julilili42/papers huggingface dataset:
Transcutaneous Immunization with a Solid Nanoscopic Imiquimod Suspension Enhances Tumor Rejection
2015-12-03

Abstract Introduction: Transcutaneous immunization (TCI) is a novel vaccination strategy to induce strong therapeutic cytotoxic T-lymphocyte (CTL) responses by directly targeting skin-resident professional antigen-presenting cells (APC). This vaccination approach is very promising to overcome current limitations of standard vaccination approaches that are mostly effective in prophylaxis, but not in the treatment of diseases. In this context, we have developed a TCI method based on a synthetic TLR7 agonist imiquimod that partial tumor protection in experimental rodent models. In our present work, we describe a novel optimized formulation of imiquimod in a solid suspension of crystalline imiquimod (IMI-Sol) that induces superior CTL responses and enhanced tumor protection. Methods: We determined imiquimod-penetration into the skin as well as systemic distribution by HPLC in C57BL/6 mice (at 6-12 weeks). For TCI, C57BL/6, Tlr7 (TLR7-/-) or Myd88 (MyD88-/-) gene deficient mice IMI-sol or imiquimod (in the commercially available formulation Aldara®) together with the CTL epitope SIINFEKL (derived from chicken ovalbumin) was applied on two consecutive days on the dorsum of the animals after hair removal by electric clippers. Induction of CTL responses was characterized after 7 days by the analysis of peptide-specific CTLs (ELISpot, tetrameric MHC I complexes) and in vivo cytolytic activity by flow cytometry. Tumor protection was assessed using the B16OVA tumor model. Results: Despite equal imiquimod penetration into the skin, we found superior vaccination capacity of IMI-Sol compared to Aldara® leading to enhanced tumor-protection. Using TLR7-/- or MyD88-/- mice, we confirmed that the induction of CTL responses with IMI-Sol was completely dependent on the TLR/MyD88 pathway. The enhanced immune response are due to more efficiently activated skin-derived DCs upon IMI-Sol treatment found in skin-draining lymph nodes. Conclusions: We developed of a superior imiquimod formulation for transcutaneous vaccination that boosts vaccination efficacy by the enhanced activation of skin-resident APCs leading to the induction of therapeutic CTL responses underscoring the importance of pharmaceutical aspects affecting vaccination potency beyond understanding of the underlying basic mechanisms. Combined efforts are necessary to establish TCI as a promising novel next generation vaccination platform that can be used for the treatment of persistent infections and cancer. Disclosures No relevant conflicts of interest to declare.
The Homeobox Gene VENTX Collaborates with AML1-ETO in Inducing an Acute Leukemia in Mice
2015-12-03

Abstract Homeobox genes are key factors in the development of acute leukemias. So far, little is known about the role of non-clustered homeobox genes such as VENTX. The Vent-like homeobox gene VENTX is a member of the Vent gene family in mammals and is the mammalian homolog of the Xenopus xvent gene. Our group has previously shown that VENTX promotes myeloid differentiation when overexpressed in normal CD34+ human stem - and progenitor cells and shows high and aberrant expression in human acute myeloid leukemia (AML) characterized by the AML1-ETO (AE) fusion (Rawat et al., PNAS 2010). To prove the functional relevance of aberrant VENTX expression in this AML genotype, we set up in vitro assays and a murine bone marrow transplantation model, mimicking high expression of VENTX by retrovirally engineered expression of the gene in murine bone marrow progenitor cells. In the CFU-S assay coexpression of both genes significantly increased the frequency of spleen colonies 14-fold and 7-fold compared to VENTX and AE, respectively (p&lt;0.02), and more than 7-fold compared to the control (p&lt;0.001). Most importantly and in contrast to AE alone, coexpression of AE and VENTX induced an acute leukemia after a median latency of 11.3 months post transplant. All leukemias evaluated were rapidly transplantable with a median latency until leukemia induced death of 40 days. The median blast percentage in primary recipient mice was 66% (range 35-100%, n=24). Of note, massive expansion of the red pulpa was documented with the appearance of erythroblasts in the spleen. Furthermore, blasts in the bone marrow expressed CD19, partly in combination with Sca1 and Gr1, pointing to an aberrant expression of this antigen in the AE/VENTX leukemias. Ex vivo, leukemic cells grew permanently generating AE/VENTX positive cell lines. RNA-Seq analyses from CD34+ cord blood transduced with VENTX documented 279 differentially expressed genes compared to the GFP control, hitting 7 pathways (Hematopoietic cell lineage, Cytokine-cytokine receptor pathway, Jak-STAT signaling pathway, Porphyrin and Chlorophyll Metabolism, Asthma, Thyroid cancer and Endocytosis) in the KEGG-Analysis and showed a downregulation of genes necessary for terminal erythroid differentiation such as the EPO-receptor and GATA1. Taken together, these data characterize VENTX as an important contributing factor to AE positive leukemias. Importantly, coexpression of AE and VENTX allows the generation of a murine AE model, which in contrast to all other published mouse models mimics aberrant expression of CD19 in human AE positive AML, a hallmark of this AML genotype. Disclosures Buske: CELLTRION, Inc.: Consultancy, Honoraria.
Attenuation of Apoptosis-Stimulating Protein of p53-2 (ASPP2) Promotes IL-3-Independent Growth and Enhances Cytogenetic Abnormalities after Gamma-Irradiation in Mouse Hematopoietic Cells - Mimicking Early Leukemogenesis of Therapy-Associated Human AML
2015-12-03

Abstract Inactivation of tp53 by chromosome/gene loss or loss-of function mutation is frequently observed in therapy-associated acute myeloid leukemia. Tp53 mutations associate with complex and monosomal karyotypes and provide the strongest predictive risk factor in this subgroup. ASPP2 is an independent tumor suppressor. We have previously shown that ASPP2 expression is attenuated in acute leukemia via methylation by unknown mechanisms. We now provide evidence that attenuation of ASPP2 expression may act as an early leukemia-initiating event - facilitating acquisition of structural chromosomal aberrations and loss of its binding partner p53. We found low ASPP2 mRNA and protein expression levels in complex karyotype AML, arguing for a role of ASPP2 loss in leukemogenesis. To evaluate for malignant transformation we engineered IL3-dependant murine Ba/F3 cells and stably silenced ASPP2 expression using retroviral-induced shRNA, Interestingly, only ASPP2(ko) cell strains could be successfully weaned from IL3 dependence - suggesting that loss of ASPP2 expression promoted cellular transformation. In contrast, empty vector control cells did not survive the weaning process. As expected, cellular proliferation was enhanced in the ASPP2(ko) cell lines as measured by standard XTT and CSFE protocols. To study how loss of ASPP2 could promote transformation, we performed cytogenetic analysis of the chromosome set of parental (+IL3) vs. gamma-irradiated empty vector controls (+IL3) and ASPP2(ko) cell strains (-IL3) with and without irradiation (+/- stress enforcement using gamma irradiation, 2 x 5Gy). Gamma irradiation did not significantly alter cytogenic analysis between the empty vector controls compared to the parental cells. Tantalizingly however, we found a consistent chromosomal loss of chr8, der8 and der19 - and, most intriguingly, loss of chromosome 11 (which locates tp53 in mice), and a monosomal karyotype in the ASPP2-attenuated cells. In addition, several marker chromosomes (6-8 in ASPP2i vs. 0-2 in empty vector controls were identified. We are now in the process of verifying our findings in knockout native human bone marrow blasts. Conclusions: Our findings provide novel evidence that loss of ASPP2 contributes to malignant transformation by promoting genomic instability as measured by increased cytogenetic abnormalities after gamma-irradiation. These findings may have important relevance for the pathogenesis of therapy-associated AML - but also of hypodiploid acute lymphoblastic leukemia, which is as well associated with tp53 loss-of-function and predicted for a dismal outcome. Studies are ongoing to elucidate the mechanisms of how loss of ASPP2 expression promotes human AML, as well as restore ASPP2 function as a novel therapeutic pathway to exploit in treating AML. Disclosures No relevant conflicts of interest to declare.
First Case of Leukemia in a Child Suffering from Cyclic Neutropenia with ELANE Mutation
2015-12-03

Congenital neutropenia (CN) and Cyclic neutropenia (CyN) are rare hematological conditions in which ELANE mutations have been found. The discrimination of Cyn from CN is based on the cycling neutrophil counts which decrease to &lt;0.2 x 109/L, usually at 21 day intervals. Infectious episodes are typically less severe in CyN compared to infections in CN patients. While in patients suffering from ELANE-CN an increased risk of leukemic transformation of more than 10 percent is well documented, no AML has been reported in the European SCNIR cohort of 38 patients with ELANE -CYN so far. Here we report the first case of AML secondary to ELANE -CyN in a 17 year old girl. Severe neutropenia was first diagnosed at the age of four weeks during a septicemia with pseudomonas, but no serial blood counts were collected. G-CSF treatment was initiated at an age of 2 years after recurrent otitis media, pneumonia and skin abscesses. Infectious events occurred infrequently under G-CSF treatment. At an age of 14 years she was referred to us after a severe infectious event (liver abscess) and an unexplained high range of ANCs (0 to more than 10x109/μl) under G-CSF treatment with varying doses. Sequential blood counts confirmed the diagnosis of CyN. Molecular genetic testing revealed a heterozygous ELANE mutation, both parents are ELANE negative. Previous bone marrows varied between a maturation arrest on the promyelocyte stage with absence of neutrophils and up to 16 percent segmented neutrophils. No cytogenetic aberrations were detected prior to AML. AML was diagnosed recently when she presented with decreasing platelets and hemoglobin. Bone marrow at this time showed 14% blasts and presence of monosomy 7 plus trisomy 21 in the cytogenetic evaluation. Molecular genetic evaluation of the bone marrow revealed presence of a CSFR3 and RUNX1 mutations in the leukemic cells. Conclusion Our patient presented with a secondary AML after ELANE positive cyclic neutropenia. Cytogenetics, CSFR3 and RUNX1 mutations in the bone marrow at the time of leukemia diagnosis have been described in the pathway of leukemogenesis in Congenital neutropenia with ELANE, HAX1 or other underlying mutation. Our patient is the first case of leukemia in ELANE -CyN and proofs that ELANE -CyN is another pre-leukemic condition. However, the risk of myeloid transformation is very low. Disclosures No relevant conflicts of interest to declare.
Activation of C/EBPa Myeloid-Specific Transcription Factor By NAMPT/SIRT1-Triggered Deacetylation
2015-12-03

Abstract Previously, we described new mechanism of G-CSF-triggered granulocytic differentiation of hematopoietic stem cells (HSCs) via activation of the enzyme Nicotinamide Phosphorybosyltransferase (NAMPT) leading to NAD+ production and activation of NAD+ -dependent protein deacetylase sirtuin 1 (SIRT1). We found, that upon stimulation of HSCs with NAMPT, SIRT1 bound to the key myeloid transcription factor C/EBPα followed by transcriptional induction of C/EBPα target genes G-CSFR and G-CSF and granulocytic differentiation. In the present work we investigated the mechanism of NAMPT/SIRT1-triggered deacetylation of C/EBPα. We found that C/EBPα is acetylated at the position Lys 161, which is evolutionarily conserved. Lys 161 is localized in the transactivation element III (TE-III) of the transactivation domain (TAD) of C/EBPα protein, which is responsible for recruitment of SWI/SNF and CDK2/CDK4. Western blot and DUOLINK analysis using rabbit polyclonal antibody specifically recognizing acetyl-Lys 161 of C/EBPα revealed predominantly nuclear localization of acetylated C/EBPα protein in acute myeloid leukemia cell lines NB4 and HL60 as well as in primary HSCs. Induction of myeloid differentiation of HSCs by treatment with G-CSF as well as ATRA-induced differentiation of NB4 cells resulted in the deacetylation of C/EBPα. NAMPT inhibition in NB4 and HL60 cell lines using specific inhibitor FK866 led to the dramatically elevated levels of acetylated C/EBPα and reduced amounts of total C/EBPα protein, which was in line with diminished mRNA expression of C/EBPα target genes (G-CSF, G-CSFR and ELANE). Interestingly, treatment of acute myeloid leukemia cell line HL60 with NAMPT or transduction of HL-60 cells with NAMPT-expressing lentiviral construct induced myeloid differentiation of these cells even without addition of ATRA. This was in line with time- and dose-dependent increase of total C/EBPα protein levels upon NAMPT treatment. Therefore, NAMPT overcomes transcriptional repression of C/EBPα in HL-60 cells by activation of positive CEBPA autoregulation. Taken together, we described a new mechanism of regulation of C/EBPα activities in hematopoiesis and leukemogenesis by its post-translational modification via NAMPT/SIRT1-triggered de-/acetylation. Disclosures No relevant conflicts of interest to declare.
Favorable Immune Signature in CLL Patients, Defined By Antigen-Specific T-Cell Responses, Might Prevent Secondary Skin Cancers
2015-12-03

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. The course of disease predisposes patients to the development of secondary malignancies, which might be due to a generalized immunosuppression induced by the disease and/or the therapies used for treatment. Skin cancer, including melanoma, is the most common secondary malignancy reported in these patients. In a recent study we reported on spontaneous memory T cell responses against CLL-associated antigens defined by HLA ligandome analysis in CLL patients. Moreover, retrospective analysis suggests a significant survival benefit for CLL patients showing immune reactions to more than one of the tested antigens (Kowalewski et. al., PNAS 2015). Here we analyzed our CLL patient cohort for the incidence of secondary malignancies, and in particular for skin cancers. A total of 15 CLL-associated HLA-peptides (6 A*02, 3 A*03, 6 B*07) were implemented for stimulation of HLA-matched PBMC obtained from CLL patients. 45 CLL patients (Binet A n=15, Binet B, n=15, Binet C n=15, median age, 33.3% (15/45) with prior CLL specific therapy) were analyzed for antigen-specific memory T-cell responses in IFNγ-ELISPOT assays. 13 patients (28.9%, response cohort) showed specific T cell response against &gt; 1 antigen, while 32 patients (71.1%, negative cohort) showed none or only one immune response. The median age was 71 in the response cohort and 73 in the negative cohort. 40.0% (6/15) of CLL patients with Binet stage A and 33.3% (5/15) with stage B were classified as responders whereas only 13.3% (2/15) of patients with stage C showed &gt;1 immune responses. Of the included CLL patients with prior therapy only one patient (8%) showed &gt; 1 immune responses. In the analyzed CLL patient cohort 7 skin cancers (melanoma: n=2, squamous cell carcinoma: n=2, basal cell carcinoma: n=3), 3 precancerous skin lesions and 9 other secondary malignancies including renal cell carcinoma (n=2), colon (n=3), breast (n=2) and prostate cancer (n=2) were observed. In the negative cohort 34.4% (11/32) of the patients developed a secondary malignancy, while only 15.4% (2/13) in the immune response cohort presented with secondary neoplasms. Strikingly all skin cancers and precancerous skin lesions were observed in the non-immune responder cohort. Furthermore multiple secondary malignancies (e.g. squamous cell carcinoma and colon cancer) were only observed in the cohort that showed no immune response. Taken together our data suggest that a favorable immune signature in CLL patients, i.e. the ability to mount T-cell responses against CLL-associated antigens not only is associated with improved overall survival but also might protect these patients from the development of secondary skin cancers. Disclosures No relevant conflicts of interest to declare.
ECM and ECM-like materials — Biomaterials for applications in regenerative medicine and cancer therapy
2015-12-04

Regenerative strategies such as stem cell-based therapies and tissue engineering applications are being developed with the aim to replace, remodel, regenerate or support damaged tissues and organs. In addition to careful cell type selection, the design of appropriate three-dimensional (3D) scaffolds is essential for the generation of bio-inspired replacement tissues. Such scaffolds are usually made of degradable or non-degradable biomaterials and can serve as cell or drug carriers. The development of more effective and efficient drug carrier systems is also highly relevant for novel cancer treatment strategies. In this review, we provide a summary of current approaches that employ ECM and ECM-like materials, or ECM-synthetic polymer hybrids, as biomaterials in the field of regenerative medicine. We further discuss the utilization of such materials for cell and drug delivery, and highlight strategies for their use as vehicles for cancer therapy.
Differential effect of DJ-1/PARK7 on development of natural and induced regulatory T cells
2015-12-04

Abstract Regulatory T cells (Tregs) are essential for maintaining an effective immune tolerance and a homeostatic balance of various other immune cells. To manipulate the immune response during infections and autoimmune disorders, it is essential to know which genes or key molecules are involved in the development of Tregs. Transcription factor Foxp3 is required for the development of Tregs and governs most of the suppressive functions of these cells. Inhibited PI3K/AKT/mTOR signalling is critical for Foxp3 stability. Previous studies have suggested that DJ-1 or PARK7 protein is a positive regulator of the PI3K/AKT/mTOR pathway by negatively regulating the activity of PTEN. Thus, we hypothesised that a lack of DJ-1 could promote the development of Tregs. As a result, loss of DJ-1 decreased the total CD4 + T cell numbers but increased the fraction of thymic and peripheral nTregs. In contrast, Foxp3 generation was not augmented following differentiation of DJ-1-deficient naïve CD4 + T cells. DJ-1-deficient-iTregs were imperfect in replication, proliferation and more prone to cell death. Furthermore, DJ-1 deficient iTregs were less sensitive to pSmad2 and pStat5 signalling but had activated AKT/mTOR signalling. These observations reveal an unexpected differential role of DJ-1 in the development of nTregs and iTregs.
Elucidation of the zinc‐dependent regulation in Amycolatopsis japonicum enabled the identification of the ethylenediamine‐disuccinate ([ S,S ]‐EDDS) genes
2015-12-04

Summary The actinomycete A mycolatopsis japonicum produces the complexing agent ethylenediamine‐disuccinate ([ S , S ]‐ EDDS ), which is an isomer of the widely industrially applied ethylenediamine‐tetraacetate ( EDTA ). In contrast to EDTA , [ S , S ]‐ EDDS is readily biodegradable and is therefore an alternative with a favourable environmental profile. Biotechnological production of [ S , S ]‐ EDDS , however, is not currently possible because its biosynthesis is inhibited by low‐micromolar zinc concentrations. Here we illustrate the development of a new strategy for identifying a biosynthetic pathway that is based on the elucidation of transcriptional regulation and the screening for binding sites of the respective regulator that controls the [ S , S ]‐ EDDS biosynthesis genes. To achieve this, we identified the zinc uptake regulator Z ur in A . japonicum and showed that it mediates the repression of the zinc uptake system ZnuABC Aj . The Z ur‐binding motif, recognized by the zinc‐bound Z ur protein in the upstream region of znu ABC Aj , was used to screen the genome, leading to the identification of the aes genes. Transcriptional analysis and shift assays reveal specific zinc‐responsive regulation of the aes genes by Z ur, and gene inactivation shows their involvement in [ S , S ]‐ EDDS biosynthesis. Z ur‐mediated zinc repression of the [ S , S ]‐ EDDS biosynthesis genes is abolished in a Δ zur mutant, which offers now the opportunity to develop a biotechnological process.
Thickness and Substrate Dependent Thin Film Growth of Picene and Impact on the Electronic Structure
2015-12-04

Thickness and substrate dependence of film growth, morphology, unit-cell structure, and electronic structures was thoroughly investigated for picene, the zigzag connected 5-ring molecule, by employing complementary techniques of in situ real-time X-ray reflectivity/diffraction, in situ electron spectroscopies, and atomic force microscopy. A different kind of thickness dependent structural transition was observed on SiO2 and graphite, resulting in a distinct electronic structure. On SiO2 picene films with 3D crystalline domains are formed with nearly upright molecular orientation from the initial growth stage. With increasing the film thickness the in-plane dimensions of the unit cell in the initially grown domains become smaller (in other words, more compressed), and, at the same time, crystalline domains with a more relaxed structure are nucleating on top of the compressed domains. In spite of such structural changes, the electronic structure, namely energy position of the highest occupied molecular orbital and threshold ionization potential (IPT), is not significantly altered. On graphite, on the other hand, we found a transition from a 2D (layer) to a 3D (island) growth mode with a variation of the molecular orientation from flat-lying to tilted one. The IPT changes significantly between the 2D and 3D growth regime in contrast to the SiO2 system. The origin of the different IPTs of these picene thin films is discussed. The present results are compared with other planar π-conjugated compounds, in particular pentacene which is a structural isomer of picene and shows electronic properties strongly different from picene thin films.
The stochastic background of gravitational waves due to thef-mode instability in neutron stars
2015-12-04

This paper presents an estimate for the spectral properties of the stochastic\\nbackground of gravitational waves emitted by a population of hot, young,\\nrapidly rotating neutron stars throughout the Universe undergoing -mode\\ninstabilities, formed through either core-collapse supernova explosions or the\\nmerger of binary neutron star systems. Their formation rate, from which the\\ngravitational wave event rate is obtained, is deduced from observation-based\\ndeterminations of the cosmic star formation rate. The gravitational wave\\nemission occurs during the spin-down phase of the -mode instability. For low\\nmagnetized neutron stars and assuming 10\\\\% of supernova events lead to -mode\\nunstable neutron stars, the background from supernova-derived neutron stars\\npeaks at for the -mode, which\\nshould be detectable by cross-correlating a pair of second generation\\ninterferometers (e.g. Advanced LIGO/Virgo) with an upper estimate for the\\nsignal-to-noise ratio of 9.8. The background from supramassive\\nneutron stars formed from binary mergers peaks at and should not be detectable, even with third generation\\ninterferometers (e.g. Einstein Telescope).\\n