julilili42/papers huggingface dataset:
Quantitative HLA Class I Expression and KIR-Mismatch between NK-Cell-Donor and Patient Predict NK Cell Mediated Lysis of Leukemic Cells from Children with Acute Lymphatic Leukemia.
2006-11-16

Abstract Relapses represent a major problem after transplantations in children with ALL. Natural Killer (NK) cells have been shown to exert remarkable Graft versus Leukemia effects after mismatched stem cell transplantation in myeloic leukemia, whereas the efficacy against lymphatic leukemia is still unclear. We therefore measured intensity of HLA class I expression on leukemic blasts by quantitative FACS analysis and investigated the impact of quantitative HLA class I expression, of several adhesion molecules and of KIR-mismatch on NK cell mediated lysis of the leukemic blasts from 21 pediatric patients with ALL. Expression of HLA class I molecules differed widely from patient to patient (range 5000–500000) and was reduced in comparison to B cells from healthy donors in 70% of cases. NK cells killed leukemic blasts very heterogeneously but a clear association between number of HLA class I molecules per cell and specific lysis (range 13–98%) was found (r2=0.68, p&lt;0.0001). For the subgroup of leukemic blasts without KIR-mismatch, this association was even stronger (r2=0.98), whereas a weak association was found for the subgroup with KIR-mismatch, since most of these targets were lysed more efficiently than one could expect according to HLA class I expression alone. KIR-mismatch alone (t-test, p=0.45) as well as different patterns of adhesion molecules (ICAM1-3, LFA1/3) and CD95 had no significant influence. However, a multivariate model taking both HLA expression and KIR-ligand-mismatch into account, provided an even stronger association (r2=0.87 p&lt;0.0001) for the whole group. Lysis was mainly dependent on HLA class I expression and in addition on KIR-mismatch for these leukemic cells. Assessment of HLA expression on leukemic blasts and KIR-receptor-ligand-mismatch between donor and recipient may be valuable to define patients who will benefit most from a NK mediated GvL effect.
Characterization of Dendritic Cell-Specific Genes p275 and p306.
2006-11-16

Abstract Dendritic cells play an inimitable role in the functioning immune system as they are the most potent antigen presenting cells being able to prime naive T-cells. Their characteristic properties that enable them to take up antigens and present them to leukocytes are due to an expression of specific genes and thus specific proteins that are unique to this subset of antigen-presenting cells. Using a substractive cDNA library based on suppression hybridization between DC cDNA and the reference monocyte cDNA, we identified in DC two differentially expressed genes p275 and p306. p275 codes for a membrane protein and represents a splice isoform of the transport protein NAT-1. The predicted structure of protein p306 is globular, suggesting that the protein is either intracellular or secreted. The expression of both genes was confirmed by RT-PCR using cDNA isolated from peripheral blood monocytes and DC, generated in vitro from monocytes or CD34+ progenitor cells. To further analyze the protein expression polyclonal antibodies were generated by immunization with synthetic peptides deduced from the identified sequences. Interestingly, inhibition of DC differentiation using IL-10 or STI571 (Imatinib) resulted in an impaired expression of both proteins. Utilizing specific primers for two recently described splice variants of p306 we identified a new splice form expressed in DC. While the gene of p306 contains eight exons, splice variant 1 consists of the exons 1,2,4,5,6, and 7 and splice variant 2 contains the exons 1,2,3,4,5,6, a shortened exon 7, and exon 8. The new identified splice form includes the exons 1–7. However, as the open reading frame starts in exon 4, the expressed protein is identical with the one corresponding to splice variant 1. Analyzing different DC populations in peripheral blood we show that p306 is expressed in plasmacytoid, but not myeloid DC. Interestingly, the activation of DC with Toll-like receptor ligands (TLRL) Pam3Cys (TLR2L), Poly I:C (TLR3L), LPS (TLR4L) and R848 (TLR7L) has no influence on the expression of p306. Although the functions of p275 and p306 in DC have yet to be determined, both genes play a role in DC differentiation and are found in different hematopoietic cell populations. Especially p306 might be an interesting marker of plasmacytoid DC as the predicted protein does not resemble any known protein structure.
The G Protein-Coupled Receptor cysLT1 Is Expressed in Acute Myeloid Leukemia Mediating Cell Migration and Survival.
2006-11-16

Abstract Previous studies have shown that the G protein-coupled receptor (GPCR) cysLT1, which recognizes inflammatory lipid mediators (cysteinyl-leukotrienes), is expressed in immature hematopoietic and tumor (eg. colon cancer) cells. In the present study, we analyzed leukemic blasts from 19 patients with newly diagnosed acute myeloid leukemia for analysis of expression and function of cysLT1. By RT-PCR, cysLT1 mRNA was found in all primary AML cells. Quantitative TaqMan PCR demonstrated high levels particularly in acute myeloblastic leukemia without maturation (FAB M1). There was also a positive correlation between the cysLT1 mRNA level and the expression of CD34 and CD-117 (c-kit), indicating that high cysLT1 expression corresponds to an AML phenotype resembling normal hematopoietic progenitor cells. CysLT1 was functionally active in AML blasts, as demonstrated by intracellular calcium fluxes and actin polymerization induced by the ligand LTD4. Similar to mRNA expression, strongest responses were seen in AML FAB M1. As LTD4 can be produced in the bone marrow by stromal cells and may contribute to bone marrow infiltration of AML, chemotaxis was analyzed. Surprisingly, already low concentrations (10nM) induced significant chemotaxis of AML blasts, while higher concentrations (up to 1 uM) were less effective in a dose-dependent manner. Incubation of myeloid leukemic cell lines (eg. KG1a) and primary AML blasts with the specific cysLT1 antagonist MK571 resulted in significantly reduced viability after 48 h in a dose-dependent manner (10nM-10uM), suggesting also an autocrine function of cysLT1 ligands. To explore signal transduction pathways involved in leukemic cell proliferation and chemotaxis, we found that in AML cell lines, LTD4 induced phosphorylation of Erk/MAP kinase, wich is related to proliferation, and Pyk2, which represents a focal adhesion kinase-like signaling molecule that links GPCRs with cell migration, while the Akt pathway was not involved. We conclude that cysLT1 is consistently expressed in acute myeloid leukemia, and mediates both chemotactic and proliferative responses. Therefore, cysLT1 antagonists, which are already used in the treatment of allergy, may improve the effectiveness of antileukemic therapy.
Down-Regulation of Glucocorticoid-Induced TNF Receptor (GITR) Ligand on Human Tumors by Proteolytic Shedding Increases Anti-Tumor Reactivity of NK Cells.
2006-11-16

Abstract Members of the TNF superfamily mediate multiple cellular functions, including cellular proliferation, differentiation and cell death. Many members of this protein family are shed from the cell surface as soluble forms, which affects cell-cell interactions by reduction of ligand densities and distally modulates effector cells bearing the respective receptor. The TNF family member Glucocorticoid-induced TNF Receptor (GITR) costimulates effector T cells, modulates apoptosis and NFkappaB and abrogates suppression of murine but not human regulatory T cells. Its cognate ligand GITRL has been found in various healthy tissues. Recently we reported that NK cells express GITR, while tumor cells express GITR ligand (GITRL), and GITR/GITRL interaction downregulates NK cell-mediated anti-tumor effector mechanisms like cytotoxicity and IFN-gamma production. Here we report that human tumor cells spontaneously release a soluble form of GITRL (sGITRL) detectable in culture supernatants by ELISA. Furthermore, we found elevated levels of sGITRL in sera of patients with various malignancies compared to healthy controls. We demonstrate that the release of GITRL from tumor cells can be blocked by inhibition of metalloproteinases, concomitantly causing accumulation of GITRL on the tumor cell surface as determined by FACS analysis. Upregulated GITRL surface expression further increased inhibition of NK cell anti-tumor effector mechanisms, while, in contrast, presence of sGITRL in cocultures of GITRL-expressing tumor cells and GITR-positive NK cells enhanced NK cell cytotoxicity and IFN-gamma production. Thus, in line with the results obtained with other TNF family members, conversion of membrane bound GITRL to its soluble form is associated with downregulation of its function, potentially due to blocking its cognate receptor. Thus, release of sGITRL substantially influences the interaction of tumor cells with NK cells. In addition, determination of sGITRL levels may be implemented as a diagnostic marker in patients with malignancies. Further prospective studies are currently being conducted addressing the value of GITRL as a tumor marker in different tumor entities.
Bone Marrow Osteoclasts Stain Positive with X-Gal: Implications for the Tracking of LacZ-Labelled Cells.
2006-11-16

Abstract The E.coli gene Lac-Z encodes for β-galactosidase and is often used to track transgenic cells after transplantation in vivo or to analyze temporospatial gene expression patterns by coupling expression of specific target genes to β-galactosidase activity. Several groups have reported endogenous β-galactosidase activity as a confounding factor in the study of different soft tissues. However, the typical background to be found on bone marrow sections when using the chromogenic substrate 5-Bromo-4-chloro-3-indolyl β-D-Galactoside (X-Gal) has not been described before. We demonstrate here that X-Gal stained wild type murine bones display a typical epiphyseal β-gal positive stain line that can already be seen on whole mount preparations. With light microscopy, the β-gal positive cells are located along the endosteum adjacent to trabecular bone and in particularly high concentrations in the epiphyseal resorption zone. In addition, the β-gal positive cells bear the typical features of osteoclasts in that they are multinucleated and display the typical foamy cytoplasm of osteoclasts. When an antibody against a well-known and previously published marker of the vasculature, pan-endothelial cell antigen (clone MECA32), was used in wild type bone, the X-Gal positive osteoclasts could be found in their typical location in epiphyseal resorption pits and closely located to the bone marrow vasculature. This provides further evidence that the X-Gal positive cells are indeed osteoclasts. Finally, double staining for tartrate resistant acid phosphatase (TRAP) and with X-Gal in wild type murine bones resulted in a complete match of the staining pattern and provided formal proof of the hypothesis that osteoclasts have strong β-galactosidase activity. In order to establish common knowledge of this potentially misleading artifact, we systematically examined the bones of different wild type mouse strains at various stages of development starting at the neonatal period. The same pattern of background staining was observed in C57bl/6, FvB/NJ, 129P3/J, and CD1 strains of mice. In addition, we examined the effect of pH and time of exposure to substrate and observed no changes in endogenous mammalian b-gal activity. In conclusion, we show that osteoclasts in bone marrow sections specifically and robustly stain blue with X-Gal. This leads to a typical background when bone marrow is examined that is present from the first day post partum throughout the adult life of experimental mice and can easily be confused with transgenic, bacterial β-galactosidase expressing hematopoietic or stromal cells. We conclude from these data that there is a field-wide need for strenuous controls and further classification when evaluating β-galactosidase positive bone marrow cells. Verifiable bacterial β-galactosidase positive bone marrow cells should be further identified using immunohistological or other approaches. Alternatively, β-galactosidase positive cells should be proven not to be osteoclasts by co-staining or by staining of adjacent sections.
Combination of Imatinib and Hypusination Inhibitors Represents a Novel Therapeutic Strategy in Bcr-Abl Positive Leukemias.
2006-11-16

Abstract In order to identify new cellular Bcr-Abl downstream targets, we have recently detected differential regulation of eukaryotic initiation factor 5A (eIF-5A) in Bcr-Abl positive cells. EIF-5A represents the only known eukaryotic protein activated by post-translational hypusination. Hypusination inhibitors (HI) GC7 and ciclopirox alone exerted an anti-proliferative effect on Bcr-Abl positive (K562, BA/F3p210) and negative human leukemia cell lines (HL-60, BA/F3) in vitro. No direct effect of HI treatment on phosphorylation of Bcr-Abl downstream targets (global p-tyrosination, p-CRKL, p-STAT5) was observed. However, the synergistic dose-effect relationship for apoptosis induction, cytotoxicity and inhibition of proliferation found for the combination of Imatinib and HI was restricted to Bcr-Abl-positive cells. Furthermore, this synergistic effect was confirmed by cytotoxicity assays, cell cycle analysis and CFSE-labeling of primary CD34+ CML cells in comparison to CD34+ cells from healthy donors. In parallel to these findings, eIF-5A mRNA is expressed at clearly (if significant then say significant and add p-value, otherwise delete clearly) elevated levels in PBMCs from CML patients at different disease stages when compared with PBMCs from healthy donors. Specificity of this effect could be demonstrated by co-treatment of K562 cells with Imatinib and a stable lentiviral siRNA against eIF-5A. Based on this observation, we observed a strong antiprolifrative effect of HI as single agents on BA/F3p210 cells, including the Imatinib-resistant mutants T315I, M351T or E255K. Furthermore, a combination of Imatinib and HI had a synergistic effect in inducing cytotoxicity, apoptosis and inhibition of proliferation in leukemic cell lines with moderate resistance to imatinib (M351T). In contrast, in BA/F3p210T315I (T315I) cells, which are resistant to Imatinib as a single agent, the co-treatment of Imatinib and HI did not lead to additive or synergistic effects. In conclusion, we have identified inhibition of eIF-5A hypusination as a promising new approach for single agent or combination therapy in Bcr-Abl
BCR-ABL Specific Sequences Are Required To Prevent Ph+ Acute Lymphoblastic Leukemia in Syngeneic Mice by DNA Vaccination.
2006-11-16

Abstract Although 70 – 80 % of children with childhood acute lymphoblastic leukemia (ALL) can be cured by poly-chemo-therapy, the prognosis of patients with Philadelphia chromosome positive (Ph+) ALL remains poor. Therefore, new relapse prevention strategies are needed for patients with Ph+ ALL during remission. We have shown previously, that vaccination of mice with leukemia cell lines modified to express costimulatory molecules and cytokines induce a systemic immunity against the syngeneic BCR-ABLp185 expressing cell line BM185. However, the difficulties to culture and transfect human leukemia cells limit the clinical application of leukemia cell based vaccines. Thus, we evaluated the pre-immunization of mice with DNA based vaccines subsequently challenged by the cell line BM185. Ballistic transfer of minimalistic immunogenically defined gene expression (MIDGE) vectors encoding a BCR-ABLp185 fusion specific peptide or GM-CSF were used for in vivo transfection of murine skin. In addition, we used double stem-loop immunomodulators (dSLIM), containing three CpG-motifs as non-specific immune adjuvant. We provide survival data that shows specific immunization and protection of mice which received the complete vaccine BCR-ABL/GM-CSF/dSLIM. Mean tumor-free survival (p=0.019) and overall survival (p=0.008) were significantly longer compared to non-vaccinated mice and 26.7% survived and never developed leukemia. In contrast, tumor-free and overall survival of mice immunized with either dSLIM or GM-CSF alone or both dSLIM and GM-CSF was not significantly longer compared to non-vaccinated mice. Similarly, substitution of BCR-ABL by irrelevant TEL-AML1 sequences abolished the vaccine efficacy of the BCR-ABL/GM-CSF/dSLIM vaccine. The biometrical data were confirmed by CTL assays which showed that specific lysis was significantly higher after vaccination with BCR-ABL/GM-CSF/dSLIM compared to GM-CSF/dSLIM (p&lt;0.05) and to naïve mice (p&lt;0.005). Previously, we have shown in T-cell depletion studies that CD8+ T cells were the effector cells in the BM185 cell vaccine model and we suggest that CD8+ T cells also play an essential role in the BM185 DNA vaccine model. Together, we provide biometrical and functional data that DNA-based vaccination with BCR-ABLp185 fusion specific sequences, GM-CSF and dSLIM can protect mice against a lethal Ph+ ALL challenge.
Zoledronic Acid Inhibits the Function of Toll-Like Receptor 4 Ligand Activated Dendritic Cells.
2006-11-16

Abstract Zoledronic acid (ZA; Zometa®) is a cyclic nitrogen-containing bisphosphonate with anti-tumor activity used to treat patients with malignant diseases. As a side effect, ZA induces inflammatory responses which are accompanied by expansion of gd T cells that probably mediate its anti-tumor activity. In our study we analyzed the effects of ZA on the function and differentiation of monocyte-derived dendritic cells (DC). We investigated the differentiation of immature and LPS-stimulated DCs incubated with different ZA concentrations which are usually achieved in treated patients. Expression of DC-specific marker molecules was assessed by FACS. Furthermore, the activation state and involvement of different signal transduction pathways were evaluated by Western blot. To monitor DC function we used a migration assay and analyzed the cytokine secretion. We found that DC activation with TLR4 ligand LPS is modulated by ZA. We show that the expression of the surface maturation markers CD80, CD86, CD40, CD54, CD83 and DC-SIGN is diminished with increasing ZA levels upon LPS activation. The migratory capacity of LPS stimulated DCs towards the chemokine CCL19/MIP-3beta and the secretion of the activatory cytokine IL-12 are reduced at higher ZA levels. Increasing ZA concentrations lead to the down-regulation MyD88 and diminish the nuclear localization of NF-kB family members RelB and c-Rel as well as interferon-regulatory factors IRF-3 and IRF-8 in LPS activated DCs. Hence, ZA concentrations used to treat patients have inhibitory effects on DC differentiation and activation. This might lead to immuno-suppression and negatively affect tumor rejection or result in infectious complications.
Expression of Glucocorticoid-Induced TNF Receptor Ligand on Acute Myeloid Leukemia Cells Mediates the Release of Immunosuppressive Cytokines and Impairs NK Cell-Mediated Immune Surveillance.
2006-11-16

Abstract NK cells play an important role in the reciprocal interaction of tumor cells with the immune system and participate in the surveillance of hematological malignancies including acute myeloid leukemia (AML). Among the molecules influencing host-tumor interaction are many members of the TNF superfamily, which mediate multiple cellular functions including cellular proliferation, differentiation and cell death. The TNF family member Glucocorticoid-induced TNF Receptor (GITR) costimulates effector T cells, modulates apoptosis and nuclear factor kappa B and abrogates suppression of murine but not human regulatory T cells. Its cognate ligand GITRL has been found in various healthy tissues. Recently we reported that NK cells express GITR, while solid tumors express GITR ligand (GITRL), and GITR/GITRL interaction downregulates NK cell cytotoxicity and IFN-γ production. Here we analyzed the role of GITR and its ligand in AML. We report for the first time that GITRL is expressed on primary AML cells in 18 of 30 patients as determined by FACS and RT-PCR analysis. Reverse signaling through GITRL using a recombinant GITR-Ig fusion protein induces the release of the immunoregulatory cytokines IL-10 and TNF as determined by ELISA. GITRL-mediated cytokine production of AML cells is abrogated by inhibition of mitogen activated protein kinase (MAPK) pathways as demonstrated by addition of the specific p38 MAPK inhibitor SB202190, the specific JNK inhibitor SP600125 and the specific ERK Inhibitor II. Furthermore, binding of AML-expressed GITRL to GITR on NK cells downregulates cellular cytotoxicity and IFN-γ production in AML-NK cell cocultures, which can be overcome by addition of GITR-blocking antibodies as determined by cytotoxicity assays and ELISA. Thus, our data indicate that GITRL expression in AML substantially influences tumor immunoediting and enables the escape of leukemia cells from NK cell-mediated immunosurveillance.
Sweeping from the Superfluid to the Mott Phase in the Bose-Hubbard Model
2006-11-17

We study the sweep through the quantum phase transition from the superfluid to the Mott state for the Bose-Hubbard model with a time-dependent tunneling rate J(t). In the experimentally relevant case of exponential decay J(t) proportional variant e -gamma t, an adapted mean-field expansion for large fillings n yields a scaling solution for the fluctuations. This enables us to analytically calculate the evolution of the number and phase variations (on-site) and correlations (off-site) for slow (gamma<<mu), intermediate, and fast (nonadiabatic gamma>>mu) sweeps, where mu is the chemical potential. Finally, we derive the dynamical decay of the off-diagonal long-range order as well as the temporal shrinkage of the superfluid fraction in a persistent ring-current setup.
A Gene Cluster for Prenylated Naphthoquinone and Prenylated Phenazine Biosynthesis in Streptomyces cinnamonensis DSM 1042
2006-11-17

Abstract Streptomyces cinnamonensis DSM 1042 produces two classes of secondary metabolites of mixed isoprenoid/nonisoprenoid origin: the polyketide–isoprenoid compound furanonaphthoquinone I (FNQ I) and several prenylated phenazines, predominantly endophenazine A. We now report the cloning and sequence analysis of a 55 kb gene cluster required for the biosynthesis of these compounds. Several inactivation experiments confirmed the involvement of this gene cluster in the biosynthesis of FNQ I and endophenazine A. The six identified genes for endophenazine biosynthesis showed close similarity to phenazine biosynthetic genes from Pseudomonas . Of the 28 open reading frames identified in the adjacent FNQ I cluster, 13 showed close similarity to genes contained in the cluster for furaquinocin—a structurally similar metabolite from another Streptomyces strain. These genes included a type III polyketide synthase sequence, a momA ‐like monooxygenase gene, and two cloQ ‐like prenyltransferase genes designated fnq26 and fnq28 . Inactivation experiments confirmed the involvement of fnq26 in FNQ I biosynthesis, whereas no change in secondary‐metabolite formation was observed after fnq28 inactivation. The FNQ I cluster contains a contiguous group of five genes, which together encode all the enzymatic functions required for the recycling of S ‐adenosylhomocysteine (SAH) to S ‐adenosylmethionine (SAM). Two SAM‐dependent methyltransferases are encoded within the cluster. Inactivation experiments showed that fnq9 is responsible for the 7‐ O ‐methylation and fnq27 for the 6‐ C ‐methylation reaction in FNQ I biosynthesis.