Dendritic cell vaccination plus low-dose doxorubicin for the treatment of spontaneous canine hemangiosarcoma.

Angiosarcoma is a deadly neoplasm of the vascular endothelium. Metastatic disease is often present at diagnosis, and 5-year survival is only 10-35%. Although there exist no immunocompetent mouse models of angiosarcoma with which to study immune-based approaches to therapy, angiosarcoma is a major killer of companion dogs, responsible for up to 2% of all canine deaths in some susceptible breeds or an estimated 120,000 per year in the US. The canine disease (HSA) often presents in the spleen as acute hemoabdomen secondary to splenic rupture. Even if life-saving splenectomy is performed, median overall survival (OS) is only 48 days, and 1-year survival is negligible. Here we report the analysis of a pilot phase I open-label trial of chemo-immunotherapy performed on consecutively presenting splenectomized canines with histologically verified HSA. Subjects received an abbreviated course of low-dose doxorubicin plus alpha interferon and an autologous dendritic cell-therapy reported to enhance durable CD8+ memory. Disease was monitored monthly by abdominal ultrasound, chest X-ray, and echocardiogram. Median OS in the per protocol population was 109 days including one of five animals that died cancer-free at 16 months after documented resolution of relapsed disease. These results indicate that therapeutic administration of chemo-immunotherapy is both feasible and safe, substantiating the rationale for additional veterinary and human clinical studies.

A novel triple purge strategy for eliminating chronic myelogenous leukemia (CML) cells from autografts.

Imatinib-refractory chronic myelogenous leukemia (CML) patients can experience long-term disease-free survival with myeloablative therapy and allogeneic hematopoietic cell transplantation; however, associated complications carry a significant risk of mortality. Transplantation of autologous hematopoietic cells has a reduced risk of complications, but residual tumor cells in the autograft may contribute to relapse. Development of methods for purging tumor cells that do not compromise the engraftment potential of the normal hematopoietic cells in the autograft has been a long-standing goal. Since primitive CML cells differentiate more rapidly in vitro than their normal counterparts and are also preferentially killed by mafosfamide and imatinib, we examined the purging effectiveness on CD34(+) CML cells using a strategy that combines a brief exposure to imatinib (0.5-1.0 microM for 72 h) and then mafosfamide (30-90 microg/ml for 30 min) followed by 2 weeks in culture with cytokines (100 ng/ml each of stem cell factor, granulocyte colony-stimulating factor and thrombopoietin). Treatment with 1.0 microM imatinib, 60 microg/ml mafosfamide and 14 days of culture with cytokines eliminated BCR-ABL(+) cells from chronic phase CML patient aphereses, while preserving normal progenitors. This novel purging strategy may offer a new approach to improving the effectiveness of autologous transplantation in imatinib-refractory CML patients.

Each mammalian mitochondrial outer membrane porin protein is dispensable: effects on cellular respiration.

Voltage-dependent anion channels (VDACs, also known as mitochondrial porins) are small pore-forming proteins of the mitochondrial outer membrane found in all eukaryotes. Mammals harbor three distinct VDAC isoforms, with each protein sharing 65-70% sequence identity. Deletion of the yeast VDAC1 gene leads to conditional lethality that can be partially or completely complemented by the mammalian VDAC genes. In vitro, VDACs conduct a variety of small metabolites and in vivo they serve as a binding site for several cytosolic kinases involved in intermediary metabolism, yet the specific physiologic role of each isoform is unknown. Here we show that mouse embryonic stem cells lacking each isoform are viable but exhibit a 30% reduction in oxygen consumption. VDAC1 and VDAC2 deficient cells exhibit reduced cytochrome c oxidase activity, whereas VDAC3 deficient cells have normal activity. These results indicate that VDACs are not essential for cell viability and we speculate that reduced respiration in part reflects decreased outer membrane permeability for small metabolites necessary for oxidative phosphorylation.

Stepping Out of the Cytosol: AIMp1/p43 Potentiates the Link Between Innate and Adaptive Immunity.

As a structural component of the multi-aminoacyl tRNA synthetase (mARS) complex, AIMp1, also known as p43, hasn't until recently been recognized for its prominent immunological functions. Together with other nonenzymatic mARS structural components AIMp2/38 and AIMp3/p18, it participates in the machinery responsible for cell-cycle control and tumor suppression. Novel studies also show that AIMp1/p43 can be released by certain cancer cells under conditions of stress. Extracellularly, AIMp1 promotes the proliferation and migration of fibroblasts/endothelial cells and importantly, pro-inflammatory gene expression in monocytes/macrophages and dendritic cells. AIMp1/p43 deficiency is also correlated with spontaneous Type-2 airway hypersensitivity in mice, indicating a potential role in skewing toward T-helper type-1 (T(H)1) immunity. Vaccination strategies in which dendritic cells receive dual MHC class I and MHC class II antigens of homologous origins (i.e., that share overlapping class I and II binding epitopes) boost downstream T(H)1 immunity in a manner that appears to be wholly dependent upon dendritic cell AIMp1 release. Here we underscore the importance of AIMp1/p43 as a pro-inflammatory cytokine when it is released from cytosol to extracellular space and discuss future directions by which the mechanisms that regulate this process might be better characterized, further elucidating the link between innate and adaptive immunity.

CD3(+) and/or CD14(+) depletion from cord blood mononuclear cells before ex vivo expansion culture improves total nucleated cell and CD34(+) cell yields.

Cord blood (CB) is used increasingly in transplant patients lacking sibling or unrelated donors. A major hurdle in the use of CB is its low cell dose, which is largely responsible for an elevated risk of graft failure and a significantly delayed neutrophil and platelet engraftment. As a positive correlation has been shown between the total nucleated cell (TNC) and CD34(+) cell dose transplanted and time to neutrophil and platelet engraftment, strategies to increase these measures are under development. One strategy includes the ex vivo expansion of CB mononuclear cells (MNC) with MSC in a cytokine cocktail. We show that this strategy can be further improved if CD3(+) and/or CD14(+) cells are first depleted from the CB MNC before ex vivo expansion. Ready translation of this depletion strategy to improve ex vivo CB expansion in the clinic is feasible as clinical-grade devices and reagents are available. Ultimately, the aim of improving TNC and CD34(+) transplant doses is to further improve the rate of neutrophil and platelet engraftment in CB recipients.

AML-loaded DC generate Th1-type cellular immune responses in vitro.

BACKGROUND: The generation of AML-specific T-lymphocyte responses by leukemia-derived DC has been documented by multiple investigators and is being pursued clinically. An obstacle to widespread use of this strategy is that it has not been possible to generate leukemic DC from all patients, and an alternative approach is needed if the majority of leukemia patients are to receive therapeutic vaccination in conjunction with other treatment protocols. METHODS: In the present study, we generated DC from CD14-selected monocytes isolated from healthy donor PBPC and loaded them with a total cell lysate from AML patient blasts. RESULTS: Immature in vitro-derived DC exhibited robust phagocytic activity, and mature DC demonstrated high expression of CD80, CD83, CD86 and the chemokine receptor CCR7, important for DC migration to local lymph nodes. Mature, Ag-loaded DC were used as APC for leukemia-specific cytotoxic T-lymphocyte (CTL) induction and demonstrated cytotoxic activity against leukemic targets. CTL lysis was Ag-specific, with killing of both allogeneic leukemic blasts and autologous DC loaded with allogeneic AML lysate. HLA-matched controls were not lysed in our system. DISCUSSION: These data support further research into the use of this strategy as an alternative approach to leukemia-derived DC vaccination.

The tissue-specific, alternatively spliced single ATG exon of the type 3 voltage-dependent anion channel gene does not create a truncated protein isoform in vivo.

Voltage-dependent anion channels (VDACs) are small, integral membrane proteins that traverse the outer mitochondrial membrane and conduct ATP and other small metabolites. They are known to bind several kinases of intermediary metabolism in a tissue-specific fashion, have been found in close association with the adenine nucleotide translocator of the inner mitochondrial membrane, and are hypothesized to form part of the mitochondrial permeability transition pore, which results in the release of cytochrome c at the onset of apoptotic cell death. VDACs are found throughout all strata of eukaryotic evolution and exhibit biophysical characteristics that are well conserved from yeast to mammals. The mammalian VDAC gene family consists of three isoforms, each of which shares approximately 70% sequence identity with the other two family members. Recently, we reported that a single codon (ATG) exon is alternatively spliced into the transcript of the type 3 voltage-dependent anion channel (VDAC3) in a tissue-specific fashion. This unusual splicing event was shown to be conserved between mouse and human, and we theorized that the spliced exon could lead to the creation of an alternative translational initiation site. Here we report that a highly specific polyclonal VDAC3 antibody was unable to detect the truncated protein isoform indicative of this putative downstream start site. From these in vivo studies, we conclude that the alternatively spliced exon results in the insertion of a single methionine residue at amino acid position 39 of the mature VDAC3 protein. Additionally, we have used a cross-species genomic sequence comparison to identify conserved regions that may be involved in the regulation of small exon splicing.

Retrotransposition of glycerol kinase-related genes from the X chromosome to autosomes: functional and evolutionary aspects.

Glycerol kinase catalyzes the metabolism of endogenously derived and dietary glycerol. GyK is a member of a small group of kinases termed ambiquitous enzymes, which are found either in the cytosol or as membrane-bound complexes associated with the voltage-dependent anion channel of the mitochondrial outer membrane. In Homo sapiens, the GyK gene family consists of an X-encoded locus and several X-linked and autosomal intronless retroposons, which, apparently, comprise both functional genes and processed pseudogenes. To study the role of the autosomal genes in mammalian physiology, we have isolated two murine GyK-like genes, determined their structures and chromosomal locations, and examined their functions. These sequences are intronless retroposons, which appear to be paralogues of the X-encoded, brain-specific GyK isoform and are expressed only in the testes. Though both retrotransposition events appear to have occurred prior to the primate-rodent divergence of some 65-80 million years ago, only one of the retrotransposed murine gene sequences, based upon its chromosomal location, is conserved with modern H. sapiens. To test the hypothesis that the murine GyK-like genes encode functional GyK activity, transient transfection of the gene sequences into COS7 cells was carried out. While in vitro translation confirmed that the transcripts could direct the synthesis of proteins of the appropriate size, no GyK activity was detected. Such data suggest that the autosomal GyK-like genes have evolved novel, testis-specific functions. A comparison of the human and mouse GyK-like gene sequences demonstrates the evolutionary relationships between each autosomal isoform and its corresponding X-linked ancestral locus.

A novel isoform of the mitochondrial outer membrane protein VDAC3 via alternative splicing of a 3-base exon. Functional characteristics and subcellular localization.

Voltage-dependent anion channels (VDACs) are pore-forming proteins found in the outer mitochondrial membrane of all eucaryotes. VDACs are the major pathway for metabolites through the outer mitochondrial membrane and, in mammals, bind several cytosolic carbohydrate kinases. Whereas yeast contain a single VDAC (YVDAC), to date three isoforms have been described in the mouse that constitute a gene family. We have observed an additional isoform of VDAC3 that appears to be generated via the tissue-specific alternative splicing of a 3-base exon (ATG). The exon is predicted to introduce a methionine 39 amino acids downstream of the amino terminus of the polypeptide. Between exons 3 and 4 is an intronic sequence that potentially encodes the exon, with flanking splice enhancer elements. Expression of this alternative form in the mouse is limited to brain, heart, and skeletal muscle. Complementation of YVDAC-deficient yeast by the two isoforms and with other sequence variants of VDAC3 suggests this residue is an important modulator of VDAC3 function. In transfected mammalian cells both isoforms localize to mitochondria. A similar variant is present in humans.

Isolation, mapping, and functional expression of the mouse X chromosome glycerol kinase gene.

Glycerol kinase (Gyk) participates in the metabolism of endogenously derived and dietary glycerol. Deficiency of the human enzyme activity is an X-linked recessive disorder with a clinical picture varying from childhood metabolic crisis to asymptomatic adults incidentally identified by hyperlipidemia screening (pseudohypertriglyceridemia). Gyk is a member of a small group of kinases termed ambiquitous enzymes that are found in the cytosol or as membrane-bound enzymes associated with the voltage-dependent anion channel of the mitochondrial outer membrane. It was recently reported that in humans there are X-linked and autosomal copies of Gyk sequences, both apparently functional genes and processed pseudogenes. To understand the role of Gyk in normal metabolism and the variable clinical features seen with Gyk deficiency, we have characterized the mouse Gyk gene. We present the sequence of a full-length mouse Gyk cDNA that is alternatively spliced in brain. The Gyk gene was mapped to the mouse X chromosome by both fluorescence in situ hybridization and an interspecies backcross panel, demonstrating conservation of synteny with dmd. To confirm the functional identity of the cDNA, transient transfection of the cDNA into COS7 cells was shown to cause a marked elevation in glycerol kinase activity.

Immotile sperm and infertility in mice lacking mitochondrial voltage-dependent anion channel type 3.

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are small channel proteins involved in the translocation of metabolites across the mitochondrial outer membrane. A single channel-forming protein is found in yeast, whereas higher eukaryotes express multiple VDACs, with humans and mice each harboring three distinct channels (VDAC1-3) encoded by separate genes. To begin to assess the functions of each of the three isoforms, the VDAC3 gene was inactivated by targeted disruption in embryonic stem cells. Here we show that mice lacking VDAC3 are healthy, but males are infertile. Although there are normal sperm numbers, the sperm exhibit markedly reduced motility. Structural defects were found in two-thirds of epididymal axonemes, with the most common abnormality being loss of a single microtubule doublet at a conserved position within the axoneme. In testicular sperm, the defect was only rarely observed, suggesting that instability of a normally formed axoneme occurs with sperm maturation. In contrast, tracheal epithelial cilia showed no structural abnormalities. In addition, skeletal muscle mitochondria were abnormally shaped, and activities of the respiratory chain complexes were reduced. These results demonstrate that axonemal defects may be caused by associated nonaxonemal components such as mitochondrial channels and illustrate that normal mitochondrial function is required for stability of the axoneme.