Cyclooxygenase-2 promoter for tumour-specific targeting of adenoviral vectors to melanoma.

Novel therapeutic strategies are warranted for the treatment of metastatic melanoma as conventional therapies are inefficient. Conceptually, these strategies should be systemic and tumour-targeted. Gene therapy and viral oncolysis represent promising new approaches for cancer treatment that allow for the incorporation of molecular targeting strategies. In this regard, we analysed cyclooxygenase-2 (cox-2) expression as a potential new target for melanoma gene therapy. By reverse transcription polymerase chain reaction analysis, we showed cox-2 mRNA expression in all of the six tested melanoma cell lines, thus establishing cox-2 as a tumour marker for melanoma of potential interest for targeted therapeutics. Next, we analysed the activity and specificity of the cox-2 promoter within adenoviral vectors by luciferase assays. For this purpose, melanoma cell lines, primary melanoma cells and normal melanocytes were infected with adenoviruses containing cox-2 promoter sequences driving the luciferase reporter gene. The results demonstrated activity of the cox-2 promoter in melanoma cell lines and primary melanoma cells, but not in non-malignant primary epidermal melanocytes. Thus, we established herein the tumour specificity of the cox-2 promoter with potential applications for transcriptional targeting of adenoviral vector-based cancer gene therapy or virotherapy to melanoma.

Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood.

It has been known for years that rodents harbor a unique population of CD4(+)CD25(+) "professional" regulatory/suppressor T cells that is crucial for the prevention of spontaneous autoimmune diseases. Here we demonstrate that CD4(+)CD25(+)CD45RO(+) T cells (mean 6% of CD4(+) T cells) are present in the blood of adult healthy volunteers. In contrast to previous reports, these CD4(+)CD25(+) T cells do not constitute conventional memory cells but rather regulatory cells exhibiting properties identical to their rodent counterparts. Cytotoxic T lymphocyte-associated antigen (CTLA)-4 (CD152), for example, which is essential for the in vivo suppressive activity of CD4(+)CD25(+) T cells, was constitutively expressed, and remained strongly upregulated after stimulation. The cells were nonproliferative to stimulation via their T cell receptor for antigen, but the anergic state was partially reversed by interleukin (IL)-2 and IL-15. Upon stimulation with allogeneic (but not syngeneic) mature dendritic cells or platebound anti-CD3 plus anti-CD28 the CD4(+)CD25(+) T cells released IL-10, and in coculture experiments suppressed the activation and proliferation of CD4(+) and CD8(+) T cells. Suppression proved IL-10 independent, yet contact dependent as in the mouse. The identification of regulatory CD4(+)CD25(+) T cells has important implications for the study of tolerance in man, notably in the context of autoimmunity, transplantation, and cancer.

Mage-3 and influenza-matrix peptide-specific cytotoxic T cells are inducible in terminal stage HLA-A2.1+ melanoma patients by mature monocyte-derived dendritic cells.

Dendritic cell (DC) vaccination, albeit still in an early stage, is a promising strategy to induce immunity to cancer. We explored whether DC can expand Ag-specific CD8+ T cells even in far-advanced stage IV melanoma patients. We found that three to five biweekly vaccinations of mature, monocyte-derived DC (three vaccinations of 6 x 106 s.c. followed by two i.v. ones of 6 and 12 x 106, respectively) pulsed with Mage-3A2.1 tumor and influenza matrix A2. 1-positive control peptides as well as the recall Ag tetanus toxoid (in three of eight patients) generated in all eight patients Ag-specific effector CD8+ T cells that were detectable in blood directly ex vivo. This is the first time that active, melanoma peptide-specific, IFN-gamma-producing, effector CD8+ T cells have been reliably observed in patients vaccinated with melanoma Ags. Therefore, our DC vaccination strategy performs an adjuvant role and encourages further optimization of this new immunization approach.

Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma.

Dendritic cells (DCs) are considered to be promising adjuvants for inducing immunity to cancer. We used mature, monocyte-derived DCs to elicit resistance to malignant melanoma. The DCs were pulsed with Mage-3A1 tumor peptide and a recall antigen, tetanus toxoid or tuberculin. 11 far advanced stage IV melanoma patients, who were progressive despite standard chemotherapy, received five DC vaccinations at 14-d intervals. The first three vaccinations were administered into the skin, 3 x 10(6) DCs each subcutaneously and intradermally, followed by two intravenous injections of 6 x 10(6) and 12 x 10(6) DCs, respectively. Only minor (less than or equal to grade II) side effects were observed. Immunity to the recall antigen was boosted. Significant expansions of Mage-3A1-specific CD8(+) cytotoxic T lymphocyte (CTL) precursors were induced in 8/11 patients. Curiously, these immune responses often declined after the intravenous vaccinations. Regressions of individual metastases (skin, lymph node, lung, and liver) were evident in 6/11 patients. Resolution of skin metastases in two of the patients was accompanied by erythema and CD8(+) T cell infiltration, whereas nonregressing lesions lacked CD8(+) T cells as well as Mage-3 mRNA expression. This study proves the principle that DC "vaccines" can frequently expand tumor-specific CTLs and elicit regressions even in advanced cancer and, in addition, provides evidence for an active CD8(+) CTL-tumor cell interaction in situ as well as escape by lack of tumor antigen expression.

Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application.

Dendritic Cell (DC)-based vaccination approaches in man require a reproducible DC generation method that can be performed in conformity with GMP (Good Manufacturing Practice) guidelines and that circumvents the need for multiple blood drawings to generate DC. To this end we modified our previously described method to generate mature DC from CD14 + monocytes by a two step method (priming in GM-SF + IL-4 followed by maturation in monocyte conditioned medium) for use with leukapheresis products as a starting population. Several adaptations were necessary. We established, for example, a modified adherence step to reliably enrich CD14 + DC precursors from apheresis mononuclear cells. The addition of GM-CSF + IL-4 at the onset of culture proved disadvantageous and was, therefore, delayed for 24 h. DC development from apheresis cells occurred faster than from fresh blood or buffy coat, and was complete after 7 days. Monocyte conditioned medium when added on day 6 resulted in fully mature and stable DC (veiled, highly migratory and T cell sensitizing cells with a characteristic phenotype such as 85% CD83 + , p55/fascin + , CD115/M-CSF-R - , CD86 + ) already after 24 h. The mature DC progeny were shown to remain stable and viable if cultured for another 1-2 days in the absence of cytokines, and to be resistant to inhibitory effects of IL-10. Freezing conditions were established to generate DC from frozen aliquots of PBMC or to freeze mature DC themselves for later use. The approach yields large numbers of standardized DC (5-10 x 10(8) mature CD83 + DC/leukapheresis) that are suitable for performing sound DC-based vaccination trials that can be compared with each other.

Heterogeneity of signal transduction mechanisms in human basophils and human skin mast cells. II. Effects of 7-O-methyl-UCN-01, NPC 15437 and bryostatin 1 and 2, four protein kinase C-modulatory agents, on mediator release.

Basophils and mast cells play a crucial role in immunological and allergic processes due to the release of inflammatory mediators such as histamine. It has been suggested for a long time that the histamine release (HR) from these cells is closely related to protein kinase (PKC) activity. However, the distinct role of PKC with its large variety of isozymes in different cell types and the actions of these isozymes in HR still remain unclear. Therefore, in the present study, we compared the effects of the two PKC inhibitors 7-O-methyl-UCN-01 (UCN-01-Me) and NPC 15437 as well as two PKC activators, bryostatin 1 and 2, on anti-IgE and Ca(2+)-ionophore-induced HR from human basophils and isolated human skin mast cells (HSMC). In both HSMC and basophils, anti-IgE-induced HR was inhibited by PKC inhibitor UCN-01-Me pre-incubation dose-dependently. In stark contrast, A23187-induced HR was unaffected by UCN-01-Me in both cell types. In our experiments, the inhibitory efficacy of the compound NPC 15437 on HR was much lower than that of UCN-01-Me and showed no statistical significance. Both bryostatins 1 and 2 produced good dose-dependent inhibition of HR from HSMC stimulated with anti-IgE, whereas HR from basophils was potentiated with these compounds. The same effects were observed with basophils stimulated with A23187, where potentiation of HR was up to fourfold of the control at the highest concentrations of bryostatins, while HSMC showed a slight decrease in HR compared to non-bryostatin-treated controls. Basophils and HSMC showed very clear differences in HR when directly stimulated with the bryostatins, since no HR was observed from HSMC while in basophils the HR increased up to 47% of total histamine at the highest concentrations of bryostatins (1 mumol/l). HR from basophils was observed to be strictly dose-dependent. The differences in the cell reactions of the two cell types incubated with these four compounds indicate distinct biochemical roles of PKC in the cascades leading to degranulation of the cells. Furthermore, the experiments with UCN-01-Me support the hypothesis of PKC-beta to play a substantial positive modulatory role for the degranulation of immunologically stimulated basophils.

Effects of basophil-priming and stimulating cytokines on histamine release from isolated human skin mast cells.

Cell priming and stimulation of different cytokines (which include chemokines and growth factors) are typical features of human basophils. Recently, it has been shown that the macrophage chemotactic protein-1 (MCP-1), RANTES and macrophage inflammatory protein-1 alpha (MIP-1 alpha) are potent direct secretagogues for human basophils and that interleukin-3 (IL-3), IL-5 and granulocyte/macrophage colony-stimulating factor (GM-CSF) are priming factors for subsequent potentiation of mediator release from basophils induced by different stimuli. This observation may be clinically important for the activation and recruitment of inflammatory cells in different immune responses of the skin (e.g. late-phase reactions). The aim of the present study was to investigate whether cytokines and chemokines are also capable of priming or stimulating isolated human skin mast cells (SMC). SMC were either stimulated directly with the cytokines alone or preincubated with these factors for 10 min before being activated with suboptimal concentrations of anti-IgE, A23187 or substance P. IL-3, IL-5, GM-CSF, platelet factor-4 (PF-4), IL-8, MCP-1 and MIP-1 alpha (each at concentrations of 1 ng/ml to 1 microgram/ml, log steps) did not significantly modulate histamine release from SMC induced by the three different secretagogues. RANTES exhibited a weak but significant potentiating effect on IgE-mediated activation. Stem cell factor (SCF) as a positive control was able to prime mast cell histamine release strongly. In addition, PF-4, MCP-1, RANTES and MIP-1 alpha were incapable of inducing direct histamine release from SMC. In experiments with isolated human peripheral basophils, however, we observed potent Fc epsilon RI-mediated priming effects evoked through IL-3, IL-5 and GM-CSF. We conclude that SMC derived from healthy donors are not targets of (immuno)modulatory factors that prime or stimulate basophils.

Heterogeneity of human skin mast cells and human basophils. I. Pharmacological experiments with activators and inhibitors of protein kinase C.

Skin mast cells and basophilic leukocytes are known as key elements of acute and subacute IgE-mediated immune responses of the skin. The present paper investigated pharmacological aspects of signal transduction pathways of both cell types using activators and inhibitors of protein kinase C (PKC). The nonselective inhibitor K252a suppressed Fc epsilon RI-mediated histamine release from basophils and skin mast cells dose-dependently with IC50 values of 0.01 and 0.28 mumol/l. However, preincubation of both cell populations with kinase inhibitors showing in vitro selectivity for PKC (Ro 31-7549, calphostin C, GF 109203X) revealed a distinct modulation of cell response: IgE-mediated mediator release was inhibited only in skin mast cells, whereas in experiments with basophils a concentration-dependent potentiation of exocytosis was observed. Further evidence for heterogenous biochemical signals following activation of both cell types derived from studies with the phorbol ester TPA. With respect to acute and late-phase IgE-mediated skin reactions, we suggest that distinct signal transduction mechanisms at the level of PKC (isozymes) in basophils and skin mast cells might reflect their functional heterogeneity.

[The role of basophilic leukocytes in inflammatory skin diseases]. / Rolle der basophilen Leukozyten bei entzündlichen Hauterkrankungen.

Basophilic leukocytes are effector cells of the peripheral blood. They have several morphological and functional characteristics in common with tissue mast cells, such as expression of the high-affinity IgE receptor (Fc epsilon RI) and a high content of histamine within their granules. Functional comparison of human basophils and human mast cells isolated from different tissues revealed marked heterogeneity of mediator release after incubation with different secretagogues. Owing to their wide range of surface receptors, their (pro)inflammatory mediators released after activation, their mobility, and their rapid turnover, basophils appear basically to be potent effector cells, which migrate transiently into the skin during IgE-mediated (and/or IgE-independent) inflammatory reactions. The present paper reviews recent findings on the possible role of basophils for different immune reactions of the skin. Inhibition of basophil (and/or mast cell) activity seems to be necessary for both effective prophylaxis and therapy of allergic and inflammatory skin diseases. To date, however, the pharmacological modulation of mediator release from basophils lacks potent clinically useful compounds that can suppress the cellular response, since mast cell stabilizers such as cromoglycate and nedocromil are not effective. With a view to the development of active compounds, further in vitro studies should focus on the mechanisms of cell activation.