT(390)IL-3 may prove highly useful for ex vivo purging of lethal malignant leukemia cells from autologous BM grafts.