Resistance of human leukocytes to vesicular stomatitis virus infection as one of the innate antiviral immune activities; participation of cell subpopulations.

Among reactions of innate immunity, resistance of human peripheral blood leukocytes (PBL) to viral infection seems important. The purpose of our study was to find, which of the subpopulations of PBL is the most responsible for the innate antiviral immunity of these cells. The innate immunity was measured by using the direct method of infection of leukocytes with vesicular stomatitis virus (VSV). The lack of VSV replication by infected leukocytes (0-1 log TCID50) was taken as an indicator for complete immunity; a low level of VSV (2-3 log) for partial immunity; and high VSV titer (more than 4 log) for no immunity. The resistance/innate immunity of whole PBL and subpopulations such as: adherent cells, fractions enriched in lymphocytes T, and lymphocytes B (separated on column with nylon wool), NK(+) and NK(-) (separated by microbeads activated cell sorting MACS) differ from each other. All fractions express higher resistance/innate immunity than the whole PBL. NK(+) cells were found the most resistant fraction of PBL to VSV infection. The results indicate that among the leukocytes in PBL the regulation mechanisms of innate immunity exist. The study on the mechanism of innate immunity regulation as well as the role of NK in innate immunity of PBL must be continued

Tissue localization of tumor antigen-loaded mouse dendritic cells applied as an anti-tumor vaccine and their influence on immune response.

The recognition, internalization and intracellular processing of antigen are the main functions of dendritic cells (DCs). In the course of these processes, DCs differentiate and acquire the ability to produce cytokines responsible for polarization of the immunological response. Therefore, vaccination with tumor antigen-loaded DCs is one of the most promising approaches to induce tumor-specific immune response. The purpose of this study was to analyze the migratory abilities, from an injection site to tumor-draining lymph nodes (tLN), of DCs applied as an anti-tumor vaccine and their capacity for immune response activation. Mouse DCs of the established JAWS II cell line transduced with EGFP gene or ex vivo bone marrow-isolated DCs (BM-DCs) stained with intravital CFDA dye were loaded with MC38 colon carcinoma tumor lysate (TAg) and then administered peritumorally to MC38 tumor-bearing C57BL/6 mice. On the first, third, fifth and seventh days after injection the tumors, tLNs and spleens were examined. The TAg-loaded DCs migrated more effectively to the tLNs than did the unloaded control DCs; however, the majority of them remained in the tumor vicinity. Immunohistological analysis of the tumor tissues demonstrated that only TAg-loaded DCs activated an immune response. Seven days after DCs vaccine administration, numerous necrotic areas and some apoptotic bodies were observed in the tumor tissue. However, the anti-MC38 tumor cytotoxic activity of spleen and tLN cells from mice treated with both TAg-loaded and unloaded DCs reached a maximum on the fifth day after DC injection. Concluding, TAg-loaded DCs migrated more efficiently to tLNs and were more effective activators of local (but not systemic) cellular immune response than were unloaded DCs. We hypothesize that only the application of TAg-loaded DCs to tumor-bearing mice as an adjuvant supporting chemotherapy may activate a more effective anti-tumor response

Clinical effects of multidrug resistance in neoplasms

Oporność na cytostatyki jest ciągle jedną z głównych przyczyn niepowodzeń systemowej terapii przeciwnowotworowej. Najlepiej poznanym mechanizmem warunkującym powstanie lekooporności jest działanie błonowych białek transportowych, które aktywnie usuwają leki z komórek nowotworowych. Nieprawidłowa, podwyższona ekspresja tych białek jest najczęściej opisywanym czynnikiem związanym z opornością nowotworów na cytostatyki. Spośród komórkowych białek transportowych najważniejszą funkcję pełni glikoproteina P (Pgp). Wzrost poziomu ekspresji tego białka uznaje się za niekorzystny czynnik rokowniczy zarówno w przypadku białaczek, jak i wielu rodzajów nowotworów litych. Znaczenie kliniczne pozostałych białek związanych z opornością wielolekową (MRP1, BCRP i LRP) jest przedmiotem intensywnych badań. W modelach doświadczalnych i próbach klinicznych stosuje się różne strategie ograniczenia ekspresji Pgp. Wyprowadzenie drugiej i trzeciej generacji modulatorów Pgp jest źródłem nadziei na ograniczenie zjawiska lekooporności głównie w nowotworach układu chłonnego i krwiotwórczego.Resistance to chemotherapy remains a major cause of the systemic anti-cancer treatment failure. The best known mechanism is often attributed to the function of drug transporter proteins in the plasma membrane, which actively remove drugs from neoplastic cells. Abnormal overexpression of these proteins is the most frequently described factor connected with cytostatics resistance. Among cellular transporter proteins the most important role plays glicoprotein P (Pgp). Increased level of this protein is considered as a poor prognostic factor both, in leukaemias and in many solid tumors. Clinical significance of other multidrug resistance proteins (MRP1, BCRP and LRP) remains subject of intensive studies. In experimental models and clinical trials different strategies are used to limit Pgp expression. Introduction of the second and third generation of Pgp blockers is a source of hope for the reversion of multidrug resistance, especially in lymphoid and blood neoplastic disorders

Resistance of human leukocytes to vesicular stomatitis virus infection as one of the innate antiviral immune activities; participation of cell subpopulations.

Among reactions of innate immunity, resistance of human peripheral blood leukocytes (PBL) to viral infection seems important. The purpose of our study was to find, which of the subpopulations of PBL is the most responsible for the innate antiviral immunity of these cells. The innate immunity was measured by using the direct method of infection of leukocytes with vesicular stomatitis virus (VSV). The lack of VSV replication by infected leukocytes (0-1 log TCID50) was taken as an indicator for complete immunity; a low level of VSV (2-3 log) for partial immunity; and high VSV titer (more than 4 log) for no immunity. The resistance/innate immunity of whole PBL and subpopulations such as: adherent cells, fractions enriched in lymphocytes T, and lymphocytes B (separated on column with nylon wool), NK(+) and NK(-) (separated by microbeads activated cell sorting MACS) differ from each other. All fractions express higher resistance/innate immunity than the whole PBL. NK(+) cells were found the most resistant fraction of PBL to VSV infection. The results indicate that among the leukocytes in PBL the regulation mechanisms of innate immunity exist. The study on the mechanism of innate immunity regulation as well as the role of NK in innate immunity of PBL must be continued

Actin in human colon adenocarcinoma cells with different metastatic potential.

Four human colon adenocarcinoma cell line variants with different metastatic potential were used to examine whether a correlation exists between actin level, state of actin polymerization and invasiveness of tumour cells. Monomeric (G), total (T) and filamentous (F) actin were determined in the cytosolic fraction of these cells. A statistically significant decrease in G actin level and increase in the state of actin polymerization (measured by F:G actin ratio) were found in the cytosol of three cell variants with higher metastatic potential and invasiveness (EB3, 3LNLN, 5W) compared with the parental cell line (LS180). Our experimental data lead to the conclusion that there is a correlation between the metastatic capacity of human colon adenocarcinoma cells and the state of actin polymerization

Antitumor effect of murine dendritic and tumor cells transduced with IL-2 gene Antitumor effect of murine dendritic and tumor cells transduced with IL-2 gene

Interleukin (IL-) 2 acts on a number of types of immune cells promoting their effector functions. To replace&lt;br /&gt;systemic administration of recombinant form of this cytokine, various genetically modified cells have been used in&lt;br /&gt;different preclinical models for tumor growth inhibition. In this study, dendritic or tumor cells transduced with retroviral&lt;br /&gt;vector carrying IL-2 gene (JAWS II/IL-2, X63/IL-2, MC38/IL-2 cells) alone or combined with tumor antigenstimulated&lt;br /&gt;dendritic cells (JAWS II/TAg) were exploited to treat colon carcinoma MC38-bearing mice. After the&lt;br /&gt;peritumoral injection of vaccine cells, the tumor growth delay and the increase in the number of tumor infiltrating&lt;br /&gt;CD4+ and CD8+ T lymphocytes were noted. A considerable increase in CD4+ cell influx into tumor tissue was observed&lt;br /&gt;when JAWS II/IL-2 cells or JAWS II/TAg with syngeneic MC38/IL-2 cells were applied. The increase in&lt;br /&gt;intensity of CD8+ cell infiltration was associated with immune reaction triggered by the same combination of applied&lt;br /&gt;cells or JAWS II/TAg with allogeneic X63/IL-2 cells. The effect observed in vivo was accompanied by MC38/0 cell&lt;br /&gt;specific cytotoxic activity of spleen cells in vitro. Thus, the application of vaccines, including IL-2-secreting cells of&lt;br /&gt;various origins, was able to induce different antitumor responses polarized by exogenous IL-2 and the encountered&lt;br /&gt;tumor antigen.<br>Interleukin (IL-) 2 acts on a number of types of immune cells promoting their effector functions. To replace&lt;br /&gt;systemic administration of recombinant form of this cytokine, various genetically modified cells have been used in&lt;br /&gt;different preclinical models for tumor growth inhibition. In this study, dendritic or tumor cells transduced with retroviral&lt;br /&gt;vector carrying IL-2 gene (JAWS II/IL-2, X63/IL-2, MC38/IL-2 cells) alone or combined with tumor antigenstimulated&lt;br /&gt;dendritic cells (JAWS II/TAg) were exploited to treat colon carcinoma MC38-bearing mice. After the&lt;br /&gt;peritumoral injection of vaccine cells, the tumor growth delay and the increase in the number of tumor infiltrating&lt;br /&gt;CD4+ and CD8+ T lymphocytes were noted. A considerable increase in CD4+ cell influx into tumor tissue was observed&lt;br /&gt;when JAWS II/IL-2 cells or JAWS II/TAg with syngeneic MC38/IL-2 cells were applied. The increase in&lt;br /&gt;intensity of CD8+ cell infiltration was associated with immune reaction triggered by the same combination of applied&lt;br /&gt;cells or JAWS II/TAg with allogeneic X63/IL-2 cells. The effect observed in vivo was accompanied by MC38/0 cell&lt;br /&gt;specific cytotoxic activity of spleen cells in vitro. Thus, the application of vaccines, including IL-2-secreting cells of&lt;br /&gt;various origins, was able to induce different antitumor responses polarized by exogenous IL-2 and the encountered&lt;br /&gt;tumor antigen

The variety of complexes formed by EcR and Usp nuclear receptors in the nuclei of living cells

International audienc

Optimization of a retroviral vector for transduction of human CD34 positive cells

Human stem and progenitor cells have recently become objects of intensive studies as an important target for gene therapy and regenerative medicine. Retroviral vectors are among the most effective tools for genetic modification of these cells. However, their transduction efficiency strongly depends on the choice of the ex vivo transduction system. The aim of this study was to elaborate a system for retroviral vector transduction of human CD34 positive cells isolated from cord blood. The retroviral vector pMINV EGFP was chosen for transduction of two human erythroblastoid cell lines: KG-1a (CD34 positive) and K562 (CD34 negative). For vector construction, three promoters and two retroviral vector packaging cell lines were used. To optimize the physicochemical conditions of the transduction process, different temperatures of supernatant harvesting, the influence of centrifugation and the presence of transduction enhancing agents were tested. The conditions elaborated with KG-1a cells were further applied for transduction of CD34 positive cells isolated from cord blood. The optimal efficiency of transduction of CD34 positive cells with pMINV EGFP retroviral vector (26% of EGFP positive cells), was obtained using infective vector with LTR retroviral promoter, produced by TE FLY GA MINV EGFP packaging cell line. The transduction was performed in the presence of serum, at 37°C, with co-centrifugation of cells with viral supernatants and the use of transduction enhancing agents. This study confirmed that for gene transfer into CD34 positive cells, the detailed optimization of each element of the transduction process is of great importance