Betulinic acid induces a novel cell death pathway that depends on cardiolipin modification.

Cancer is associated with strong changes in lipid metabolism. For instance, normal cells take up fatty acids (FAs) from the circulation, while tumour cells generate their own and become dependent on de novo FA synthesis, which could provide a vulnerability to target tumour cells. Betulinic acid (BetA) is a natural compound that selectively kills tumour cells through an ill-defined mechanism that is independent of BAX and BAK, but depends on mitochondrial permeability transition-pore opening. Here we unravel this pathway and show that BetA inhibits the activity of steroyl-CoA-desaturase (SCD-1). This enzyme is overexpressed in tumour cells and critically important for cells that utilize de novo FA synthesis as it converts newly synthesized saturated FAs to unsaturated FAs. Intriguingly, we find that inhibition of SCD-1 by BetA or, alternatively, with a specific SCD-1 inhibitor directly and rapidly impacts on the saturation level of cardiolipin (CL), a mitochondrial lipid that has important structural and metabolic functions and at the same time regulates mitochondria-dependent cell death. As a result of the enhanced CL saturation mitochondria of cancer cells, but not normal cells that do not depend on de novo FA synthesis, undergo ultrastructural changes, release cytochrome c and quickly induce cell death. Importantly, addition of unsaturated FAs circumvented the need for SCD-1 activity and thereby prevented BetA-induced CL saturation and subsequent cytotoxicity, supporting the importance of this novel pathway in the cytotoxicity induced by BetA.

Betulinic acid-induced mitochondria-dependent cell death is counterbalanced by an autophagic salvage response.

Betulinic acid (BetA) is a plant-derived pentacyclic triterpenoid that exerts potent anti-cancer effects in vitro and in vivo. It was shown to induce apoptosis via a direct effect on mitochondria. This is largely independent of proapoptotic BAK and BAX, but can be inhibited by cyclosporin A (CsA), an inhibitor of the permeability transition (PT) pore. Here we show that blocking apoptosis with general caspase inhibitors did not prevent cell death, indicating that alternative, caspase-independent cell death pathways were activated. BetA did not induce necroptosis, but we observed a strong induction of autophagy in several cancer cell lines. Autophagy was functional as shown by enhanced flux and degradation of long-lived proteins. BetA-induced autophagy could be blocked, just like apoptosis, with CsA, suggesting that autophagy is activated as a response to the mitochondrial damage inflicted by BetA. As both a survival and cell death role have been attributed to autophagy, autophagy-deficient tumor cells and mouse embryo fibroblasts were analyzed to determine the role of autophagy in BetA-induced cell death. This clearly established BetA-induced autophagy as a survival mechanism and indicates that BetA utilizes an as yet-undefined mechanism to kill cancer cells.

Identification of T-cell epitopes for cancer immunotherapy.

The effectiveness of T-cell-mediated immunotherapy of cancer depends on both an optimal immunostimulatory context of the therapy and the proper selection with respect to quality and quantity of the targeted tumor-associated antigens (TAA), and, more precisely, the T-cell epitopes contained in these tumor proteins. Our progressing insight in human leukocyte antigen (HLA) class I and class II antigen processing and presentation mechanisms has improved the prediction by reverse immunology of novel cytotoxic T lymphocyte and T-helper cell epitopes within known antigens. Computer algorithms that in silico predict HLA class I and class II binding, proteasome cleavage patterns and transporter associated with antigen processing translocation are now available to expedite epitope identification. The advent of genomics allows a high-throughput screening for tumor-specific transcripts and mutations, with that identifying novel shared and unique TAA. The increasing power of mass spectrometry and proteomics will lead to the direct identification from the tumor cell surface of numerous novel tumor-specific HLA class I and class II presented ligands. Together, the expanded repertoire of tumor-specific T-cell epitopes will enable more precise immunomonitoring and the development of effective epitope-defined adoptive T-cell transfer and multi-epitope-based vaccination strategies targeting epitopes derived from a wider diversity of TAA presented in a broader array of HLA molecules.

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

"Reference Biospheres" for solid radioactive waste disposal: the BIOMASS methodology.

The BIOMASS Theme 1 project has developed a methodology for the logical and defensible construction of 'assessment biospheres': mathematical representations of biospheres used in the total system performance assessment of radioactive waste disposal. The BIOMASS Methodology provides a systematic approach to decision making, including decisions on how to address biosphere change. The BIOMASS Methodology was developed through consultation and collaboration with many relevant organisations, including regulators, operators and a variety of independent experts. It has been developed to be practical and to be consistent with recommendations from ICRP and IAEA on radiation protection in the context of the disposal of long-lived solid radioactive wastes. The five main steps in the methodology are described in this paper. The importance of a clear assessment context, to clarify intentions and to support a coherent biosphere assessment process within an overall repository performance assessment, is strongly emphasised. A well described assessment context is an important tool for ensuring consistency across the performance assessment as a whole. The use of interaction matrices has been found to be helpful in clarifying the interactions between different habitats within the biosphere system and the significant radionuclide transfer pathways within the biosphere system. Matrices also provide a useful means of checking for consistency.

Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis.

We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A(*)0201-presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved "reverse immunology" strategy. Next to motif-based HLA-A(*)0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A(*)0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA(100-108); SLYSFPEPEA, PRA(142-151); ALYVDSLFFL, PRA(300-309); and SLLQHLIGL, PRA(425-433)) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A(*)0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.

BCR-ABL directed immunotherapy: a virtual reality?

Nearly ten years of research on the feasibility of specific immunotherapy targeting the junctional regions of BCR-ABL has considerably increased our knowledge of which MHC alleles might present BCR-ABL peptides, yet has failed to provide us with definite proof of appropriate processing of the hybrid oncoprotein into such antigenic peptides. This paper intends to provide an overview of the current state of affairs as well as to delineate limitations and future directions of this line of research.

A BCR-ABL oncoprotein p210b2a2 fusion region sequence is recognized by HLA-DR2a restricted cytotoxic T lymphocytes and presented by HLA-DR matched cells transfected with an Ii(b2a2) construct.

Peptides corresponding to the fusion site in 210 kD BCR-ABL protein b3a2 (p210b3a2) were previously shown to bind to several HLA class I and II alleles. We have found that b3a2 peptide-specific CD4-positive T-helper cells were able to recognize p210b3a2-positive chronic myelogenous leukemia (CML) blasts in a DR4 restricted manner. Until now, there were no reports of b2a2 breakpoint-specific human T-cell responses. Here we show that repetitive stimulation of T lymphocytes with a 17mer peptide covering the fusion region in p210b2a2 also leads to specific T-cell responses. CD4 and CD4/CD8 double-positive clones obtained from a b2a2 peptide-specific cell line were cytotoxic and proliferative in an HLA-DR2a (DRB5*0101) restricted fashion. Autologous Epstein-Barr virus (EBV) transformed cells, expressing BCR-ABL(b2a2) on transfection, and allogeneic HLA-DR matched p210b2a2-positive cells from CML patients were, however, not lysed. BCR-ABL peptide-specific T-cell clones did respond to autologous EBV cells transfected with invariant chain (li) cDNA in which the HLA class II-associated invariant chain peptide (CLIP) was replaced by a BCR-ABL b2a2 fusion oligonucleotide sequence, illustrating the potential of these T cells to recognize an endogenous BCR-ABL(b2a2) ligand.

Total system performance assessment for waste disposal using a logic tree approach.

The Electric Power Research Institute (EPRI) has sponsored the development of a model to assess the long-term, overall "performance" of the candidate spent fuel and high-level radioactive waste (HLW) disposal facility at Yucca Mountain, Nevada. The model simulates the processes that lead to HLW container corrosion, HLW mobilization from the spent fuel, and transport by groundwater, and contaminated groundwater usage by future hypothetical individuals leading to radiation doses to those individuals. The model must incorporate a multitude of complex, coupled processes across a variety of technical disciplines. Furthermore, because of the very long time frames involved in the modeling effort (>> 10(4) years), the relative lack of directly applicable data, and many uncertainties and variabilities in those data, a probabilistic approach to model development was necessary. The developers of the model chose a logic tree approach to represent uncertainties in both conceptual models and model parameter values. The developers felt the logic tree approach was the most appropriate. This paper discusses the value and use of logic trees applied to assessing the uncertainties in HLW disposal, the components of the model, and a few of the results of that model. The paper concludes with a comparison of logic trees and Monte Carlo approaches.

BCR-ABL oncoprotein is expressed by platelets from CML patients and associated with a special pattern of CrkL phosphorylation.

Constitutive tyrosine phosphorylation of CrkL was recently demonstrated in platelets from chronic myelogenous leukaemia (CML) patients but BCR-ABL tyrosine kinase could not be detected in the platelet lysates. We studied platelets from 14 CML patients with different types of BCR-ABL mRNA and with maximal platelet counts ranging from 149 to 3069 x 10(9)/l. P210BCR-ABL protein was detected by Western blotting in platelet lysates of 12/13 CML patients with active disease but not in the lysate of platelets from a Ph-positive acute lymphoblastic leukaemia (ALL) patient in remission or eight BCR-ABL-negative controls including one essential thrombocythaemia (ET) patient. Immunoblotting of p210BCR-ABL-positive platelets lysates with anti-CrkL antibody revealed a CrkL triplet consisting of one unphosphorylated and two phosphorylated forms of the protein. This CrkL phosphorylation pattern was not observed in normal platelets or CML platelets treated with ABL tyrosine kinase inhibitor CGP57148B. The presence of BCR-ABL provides an explanation for the constitutive tyrosine phosphorylation of CrkL in CML platelets. As no correlation was observed between platelet counts and platelet BCR-ABL protein expression, thrombocytosis or thrombocythaemia in CML cannot be explained by constitutive BCR-ABL-mediated CrkL tyrosine phosphorylation.

Recognition of BCR-ABL positive leukemic blasts by human CD4+ T cells elicited by primary in vitro immunization with a BCR-ABL breakpoint peptide.

In chronic myeloid leukemia (CML) the classical 9;22 translocation results in a BCR-ABL fusion gene, which encodes chimeric BCR-ABL fusion 210 kD oncoproteins (p210BCR-ABL). The two main p210BCR-ABL fusion variants in CML, b2a2 and b3a2 are examples of well characterized antigens expressed by malignant cells. The possibility of an immunotherapeutic approach involving the fusion part of p210BCR-ABL in CML has previously been illustrated by observed peptide binding to major histocompatibility complex (MHC) class I alleles and by demonstrating the immunogenicity of p210BCR-ABL breakpoint peptides. In this report we show that in vitro immunization of human T cells with a 17 amino acid (aa) peptide representing the p210BCR-ABL fusion region resulted in peptide specific CD4+ T-cell lines designated P4, P6, and P7. HLA DR4 (DRB1*0401) restricted T-cell line P4 and several subsequently derived clones recognized HLA-DRB1*0401 and p210b3a2-mRNA expressing blasts from an allogeneic patient with CML in blast crisis. Recognition appeared DR expression-dependent. No responses were observed with DR4 positive p210BCR-ABL negative cells or with p210b3a2 leukemic cells with absent or insufficient expression of DR4. These observations indicate that oncoprotein p210b3a2 can be degraded and processed for presentation by MHC class II molecules at the surface of leukemic cells. The BCR-ABL fusion region is in all likelihood presented as peptides by HLA DR and thus capable to act as a distinctive tumor antigen to peptide specific CD4+ T cells.

Treatment of prehospital refractory ventricular fibrillation with bretylium tosylate.

Among 218 patients treated for prehospital arrest during an eight-month baseline period prior to addition of bretylium tosylate to the paramedic protocol in Columbus, 16 (7.3%) were seen with refractory ventricular fibrillation (RVF). These patients failed to respond to multiple countershocks, lidocaine, bicarbonate and epinephrine, and either were transported in arrest during cardiopulmonary resuscitation (CPR)(14) or were pronounced dead at the scene (2). A single patient was eventually resuscitated in and discharged from the hospital. During the subsequent 16 1/2-month experience with bretylium used only for prehospital RVF, 421 patients with prehospital arrest were seen, 35 of whom (8.3%) had RVF. All but five patients were defibrillated successfully, and 14 (40%) were converted to a rhythm sufficient to obviate CPR during transportation. Eleven patients (31%) survived to be admitted to the hospital, and eight of 35 (23% vs 1/16 or 6.2% above, P less than .05) were discharged and remained well three to 17 months later. Bretylium tosylate may provide life-saving therapy for refractory prehospital ventricular fibrillation so that survival from an almost uniformly fatal condition is improved. While patients with persistent arrest generally should be transported to the hospital, such patients should not be subjected to the difficulties of CPR in transit unless they are first given bretylium if RVF is present.