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Y Wan

Publications and source records attributed to Y Wan.

At least 91 records · Page 5Linked to original sources
PubMed1999

Effects of stuffer DNA on transgene expression from helper-dependent adenovirus vectors.

We have analyzed transgene (lacZ) expression from a first-generation adenovirus (Ad) vector in comparison to helper-dependent (hd) Ads deleted for various portions of the viral coding sequences and generated by using the Cre/loxP helper-dependent system (R. J. Parks et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996). An hd vector deleted for approximately 70% of the Ad genome (AdRP1001) provided levels and durations of transgene expression similar to those of a control first generation Ad vector containing an identical expression cassette. Deletion of all Ad sequences from the hdAd and replacement with a approximately 22-kb fragment of lambda DNA resulted in a decrease in the level and duration of lacZ expression which could not be reversed by the inclusion of a matrix attachment region. However, substitution of the lambda stuffer in the fully deleted hdAd with sequences from the human hypoxanthine-guanine phosphoribosyltransferase gene resulted in significantly improved transgene expression. In vitro assays for cytotoxic T lymphocytes (CTL) directed against putative peptides encoded by the vector backbone showed that, although CTL were generated against the vector containing the lambda DNA, no such CTL were generated against the vector containing the hypoxanthine-guanine phosphoribosyltransferase (HPRT) sequences. Surprisingly, the rate of loss of the HPRT- and lambda-containing vectors from mouse liver was similar, despite the differences in expression kinetics, indicating that the lambda stuffer-directed CTL were inefficient at eliminating the transduced cells. Thus, the nature of the DNA backbone of hdAds can have important effects on the functioning of the vector. Since most fully deleted vectors require "stuffer" DNA as part of the vector backbone to maintain optimum vector size, these observations must be taken into account in the design of hdAd vectors.

Adenoviridae↗
PubMed1999

Testing for linkage under robust genetic models.

Robust genetic models are used to assess linkage between a quantitative trait and genetic variation at a specific locus using allele-sharing data. Little is known about the relative performance of different possible significance tests under these models. Under the robust variance components model approach there are several alternatives: standard Wald and likelihood ratio tests, a quasilikelihood Wald test, and a Monte Carlo test. This paper reports on the relative performance (significance level and power) of the robust sibling pair test and the different alternatives under the robust variance components model. Simulations show that (1) for a fixed sample size of nuclear families, the variance components model approach is more powerful than the robust sibling pair approach; (2) when the number of nuclear families is at least approximately 100 and heritability at the trait locus is moderate to high (>0.20) all tests based on the variance components model are equally effective; (3) when the number of nuclear families is less than approximately 100 or heritability at the trait locus is low (<0. 20), on balance, the Monte Carlo test provides the best power and is the most valid. The different testing procedures are applied to determine which are able to detect the known association between low density lipoprotein cholesterol and the common genotypes at the locus encoding apolipoprotein E. Results from this application show that the robust sibling pair method may be more effective in practice than that indicated by simulations.

Alleles↗
PubMed1999

Murine dendritic cells transduced with an adenoviral vector expressing a defined tumor antigen can overcome anti-adenovirus neutralizing immunity and induce effective tumor regression.

In this study transduced dendritic cells (DCs) were used to enhance immunogenecity of a specific tumor antigen. Using a polyoma middle T (PyMT) transgenic mammary carcinoma model we found that injections of DCs transduced with an adenoviral (Ad) vector expressing PyMT (DCAd-PymT) led to potent specific anti-tumor immunity. Efficacy was not affected by neutralizing Abs (high or low titers) and naive animals did not produce detectable anti-Ad Abs following two injections of transduced DCs. Repeated injections of transduced DCs significantly improved therapeutic efficacy in mice with established lung metastases. These data emphasize the ability of Ad-infected DCs to: i) minimize anti-Ad Ab production, ii) overcome pre-existing anti-Ad humoral immunity, and iii) improve vaccination efficacy when injected more than once.

Adenoviridae↗
PubMed1998

Phenotype, function, and in vivo migration and survival of allogeneic dendritic cell progenitors genetically engineered to express TGF-beta.

BACKGROUND: Administration of donor bone marrow (BM)-derived dendritic cell (DC) progenitors (DCp) that are major histocompatibility complex (MHC) class II+ but costimulatory molecule (CD40, CD80, CD86)-deficient can prolong mouse heart allograft survival This is associated with microchimerism and inhibition of antidonor cytotoxic T lymphocyte (CTL) activity. Genetic modification of these donor antigen-presenting cells to express an immunosuppressive molecule(s) may enhance their in vivo survival and potential tolerogenicity. METHODS: The surface phenotype of B10(H-2b) DCp before and after gene transfer using replication-deficient adenoviral (Ad) vectors was determined by monoclonal antibody (mAb) staining and flow cytometry. Transforming growth factor-beta (TGF-beta) production was quantitated by enzyme-linked immunosorbent assay. Allostimulatory activity of the gene-transduced DCp was ascertained by mixed leukocyte reaction (MLR) and CTL induction. To assess their in vivo migratory activity and survival, the transduced cells were injected subcutaneously into one hind footpad of C3H (H-2k) mice. Tissues (draining popliteal lymph nodes [LN], spleens, and thymi) were removed 1, 2, 7, and 14 days later and stained for donor MHC class II using anti-LA(b) mAb in an immunohistochemical procedure. The mean number of IAb+ cells per unit area was determined. RESULTS: Transduction with a control Ad vector (Ad-LacZ) at 50 multiplicity of infection slightly increased CD40 and CD86 expression and up-regulated the poor allostimulatory activity of the DCp assessed by MLR and CTL responses. These effects on function were negated in Ad-TGF-beta1-transduced cells. After their injection into mouse footpads, the gene-transduced IAb+ cells were observed in maximal numbers in the popliteal LN at day 1 and in marginal zones and T-dependent areas of spleens (peak at day 7) but were rare in thymi. Transduction with Ad-LacZ reduced the numbers of IAb+ cells identified in both LN and spleens at all time points postinjection, suggesting that the vector alone affected DC life span in allogeneic recipients. TGF-beta1 transgene expression not only fully prevented the reduction in DC induced by Ad transduction alone, but also increased numbers and prolonged the survival of donor cells in the spleen, as shown by a two-to fivefold increase in IAb+ cells at days 2-14 compared with control (Ad-LacZ-transduced) DC. CONCLUSION: BM-derived DCp can be transduced efficiently to express TGF-beta1 using an Ad vector. They exhibit very poor allostimulatory activity and similar migration characteristics in vivo to unmodified DCp. Survival of TGF-beta gene-transduced DC, however, is enhanced significantly compared with unmodified and (especially) control Ad-LacZ gene-transduced DC. Genetic engineering of donor DC to express the immunosuppressive molecule TGF-beta promotes their survival in allogeneic hosts and may potentiate their previously reported tolerogenicity.

Adenoviridae↗
PubMed1998

The G protein G alpha12 stimulates Bruton's tyrosine kinase and a rasGAP through a conserved PH/BM domain.

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are signal transducers that relay messages from many receptors on the cell surface to modulate various cellular processes. The direct downstream effectors of G proteins consist of the signalling molecules that are activated by their physical interactions with a G alpha or Gbetagamma subunit. Effectors that interact directly with G alpha12 G proteins have yet to be identified. Here we show that G alpha12 binds directly to, and stimulates the activity of, Bruton's tyrosine kinase (Btk) and a Ras GTPase-activating protein, Gap1m, in vitro and in vivo. G alpha12 interacts with a conserved domain, composed of the pleckstrin-homology domain and the adjacent Btk motif, that is present in both Btk and Gap1m. Our results are, to our knowledge, the first to identify direct effectors for G alpha12 and to show that there is a direct link between heterotrimeric and monomeric G proteins.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

OFQ reverses the kappa-opioid receptor-mediated depression of calcium current in rat dorsal root ganglion neurons.

Since the characterization of orphanin FQ (OFQ), the endogenous ligand of ORL1 receptor, much work has focused on its physiological functions. OFQ was reported to antagonize the effect of opioid-induced antinociception, although its mechanism remains obscure. In the present study, whole-cell patch clamp recording technique was used to observe if OFQ can reverse the inhibition of calcium current produced by the kappa-opioid agonist U50,488H (U50) in acutely dissociated rat DRG neurons. The concentrations of OFQ and U50 were 50 nM and 10 microM, respectively. Among 49 cells recorded, the calcium channel currents of 37 (75.5%) cells were inhibited by U50, of which 30 (81.1%) cells could be reversed by OFQ. It was interesting to note the similarity between OFQ and the well characterized anti-opioid peptide CCK-8 in that it reversed kappa-opioid receptor agonist induced suppression on calcium channel current, while by itself showed a calcium channel suppressive effect. Thus OFQ may be regarded as another anti-opioid peptide.

Animals↗
PubMed1998

Analysis of the Gs/mitogen-activated protein kinase pathway in mutant S49 cells.

Heterotrimeric G protein-coupled receptors can activate the mitogen-activated protein kinase (MAPK) cascade. Recent studies using pharmacological inhibitors or dominant-negative mutants of signaling molecules have advanced our understanding of the pathways from G protein-coupled receptors to MAPK. However, molecular genetic analysis of these pathways is inadequate in mammalian cells. Here, using the well characterized Gsalpha- and protein kinase A-deficient S49 mouse lymphoma cells, we provide the molecular genetic evidence that Gsalpha is responsible for transducing the beta-adrenergic receptor signal to MAPK in a protein kinase A-dependent pathway involving Rap1 and Raf (but not Ras) molecules.

Animals↗
PubMed1998

Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo.

Human skin is exposed daily to solar ultraviolet (UV) radiation. UV induces the matrix metalloproteinases collagenase, 92-kD gelatinase, and stromelysin, which degrade skin connective tissue and may contribute to premature skin aging (photoaging). Pretreatment of skin with all-trans retinoic acid (tRA) inhibits UV induction of matrix metalloproteinases. We investigated upstream signal transduction pathways and the mechanism of tRA inhibition of UV induction of matrix metalloproteinases in human skin in vivo. Exposure of human skin in vivo to low doses of UV activated EGF receptors, the GTP-binding regulatory protein p21Ras, and stimulated mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38. Both JNK and p38 phosphorylated, and thereby activated transcription factors c-Jun and activating transcription factor 2 (ATF-2), which bound to the c-Jun promoter and upregulated c-Jun gene expression. Elevated c-Jun, in association with constitutively expressed c-Fos, formed increased levels of transcription factor activator protein (AP) 1, which is required for transcription of matrix metalloproteinases. Pretreatment of human skin with tRA inhibited UV induction of c-Jun protein and, consequently, AP-1. c-Jun protein inhibition occurred via a posttranscriptional mechanism, since tRA did not inhibit UV induction of c-Jun mRNA. These data demonstrate, for the first time, activation of MAP kinase pathways in humans in vivo, and reveal a novel posttranscriptional mechanism by which tRA antagonizes UV activation of AP-1 by inhibiting c-Jun protein induction. Inhibition of c-Jun induction likely contributes to the previously reported prevention by tRA of UV induction of AP-1-regulated matrix-degrading metalloproteinases in human skin.

Activating Transcription Factor 2↗
PubMed1998

Case-control study of the D2 dopamine receptor gene and smoking status in lung cancer patients.

BACKGROUND: Interindividual differences in the structure and expression of the dopamine receptor genes affect dopamine availability and may be the genetic basis for variation in vulnerability to tobacco smoking. In this study, prevalences of polymorphisms in the TaqIA allele (A1 and A2) and the TaqIB allele (B1 and B2) of the D2 dopamine receptor gene in 157 lung cancer case patients and 126 control subjects were determined to assess whether individuals homozygous or heterozygous for the less common A1 and B1 alleles are more vulnerable to nicotine addiction. METHODS: Case and control subjects were accrued from an ongoing epidemiologic study. Blood samples were collected from them and subjected to molecular genetic analyses. Subjects were interviewed to obtain relevant information. Current and former smokers were administered a questionnaire to quantify their addiction to nicotine. RESULTS: The combined B1B2 genotypes appeared to be more prevalent in ever smokers than in never smokers among case patients (30.3% versus 13.3%; two-sided P = .233) and among control subjects (30.9% and 0%; two-sided P = .02); statistically significant differences were not observed among those with A1 genotypes. Statistically significant correlations between the presence of the A1 and B1 alleles were observed (r = .73 for case subjects and r = .76 for control subjects; two-sided P<.001). Individuals with rarer genotypes reported having been substantially younger at the time of smoking initiation (statistically significant for both A1 and B1) and having attempted to quit smoking fewer times (statistically significant for only A1). CONCLUSION: Variant alleles of the D2 dopamine receptor gene may play a role in determining nicotine addiction, although the associations between the at-risk genotypes and measures of nicotine addiction were not entirely consistent.

Aged↗
PubMed1998

A double recombinant adenovirus expressing the costimulatory molecule B7-1 (murine) and human IL-2 induces complete tumor regression in a murine breast adenocarcinoma model.

Tumors that express tumor-specific antigens can maintain growth in an immunocompetent organism. Current hypotheses tend toward T cell anergy as a key component for the inhibition of immunoreactivity against such tumors. Anergy is thought to occur from hyperactive stimulation of the TCR in the absence of costimulation (costimulation leads to proliferation via IL-2 production). Subcutaneous injection of transgenic polyoma middle T transformed breast adenocarcinoma tumor cells (PyMT) in the hind flank of FVB/n mice results in the formation of tumor nodules at this site. We determined the MHC class I and class II, B7-1, and B7-2 expression in the tumor cells by flow cytometry and showed positive staining for only MHC class I. We show that a single E1-deleted adenovirus constructed to express both the costimulatory molecule B7-1 (murine) and human IL-2 genes (Ad5E1 mB7-1/human IL-2) elicits a very potent antitumor response when administered intratumorally. Ad5E1 mB7-1/human IL-2 induced rapid and complete regression (100%) of all tumors compared with Ad5 E1 mB7-1 (38%), Ad CAIL-2 (42%), and Ad5E1 dl70-3 (control vector) (0%). All mice that exhibited complete tumor regression were fully protected in tumor cell challenge experiments. The systemic immunity generated by intratumoral administration of the Ad vectors was associated with a strong anti-PyMT CTL response. These observations indicate that augmenting the immunogenicity of the tumor with coincident expression of B7-1 in combination with IL-2 may prove beneficial in direct tumor immunotherapy.

Adenocarcinoma↗
PubMed1998

Effect of estrogen on vascular smooth muscle cells is dependent upon cellular phenotype.

To investigate the growth-regulating action of estrogen on vascular smooth muscle cells (SMC), effects of beta-17-estradiol (beta-E2) on phenotypic modulation and proliferation of rabbit aortic SMC were observed in vitro. At 10(-8)M, beta-E2 significantly slowed the decrease in volume fraction of myofilaments (Vv myo) of freshly dispersed SMCs in primary culture, indicating an inhibitory effect of beta-E2 on spontaneous phenotypic modulation of SMC from a contractile to a synthetic phenotype. Freshly dispersed SMCs treated with beta-E2 also had a relatively longer quiescent phase than control cells before intense proliferation occurred. This was in contrast to SMCs in passage 2 3 (synthetic state), where beta-E2-treated cells replicated significantly faster than untreated cells. beta-E2 also markedly enhanced the serum-induced DNA synthesis of synthetic SMCs in a concentration-dependent manner within physiological range (10(-10)to 10(-8)M). These findings indicate that the growth-regulating effect of estrogen on vascular SMC is dependent on the cell's phenotypic state. It delays the cell cycle re-entry of the contractile SMCs by retarding their phenotypic modulation: however, once cells have modulated to the synthetic phenotype, it promotes their replication.

Animals↗
PubMed1998

Identification of a novel point mutation (Leu72Pro) in the NADH-cytochrome b5 reductase gene of a patient with hereditary methaemoglobinaemia type I.

Utilizing polymerase chain reaction (PCR) related technology, we investigated the b5R gene of a Chinese patient with hereditary methaemoglobinaemia type I and found a novel missense mutation (CTC-CCC) at codon 72 in exon 3 of the gene. As the mutation generates an Apa I recognition site, homozygosity for the mutation was confirmed by restriction analysis of PCR-amplified fragments from the patient's genomic DNA. We predicted that the residue replacement of Leu with Pro of the mutant enzyme would account for the b5R deficiency in the patient. The results further confirm the genetic polymorphism of b5R gene mutations found in the RCM type I.

Child, Preschool↗
PubMed1998

Genetic linkage analysis using lognormal variance components.

Typically genetic studies of continuous traits such as cholesterol levels or blood pressure assume that interindividual variability follows a normal distribution. Here we develop methods to analyze positively skewed data by assuming a lognormal distribution. We develop a variance components approach for identifying such effects from a major gene, residual polygenic factors and nongenetic factors. We compare by a simulation study results from fitting this lognormal model with either applying the log transformation or not transforming the data. We found that the lognormal model provided more precise estimates and more powerful tests than a simple log transformation when analyzing lognormally distributed data. Power varied with sibship size. For the same total number of nonindependent sibpairs, larger sibships were less powerful. However, larger sibships are more economical because they require a smaller sample size to obtain a specified power. To illustrate the application of this lognormal model to real data, we studied evidence for linkage between triglycerides and the lipoprotein lipase gene.

Genetic Linkage↗
PubMed1998

Linkage of familial Hibernian fever to chromosome 12p13.

Autosomal dominant periodic fevers are characterized by intermittent febrile attacks of unknown etiology and by recurrent abdominal pains. The biochemical and molecular bases of all autosomal dominant periodic fevers are unknown, and only familial Hibernian fever (FHF) has been described as a distinct clinical entity. FHF has been reported in three families-the original Irish-Scottish family and two Irish families with similar clinical features. We have undertaken a genomewide search in these families and report significant multipoint LOD scores between the disease and markers on chromosome 12p13. Cumulative multipoint linkage analyses indicate that an FHF gene is likely to be located in an 8-cM interval between D12S77 and D12S356, with a maximum LOD score (Z max) of 3.79. The two-point Z max was 3.11, for D12S77. There was no evidence of genetic heterogeneity in these three families; it is proposed that these markers should be tested in other families, of different background, that have autosomal dominant periodic fever, as a prelude to identification of the FHF-susceptibility gene.

Chromosome Mapping↗
PubMed1998

Enhanced interleukin-2 gene transfer immunotherapy of breast cancer by coexpression of B7-1 and B7-2.

The capability of B7-1 to augment the antitumor activity of some cytokines has been shown primarily for such cytokines as interleukin-12 (IL-12), IL-7, and to a lesser extent IL-2. In this study, we investigate the ability of B7-1 and B7-2 to augment the antitumor activity of IL-2. Considering the affinity of both molecules for CD28 (T cell receptor for B7-1 and B7-2), we postulated that their potential to augment IL-2 antitumor activity would be similar. Two murine transgenic adenocarcinoma models were chosen to investigate the activity of adenoviral vectors constructed to express either B7-1 and IL-2 or B7-2 and IL-2. Before administering the vector intratumorally to tumor-bearing mice, we determined the expression of B7-1, B7-2, MHC I, and MHC II on these tumor cells and demonstrated positive expression of only MHC I. Intratumoral injection of the vector expressing B7-1 and IL-2 resulted in complete regression of all tumors treated. In contrast, the vector expressing B7-2 and IL-2 was significantly less effective at regressing PyMT tumors, whereas both double recombinant vectors demonstrated similar levels of complete regression in the Neu (NDL 8142) model. Regressed mice were all protected for rechallenge in both models and demonstrated antigen-specific cytotoxic T lymphocytes (CTL) in the PyMT model. These findings indicate that the combination of IL-2 with B7-1 or B7-2 significantly enhances the antitumor activity of IL-2.

Adenocarcinoma↗
PubMed1998

[Anti-tumor effect of cytotoxic T lymphocytes induced by soluble antigen of ovarian carcinoma and anti-CD3 monoclonal antibody on ovarian carcinoma].

OBJECTIVE: To evaluate the therapeutic effect of the new method--adoptive cell immunotherapy on ovarian carcinoma. METHODS: Tumor soluble antigen (TSA) was extracted from cells of ovarian carcinoma (COC1 and COC2n). To generate cytotoxic T lymphocytes (CTL), peripheral blood mononuclear cells (PBMC) collected from healthy persons were induced by TSA and anti-CD3 monoclonal antibody (CD3-McAb). The antitumor effect of CTL on COC1 cells in vitro was compared with LAK and CD3-AK, whereas the growth inhibitory effect of CTL on COC2n transplantation tumors in nude mice was compared with CD3-AK. RESULTS: The cytotoxicity of CTL, CD3-AK and LAK was 79.4%, 52.1% and 51.7% respectively (P < 0.01). Mean volume of tumor in nude mice on the 9th day after ovarian carcinoma cells transplanted was 44.4 +/- 24.2 mm3 in CTL group, 118.8 +/- 40.0 mm3 in CD3-AK group and 443.0 +/- 158.7 mm3 in control (no treatment) (P < 0.01), whereas mean duration of survival was 28.5 days, 25.5 days and 17 days respectively (P < 0.01). CONCLUSION: The results indicated that CTL exhibited higher cytotoxic effect in vitro and greater inhibitory effect on tumor growth resulting in longer survival in vivo. It suggested that the adoptive cell immunotherapy might provide a new idea for treatment of ovarian carcinoma.

Animals↗