Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy.

Many tumor-associated Ags represent tissue differentiation Ags that are poorly immunogenic. Their weak immunogenicity may be due to immune tolerance to self-Ags. Prostatic acid phosphatase (PAP) is just such an Ag that is expressed by both normal and malignant prostate tissue. We have previously demonstrated that PAP can be immunogenic in a rodent model. However, generation of prostate-specific autoimmunity was seen only when a xenogeneic homolog of PAP was used as the immunogen. To explore the potential role of xenoantigen immunization in cancer patients, we performed a phase I clinical trial using dendritic cells pulsed with recombinant mouse PAP as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly vaccinations of xenoantigen-loaded dendritic cells with minimal treatment-associated side effects. All patients developed T cell immunity to mouse PAP following immunization. Eleven of the 21 patients also developed T cell proliferative responses to the homologous self-Ag. These responses were associated with Ag-specific IFN-gamma and/or TNF-alpha secretion, but not IL-4, consistent with induction of Th1 immunity. Finally, 6 of 21 patients had clinical stabilization of their previously progressing prostate cancer. All six of these patients developed T cell immunity to human PAP following vaccination. These results demonstrate that xenoantigen immunization can break tolerance to a self-Ag in humans, resulting in a clinically significant antitumor effect.

Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells.

PURPOSE: Provenge (Dendreon Corp, Seattle, WA) is an immunotherapy product consisting of autologous dendritic cells loaded ex vivo with a recombinant fusion protein consisting of prostatic acid phosphatase (PAP) linked to granulocyte-macrophage colony-stimulating factor. Sequential phase I and phase II trials were performed to determine the safety and efficacy of Provenge and to assess its capacity to break immune tolerance to the normal tissue antigen PAP. PATIENTS AND METHODS: All patients had hormone-refractory prostate cancer. Dendritic-cell precursors were harvested by leukapheresis in weeks 0, 4, 8, and 24, loaded ex vivo with antigen for 2 days, and then infused intravenously over 30 minutes. Phase I patients received increasing doses of Provenge, and phase II patients received all the Provenge that could be prepared from a leukapheresis product. RESULTS: Patients tolerated treatment well. Fever, the most common adverse event, occurred after 15 infusions (14.7%). All patients developed immune responses to the recombinant fusion protein used to prepare Provenge, and 38% developed immune responses to PAP. Three patients had a more than 50% decline in prostate-specific antigen (PSA) level, and another three patients had 25% to 49% decreases in PSA. The time to disease progression correlated with development of an immune response to PAP and with the dose of dendritic cells received. CONCLUSION: Provenge is a novel immunotherapy agent that is safe and breaks tolerance to the tissue antigen PAP. Preliminary evidence for clinical efficacy warrants further exploration.

Priming tissue-specific cellular immunity in a phase I trial of autologous dendritic cells for prostate cancer.

We attempted to induce therapeutic immunity against prostate-derived tissues in patients suffering from progressive hormone-refractory metastatic prostate carcinoma. Thirteen patients were treated with two infusions, 1 month apart, of autologous dendritic cells (APC8015) preexposed ex vivo to PA2024, a fusion protein consisting of human granulocyte/macrophage-colony stimulating factor (GM-CSF) and human prostatic acid phosphatase (PAP). The infusions were followed by three s.c. monthly doses of PA2024 without cells. Three groups of patients each received PA2024 at 0.3, 0.6, or 1.0 mg/injection. All Ps were two-sided. Treatment was well tolerated. After infusions of APC8015, patients experienced only mild (grade 1-2) short-lived fever and/or chills, myalgia, pain, and fatigue. One patient developed grade 3 fatigue. Four patients developed mild local reactions to s.c. PA2024. Twelve patients were evaluable for response to treatment. Circulating prostate-specific antigen levels dropped in three patients. T cells, drawn from patients after infusions of APC8015, but not before, could be stimulated in vitro by GM-CSF (P = 0.0004) and PAP (P = 0.0001), demonstrating broken immune tolerance against these two normal proteins. Injections of PA2024 did not influence the reactivity of T cells against PAP and GM-CSF. However, antibodies to GM-CSF and, to a much lesser extent, to PAP reached maximum titers only after two or even three injections of PA2024, showing that directly injected PA2024 was involved in stimulation of humoral immunity. Dendritic cells exposed to antigen ex vivo can induce antigen-specific cellular immunity in prostate cancer patients, warranting further studies of this mode of immunotherapy.

The application of multiwavelet filterbanks to image processing.

Multiwavelets are a new addition to the body of wavelet theory. Realizable as matrix-valued filterbanks leading to wavelet bases, multiwavelets offer simultaneous orthogonality, symmetry, and short support, which is not possible with scalar two-channel wavelet systems. After reviewing this theory, we examine the use of multiwavelets in a filterbank setting for discrete-time signal and image processing. Multiwavelets differ from scalar wavelet systems in requiring two or more input streams to the multiwavelet filterbank. We describe two methods (repeated row and approximation/deapproximation) for obtaining such a vector input stream from a one-dimensional (1-D) signal. Algorithms for symmetric extension of signals at boundaries are then developed, and naturally integrated with approximation-based preprocessing. We describe an additional algorithm for multiwavelet processing of two-dimensional (2-D) signals, two rows at a time, and develop a new family of multiwavelets (the constrained pairs) that is well-suited to this approach. This suite of novel techniques is then applied to two basic signal processing problems, denoising via wavelet-shrinkage, and data compression. After developing the approach via model problems in one dimension, we apply multiwavelet processing to images, frequently obtaining performance superior to the comparable scalar wavelet transform.

Immunogenic characterization of a tissue culture-derived vaccine that affords partial protection against avian coccidiosis.

The immunogenicity of a tissue culture-derived vaccine generated from an Eimeria tenella-infected cell line in a serologically defined bird line, and the ability to confer protection against homologous challenge in young chicks was examined. The cell line, SB-CEV-1/F7, was infected with E. tenella sporozoites and the resulting 72-h postinfection cell-free supernatants were adjuvanted and used to immunize Leghorn chicks homozygous for the B19 haplotype. Peripheral blood and splenic lymphocytes from these immunized birds proliferated in vitro in response to both sporozoite and SB-CEV-1/F7 tissue culture-derived parasite antigens. In addition, splenic immune lymphocytes obtained from birds previously exposed to E. tenella in vivo responded to these tissue culture-derived parasite antigens in vitro. To evaluate the efficacy of the vaccine, B19B19 chicks were vaccinated s.c. with adjuvanted 72-h postinfection cell-free supernatants or an ammonium sulfate precipitate derivative thereof, orally boosted, and then subjected to homologous parasite challenge at 10 d of age. The level of protection (body weight gain, cecal lesions) was assessed 6 d after challenge. Performance results from four battery trials demonstrated that vaccinated birds were significantly protected against weight loss compared to unimmunized, challenged controls. In addition, in two of the four trials, vaccinated birds were significantly protected against lesions. These results provide strong evidence that tissue culture-derived parasite antigens obtained from the E. tenella-infected SB-CEV-1/F7 cell line are immunogenic in birds and can provide partial protection against E. tenella clinical coccidiosis.

Biological properties of dendritic cells: implications to their use in the treatment of cancer.

Dendritic cells are the principal initiators of antigen-specific immune responses. The mechanisms by which they activate resting, naive T cells are increasingly well understood. Dendritic cells have several molecules on their surface that are critical for T-cell activation; they secrete multiple soluble factors that are important for T-cell differentiation and growth; and they home to areas in lymphoid organs that are rich in effector T cells. This knowledge has led to the belief that delivery of antigens with dendritic cells that have been manipulated ex vivo could stimulate powerful cellular immune responses against tumors. Pilot clinical trials indicate that dendritic cell vaccines can induce efficacious tumor-specific immune responses.

Human T cell responses to human papillomavirus type 16 L1 and E6 synthetic peptides: identification of T cell determinants, HLA-DR restriction and virus type specificity.

Four T cell determinants in the major capsid protein of human papillomavirus (HPV) type 16 L1 and one in the E6 protein associated with cellular transformation were defined using synthetic peptides to stimulate peripheral blood mononuclear cells from asymptomatic individuals. HLA-DR restriction was defined using murine L cells transfected with HLA-DR genes to present antigen. Responses to two of the five determinants by T cell lines and clones were shown to be specific for HPV-16 based on the lack of cross-recognition of the corresponding sequences of other known papillomavirus sequences (types 1a, 5, 6b, 8, 11, 18 and 33). The T cells raised against two of the other peptides cross-reacted with corresponding peptides from other strains to varying extents, depending on their structural homology. The implications of these results regarding the prevalence of HPV-16 infection in the population and the possible diagnostic role of these responses in papillomavirus infection is discussed.

Degenerate binding of immunogenic peptides to HLA-DR proteins on B cell surfaces.

Binding of linear fragments of protein antigens to class I or class II molecules of the MHC is necessary for the stimulation of a cellular immune response. This report describes the binding of a biotinylated T cell determinant from influenza hemagglutinin to class II proteins on the surface of Epstein-Barr virus-transformed B lymphocytes. The rapid, simple, and quantitative binding assay involves flow cytometric analysis of transformed B cells stained with fluoresceinated streptavidin following incubation with the biotinylated peptide. Binding of the biotinylated peptide required cell surface expression of human class II molecules, and was inhibited by an anti-HLA-DR monoclonal antibody as well as the unbiotinylated natural determinant. Rates of association and dissociation of the peptide were similar to those reported for purified MHC class II proteins, and the peptide bound only approximately 1% of the DR molecules expressed on the cell surface. When assayed on many different DR-homozygous B cell lines, the biotinylated hemagglutinin T cell determinant bound to HLA-DR on each cell line. The degeneracy of peptide binding to B cell lines was not unique to the hemagglutinin peptide because three other biotinylated T cell determinants failed to bind to class II deficient B-lymphoblastoid cells but bound to varying degrees to multiple DR-homozygous lines.

In vitro expansion of Epstein-Barr virus-specific HLA-restricted cytotoxic T cells direct from the blood of infectious mononucleosis patients.

The cytotoxic T-cell response induced by primary Epstein-Barr virus (EBV) infection and detectable in the blood of infectious mononucleosis (IM) patients shows several unusual features when tested in in vitro assays. Lysis of EBV-transformed target lines occurs with no apparent HLA restriction, and the putative EBV specificity of the response has been seriously questioned. In the present work we show that the primary T-cell response in IM is polyclonal and indeed does contain a virus-specific HLA class I antigen-restricted component, which can be selectively expanded in vitro in the presence of appropriate stimulator cells and IL-2. This allows functional analysis of the virus-specific component of the response in the absence of co-resident reactivities. Studies on blood samples taken from individuals in the acute phase of IM and again post-convalescence suggest that functionally similar populations of HLA class I-restricted cytotoxic T cells are involved in the control of both the primary and persistent phase of EBV infection.

Multiple HLA class I-dependent cytotoxicities constitute the "non-HLA-restricted" response in infectious mononucleosis.

Primary Epstein-Barr virus (EBV) infection, when manifest as infectious mononucleosis (IM), induces a broad-ranging and apparently non-HLA-restricted cytotoxic response whose nature has not been resolved. In the present experiments the ability to cryo-preserve IM mononuclear cell preparations, after depletion of CD16+ natural killer cells, has allowed detailed analysis of the response on appropriately constructed target cell panels. The results show that IM effector preparations are polyclonal with separate HLA class I antigen-dependent reactivities against the autologous EBV-transformed lymphoblastoid cell line (LCL) and particular HLA class I-mismatched LCLs. The autologous LCL-directed response shows the hallmarks of immunologically specific T cell cytotoxicity; only EBV+ B cell blasts are recognized and the interaction can be blocked by monoclonal antibodies to CD3 and CD8 on the effector cell surface and to HLA class I antigens on the target cell. Such findings demonstrate, for the first time, that the primary cytotoxic response to EBV infection includes a virus-specific HLA-restricted component like that found in the T cell memory of persistently infected individuals. Separate components of the response are preferentially active against some (but not all) HLA-mismatched LCLs, the pattern of reactivity being distinct for each individual IM patient and reproducible on repeated testing. Monoclonal antibody blocking experiments show that these HLA-mismatched interactions also involve CD3 and CD8 antigens on the effector cell and HLA class I antigens on the target cell. Where tested, such lysis affected both EBV+ and EBV- B cell blasts from the relevant HLA-mismatched donors. We postulate that a widespread primary infection of the B cell system by EBV leads to a generalized expansion not just of the virus-specific response but also of other T cell responses coincidentally active at the time. The unusual activity of IM effector preparations against HLA-mismatched LCLs arises from fortuitous cross-recognition of allogeneic cells by immunologically specific cytotoxic T cell clones coincidentally expanded in vivo alongside the EBV-specific response.

Cyclic AMP: a mitogenic signal for Swiss 3T3 cells.

Addition of cholera toxin (100 ng/ml) to quiescent cultures of Swiss 3T3 cells acts synergistically with serum (2-4%), insulin, phorbol esters, epidermal growth factor, and fibroblast-derived growth factor to stimulate DNA synthesis. In the presence of insulin, cholera toxin caused a dose-dependent increase in cumulative [3H]thymidine incorporation into acid-insoluble material and in the intracellular cyclic AMP (cAMP) level. The dose--response curves for the two processes were similar. Furthermore, addition of 1-methyl-3-isobutylxanthine (15--500 microM) or of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (5--100 microM), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase which are potent inhibitors of cyclic nucleotide phosphodiesterase activity, stimulated DNA synthesis and increased cAMP levels in Swiss 3T3 cells. These compounds strikingly potentiated the effect of cholera toxin on DNA synthesis and on cAMP levels. When quiescent Swiss 3T3 cells were exposed to cholera toxin (100 ng/ml) and insulin at 10 micrograms/ml (4- to 7-fold increase in cAMP level) or to these agents and 1-methyl-3-isobutyl xanthine at 50 microM (35-fold increase in cAMP level), DNA synthesis began after a lag of 16 hr. These results indicate that cAMP acts as a mitogenic signal for Swiss 3T3 cells and differ from the widely held view that cyclic AMP inhibits the proliferation of fibroblast cells.

Benign gastrocolic fistula healing with conservative management.

We report a case of gastrocolic fistula resulting from a benign gastric ulcer, diagnosed by barium meal and endoscopy. The fistula healed with conservative management and treatment with carbenoxolone sodium. To our knowledge this is the first reported case of successful conservative treatment of a benign gastrocolic fistula using carbenoxolone.