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.     

Homeobox Genes in the Rodent Pineal Gland: Roles in Development and Phenotype Maintenance

The pineal gland is a neuroendocrine gland responsible for nocturnal synthesis of melatonin. During early development of the rodent pineal gland from the roof of the diencephalon, homeobox genes of the orthodenticle homeobox (Otx)- and paired box (Pax)-families are expressed and are essential for normal pineal development consistent with the well-established role that homeobox genes play in developmental processes. However, the pineal gland appears to be unusual because strong homeobox gene expression persists in the pineal gland of the adult brain. Accordingly, in addition to developmental functions, homeobox genes appear to be key regulators in postnatal phenotype maintenance in this tissue. In this paper, we review ontogenetic and phylogenetic aspects of pineal development and recent progress in understanding the involvement of homebox genes in rodent pineal development and adult function. A working model is proposed for understanding the sequential action of homeobox genes in controlling development and mature circadian function of the mammalian pinealocyte based on knowledge from detailed developmental and daily gene expression analyses in rats, the pineal phenotypes of homebox gene-deficient mice and studies on development of the retinal photoreceptor; the pinealocyte and retinal photoreceptor share features not seen in other tissues and are likely to have evolved from the same ancestral photodetector cell.     

The Perivascular Phagocyte of the Mouse Pineal Gland: an Antigen‐Presenting Cell

The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal gland of mice. Actively phagocytosing cells with a prominent lysosomal system were found in the pericapillary spaces of the mouse pineal gland following intravenous injection of horseradish peroxidase. The cells also exhibited strong acid phosphatase activity. Perivascular cells were immunopositive for MHC class II protein and for CD68, a marker of monocytes/phagocytes. This study verifies that perivascular phagocytes with antigen‐presenting properties are present in the mouse pineal gland.     

NetMHCIIpan-3.0, a common pan-specific MHC class II prediction method including all three human MHC class II isotypes, HLA-DR, HLA-DP and HLA-DQ

Major histocompatibility complex class II (MHCII) molecules play an important role in cell-mediated immunity. They present specific peptides derived from endosomal proteins for recognition by T helper cells. The identification of peptides that bind to MHCII molecules is therefore of great importance for understanding the nature of immune responses and identifying T cell epitopes for the design of new vaccines and immunotherapies. Given the large number of MHC variants, and the costly experimental procedures needed to evaluate individual peptide–MHC interactions, computational predictions have become particularly attractive as first-line methods in epitope discovery. However, only a few so-called pan-specific prediction methods capable of predicting binding to any MHC molecule with known protein sequence are currently available, and all of them are limited to HLA-DR. Here, we present the first pan-specific method capable of predicting peptide binding to any HLA class II molecule with a defined protein sequence. The method employs a strategy common for HLA-DR, HLA-DP and HLA-DQ molecules to define the peptide-binding MHC environment in terms of a pseudo sequence. This strategy allows the inclusion of new molecules even from other species. The method was evaluated in several benchmarks and demonstrates a significant improvement over molecule-specific methods as well as the ability to predict peptide binding of previously uncharacterised MHCII molecules. To the best of our knowledge, the NetMHCIIpan-3.0 method is the first pan-specific predictor covering all HLA class II molecules with known sequences including HLA-DR, HLA-DP, and HLA-DQ. The NetMHCpan-3.0 method is available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.0.     

Anther extrusion and plant height are associated with Type I resistance to Fusarium head blight in bread wheat line ‘Shanghai-3/Catbird’

Fusarium head blight (FHB) is a destructive wheat disease of global importance. Resistance breeding depends heavily on the Fhb1 gene. The CIMMYT line Shanghai-3/Catbird (SHA3/CBRD) is a promising source without this gene. A recombinant inbred line (RIL) population from the cross of SHA3/CBRD with the German spring wheat cv. Naxos was evaluated for FHB resistance and related traits in field trials using spray and spawn inoculation in Norway and point inoculation in China. After spray and spawn inoculation, FHB severities were negatively correlated with both anther extrusion (AE) and plant height (PH). The QTL analysis showed that the Rht-B1b dwarfing allele co-localized with a QTL for low AE and increased susceptibility after spawn and spray inoculation. In general, SHA3/CBRD contributed most of the favorable alleles for resistance to severity after spray and spawn inoculation, while Naxos contributed more favorable alleles for reduction in FDK and DON content and resistance to severity after point inoculation. SHA3/CBRD contributed a major resistance QTL close to the centromere on 2DLc affecting FHB severity and DON after all inoculation methods. This QTL was also associated with AE and PH, with high AE and tall alleles contributed by SHA3/CBRD. Several QTL for AE and PH were detected, and low AE or reduced PH was always associated with increased susceptibility after spawn and spray inoculation. Most of the other minor FHB resistance QTL from SHA3/CBRD were associated with AE or PH, while the QTL from Naxos were mostly not. After point inoculation, no other QTL for FHB traits was associated with AE or PH, except the 2DLc QTL which was common across all inoculation methods. Marker-assisted selection based on the 2DLc QTL from SHA3/CBRD combined with phenotypic selection for AE is recommended for resistance breeding based on this valuable source of resistance.     

QTL for spot blotch resistance in bread wheat line Saar co-locate to the biotrophic disease resistance loci Lr34 and Lr46

Spot blotch caused by Bipolaris sorokiniana is a major disease of wheat in warm and humid wheat growing regions of the world including south Asian countries such as India, Nepal and Bangladesh. The CIMMYT bread wheat line Saar which carries the leaf tip necrosis (LTN)-associated rust resistance genes Lr34 and Lr46 has exhibited a low level of spot blotch disease in field trials conducted in Asia and South America. One hundred and fourteen recombinant inbred lines (RILs) of Avocet (Susceptible) × Saar, were evaluated along with parents in two dates of sowing in India for 3 years (2007–2008 to 2009–2010) to identify quantitative trait loci (QTL) associated with spot blotch resistance, and to determine the potential association of Lr34 and Lr46 with resistance to this disease. Lr34 was found to constitute the main locus for spot blotch resistance, and explained as much as 55 % of the phenotypic variation in the mean disease data across the six environments. Based on the large effect, the spot blotch resistance at this locus has been given the gene designation Sb1. Two further, minor QTL were detected in the sub-population of RILs not containing Lr34. The first of these was located about 40 cM distal to Lr34 on 7DS, and the other corresponded to Lr46 on 1BL. A major implication for wheat breeding is that Lr34 and Lr46, which are widely used in wheat breeding to improve resistance to rust diseases and powdery mildew, also have a beneficial effect on spot blotch.     

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.     

Observations on Chrysophyceae from a Norwegian mountain lake

Chrysosphaerella coronasircumspina Wujek & Kristiansen and Spiniferomonas bilacunosa Takahashi have been shown by electron microscopy to be regular members of the phytoplankton society in an oligotrophic Norwegian mountain lake. This is the first published record of these species in Norway.     

The development and characterization of a competitive ELISA for measuring active ADAMTS-4 in a bovine cartilage ex vivo model

ADAMTS-4 (aggrecanase1) is believed to play an important role in the degradation of aggrecan during the progression of joint diseases. ADAMTS-4 is synthesized as a latent pro-enzyme that requires the removal of the pro-domain, exposing the N-terminal neoepitope, to achieve activity. We developed a monoclonal antibody against this neoepitope of active ADAMTS-4. Furthermore, we established and characterized a competitive ELISA for measuring active ADAMTS-4 form applying the specific antibody. We used this assay to profile the presence of active ADAMTS-4 and its aggrecan degradation product (NITEGE³⁷³) in a bovine cartilage ex vivo model. We found that after stimulation with catabolic factors, the cartilage initially released high levels of aggrecanase-derived aggrecan fragments into supernatant but subsequently decreased to background levels. The level of active ADAMTS-4 released into the supernatant and retained in the cartilage matrix increased continuously throughout the 21 days of the study. The activity of ADAMTS-4 on the last day of catabolic stimulation was verified in vitro by adding deglycosylated or native aggrecan to the conditioned medium. Samples of human cartilage affected by varying degrees of osteoarthritis stained strongly for active ADAMTS-4 where surface fibrillation and clustered chondrocytes were observed. This assay could be an effective tool for studying ADAMTS-4 activity and for screening drugs regulating ADAMTS-4 activation. Moreover, it could be a potential biomarker for degenerative joint disease.