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  • ASSAY  (4)
  • 1
    Keywords: CANCER ; CELLS ; EXPRESSION ; IN-VITRO ; proliferation ; tumor ; BLOOD ; CELL ; Germany ; human ; VITRO ; NEW-YORK ; POPULATION ; PROTEIN ; cell line ; LINES ; PATIENT ; RESPONSES ; IFN-GAMMA ; DONOR ; ANTIGENS ; DENDRITIC CELLS ; T cell ; T cells ; T-CELL ; T-CELLS ; CELL-LINES ; E7 ; papillomavirus ; LIMITATION ; STIMULATION ; TARGET ; ASSAY ; cervical cancer ; CERVICAL-CANCER ; human papillomavirus ; TYPE-16 ; GENOTYPES ; HPV ; E6 ; HPV16 ; HUMAN-PAPILLOMAVIRUS ; POPULATIONS ; ONCOPROTEIN ; VACCINE ; CANCER-PATIENTS ; CD8(+) ; ELISPOT ; EPITOPE ; EPITOPES ; IMMUNOTHERAPY ; intraepithelial neoplasia ; HUMAN-PAPILLOMAVIRUS TYPE-16 ; E7 ONCOPROTEIN ; IMMUNOGENICITY ; TARGETS ; INTERFERON-GAMMA ; 14 HIGH-RISK ; ENZYME-IMMUNOASSAY ; HUMAN BLOOD ; immunotherapy,T cell response,cytotoxic T cell,uterine cancer,tumor infiltrating lymphocytes ; INFILTRATING LYMPHOCYTES
    Abstract: Purpose. Human papillomavirus (HPV) type 16 and 18 are the most prevalent genotypes in cervical cancers. The viral oncoproteins E6 and E7 are considered to be tumor-specific targets for immunotherapy. HPV E7 antigen-loaded dendritic cells (DC) were evaluated as cellular tumor vaccine. Methods. Autologous monocyte-derived DCs loaded with recombinant HPV16 or HPV18 E7 oncoprotein were used to induce in vitro a specific T cell response. Specificities of activated T cells were determined. Results. E7-specific T cells could be identified in 18/20 T cell lines from healthy blood donors. CD4(+) T cell responses (13/16) were found by proliferation assay. CD8(+) CTLs (12/18) were detectable by interferon-gamma (IFN-gamma) ELISpot analysis. Seven donors reacted in both assays and only 2/20 T cell lines did not react in any assay. Thus, specific T cells could be activated in 〉80% of healthy individuals. T cell lines from suitable donors were specific for HLA-A*0201-restricted epitopes. Furthermore, HPV E7 antigen-loaded DC stimulated specific responses in freshly isolated tumor infiltrating lymphocyte (TIL) populations of cervical cancer patients. Conclusion. Autologous dendritic cells loaded with HPV E7 protein can induce T cell responses in healthy individuals by in vitro stimulation and evoke responses in TIL from cervical cancer biopsies. Since there are no limitations with respect to specific HLA-haplotypes, these findings may be a basis for the development of a therapeutic protein-based DC tumor vaccine against cervical cancer for HPV16- and HPV18-positive patients
    Type of Publication: Journal article published
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  • 2
    Keywords: CANCER ; CELLS ; IN-VITRO ; tumor ; Germany ; human ; IN-VIVO ; THERAPY ; VITRO ; SYSTEM ; DISEASE ; RISK ; GENE ; GENE-TRANSFER ; DNA ; INFECTION ; INDUCTION ; ANTIGEN ; DENDRITIC CELLS ; T cells ; T-CELLS ; E7 ; OPEN READING FRAME ; papillomavirus ; HUMANS ; ASSAY ; WOMEN ; cervical cancer ; CERVICAL-CANCER ; human papillomavirus ; HIGH-RISK ; ONCOGENE ; TRANSFORMATION ; HUMAN-PAPILLOMAVIRUS ; VACCINE ; SAFETY ; EPITOPE ; IMMUNOTHERAPY ; PLASMID DNA ; IMMUNODEFICIENCY-VIRUS TYPE-1 ; IMMUNIZATION ; MUTATIONAL ANALYSIS ; ASSAYS ; cytotoxic T lymphocyte ; BACTERIAL-DNA ; RISK-FACTOR ; in vivo ; tumor regression ; tumor protection ; ENHANCED IMMUNOGENICITY ; gene shuffling ; HPV16 E7 ; in vitro immunization
    Abstract: Anew and very promising approach in vaccine development is the application of naked DNA. In comparison to conventional vaccines it offers several advantages, especially if there is a need for the development of low cost vaccines. Infection with high-risk human papillomaviruses (hr-HPVs) is the major risk factor for the development of cervical cancer (cc), the third most common cancer in women worldwide. The HPV E7 oncogene is constitutively expressed in HPV-infected cells and represents an excellent target for immune therapy of HPV-related disease. Therefore, we chose the HPV-16 E7 as model antigen in the development of a therapeutic DNA vaccine candidate. For safety reasons the use of a transforming gene like the HPV-16 E7 for DNA vaccination is not feasible in humans. In consequence we have generated an artificial ("shuffled") HPV-16 E7-gene (HPV-16 E7SH), containing all putative cytotoxic T-lymphocyte (CTLs) epitopes and exhibiting high safety features. Here, we show the induction of a strong E7-wildtype (E7WT) directed cellular and humoral immune response including tumor protection and regression after in Vivo immunization in the murine system. Moreover, the vaccine candidate demonstrated immunogenicity in humans, demonstrated by priming of antigen-specific T cells in vitro. Importantly, the artificial HPV-gene has completely lost its transforming properties as measured in soft agar transformation assays. These results may be of importance for the development of vaccines based on oncogenes or oncoproteins. (c) 2006 Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16472545
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  • 3
    Keywords: EXPRESSION ; Germany ; IN-VIVO ; PATHWAY ; PATHWAYS ; VIVO ; PROTEIN ; FAMILY ; DOMAIN ; BINDING ; ASSAY ; PLASMA ; PLASMA-MEMBRANE ; DEGRADATION ; INVOLVEMENT ; KINASE-C ; DOMAINS ; INTERCELLULAR COMMUNICATION ; CARDIAC MYOCYTES ; ASSAYS ; in vivo ; PROLINE ; JUNCTION ; connexin ; EPITHELIAL NA+ CHANNEL ; GAMMA-ENAC ; gap junction ; GAP-JUNCTION PROTEIN ; PY motif ; ubiquitylation ; WW DOMAINS
    Abstract: Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization
    Type of Publication: Journal article published
    PubMed ID: 16931598
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  • 4
    Keywords: CANCER ; CELLS ; EXPRESSION ; proliferation ; radiotherapy ; tumor ; CELL ; Germany ; human ; TOXICITY ; NEW-YORK ; PROTEIN ; TUMORS ; PATIENT ; RESPONSES ; IFN-GAMMA ; INDUCTION ; DENDRITIC CELLS ; T cell ; T-CELL ; T-CELLS ; E7 ; papillomavirus ; IMMUNE-RESPONSES ; TARGET ; STAGE ; ASSAY ; cervical cancer ; CERVICAL-CANCER ; MARKERS ; human papillomavirus ; TYPE-16 ; GENOTYPES ; HPV ; E6 ; HPV16 ; HUMAN-PAPILLOMAVIRUS ; ONCOPROTEIN ; VACCINE ; CANCER-PATIENTS ; SAFETY ; ELISPOT ; immune response ; IMMUNE-RESPONSE ; IMMUNOTHERAPY ; vaccination ; HUMAN-PAPILLOMAVIRUS TYPE-16 ; SERIES ; CARCINOMA SCC ANTIGEN ; DE-NOVO ; E7 ONCOPROTEIN ; HLA ; IMMUNOGENICITY ; immunotherapy,ELISpot,T lymphocytes,CTL,uterine cancer ; STAGE IB ; TARGETS ; tumor marker
    Abstract: Purpose. Human papillomavirus (HPV) type 16 and 18 are the most prevalent genotypes in cervical cancer. The viral oncoproteins E6 and E7 are considered to be tumor-specific targets for immunotherapy. HPV E7 antigen-loaded autologous dendritic cells (DC) were evaluated as cellular tumor vaccine in a case series of cervical cancer patients. Methods. Autologous monocyte-derived DCs were pulsed with recombinant HPV16 E7 or HPV18 E7 oncoprotein and administered to 15 stage IV cervical cancer patients. Safety, toxicity, and induction of serological and cellular immune responses were monitored. Results. The vaccine was well-tolerated and no local or systemic side effects or toxicity were recorded. A specific serologic response was seen in 3/11 evaluated patients. Specific cellular immune responses (4/11) were detected with 2/10 positive de novo reactions plus one boosted preexistent response in proliferation assays and 3/11 in IFN-gamma ELISpot assays. A transient drop in tumor marker SCC was observed in 5/9 evaluable patients but did not correlate with markers of the immune response. No objective clinical response was observed. Tumor biopsies available from four patients showed severe or complete loss of HLA expression in three of the advanced tumors. Conclusion. Autologous dendritic cells pulsed with HPV E7 protein can induce T cell responses in a portion of late stage cervical cancer patients. Boosting of immune responses by adjuvants and vaccination of tumor HLA-positive patients will be mandatory in future trials
    Type of Publication: Journal article published
    PubMed ID: 12898233
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