AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutio...AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the “a” region.METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the “a” determinant at positions 120 (P→S), 123(T→N) and 161(M→T) were found to affect reactivity of these mAbs.CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.展开更多
In this In vitro study, T cell responses induced by breast tumor cell lysate pulsed monocyte-derived DCs were analyzed in terms of proliferation, specific cytotoxicity and cytokine-release in order to use in immunothe...In this In vitro study, T cell responses induced by breast tumor cell lysate pulsed monocyte-derived DCs were analyzed in terms of proliferation, specific cytotoxicity and cytokine-release in order to use in immunotherapeutic settings. Nylon wool enriched T lymphocytes from 5 patients with breast cancer stimulated In vitro with tumor cell lysate pulsed monocyte-derived DCs and their proliferation response were analyzed by [3H] thymidine uptake test. Specific cytotoxic activity of tumor antigen primed T cells after three rounds weekly stimulation was evaluated by flow cytometry, and interferon-γ (IFN-γ) and interleukin-4 (IL-4) cytokines release assay was carried out 24 hours after last stimulation in the supernatant of primed T cells using commercially available ELI-SA kits. T cell proliferation assay revealed that tumor cell lysate pulsed DCs could stimulate autologous T cell proliferation response with stimulation indices 4.9 - 30. T cell mediated cytotoxicity assay demonstrated that tumor antigen primed T cells could significantly kill autologous tumor cells more than normal cells (P γ and IL-4 in response to restimulation by antigen pulsed DCs which were dominated by IFN-γ production in 2 and IL-4 production in 3 out of 5 patients. Our result suggested that breast tumor antigen pulsed DCs could elicit effective specific antitumor T cell responses In vitro, therefore, tumor antigen pulsed DC vaccination may be considered as a novel strategy for immunotherapy of patients with breast cancer refractory to standard modalities.展开更多
基金Supported by Tehran University of Medical Sciences
文摘AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the “a” region.METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the “a” determinant at positions 120 (P→S), 123(T→N) and 161(M→T) were found to affect reactivity of these mAbs.CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.
文摘In this In vitro study, T cell responses induced by breast tumor cell lysate pulsed monocyte-derived DCs were analyzed in terms of proliferation, specific cytotoxicity and cytokine-release in order to use in immunotherapeutic settings. Nylon wool enriched T lymphocytes from 5 patients with breast cancer stimulated In vitro with tumor cell lysate pulsed monocyte-derived DCs and their proliferation response were analyzed by [3H] thymidine uptake test. Specific cytotoxic activity of tumor antigen primed T cells after three rounds weekly stimulation was evaluated by flow cytometry, and interferon-γ (IFN-γ) and interleukin-4 (IL-4) cytokines release assay was carried out 24 hours after last stimulation in the supernatant of primed T cells using commercially available ELI-SA kits. T cell proliferation assay revealed that tumor cell lysate pulsed DCs could stimulate autologous T cell proliferation response with stimulation indices 4.9 - 30. T cell mediated cytotoxicity assay demonstrated that tumor antigen primed T cells could significantly kill autologous tumor cells more than normal cells (P γ and IL-4 in response to restimulation by antigen pulsed DCs which were dominated by IFN-γ production in 2 and IL-4 production in 3 out of 5 patients. Our result suggested that breast tumor antigen pulsed DCs could elicit effective specific antitumor T cell responses In vitro, therefore, tumor antigen pulsed DC vaccination may be considered as a novel strategy for immunotherapy of patients with breast cancer refractory to standard modalities.
