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Biomedical subjects

E Wong

Publications and source records attributed to E Wong.

At least 19 recordsLinked to original sources
PubMed1997

Hemispheric asymmetries in global and local processing: evidence from fMRI.

Functional magnetic resonance imaging (fMRI) was used to explore the brain substrate associated with global and local processing of visuospatial patterns. Systematic differences in activation, consistent with differences observed in reaction time data collected under conditions of visual hemifield presentation, were found in occipitotemporal regions of the right and left hemispheres. Within the right hemisphere, area of activation and fractional signal changes were greater under conditions of global processing than under local processing conditions. In the left hemisphere, activation to global and local input was high and comparable.

Adult↗
PubMed1997

Conversion of prostaglandin G/H synthase-1 into an enzyme sensitive to PGHS-2-selective inhibitors by a double His513 --> Arg and Ile523 --> val mutation.

Modeling of the active site of prostaglandin G/H synthase-2 (PGHS-2) onto PGHS-1 utilizing the known crystal structure of PGHS-1 shows that the only residues impinging directly on the active site that were not conserved in the two enzymes are His513 and Ile523 of PGHS-1 (Arg499 and Val509 of PGHS-2). These residues of human PGHS-1 were each mutated to the corresponding PGHS-2 residues (His513 --> Arg and Ile523 --> Val) and a double mutant (His513 --> Arg,Ile523 --> Val) containing both residues was also constructed. The mutant enzyme forms were expressed in COS-7 cells, and their properties were compared with those of the normal isoforms using microsomal membranes. The mutated enzyme forms all had apparent Km values within 1.4-fold that of the wild type enzyme, and the specific activity of the mutants were within 2-fold of that of PGHS-1. DuP697, NS-398, DFU, and SC-58125 are selective PGHS-2 inhibitors that act as time-dependent inhibitors of PGHS-2 and rapidly reversible competitive inhibitors of PGHS-1. The single Ile523 --> Val mutation increased the sensitivity to each of these selective inhibitors with most of the effect detected using instantaneous inhibition assays, except for DuP697, whose potency was further increased by preincubation with the enzyme. The double PGHS-1 His513 --> Arg, Ile523 --> Val mutant became more sensitive to inhibition by NS-398 and DFU than the single IV mutant, and time-dependent inhibition was observed. In contrast, the single HR mutation did not increase the sensitivity to inhibition by the selective PGHS-2 inhibitors. The potency of a selective PGHS-1 inhibitor, L-745,296, was decreased 5- and 13-fold in the HR and HR-IV mutants, respectively. All the results indicate that mutations of His513 and Ile523 residues of PGHS-1 can strongly increase sensitivity to selective PGHS-2 inhibition and restore time-dependent inhibition. They also suggest that the corresponding Arg499 and Val509 residues of PGHS-2 are essential determinants in differentiating between the interaction of nonselective NSAIDs and selective PGHS-2 inhibitors and their mechanism of action.

Amino Acid Sequence↗
PubMed1997

Cyclic AMP regulation of mouse proline-rich protein gene expression: isoproterenol induction of AP-1 transcription factors in parotid glands.

Proline-rich protein mRNAs are increased dramatically in the salivary glands of rats, mice, and hamsters upon treatment with the beta-agonist isoproterenol. Sequence comparisons between mice and hamster proline-rich protein genes identified conserved regions upstream from the transcription start site. Reporter plasmids containing these 5'-flanking sequences from a mouse proline-rich protein gene, MP2, were constructed and tested for transcriptional regulation by cAMP. Transient transfection experiments in mouse L-M cells showed that the upstream region -702 to -322 bp relative to the transcription start site is sufficient to confer cAMP induction on a heterologous promoter. Multiple copies of the AP-1 sequence elements within this region (-625 to -551) mediate the cAMP transcriptional response of reporter gene expression in L-M cells. L-M cell nuclear proteins and purified human c-jun protein bind to these upstream elements as determined by DNase I footprint analysis. Nuclear proteins isolated from mouse parotid glands protected the consensus AP-1 binding site 5'-TGAGTCA-3' (-592 to -586). The nuclear proteins interacting at this site were increased about sixfold in glands isolated from isoproterenol-treated mice when compared with glands from untreated mice. These results suggest that induction of AP-1 transcription factors in the parotid gland control the upregulation of some mouse salivary proline-rich proteins.

Adrenergic beta-Agonists↗
PubMed1997

Adenomatous polyposis coli tumor suppressor protein has signaling activity in Xenopus laevis embryos resulting in the induction of an ectopic dorsoanterior axis.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are linked to both familial and sporadic human colon cancer. So far, a clear biological function for the APC gene product has not been determined. We assayed the activity of APC in the early Xenopus embryo, which has been established as a good model for the analysis of the signaling activity of the APC-associated protein beta-catenin. When expressed in the future ventral side of a four-cell embryo, full-length APC induced a secondary dorsoanterior axis and the induction of the homeobox gene Siamois. This is similar to the phenotype previously observed for ectopic beta-catenin expression. In fact, axis induction by APC required the availability of cytosolic beta-catenin. These results indicate that APC has signaling activity in the early Xenopus embryo. Signaling activity resides in the central domain of the protein, a part of the molecule that is missing in most of the truncating APC mutations in colon cancer. Signaling by APC in Xenopus embryos is not accompanied by detectable changes in expression levels of beta-catenin, indicating that it has direct positive signaling activity in addition to its role in beta-catenin turnover. From these results we propose a model in which APC acts as part of the Wnt/beta-catenin signaling pathway, either upstream of, or in conjunction with, beta-catenin.

Adenomatous Polyposis Coli Protein↗
PubMed1997

Characterization of autocrine inducible prostaglandin H synthase-2 (PGHS-2) in human osteosarcoma cells.

The human osteosarcoma 143.98.2 cell line was found to express high levels of prostaglandin synthase-2 (PGHS-2) without detectable levels of prostaglandin synthase-1 (PGHS-1) as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Maximal levels of PGHS-2 induction were attained when the cells were grown beyond confluence. The osteosarcoma cells also secrete IL-1 alpha, IL-1 beta and TNF alpha in the culture medium. PGHS-2 expression was inducible by the exogenous addition of these cytokines as well as conditioned media from auto-induced cultures and inhibitable by treatment with dexamethasone. In contrast, undifferentiated U937 cells selectively express PGHS-1 as analyzed by RT-PCR and Western blotting. The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on the cellular PGE2 production mediated by each isoform of human PGHS were determined using osteosarcoma and undifferentiated U937 cells. When cells were preincubated with inhibitors to allow time-dependent inhibition prior to arachidonic acid stimulation, NS-398, CGP 28238, L-745,337, SC-58125 all behaved as potent (IC50 = 1-30 nM) and selective inhibitors of PGHS-2, in contrast to indomethacin, flurbiprofen or diclofenac which are potent inhibitors of enzymes. DuP-697 and sulindac sulfide were also potent inhibitors of PGHS-2 but both compounds inhibited cellular PGHS-1 activity at higher doses (IC50 = 0.2-0.4 microM). Time-dependent inhibition of PGE2 production in osteosarcoma cells was observed for indomethacin, diclofenac and etodolac. The synthesis of PGE2 by U937 cells was strongly dependent on exogenous arachidonic acid (100-fold stimulation) whereas confluent osteosarcoma cells also produced PGE2 without exogenous stimulus (7-fold stimulation by arachidonic acid). Osteosarcoma cells grown beyond confluence released more PGE2 from endogenous substrate than arachidonic acid stimulated undifferentiated U937 cells. These results indicate that osteosarcoma cells selectively express PGHS-2 with an autocrine regulation and effective utilization of endogenous arachidonic acid for PGE2 synthesis.

Arachidonic Acid↗
PubMed1997

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor.

1. DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furan one) was identified as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid-dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX-2 (IC50 = 41 +/- 14 nM) over COX-1 (IC50 > 50 microM). Indomethacin was a potent inhibitor of both COX-1 (IC50 = 18 +/- 3 nM) and COX-2 (IC50 = 26 +/- 6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B2 (TXB2) by Ca2+ ionophore-challenged human platelets (IC50 > 50 microM and 4.1 +/- 1.7 nM, respectively). 3. DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a Ki, value of 140 +/- 68 microM for the initial reversible binding to enzyme and a kappa 2 value of 0.11 +/- 0.06 s-1 for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62 +/- 26 microM and 0.06 +/- 0.01 s-1, respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1:1 stoichiometry and to dissociate only very slowly (t1/2 = 1-3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4. Inhibition of purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC50 = 63 +/- 5 microM at 0.1 microM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5. DFU inhibited lipopolysaccharide (LPS)-induced PGE2 production (COX-2) in a human whole blood assay with a potency (IC50 = 0.28 +/- 0.04 microM) similar to indomethacin (IC50 = 0.68 +/- 0.17 microM). In contrast, DFU was at least 500 times less potent (IC50 > 97 microM) than indomethacin at inhibiting coagulation-induced TXB2 production (COX-1) (IC50 = 0.19 +/- 0.02 microM). 6. In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 microM), DFU inhibited COX-1 with an IC50 value of 13 +/- 2 microM as compared to 20 +/- 1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac > indomethacin approximately naproxen > nimesulide approximately meloxicam approximately piroxicam > NS-398 approximately SC-57666 > SC-58125 > CGP 28238 approximately etodolac > L-745,337 > DFU. 7. DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED50 of 1.1 mg kg-1 vs 2.0 mg kg-1 for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg-1 vs 1.5 mg kg-1 for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED50 = 0.76 mg kg-1 vs 1.1 mg kg-1 for indomethacin). 8. In a sensitive model in which 51Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg-1, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg-1), meloxicam (3 mg kg-1) or etodolac (10-30 mg kg-1). A 5 day administration of DFU in squirrel monkeys (100 mg kg-1) did not affect chromium leakage in contrast to diclofenac (1 mg kg-1) or naproxen (5 mg kg-1). 9. The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX-1, a consistent high selectivity of inhibition of COX-2 over COX-1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX-1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti-inflammatory effect to gastropathy can be achieved with a selective COX-2 inhibitor.

Administration, Oral↗
PubMed1997

Seroprevalence of and risk factors for hepatitis A infection among young homosexual and bisexual men.

To evaluate hepatitis A infection among young homosexual and bisexual men, 411 men aged 17-22 years were surveyed at 26 public venues in San Francisco and Berkeley. Seroprevalence of hepatitis A infection was 28.0% (95% confidence interval [CI], 23.7%-32.6%). Recent infection was evident in 3.3% of susceptible men (95% CI, 1.6%-5.9%). Independent predictors of hepatitis A infection were Latino ethnicity (odds ratio [OR] = 5.3; 95% CI, 3.1-8.9), having > or = 50 lifetime male sex partners (OR = 1.8; 95% CI, 1.1-3.0), less than high school education (OR = 2.2; 95% CI, 1.2-4.1), and being a high school graduate (OR = 1.7; 95% CI, 1.0-2.9). Independent predictors of recent infection were less than high school graduate (OR = 7.6; 95% CI, 1.9-30.5), insertive anal intercourse (OR = 5.6; 95% CI, 1.0-32.8), and sharing needles without cleaning them (OR = 32.1; 95% CI, 3.0-346). Hepatitis A is a common infection in young homosexual men and is associated with sexual and drug-using behaviors.

Adolescent↗
PubMed1997

Determinants of peak bone mass in Chinese women aged 21-40 years. III. Physical activity and bone mineral density.

Previous studies on the relation between moderate physical activity and bone mass have observed conflicting results. Many of these studies have not dissociated the role of physical activity by age groups and in relation to the period of peak bone mass formation. Our cross-sectional analysis of the baseline data of a longitudinal study of 273 women aged 21-40 attempted to evaluate the role of moderate physical activity on bone mass around the period of peak bone mass attainment. The analyses were carried out separately for the two age groups--21-30 and 31-40--and had also taken into account the effects of age, dietary calcium intake, and lean body mass on bone mineral density (BMD). The total metabolic equivalent values (MET) of leisure time physical activity was based on the MET values for each activity and the reported time spent on each activity in the past year. The results indicated that among the younger group of women, high level of leisure time physical activity was associated with higher bone mass at both the spine and the hip. Additive effects of physical activity and dietary calcium intake on the spine and the hip BMD were observed. Together with age and lean body mass, physical activity and dietary calcium intake accounted for 19% of the variances of bone mineral at the spine and 9-11% at the hip. Among women aged 31-40, presumably after the peak bone mass formation, lean body mass as well as fat mass have independent strong association with BMD. Physical activity was not associated with bone mass in this age group.

Absorptiometry, Photon↗
PubMed1997

Comparison of the cyclooxygenase-1 inhibitory properties of nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors, using sensitive microsomal and platelet assays.

Two forms of cyclooxygenase (COX) activity are involved in the synthesis of prostaglandins, prostacyclins, and thromboxanes in mammalian cells. There is now convincing evidence, obtained with a number of structurally distinct inhibitors, that selective COX-2 inhibitors possess anti-inflammatory effects with an improved gastrointestinal tolerability compared with conventional nonsteroidal anti-inflammatory drugs (NSAIDs) affecting both COX-1 and COX-2. As more selective COX-2 inhibitors are being developed, assays with a high degree of sensitivity to inhibition are needed to compare the relative effects of compounds on COX-1 activity. In the present report, we describe a sensitive assay for the inhibition of human COX-1 based on the production of prostaglandin E2 by microsomes from U937 cells incubated with a subsaturating concentration of arachidonic acid. More than 45 NSAIDs and selective COX-2 inhibitors were tested in this assay. IC50 values ranged from 1 nM for flunixin and flurbiprofen to about 200-500 microM for salicylate and acetaminophen. Potent and nonselective NSAIDs such as sulindac sulfide, diclofenac, and indomethacin showed IC50 values of < 20 nM. Among the compounds that have been reported to show selectivity for COX-2, the rank order of potency against COX-1 was DuP 697 > SC-58451 > celecoxib > nimesulide-meloxicam-piroxicam-NS-398-RS-57067 > SC-57666 > SC-58125 > flosulide > etodolac > L-745,337 > DFU-T-614, with IC50 values ranging from 7 nM to 17 microM. A good correlation was obtained between the IC50 values for the inhibition of microsomal COX-1 and both the inhibition of TXB2 production by Ca2+ ionophore challenged platelets and the inhibition of prostaglandin E2 production by CHO cells stably expressing human COX-1. However, the microsomal assay was more sensitive to inhibition than cell-based assays and allowed the detection of inhibitory effects on COX-1 for all NSAIDs and selective COX-2 inhibitors examined with discrimination of their potency under conditions of limited availability of arachidonic acid.

Animals↗
PubMed1996

Meta-analysis of respiratory rehabilitation in chronic obstructive pulmonary disease.

BACKGROUND: Respiratory rehabilitation is increasingly recognised as an important part of the management of patients with chronic obstructive pulmonary disease (COPD). The widespread application of such programmes should be preceded by evidence of directly attributable improvements in function. We assessed the effect of respiratory rehabilitation on exercise capacity and health-related quality of life (HRQL) in patients with COPD. METHODS: We carried out a meta-analysis of randomised controlled trials of respiratory rehabilitation in patients with COPD that assessed functional or maximal exercise capacity, HRQL, or both. Respiratory rehabilitation was defined as exercise training (for at least 4 weeks) with or without education, psychological support, or both. The most commonly used measure for HRQL was the chronic respiratory questionnaire, in which responses were presented on a 7-point scale. The control groups received no rehabilitation. Within each trial and for each outcome an effect size was calculated; the effect sizes were then pooled by a random-effects model. The overall effect of treatment was compared with its minimum clinically important difference (MCID)--defined as the smallest difference perceived as important by the average patient. FINDINGS: We included 14 trials. Significant improvements were found for all the outcomes. For two important features of HRQL, dyspnoea and mastery, the overall treatment effect was larger than the MCID: 1.0 (95% CI 0.6-1.5) and 0.8 (0.5-1.2), respectively, compared with an MCID of 0.5. For functional exercise capacity (6-min walk test), the overall effect was 55.7 m (27.8-92.8), and for maximum exercise capacity (incremental cycle ergometer test), 8.3 W (2.8-16.5). Functional exercise capacity showed heterogeneity that could not be explained by the sensitivity analyses. INTERPRETATION: Respiratory rehabilitation relieves dyspnoea and improves the control over COPD. These improvements are clinically important. The value of the improvement in exercise capacity is not clear. Respiratory rehabilitation is an effective part of care in patients with COPD.

Exercise Therapy↗
PubMed1996

Mechanism of selective inhibition of human prostaglandin G/H synthase-1 and -2 in intact cells.

Selective inhibitors of prostaglandin synthase-2 (PGHS-2) possess potent anti-inflammatory, antipyretic, and analgesic properties but demonstrate reduced side-effects (e.g. gastrotoxicity) when compared with nonselective inhibitors of PGHS-1 and -2. We investigated the mechanism of the differential inhibition of human PGHS-1 (hPGHS-1) and -2 (hPGHS-2) in intact cells by nonsteroidal anti-inflammatory drugs (NSAIDs) and examined factors that contribute to the increased potency of PGHS inhibitors observed in intact cells versus cell-free systems. In intact Chinese hamster ovary (CHO) cell lines stably expressing the hPGHS isozymes, both PGHS isoforms exhibited the same affinity for arachidonic acid. Exogenous and endogenous arachidonic acid were used as substrates by both CHO [hPGHS-1] and CHO [hPGHS-2] cell lines. However, differences were observed in the ability of the hPGHS isoforms to utilize endogenous arachidonic acid released intracellularly following calcium ionophore stimulation or released by human cytosolic phospholipase A2 transiently expressed in the cells. Cell-based screening of PGHS inhibitors demonstrated that the selectivities and potencies of PGHS inhibitors determined using intact cells are affected by substrate concentration and differ from that determined in cell-free microsomal or purified enzyme preparations of PGHS isozymes. The mechanism of inhibition of PGHS isozymes by NSAIDs in intact cells involved difference in their time-dependent inhibition. Indomethacin displayed time-dependent inhibition of cellular hPGHS-1 and -2. In contrast, the selective PGHS-2 inhibitor NS-398 exhibited time-independent inhibition of hPGHS-1 but time-dependent inhibition of hPGHS-2 in intact cells. Reversible inhibition of cellular CHO [hPGHS-1] and CHO [hPGHS-2] was observed with the nonselective NSAIDs ibuprofen and indomethacin, whereas inhibition by the selective PGHS-2 inhibitor DuP-697 was reversible against hPGHS-1 but irreversible against hPGHS-2.

Animals↗
PubMed1996

Effect of oral estradiol on Lp(a) and other lipoproteins in postmenopausal women. A randomized, double-blind, placebo-controlled, crossover study.

BACKGROUND: Lp(a) lipoprotein level is an independent risk factor for premature coronary artery disease and cerebrovascular accident. Concentrations of this lipoprotein tend to increase after menopause. OBJECTIVE: To determine whether oral estrogen was effective in lowering concentrations of Lp(a) lipoprotein in postmenopausal women. METHODS: A double-blind, placebo-controlled, cross-over study was conducted during a 12-month period in 100 postmenopausal women who had undergone hysterectomy. They were randomized into two groups: group 1 received oral estradiol, 2 mg/d, for the first 6 months and placebo for the second, and group 2 received these treatments in the reverse order. After completion of the crossover study, the effect of prolonged administration of oral estradiol was examined by placing all patients on active treatment and repeating the lipoprotein measurements approximately 12 months later. RESULTS: No significant differences were noted between the two groups at the commencement of the study (median concentration of Lp[a] lipoprotein, 10.78 mg/dL [range, 2.2 to 108.5 mg/dL] in group 1 and 12.74 mg/dL [range, 0.8 to 98.1 mg/dL] in group 2). Crossover analysis showed a 9.62% reduction in values of Lp(a) lipoprotein with estradiol treatment compared with placebo during 12 months of treatment (P < .001). With prolonged treatment, the median concentration of Lp(a) lipoprotein for those in group 1 decreased from 8.12 mg/dL (range, 1.05 to 57.4 mg/dL) to 5.77 mg/dL (range, 0.84 to 75.39 mg/dL) (P < .001). In group 2, the median concentration decreased from 8.19 mg/dL (range, 2.52 to 99.82 mg/dL) to 7.07 mg/dL (range, 0.70 to 48.79 mg/dL), but this difference was not significant (P = .63). CONCLUSIONS: The results of this study confirm the beneficial effect of oral estradiol on the basic lipoprotein pattern and demonstrate that this treatment is effective in reducing concentrations of Lp(a) lipoprotein in postmenopausal women.

Administration, Oral↗
PubMed1996

Nitric oxide synthase in the olfactory mucosa of the larval sea lamprey (Petromyzon marinus).

The use of nitric oxide, a product of enzymatic reduction of L-arginine by nitric oxide synthase, as a modulator of processes within the olfactory mucosa was investigated in larval sea lampreys, extant fish of ancient vertebrate origin. In the present study, we demonstrated that the sea lamprey olfactory mucosa is specifically sensitive to L-arginine, that the L-arginine responses are inhibited by an inhibitor of nitric oxide synthase, N omega-nitro-L-arginine, and that nitric oxide synthase is present in olfactory receptor cells, sustentacular cells, and basal cells. Electron microscopic examination using NADPH-diaphorase histochemistry revealed intense labeling within secretory vesicles of sustentacular cells and in proximity to mitochondria within olfactory receptor cell dendrites and sustentacular cells. At the base of the olfactory epithelium, NADPH-diaphorase staining was intense in the perinuclear cytoplasm of a subpopulation of basal cells, moderate in sustentacular cell foot processes, and scattered in olfactory receptor cell axons. Throughout axons in the olfactory epithelium and the lamina propria, labeling predominated in axonal profiles with mitochondria. These physiological and ultrastructural studies imply that in sea lamprey larvae, nitric oxide modulates peri-receptor events of L-arginine chemostimulation, olfactory receptor cell axonal activity, and developmental processes.

Animals↗
PubMed1996

Transient protection of human T-cells from human immunodeficiency virus type 1 infection by transduction with adeno-associated viral vectors which express RNA decoys.

RNA decoys are oligonucleotides corresponding to the TAR and RRE sequences of HIV which inhibit the HIV-encoded regulatory proteins Tat and Rev, respectively. Adeno-associated viral vectors encoding RNA decoys stably transduced into the human T-cell line CEM-SS expressed transactivating region (TAR) and Rev-responsive element (RRE) RNA decoys from tRNA polIII promoters at high levels, without any apparent deleterious effects on cell growth or expression of CD4. DNA blot analysis indicated that RNA decoy-encoding vectors were not rearranged and were integrated into the genomic DNA of selected cell lines. Vector DNA with the appropriate TAR and RRE sequences was isolated from transduced cell lines after prolonged growth in culture, further confirming that the vector DNA was present in a stable form through multiple cell cycles. Cell lines expressing TAR and RRE decoys transiently inhibited HIV gene expression and replication by 70-99% as determined by measurement of intracellular and extracellular HIV p24 production. Adeno-associated vectors encoding RNA decoys may be useful for gene therapy of HIV infection.

Cell Line↗
PubMed1996

Newly established MST-1 tumour cell line and tumour-infiltrating lymphocyte culture from a patient with soft tissue melanoma (clear cell sarcoma) and their potential applications to patient immunotherapy.

The establishment and characterisation of paired autologous tumour cell line (MST-1) and tumour-infiltrating lymphocyte (TIL) culture from a tumour mass of a 14-year-old Taiwanese girl with soft tissue melanoma are described. MST-1 cells grown in vitro were heterogeneous in morphology, ranging from floating round cells, loosely attached round/oval or elongated cells with prominent pseudopod-like processes, to well-attached spindle and elongated dendritic cells without obvious pseudopods. Immunostaining revealed that major melanoma-associated antigens, such as S100 protein, HMB-45, melanotransferrin, chondroitin sulphate proteoglycan, and the gangliosides GD2 and GD3, were consistently expressed by the tumour tissue, severe combined immunodeficiency (SCID) mouse xenograft and derived cell lines. Flow cytometric analysis of the tumour DNA content showed an index of 1.8 relative to normal peripheral blood lymphocyte DNA. Chromosome analysis revealed all cells at a hypotetraploid level with several clonal chromosome aberrations, including deletions at 10p and 12q, an addition at 12q, translocations t(1;14) and t(5;6). Electron microscopy showed melanosome structures. This observation and the expression of the major melanoma-associated antigens were all indicative of the melanocytic origin of MST-1 tumour. Interleukin-2 (IL-2) expanded TILs had the predominant CD8+ phenotype and the capacity to lyse cells of the cultured autologous tumour. The availability of the soft tissue melanoma cell line, the SCID mouse xenograft tumour system as well as autologous TILs described herein would provide useful materials for identifying T-cell-defined antigens as well as a model system for devising individualised cancer biotherapeutic strategies. This cell line can also be used for further studies aimed at uncovering the histogenesis of this rare cancer.

Adolescent↗