Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

Akt signaling pathway in pacing-induced heart failure

Marked changes in energy substrate utilization occur during the progression of congestive heart failure (CHF) where fatty acid utilization, as the primary source of cardiac energy, is severely diminished, oxidative phosphorylation is down-regulated, and glucose uptake and utilization increase. Neither the signaling events or the molecular basis for the shift in substrate utilization have yet been elucidated. This study was designed to examine in the canine model of paced-induced CHF, the potential role of the Akt pathway in signaling the metabolic transitions central to progression to heart failure. Myocardial Akt levels were elevated in early heart failure (after 1–2 weeks of pacing) accompanied by increased levels of oxidative stress, cytokine tumor necrosis factor- (TNF-) and free fatty acid accumulation, reduced activity levels of mitochondrial respiratory complexes III and V and apoptosis initiation. At severe heart failure (3–4 weeks of pacing), there was significant further increase in myocardial apoptosis, with pronounced decline in myocardial Akt kinase activity. At this later stage, there were no further changes in free fatty acid accumulation, complex V activity or in oxidative stress levels indicating that these changes primarily occurred in the earlier stage of evolving heart failure. In contrast, during severe heart failure, both the reduction in complex III activity and increase in TNF- level became more pronounced. Our data provide critical support for the hypothesis that the Akt signaling pathway is a contributory element in the early signaling events leading to the progression of pacing-induced heart failure, accompanying the shift in substrate utilization. (Mol Cell Biochem 268: 103–110, 2005)     

Correlating a protein structure with function of a bacterial mechanosensitive channel

MscL, a mechanosensitive channel found in many bacteria, protects cells from hypotonic shock by reducing intracellular pressure through release of cytoplasmic osmolytes. First isolated from Escherichia coli, this protein has served as a model for how a protein senses and responds to membrane tension. Recently the structure of a functionally uncharacterized MscL homologue from Mycobacterium tuberculosis was solved by x-ray diffraction to a resolution of 3.5 A. Here we demonstrate that the protein forms a functional MscL-like mechanosensitive channel in E. coli membranes and azolectin proteoliposomes. Furthermore, we show that M. tuberculosis MscL crystals, when re-solubilized and reconstituted, yield wild-type channel currents in patch clamp, demonstrating that the protein does not irreversibly change conformation upon crystallization. Finally, we apply functional clues acquired from the E. coli MscL to the M. tuberculosis channel and show a mechanistic correlation between these channels. However, the inability of the M. tuberculosis channel to gate at physiological membrane tensions, demonstrated by in vivo E. coli expression and in vitro reconstitution, suggests that the membrane environment or other additional factors influence the gating of this channel.     

A crucial requirement for Hedgehog signaling in small cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.     

The WISER metadatabase: the key to more than 100 ecological datasets from European rivers, lakes and coastal waters

In ecological sciences, the role of metadata (i.e. key information about a dataset) to make existing datasets visible and discoverable has become increasingly important. Within the EU-funded WISER project (Water bodies in Europe: Integrative Systems to assess Ecological status and Recovery), we designed a metadatabase to allow scientists to find the optimal data for their analyses. An online questionnaire helped to collect metadata from the data providers and an online query tool (http://www.wiser.eu/results/meta-database/) facilitated data evaluation. The WISER metadatabase currently holds information on 114 datasets (22 river, 71 lake, 1 general freshwater and 20 coastal/transitional datasets), which also can be accessed by external scientists. We evaluate if generally used metadata standards (e.g. Darwin Core, ISO 19115, CSDGM, EML) are suitable for such specific purposes as WISER and suggest at least the linkage with standard metadata fields. Furthermore, we discuss whether the simple metadata documentation is enough for others to reuse a dataset and why there is still reluctance to publish both metadata and primary research data (i.e. time and financial constraints, misuse of data, abandoning intellectual property rights). We emphasise that metadata publication has major advantages as it makes datasets detectable by other scientists and generally makes a scientist’s work more visible.     

A new record of Amphitetranychus quercivorus (Acari: Tetranychidae) in Korea and molecular comparison with A. viennensis

Amphitetranychus quercivorus (Ehara and Gotoh, 1990) was first identified in Mongolian oak tree Quercus mongolica in Daegu, Korea. In comparison to A. viennensis (Zacher), A. quercivorus was much shorter in the distal, dorsally directed portion of the male aedeagus, as well as smaller and less folded in the distal anastomosed portion of the female peritreme. Comparison of the nucleotide sequences of ITS2 and COI revealed 90% and 89% similarities, respectively, between the two species. In addition, species-specific primer sets for each species were designed using unique ITS2 sequences and then used to diagnose these two species under plant quarantine. We report the Korean name of this species as “Sin-gal-na-mu-eung-ae”.     

Functional design of bacterial mechanosensitive channels. Comparisons and contrasts illuminated by random mutagenesis

MscS and MscL are mechanosensitive channels found in bacterial plasma membranes that open large pores in response to membrane tension. These channels function to alleviate excess cell turgor invoked by rapid osmotic downshock. Although much is known of the structure and molecular mechanisms underlying MscL, genes correlating with MscS activity have only recently been identified. Previously, it was shown that eliminating the expression of Escherichia coli yggB removed a major portion of MscS activity. YggB is distinct from MscL by having no obvious structural similarity. Here we have reconstituted purified YggB in proteoliposomes and have successfully detected MscS channel activity, confirming that purified YggB protein encodes MscS activity. Additionally, to define functional regions of the channel protein, we have randomly mutagenized the structural gene and isolated a mutant that evokes a gain-of-function phenotype. Physiological experiments demonstrate that the mutated channel allows leakage of solutes from the cell, suggesting inappropriate channel opening. Interestingly, this mutation is analogous in position and character to mutations yielding a similar phenotype in MscL. Hence, although MscS and MscL mechanosensitive channels are structurally quite distinct, there may be analogies in their gating mechanisms.     

Actinomyces naturae sp. nov., the first Actinomyces sp. isolated from a non-human or animal source

Three facultatively anaerobic, Gram-positive staining, rod-shaped, non-spore forming, flagellated bacterial strains, BL-75, BL-79^T and BL-104, were isolated from chlorinated solvent-contaminated groundwater. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed them to represent a distinct lineage within the genus Actinomyces with sequence identities in the range of <88–95.4% with previously described Actinomyces species. The strains were oxidase and catalase negative. Nitrate was not reduced. Esculin was hydrolyzed. Growth occurred in the temperature range of 20–43°C (optimum 30–37°C) and pH range 4.5–9.0 (optimum pH 6.5). Substrates supporting growth included various mono-, di-, and tri-saccharides. The end products of glucose fermentation were acetate, lactate, succinate and formate. Fermentative growth was observed in the presence of near saturation concentrations of perchloroethene (PCE) and toluene and in the presence of 1,2-dichloroethane and 1,1,2-trichloroethane at concentrations up to at least 24.4 mM and 11.2 mM, respectively. The dominant cellular fatty acids when grown in peptone/yeast extract/glucose (PYG) medium were C_18:1 ω9c, C_16:0, and C_14:0. The peptidoglycan was found to contain the amino acids alanine, glutamic acid, lysine, and ornithine at approximate molar ratios of 1.7 Ala: 2.3 Glu: 1.3 Lys: 1.0 Orn. The cell wall sugars were found to include rhamnose and mannose. The polar lipids were found to include diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phospholipid (PL), phosphoglycolipids (PGL), and glycolipids (GL). The main respiratory quinone of strain BL-79^T was MK-9(H₄), with minor components MK-10(H₄) and MK-8(H₄). The DNA mol% G+C content of the type strain is 69.8%. On the basis of phylogenetic and phenotypic characteristics, these strains could be differentiated from previously described species of the genus Actinomyces. Strains BL-75, BL-79^T and BL-104 are designated as a novel species, for which the name Actinomyces naturae sp. nov. is proposed. This is the first Actinomyces species isolated from an environmental rather than human or animal sources. The type strain of Actinomyces naturae is BL-79^T (= CCUG 56698^T = NRRL B-24670^T).     

Regulation of fibroblast growth factor-23 signaling by klotho

The aging suppressor gene Klotho encodes a single-pass transmembrane protein. Klotho-deficient mice exhibit a variety of aging-like phenotypes, many of which are similar to those observed in fibroblast growth factor-23 (FGF23)-deficient mice. To test the possibility that Klotho and FGF23 may function in a common signal transduction pathway(s), we investigated whether Klotho is involved in FGF signaling. Here we show that Klotho protein directly binds to multiple FGF receptors (FGFRs). The Klotho-FGFR complex binds to FGF23 with higher affinity than FGFR or Klotho alone. In addition, Klotho significantly enhanced the ability of FGF23 to induce phosphorylation of FGF receptor substrate and ERK in various types of cells. Thus, Klotho functions as a cofactor essential for activation of FGF signaling by FGF23.