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Biomedical subjects

C Nicolette

Publications and source records attributed to C Nicolette.

7 recordsLinked to original sources
PubMed2000

In vitro priming with adenovirus/gp100 antigen-transduced dendritic cells reveals the epitope specificity of HLA-A*0201-restricted CD8+ T cells in patients with melanoma.

Replication-deficient recombinant adenovirus (Ad) encoding human gp100 or MART-1 melanoma Ag was used to transduce human dendritic cells (DC) ex vivo as a model system for cancer vaccine therapy. A second generation E1/E4 region deleted Ad which harbors the CMV immediate-early promoter/enhancer and a unique E4-ORF6/pIX chimeric gene was employed as the backbone vector. We demonstrate that human monocyte-derived DC are permissive to Ad infection at multiplicity of infection between 100 and 500 and occurs independent of the coxsackie Ad receptor. Fluorescent-labeled Ad was used to assess the kinetics and distribution of viral vector within DC. Ad-transduced DC show peak transgene expression at 24-48 h and expression remains detectable for at least 7 days. DC transduced with replication-deficient Ad do not exhibit any unusual phenotypic characteristics or cytopathic effects. DC transduced with Ad2/gp100v2 can elicit tumor-specific CTL in vitro from patients bearing gp100+ metastatic melanoma. Using a panel of gp100-derived synthetic peptides, we show that Ad2/gp100v2-transduced DC elicit Ag-specific CTL that recognize only the G209 and G280 epitopes, both of which display relatively short half-lives ( approximately 7-8 h) on the surface of HLA-A*0201+ cells. Thus, patients with metastatic melanoma are not tolerant to gp100 Ag based on the detection of CD8+ T cells specific for multiple HLA-A*0201-restricted, gp100-derived epitopes.

Adenoviruses, Human↗
PubMed2000

Adenoviral vector expressing ICP47 inhibits adenovirus-specific cytotoxic T lymphocytes in nonhuman primates.

Studies from several laboratories have shown that administration of E1-deleted Ad vectors results only in transient transgene expression in the lungs of immunocompetent animals. This is due, at least in part, to destruction of vector-transduced cells by host cellular immune responses (predominantly CD8(+) CTLs) directed against viral proteins and/or immunogenic transgene products. We have previously demonstrated that E1-deleted Ad vectors can lead to persistent expression of human cystic fibrosis transmembrane conductance regulator (hCFTR) in the lungs of several strains of immunocompetent mice, despite the presence of Ad-specific CTLs. However, we found that these same vectors gave rise only to transient hCFTR expression in the lungs of rhesus monkeys. We have constructed new Ad vectors that coexpress both hCFTR and the ICP47 gene from herpes simplex virus. ICP47 has been shown to inhibit the transporter associated with antigen presentation, thus blocking major histocompatibility antigen I (MHC class I)-mediated antigen presentation to CD8(+) T cells. The Ad/hCFTR/ICP47 vector decreased levels of cell-surface MHC class I molecules on infected monkey and human cell lines. Similar results were obtained with primary human cells and primary monkey airway epithelial cells. In vitro studies showed that the Ad/hCFTR/ICP47 vector decreased cytolysis by both monkey and human CTLs. When Ad/hCFTR/ICP47 was administered to the lungs of rhesus monkeys, it inhibited the generation of Ad-specific CTLs. However, natural killer cell activity was enhanced in monkeys treated with the Ad/hCFTR/ICP47 vector.

Adenoviridae↗
PubMed1995

Multiple Ras functions can contribute to mammalian cell transformation.

We have developed a generalized approach, using two hybrid interactions, to isolate Ha-Ras effector loop mutations that separate the ability of Ha-Ras to interact with different downstream effectors. These mutations attenuate or eliminate Ha-ras(G12V) transformation of mammalian cells, but retain complementary activity, as demonstrated by synergistic induction of foci of growth-transformed cells, and by the ability to activate different downstream components. The transformation defect of Ha-ras(G12V, E37G) is rescued by a mutant, raf1, that restores interaction. These results indicate that multiple cellular components, including Raf1, are activated by Ha-Ras and contribute to Ha-Ras-induced mammalian cell transformation.

Animals↗
PubMed1991

Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains expressing the activated RAS2Val19 gene or lacking both cAMP phosphodiesterase genes, PDE1 and PDE2, have impaired growth control and display an acute sensitivity to heat shock. We have isolated two classes of mammalian cDNAs from yeast expression libraries that suppress the heat shock-sensitive phenotype of RAS2Val19 strain. Members of the first class of cDNAs also suppress the heat shock-sensitive phenotype of pde1- pde2- strains and encode cAMP phosphodiesterases. Members of the second class fail to suppress the phenotype of pde1- pde2- strains and therefore are candidate cDNAs encoding proteins that interact with RAS proteins. We report the nucleotide sequence of three members of this class. Two of these cDNAs share considerable sequence similarity, but none are clearly similar to previously isolated genes.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Mutagenesis of the regulatory subunit of yeast cAMP-dependent protein kinase. Isolation of site-directed mutants with altered binding affinity for catalytic subunit.

Oligonucleotide-directed mutagenesis was used to produce mutants in the hinge region of the regulatory subunit (R) of the Saccharomyces cerevisiae cAMP-dependent protein kinase. The mutant proteins were expressed in Escherichia coli, purified, urea treated to produce cAMP-free regulatory (R), and analyzed in vitro for catalytic (C) subunit inhibitory activity in the presence and absence of cAMP. When assayed in the absence of cAMP, wild type R dimer inhibited C with an IC50 of 40 nM. Replacement of amino acid residue Ser-145 (the autophosphorylation site of yeast R) with Ala or Gly produced mutants which were 2-10-fold better inhibitors of C, while replacement with Glu, Asp, Lys, or Thr produced mutants which were 2-5-fold worse inhibitors of C relative to wild type R. When assayed in the presence of cAMP, all R subunits had a decreased affinity for C subunit, with Ser-145 and Thr-145 undergoing autophosphorylation. These results suggest that the amino acid at position 145 of R contributes to R-C interaction and therefore influences the equilibrium of yeast protein kinase subunits in vitro.

Amino Acid Sequence↗