Effects of flunarizine on spontaneous synaptic currents in rat neocortex.

Flunarizine, a non-selective blocker of voltage-dependent Ca(2+) and Na(+) channels, is clinically effective against several neurological disorders, including epilepsy, migraine, and alternating hemiplegia of childhood. We examined the effects of flunarizine on spontaneous post-synaptic currents in acute brain slices maintained in vitro using patch-clamp electrophysiology. Flunarizine significantly attenuated the amplitude of spontaneous currents in pyramidal neurons from juvenile rat neocortex. Flunarizine had no effect on miniature spontaneous events recorded in the presence of tetrodotoxin, a blocker of voltage-dependent sodium channels. In high (9 mM) extracellular potassium, flunarizine reduced the amplitude and frequency of the spontaneous currents. Additionally, dimethyl sulfoxide, the solvent used in our experiments, reduced the amplitude of spontaneous currents, but only in high extracellular potassium. Our data suggest that the clinical activity of flunarizine may in part be a consequence of reducing spontaneous synaptic currents in the neocortex, especially under conditions of heightened neuronal activity.

Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens.

Many tumors express tumor-specific antigens capable of being presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules. Antigen presentation models predict that the tumor cell itself should present these antigens to T cells. However, when conditions for the priming of tumor-specific responses were examined in mice, no detectable presentation of MHC class I-restricted tumor antigens by the tumor itself was found. Rather, tumor antigens were exclusively presented by host bone marrow-derived cells. Thus, MHC class I-restricted antigens are efficiently transferred in vivo to bone marrow-derived antigen-presenting cells, which suggests that human leukocyte antigen matching may be less critical in the application of tumor vaccines than previously thought.

Bone marrow-derived cells present MHC class I-restricted tumour antigens in priming of antitumour immune responses.

Many tumours express tumour-specific antigens capable of being presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules. Current models of antigen presentation predict that the tumour cell itself should present its own MHC class I-restricted antigens to T cells. Earlier cross-priming experiments have demonstrated that at least some MHC class I-restricted antigens may also be presented by bystander cells. There is no detectable presentation of MHC class I-restricted tumour antigens by the tumour itself during priming of tumour-specific responses. The tumour antigens are presented exclusively by host bone marrow-derived cells. These results imply that an efficient mechanism exists in vivo for transfer of MHC I-restricted antigens to bone marrow-derived antigen presenting cells. They also suggest that HLA matching may not be critical in the clinical application of allogeneic tumour vaccines.

Controlled release, biodegradable cytokine depots: a new approach in cancer vaccine design.

Experimental studies using murine tumor models have demonstrated that potent systemic immunity can be generated using tumor vaccines engineered by gene transfer to secrete certain cytokines. The underlying physiological principle behind these strategies involves the sustained release of high doses of cytokine at the site of the tumor. In some cases, this paracrine approach appears to enhance tumor antigen presentation and avoids systemic cytokine toxicity. The widespread clinical use of autologous cytokine gene transduced tumor vaccines may be limited by the technical difficulty and labor intensity of individualized gene transfer. We have therefore explored an alternate approach to generating sustained release of cytokines local to the tumor cells. High doses of granulocyte-macrophage colony-stimulating factor encapsulated in cell-sized gelatin-chondroitin sulfate microspheres were mixed with irradiated tumor cells prior to s.c. injection. This vaccination scheme resulted in systemic anti-tumor immune responses comparable to granulocyte-macrophage colony-stimulating factor gene transduced tumor vaccines.

Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity.

To compare the ability of different cytokines and other molecules to enhance the immunogenicity of tumor cells, we generated 10 retroviruses encoding potential immunomodulators and studied the vaccination properties of murine tumor cells transduced by the viruses. Using a B16 melanoma model, in which irradiated tumor cells alone do not stimulate significant anti-tumor immunity, we found that irradiated tumor cells expressing murine granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated potent, long-lasting, and specific anti-tumor immunity, requiring both CD4+ and CD8+ cells. Irradiated cells expressing interleukins 4 and 6 also stimulated detectable, but weaker, activity. In contrast to the B16 system, we found that in a number of other tumor models, the levels of anti-tumor immunity reported previously in cytokine gene transfer studies involving live, transduced cells could be achieved through the use of irradiated cells alone. Nevertheless, manipulation of the vaccine or challenge doses made it possible to demonstrate the activity of murine GM-CSF in those systems as well. Overall, our results have important implications for the clinical use of genetically modified tumor cells as therapeutic cancer vaccines.

The antitumor immune response as a problem of self-nonself discrimination: implications for immunotherapy.

Much evidence now exists that tumors possess specific antigens recognizable by T cells. The goal of immunotherapy is to break tolerance to these antigens while preserving self-tolerance. Recently, newer approaches have been developed in animal systems that modify tumor cells genetically so that they express new antigens or secrete certain cytokines. Engineering tumor cells to secrete cytokines in a paracrine fashion can induce powerful local cytokine effects which, in addition to inducing local inflammation, can alter the presentation of tumor antigens or the activation of tumor-antigen-specific T lymphocytes, resulting in systemic antitumor immunity.

Herpes simplex-1 virus thymidine kinase gene is unable to completely eliminate live, nonimmunogenic tumor cell vaccines.

Recent experiments with genetically engineered tumors have generated renewed interest in active cellular immunotherapy as a cancer treatment modality. In order to consider the use of live tumor cells for immunotherapy in human cancer patients, it will be important to ensure that these cells do not themselves produce morbidity in the event the immune system fails to eliminate them. Toward this end, we have examined a strategy for eliminating genetically manipulated nonimmunogenic tumors in vivo. When B16F10 melanoma cells were transfected with the Herpes simplex virus 1 thymidine kinase (HSV-TK) gene, cells were rendered susceptible to killing by the nucleoside analogs acyclovir (ACV) and ganciclovir (GCV). B16-HSV-TK+ tumors established in C57BL6 mice were successfully "suicided" in vivo when GCV was administered by continuous infusion. However, late recurrences were observed even after 1 month of continuous GCV treatment. In vivo growth kinetics suggested that the recurrences resulted from a tiny number (< 20) of cells that had survived the GCV treatment. Interestingly, recurrent tumors were as sensitive to GCV as the parental B16-HSV-TK+ line. While these results demonstrate potential feasibility of the suicide gene strategy for active immunotherapy with live tumor cells, they also illustrate that approaches dependent on the intracellular generation of cell cycle-dependent toxins may fail to eliminate small numbers of cells that temporarily exit cell cycle or that are pharmacologically sequestered.

Treatment of established renal cancer by tumor cells engineered to secrete interleukin-4.

The generation of antigen-specific antitumor immunity is the ultimate goal in cancer immunotherapy. When cells from a spontaneously arising murine renal cell tumor were engineered to secrete large doses of interleukin-4 (IL-4) locally, they were rejected in a predominantly T cell-independent manner. However, animals that rejected the IL-4-transfected tumors developed T cell-dependent systemic immunity to the parental tumor. This systemic immunity was tumor-specific and primarily mediated by CD8+ T cells. Established parental tumors could be cured by the systemic immune response generated by injection of the genetically engineered tumors. These results provide a rationale for the use of lymphokine gene-transfected tumor cells as a modality for cancer therapy.

The fate of CD4-8- T cell receptor-alpha beta+ thymocytes.

CD4-8- TCR-alpha beta+ thymocytes represent a distinct population whose fate and function have remained a mystery. We show here that this thymocyte subset bears NK1, a surface Ag previously thought to be expressed exclusively by TCR- NK cells. Analysis of peripheral lymphocytes for the coexpression of TCR-alpha beta and NK1 revealed a subset with similar characteristics to the NK1+ thymocytes: a large fraction that are CD4-8- and a skewed TCR repertoire in which V beta 8 is overrepresented. Thymus transplant experiments into congenically marked athymic (nude) mice revealed that the NK1+TCR alpha beta+ subset was exclusively thymus derived and represented a distinct subset from the thymus-independent NK1+TCR- population. Finally, the NK1+TCR alpha beta+ population preferentially localizes to the bone marrow. These results demonstrate that this T cell subset is exported to the periphery after developing in the thymus. Their unique surface Ag expression and tissue localization suggest an immune function distinct from classical T cells.

Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response.

A poorly immunogenic murine colon cancer was used to investigate mechanisms of antitumor immunity. Injection of tumor cells engineered by gene transfection to secrete IL-2 stimulated an MHC class I-restricted cytolytic T lymphocyte (CTL) response against the parental tumor. The tumor cells secreting IL-2 produced an antitumor response in vivo, even in the absence of CD4+ T cells. Animals immunized with the engineered cells were protected against subsequent challenge with the parental tumor cell line. Similar findings were demonstrated for other tumor types. Thus, provision of a helper lymphokine in a paracrine fashion induced a tumor-specific immune response involving activation of endogenous CTLs and other immune effector cells. These findings demonstrate that the failure of an effective antitumor immune response may be primarily due to a helper arm deficiency of the immune system rather than a paucity of tumor-specific cytotoxic effector cells. Furthermore, they outline a novel strategy for augmenting tumor immunity.

Point mutations and small substitution mutations in three different upstream elements inhibit the activity of the mouse alpha 2(I) collagen promoter.

Point mutations and small substitution mutations were introduced in three different promoter segments of the mouse alpha 2(I) collagen gene that are binding sites for specific DNA-binding proteins. The first of these promoter elements contains a CCAAT motif between -80 and -84 and binds a novel factor consisting of two different components which are both needed for DNA binding (Hatamochi, A., Golumbek, P.T., Van Schaftingen, E., and de Crombrugghe, B. (1988) J. Biol. Chem. 263, 5940-5947). Four different point mutations in the CCAAT motif, which strongly inhibit binding of this heterodimeric CCAAT-binding factor, decrease the activity of the promoter at least 8-fold when assayed by DNA transfection of NIH 3T3 fibroblasts. The second cis-acting element is located around -250. Two mutations in this element, one a 3-base pair (bp), another a 4-bp substitution mutation, decrease the activity of the promoter in DNA transfection experiments by about 8- and 12-fold, respectively. The third element is located between -315 and -295 and binds nuclear factor 1 (NF1). Both 1- and 2-bp mutations which strongly inhibit binding of NF1 to this site decrease the activity of the promoter 8-10-fold in DNA transfection assays whereas another mutation in this site shows less inhibition in the activity of the promoter. Transfer of this NF1-binding site 5' to the SV40 early promoter increases the activity of this promoter more than 10-fold. These results indicate that the integrity of each of these three cis-acting elements is required for optimal activity of the alpha 2(I) collagen promoter and suggest a model whereby the factors which bind to these sequences cooperate in the formation of a transcription initiation complex.

Selective activation of transcription by a novel CCAAT binding factor.

A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the alpha 2(I) collagen, the alpha 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.

A CCAAT DNA binding factor consisting of two different components that are both required for DNA binding.

A CCAAT-binding activity present in nuclear extracts of rat liver and NIH 3T3 fibroblasts was purified using, as assay, DNA binding to a segment of the mouse alpha 2(I) collagen promoter. The activity consists of two components, designated factors A and B, which are separated by ion exchange chromatography on either Mono Q or Mono S columns. Factor A is heat-sensitive, whereas factor B is heat-resistant. Both factors are required for DNA binding and both are present in the DNA protein complex. The A + B complex was extensively purified by heparin-agarose and sequence-specific affinity chromatography. The Mr of factor A is 39,000, whereas the Mr of factor B is 41,000 as determined by renaturation of a highly purified preparation after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Competition experiments indicate that this CCAAT-binding complex has a DNA sequence specificity that is different from those of other CCAAT-binding proteins.