Gaute Brede

Y-Chromosomal Diversity in Europe Is Clinal and Influenced Primarily by Geography, Rather than by Language

Clinal patterns of autosomal genetic diversity within Europe have been interpreted in previous studies in terms of a Neolithic demic diffusion model for the spread of agriculture; in contrast, studies using mtDNA have traced many founding lineages to the Paleolithic and have not shown strongly clinal variation. We have used 11 human Y-chromosomal biallelic polymorphisms, defining 10 haplogroups, to analyze a sample of 3,616 Y chromosomes belonging to 47 European and circum-European populations. Patterns of geographic differentiation are highly nonrandom, and, when they are assessed using spatial autocorrelation analysis, they show significant clines for five of six haplogroups analyzed. Clines for two haplogroups, representing 45% of the chromosomes, are continentwide and consistent with the demic diffusion hypothesis. Clines for three other haplogroups each have different foci and are more regionally restricted and are likely to reflect distinct population movements, including one from north of the Black Sea. Principal-components analysis suggests that populations are related primarily on the basis of geography, rather than on the basis of linguistic affinity. This is confirmed in Mantel tests, which show a strong and highly significant partial correlation between genetics and geography but a low, nonsignificant partial correlation between genetics and language. Genetic-barrier analysis also indicates the primacy of geography in the shaping of patterns of variation. These patterns retain a strong signal of expansion from the Near East but also suggest that the demographic history of Europe has been complex and influenced by other major population movements, as well as by linguistic and geographic heterogeneities and the effects of drift.

Bone morphogenetic proteins induce apoptosis in multiple myeloma cells by Smad-dependent repression of MYC

Bone morphogenetic proteins (BMPs) have been shown to induce apoptosis and growth arrest in myeloma cells. However, the molecular mechanisms behind these events are not known. The MYC oncogene is a master regulator of cell growth and protein synthesis and MYC overexpression has been proposed to be associated with the progression of multiple myeloma. Here, we show that BMP-induced apoptosis in myeloma cells is dependent on downregulation of MYC. Moreover, the results suggest that targeting the MYC addiction in multiple myeloma is an efficient way of killing a majority of primary myeloma clones. We also found that myeloma cells harboring immunoglobulin (IG)-MYC translocations evaded BMP-induced apoptosis, suggesting a novel way for myeloma cells to overcome potential tumor suppression by BMPs.

The gene cluster containing the LCAT gene is conserved between human and pig.

A pooled DNA probe from P1 artificial chromosome clones (PACs) containing the human lecithin:cholesterol acyl transferase (LCAT) gene cluster was used in fluorescence in situ hybridization (FISH) experiments assigning the genes to pig chromosome 6p13. In addition, probes derived from the coding regions in the human gene cluster were used in long range mapping experiments to show that the overall structures of the human and porcine LCAT gene clusters are identical. Both the linear order and the close physical distance of five apparently unrelated genes have been maintained throughout 90 million years of divergent evolution between human and pig. The extremely dense clustering of the genes in the LCAT gene cluster suggests that this gene organization has biological significance. The conservation of the gene cluster between human and pig supports this suggestion.

Physical linkage of the gene cluster containing the LCAT gene to the DNA marker D16S124 at human chromosome region 16q22.1

Pulsed-field gel electrophoresis has been used to construct a long-range restriction map spanning more than 900 kb in the q22.1 region of human chromosome 16. The gene cluster containing the lecithin:cholesterol acyl transferase (LCAT) gene is located less than 480 kb from the anonymous DNA marker D16S124 in this map. The results suggest three putative CpG islands within 125 kb, in addition to the island previously shown to be located within the gene cluster. This implies a clustering of both genes and CpG islands in this chromosomal region.

Human Breast Milk miRNA, Maternal Probiotic Supplementation and Atopic Dermatitis in Offspring

Background Perinatal probiotic ingestion has been shown to prevent atopic dermatitis (AD) in infancy in a number of randomised trials. The Probiotics in the Prevention of Allergy among Children in Trondheim (ProPACT) trial involved a probiotic supplementation regime given solely to mothers in the perinatal period and demonstrated a ~40% relative risk reduction in the cumulative incidence of AD at 2 years of age. However, the mechanisms behind this effect are incompletely understood. Micro-RNAs (miRNA) are abundant in mammalian milk and may influence the developing gastrointestinal and immune systems of newborn infants. The objectives of this study were to describe the miRNA profile of human breast milk, and to investigate breast milk miRNAs as possible mediators of the observed preventative effect of probiotics. Methods Small RNA sequencing was conducted on samples collected 3 months postpartum from 54 women participating in the ProPACT trial. Differential expression of miRNA was assessed for the probiotic vs placebo and AD vs non-AD groups. The results were further analysed using functional prediction techniques. Results Human breast milk samples contain a relatively stable core group of highly expressed miRNAs, including miR-148a-3p, miR-22-3p, miR-30d-5p, let-7b-5p and miR-200a-3p. Functional analysis of these miRNAs revealed enrichment in a broad range of biological processes and molecular functions. Although several miRNAs were found to be differentially expressed on comparison of the probiotic vs placebo and AD vs non-AD groups, none had an acceptable false discovery rate and their biological significance in the development of AD is not immediately apparent from their predicted functional consequences. Conclusion Whilst breast milk miRNAs have the potential to be active in a diverse range of tissues and biological process, individual miRNAs in breast milk 3 months postpartum are unlikely to play a major role in the prevention of atopic dermatitis in infancy by probiotics ingestion in the perinatal period. Trial Registration ClinicalTrials.gov NCT00159523

Erythropoietin (EPO)-receptor signaling induces cell death of primary myeloma cells in vitro

BackgroundMultiple myeloma is an incurable complex disease characterized by clonal proliferation of malignant plasma cells in a hypoxic bone marrow environment. Hypoxia-dependent erythropoietin (EPO)-receptor (EPOR) signaling is central in various cancers, but the relevance of EPOR signaling in multiple myeloma cells has not yet been thoroughly investigated.MethodsMyeloma cell lines and malignant plasma cells isolated from bone marrow of myeloma patients were used in this study. Transcript levels were analysed by quantitative PCR and cell surface levels of EPOR in primary cells by flow cytometry. Knockdown of EPOR by short interfering RNA was used to show specific EPOR signaling in the myeloma cell line INA-6. Flow cytometry was used to assess viability in primary cells treated with EPO in the presence and absence of neutralizing anti-EPOR antibodies. Gene expression data for total therapy 2 (TT2), total therapy 3A (TT3A) trials and APEX 039 and 040 were retrieved from NIH GEO omnibus and EBI ArrayExpress.ResultsWe show that the EPOR is expressed in myeloma cell lines and in primary myeloma cells both at the mRNA and protein level. Exposure to recombinant human EPO (rhEPO) reduced viability of INA-6 myeloma cell line and of primary myeloma cells. This effect could be partially reversed by neutralizing antibodies against EPOR. In INA-6 cells and primary myeloma cells, janus kinase 2 (JAK-2) and extracellular signal regulated kinase 1 and 2 (ERK-1/2) were phosphorylated by rhEPO treatment. Knockdown of EPOR expression in INA-6 cells reduced rhEPO-induced phospo-JAK-2 and phospho-ERK-1/2. Co-cultures of primary myeloma cells with bone marrow-derived stroma cells did not protect the myeloma cells from rhEPO-induced cell death. In four different clinical trials, survival data linked to gene expression analysis indicated that high levels of EPOR mRNA were associated with better survival.ConclusionsOur results demonstrate for the first time active EPOR signaling in malignant plasma cells. EPO-mediated EPOR signaling reduced the viability of myeloma cell lines and of malignant primary plasma cells in vitro. Our results encourage further studies to investigate the importance of EPO/EPOR in multiple myeloma progression and treatment.Trial registration[Trial registration number for Total Therapy (TT) 2: NCT00083551 and TT3: NCT00081939].

Allelic mutations in noncoding genomic sequences construct novel transcription factor binding sites that promote gene overexpression.

The growth and survival factor hepatocyte growth factor (HGF) is expressed at high levels in multiple myeloma (MM) cells. We report here that elevated HGF transcription in MM was traced to DNA mutations in the promoter alleles of HGF. Sequence analysis revealed a previously undiscovered single‐nucleotide polymorphism (SNP) and crucial single‐nucleotide variants (SNVs) in the promoters of myeloma cells that produce large amounts of HGF. The allele‐specific mutations functionally reassembled wild‐type sequences into the motifs that affiliate with endogenous transcription factors NFKB (nuclear factor kappa‐B), MZF1 (myeloid zinc finger 1), and NRF‐2 (nuclear factor erythroid 2‐related factor 2). In vitro, a mutant allele that gained novel NFKB–binding sites directly responded to transcriptional signaling induced by tumor necrosis factor alpha (TNFα) to promote high levels of luciferase reporter. Given the recent discovery by genome‐wide sequencing (GWS) of numerous non‐coding mutations in myeloma genomes, our data provide evidence that heterogeneous SNVs in the gene regulatory regions may frequently transform wild‐type alleles into novel transcription factor binding properties to aberrantly interact with dysregulated transcriptional signals in MM and other cancer cells.

Photochemical internalization of peptide antigens provides a novel strategy to realize therapeutic cancer vaccination

Effective priming and activation of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) is crucial for realizing the potential of therapeutic cancer vaccination. This requires cytosolic antigens that feed into the MHC class I presentation pathway, which is not efficiently achieved with most current vaccination technologies. Photochemical internalization (PCI) provides an emerging technology to route endocytosed material to the cytosol of cells, based on light-induced disruption of endosomal membranes using a photosensitizing compound. Here, we investigated the potential of PCI as a novel, minimally invasive, and well-tolerated vaccination technology to induce priming of cancer-specific CTL responses to peptide antigens. We show that PCI effectively promotes delivery of peptide antigens to the cytosol of antigen-presenting cells (APCs) in vitro. This resulted in a 30-fold increase in MHC class I/peptide complex formation and surface presentation, and a subsequent 30- to 100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. The effect was found to be highly dependent on the dose of the PCI treatment, where optimal doses promoted maturation of immature dendritic cells, thus also providing an adjuvant effect. The effect of PCI was confirmed in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination using PCI technology. We thus show new and strong evidence that PCI technology holds great potential as a novel strategy for improving the outcome of peptide vaccines aimed at triggering cancer-specific CD8+ CTL responses.

A-kinase anchoring protein AKAP95 is a novel regulator of ribosomal RNA synthesis.

The RNA polymerase I transcription apparatus acquires and integrates the combined information from multiple cellular signalling cascades to regulate ribosome production essential for cell growth and proliferation. In the present study, we show that a subpopulation of A‐kinase anchoring protein 95 (AKAP95) targets the nucleolus during interphase and is involved in regulating rRNA production. We show that AKAP95 co‐localizes with the nucleolar upstream binding factor, an essential rRNA transcription factor. Similar to other members of the C2H2‐zinc finger family, we show, using systematic selection and evolution of ligands by exponential enrichment and in vitro binding analysis, that AKAP95 has a preference for GC‐rich DNA in vitro, whereas fluorescence recovery after photobleaching analysis reveals AKAP95 to be a highly mobile protein that exhibits RNA polymerase I and II dependent nucleolar trafficking. In line with its GC‐binding features, chromatin immunoprecipitation analysis revealed AKAP95 to be associated with ribosomal chromatin in vivo. Manipulation of AKAP95‐expression in U2OS cells revealed a reciprocal relationship between the expression of AKAP95 and 47S rRNA. Taken together, our data indicate that AKAP95 is a novel nucleolus‐associated protein with a regulatory role on rRNA production.

Abstract A008: Photochemical internalization: Light-induced enhancement of MHC Class I antigen presentation, giving strong enhancement of cytotoxic T-cell responses to vaccination

For cancer vaccination and immunotherapy it is essential to stimulate cytotoxic T-cells (CTLs) to recognize and kill the tumor cells. Priming of CTLs is generally mediated through MHC Class I antigen presentation by antigen presenting cells (APCs). Since the MHC class I presentation machinery is localised in the cytosol, MHC class I presentation typically requires cytosolic delivery of the antigen. Unfortunately, this is often difficult to achieve with exogenously added peptide or protein antigens, since such antigens are primarily taken up into endocytic vesicles, and then “by default” are routed for MHC Class II presentation. Photochemical internalisation (PCI) is a technology that can help solving this problem by inducing an illumination-mediated permeabilisation of the membranes of endocytic vesicles, thereby releasing endocytosed antigens into the cytosol. This is achieved by employing a photosensitising molecule that is designed to localise specifically in endocytic membranes. Upon illumination, the photosensitiser induces photochemical reactions that make the membranes leaky, thereby releasing endocytosed molecules into the cytosol. In vitro it has been shown that the use of PCI leads to strongly increases MHC Class I presentation APCs. In vivo PCI-mediated vaccination is performed by injecting a mixture of vaccine and photosensitiser intradermally, followed by illumination of the injection site; and with this regimen, PCI substantially enhances immune responses to various types of polypeptide- based antigens. Thus, with short peptide antigens enhancement of CD8+ T-cell responses of up to 100 times have been observed when PCI is used in combination with the poly(IC) adjuvant; with a strong synergy between PCI and the adjuvant. With an HPV long peptide and with several protein antigens in addition to the CD8+ T-cell response a significant stimulation of CD4+ T-cell responses can also be observed, and in some cases also an increase in antibody production. In vivo studies with therapeutic peptide antigen vaccination in a mouse model for HPV-induced cancer show that the use of PCI strongly enhances anti-tumor responses to the vaccine, both when the vaccination is performed intradermally and when intratumoral administration is employed. The TPCS2a photosensitiser used in PCI is cheap to produce, withstands autoclavation and is stable for several years at ambient temperatures. TPCS2a is currently in clinical development for enhancement of the effect of cytotoxic anti-cancer drugs, and it has been shown that TPCS2a can be administered safely to patients in much higher doses than what is needed for the use in immunotherapy. In conclusion, PCI has a completely new mechanism of action as a vaccination enhancement technology, representing a new and potent tool for stimulation of CTL and in some cases also other types of immune responses. Preparations for a clinical study with PCI-mediated vaccination is on-going. Citation Format: Tone Otterhaug, Markus Haug, Gaute Brede, Monika Hakerud, Anne Grete Nedberg, Victoria Edwards, Pal Kristian Selbo, Pal Johansen, Oyvind Halaas, Anders Hogset. Photochemical internalization: Light-induced enhancement of MHC Class I antigen presentation, giving strong enhancement of cytotoxic T-cell responses to vaccination [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A008.