julilili42/papers huggingface dataset:
Vaccination with the Heptavalent Pneumococcal Conjugate Vaccine Prevenar™ Provides Early Protective Antibody Responses in Children after Allogeneic Stem Cell Transplantation.
2005-11-16

Abstract Children undergoing allogeneic hematopoietic stem cell transplantation (HSCT) are at significant risk for life-threatening infections with Streptococcus pneumoniae especially in the first two years after transplantation. Due to immaturity of the immune system, immunization with conventional pneumococcal polysaccharide vaccine fails to elicit protective immunity in the majority of pediatric HSCT recipients. The heptavalent pneumococcal conjugate vaccine PrevenarTM (PCV7; Wyeth Pharma) provides effective protection from invasive disease in infants, who like aHSCT recipients are unable to respond to polysaccharide vaccines due to immunologic immaturity. Currently there is a dearth of data on the safety and efficacy of PCV7 vaccination in pediatric transplant recipients. Here we report tolerability and profound immunogenicity of PCV7 in children after HSCT and show for the first time that PCV7 provides early protective anti-pneumococcal antibody responses in a homogeneously treated cohort of pediatric HSCT recipients recruited into a prospective, multicenter vaccination trial. 53 children (median age 8,3 (1,416,9) years), who underwent HSCT from related (n=24) or unrelated (n=29) donors were immunized with three doses of PCV7 in monthly intervals starting at 6–9 months after HSCT. PCV7 was administered in combination with a hexavalent vaccine against Tetanus, Diphtheria, Pertussis, Poliovirus, Haemophilus influenzae type B and Hepatitis B (Infanrix hexaTM; GlaxoSmithKline Pharma). Serum was obtained at baseline and 4–6 weeks after the 2nd and 3rd vaccine and local and systemic adverse events were recorded for 28 days after each vaccination. Responses, defined as seroconversion to protective serotype-specific antibody concentrations (Ab conc.) of 0,5 mg/ml or &amp;gt;2-fold increase were detected in 41.9% – 86.0% of patients (pts) after the 2nd vaccine and in 58.1% – 90.7% of pts after the 3rd vaccine (n=43). As a result of a highly significant increase in mean geometric Ab conc. (GMCs) (p&amp;lt;0.001) protection to all 7 vaccine serotypes was achieved in 56% and 74% of pts after the 2nd and 3rd application of PCV7 at a median of 10 and 11 months after transplantation, respectively. Even in a small subgroup of pts suffering from chronic GvHD 5/7 pts attained protective Ab conc. to 6/7 serotypes (n=7). Local and systemic tolerability was very good and no vaccine-related severe adverse events were observed. In conclusion, our data show that vaccination with PCV7 elicits highly protective anti-pneumococcal antibody responses in pediatric HSCT recipients within the 1st year following aHSCT. Therefore, PCV7 should be included in routine revaccination programs for pediatric HSCT recipients.
The Target Receptor Siglec-3 (CD33) Is Expressed on AML Stem Cells in a Majority of All Patients with AML.
2005-11-16

Abstract The cell surface antigen Siglec-3 = CD33 is becoming increasingly important as target of therapy in acute myeloid leukemia (AML). In particular, a conjugate consisting of the humanized CD33 antibody P67.6 (gemtuzumab) and the cytostatic drug calicheamicin has been developed for clinical use and was found to work as an effective antileukemic agent (Mylotarg®) in patients with CD33+ AML. In normal myelopoiesis, expression of CD33 is restricted to advanced stages of differentiation, whereas primitive stem cells do not express CD33 (Siglec-3). In line with this notion, CD33-targeting therapy is a non-myeloablative approach. In the present study, we asked whether leukemic stem cells in patients with AML express CD33. For this purpose, a multicolor-staining technique was applied in eleven patients with AML. Leukemic stem cells were defined as CD34+/CD38−/CD123+ cells. In all patients in whom the majority of myeloblasts expressed CD33 (=CD33+ AML, n=8), the AML progenitor cells reacted with the CD33 antibody P67.6. Repopulation experiments utilizing NOD/SCID mice confirmed that the AML stem cells in these patients reside within the CD33+ subpopulation of leukemic cells. Moreover, AML stem cells (CD34+/CD38−/CD123+ cells) highly purified (&amp;gt;98% purity) from patients with (CD33+) AML by cell sorting, were found to express CD33 mRNA in RT-PCR analyses. To demonstrate that AML stem cells can also reside within the CD33-negative fraction of the AML clone, we purified CD33-negative cells in a patient with AML in whom a majority of leukemic stem cells were found to lack CD33. In this particular patient, the CD33-negative cells were found to repopulate NOD/SCID mice with leukemias in the same way as the entire leukemic clone did. The CD33 antigen was neither detectable on CD34+/CD38− cells in the normal bone marrow nor on leukemic stem cells in patients with CD33-negative AML. In summary, our data show that leukemic stem cells in patients with CD33+ AML frequently express the target receptor CD33. This observation is in favor of novel treatment concepts employing CD33-targeting antibodies (Mylotarg®) in acute myeloid leukemia.
hDectin-1 Is Involved in the Uptake of Apoptotic and Infected Cells and Mediates Cross-Presentation of Engulfed Antigens.
2005-11-16

Abstract Dectin-1 is a member of the c-type-lectin-like receptor family that was shown to be the major receptor for fungal beta-glucans and to play an important role in the cellular responses mediated by these carbohydrates. In the present study we identified the hDectin-1b splice variant as the major isoform expressed in in vitro generated mDC using a quantitative RT-PCR and western blot analysis. Interestingly, stimulation of immature DC with the toll like receptor ligands Poly I:C (TLR3) or LPS (TLR4) but not with TLR ligands 2 and 7 or TNF-a led to a dramatic down regulation of hDectin-1 protein expression. To further analyze the possible involvement of Dectin-1 in the phagocytosis of cellular material we recombinantly expressed the extracellular domain (ECD) of hDectin-1b and used it to stain tumor cells. The recombinant ECD showed a specific binding to several human tumor cell lines that could be increased by induction of apoptosis in malignant cells and inhibited by incubation of the extracellular domain with zymosan, a crude cell wall extract of saccharomyces cerevisiae. Furthermore, uptake of tumor cells by immature mDC was reduced by zymosan or the presence of the recombinant Dectin-1 in phagocytosis assays suggesting that Dectin-1 is involved in the engulfement of tumor cells by DC. In line with the expression analysis of hDectin-1, we found that phagocytosis of apoptotic cells was dramatically reduced upon stimulation of DCs with Poly I:C or LPS as compared to immature DC or DC activated with TLR2 or 7 ligands. We next analysed the role of Dectin-1 in the cross-presentation of cell derived antigens and used DC that were incubated with CMV infected fibroblasts and an autologous CTL line specific for the HLA-A2 binding pp65 peptide. Stimulation of CMV peptide specific CTL in ELIspot assays by DC that were incubated with CMV infected fibroblasts efficiently stimulated IFN-gamma secretion that could be inhibited by zymosan and the extracellular domain of Dectin-1. In line with the results from phagocytosis assays stimulation of CMV specific CTL was reduced by stimulation of DC with TLR2 and 4 ligands. Our results identify hDectin-1 as a new receptor for endogenous ligands on mammalian cells and indicate an important role of this molecule in the clearing of apoptotic and infected cells and cross-presentation of cell derived antigens..
Acquired Bortezomib (Velcade®)-Resistance Is Accompanied by Downmodulation of Proteasome Activity and Upregulation of Tripeptidyl Peptidase II (TPPII) and a Ubiquitin-Specific Protease.
2005-11-16

Abstract Because cell lines can be adapted to proteasome inhibition in vitro (Pfeifer G. et al., Science 283: 978–981, 1999.), secondary resistance of malignant cells towards Bortezomib (Velcade®) in vivo is a likely scenario. We have succeeded to adapt the human AML cell line HL-60 to Bortezomib in vitro, so that the adapted subline (HL-60a) shows normal viability and growth rate at 40 nM Velcade®, while cell death is induced above 10nM in the parental line. We hypothesized that alternative proteolytic pathways might allow the continued proliferation and survival of Bortezomib-adapted cells, and assessed the activity-profiles of proteasomal subunits, ubiquitin-specific proteases and lysosomal cysteine proteases in a functional proteomics approach, using recently developed synthetic affinity probes. These tools for the first time allow semiquantitative visualization of the individual members of these protease families, based on their activity, in contrast to western blot which lacks activity information or to the turnover of fluorogenic substrates, which is not truly protease-specific. After 72h of culture in Bortezomib-free medium (wash out phase), HL-60a cells contained significantly reduced levels of active proteasomal β1,β2, and β5 subunits, compared to the parental line, as confirmed by a reduced turnover of the β5-selective fluorogenic substrate Suc-LLVY-AMC. A panel of 7 different ubiquitin-specific proteases (USP) was visualized in both types of cells, using the affinity probe HAUbVS. Of these, a 97 kD USP that we have identified as USP14 by mass spectrometry-based sequencing, was significantly upregulated in HL-60a cells. By contrast, the activity of lysosomal cysteine proteases remained unchanged in HL-60a cells. Because the cytosolic protease tripeptidyl peptidase II (TPPII) can partially substitute for proteasome activity in lactacystine-treated cell lines, we assessed TPPII-activity using a fluorogenic substrate. We observed a significant upregulation of TPPII-activity in HL-60a cells, compared to non-adapted controls. Inhibition of TPPII, however, was not sufficient to restore Bortezomib-sensitivity in HL-60a cells. Interestingly, the combination of Bortezomib with the HIV protease inhibitor Ritonavir induced synergistic cytotoxicity both in HL-60 and HL-60a cells. Thus, Bortezomib-resistance is accompabied by upregulation of protease activities in alternative pathways. Combining different protease inhibitors like Bortezomib and Ritonavir might be a promising option to overcome primary or secondary Bortezomib resistance.
Depletion of the Leukemic Fusion Protein MLL-AF4 with Short Interfering RNAs (siRNAs) Affects Post-Translational Modification of Aldolase A.
2005-11-16

Abstract The chromosomal translocation t(4;11) marks a therapy-resistant infant leukemia with very poor prognosis. It results in the expression of two fusion-proteins, MLL-AF4 and AF4-MLL. We addressed the role of MLL-AF4 in t(4;11) positive SEM cells by siRNA-mediated suppression. Depletion of MLL-AF4 results in induction of apoptosis, inhibition of proliferation, decrease in colony formation and diminished leukemic engraftment in vivo. Currently, we are analyzing global changes in protein expression. For that, we compare the proteome of MLL-AF4 depleted SEM cells with those of control cells. The analysis is performed by 2D-gelelectrophoresis followed by mass spectrometry identification and immunoblot validation of differentially expressed spots. One of these spots was identified as Aldolase A. Comparison of MLL-AF4 depleted SEM cells with control cells showed neither change in mRNA levels nor in absolute protein levels of Aldolase A. Two-dimensional western blotting, however, revealed differences in the protein pattern, suggesting changes in Aldolase A modifications upon MLL-AF4 depletion. These analyses will provide us with a better insight into the effects of siRNA-mediated MLL-AF4 knockdown on the proteome, and may enable us to identify new targets for molecular therapeutic approaches.
Induction of Localized TNF Activity Against Tumors Using Bispecific Antibodies.
2005-11-16

Abstract Tumor necrosis factor (TNF) is a highly effective cytokine with considerable anti tumor activity not only because of direct tumoricidal effects but also due to its strong inflammatory and anti-angiogenic properties. However, while impressive clinical responses were obtained by locoregional application, e.g. in isolated limb perfusion, systemic application of TNF causes intolerable side effects. Therefore, multiple attempts have aimed for targeted delivery of TNF to maximize therapeutic efficiency and sparing normal tissue from detrimental effects, e.g. by generating antibody-derived fusion proteins with human TNF replacing the IgG1 CH2/CH3 Fc domain. However, a highly selective and thus clinically applicable drug for stimulation of TNF-receptors (TNF-R) at the tumor site with minimal unspecific TNF-R stimulation is still not available. Here we report the generation of a bispecific EpCAM X TNFR1 Fab2 antibody (bsFab2). The Fab fragments of an agonistic antibody to mouse TNFR1 were chemically hybridized to anti-EpCAM Fab fragment which served as a targeting molecule on EpCAM transfected mouse melanoma cells. Bound to such cells the bsFab2 exerted strong TNF activity as measured by killing of TNF sensitive WEHI-164 cells at concentrations as low as 0.1ng/ml. In the presence of untransfected tumor cells weak killing was detectable only at antibody concentrations as high as 1000ng/ml. Even more, killing of WEHI-164 cells in the absence of tumor target cells was undetectable. This demonstrates that TNF activity is induced by the bsFab2 in a target cell restricted manner. Target cell restriction was also confirmed when caspase activation rather than cell death was measured. We also demonstrate that the bsFab2 is able to induce target antigen restricted cellular inflammatory responses like monocyte/macrophage cytokine production. Next we investigated the activity of the bsFab2 in the B16 melanoma mouse model with syngeneic, immunocompetent mice. C57Bl/6 mice were inoculated 2500 EpCAM transfected B16 melanoma cells and treated with 6x20μg of the bsFab2. While all of six treated mice survived without developing a tumor, in the control group five of six mice died within 10 weeks. No significant side effects were observed. Our data demonstrate that the TNF activity of the EpCAM X TNFR1-bsFab2 is strictly depending on the presence of EpCAM positive tumor cells, stimulates cellular inflammatory responses in immediate vicinity of target antigen expressing tumors and provide the rationale for further studies evaluating the clinical potential of selective TNFR activation with bispecific antibodies in humans.
The G Protein-Coupled Receptor cysLT1 Signals Via ERK/MAP-Kinase and Pyk2, but Not NF-kappa B, in CD34+ Progenitor Cells, Explaining Its Dual Effect on Both, Cell Migration and Proliferation.
2005-11-16

Abstract The G protein-coupled receptor (GPCR) cysLT1 is expressed in CD34+ cell lines and immature hematopoietic progenitor cells (HPC), while its ligand leukotriene D4 (LTD4) is produced by bone marrow endothelium and stromal cells when deprived of hematopoietic cells. In the present study, we analyzed the signal transduction pathways of cysLT1 in progenitor cell lines and primary CD34+ HPC isolated from the mobilized peripheral blood. Of note, the effects of LTD4 on intracellular calcium fluxes, a typical event related to signaling of Gi-coupled GPCR, were substantially stronger than those observed after stimulation of the HPC-associated chemokine receptor CXCR4 with optimal doses of the corresponding ligand stromal cell-derived factor-1 (SDF-1, CXCL12). In cytokine-supplemented liquid cultures in vitro (containing interleukin-3, stem cell factor, and FLT3-ligand), addition of LTD4 resulted in a significantly greater expansion of both, committed and immature CD34+ progenitors. The effect of LTD4 in these cultures was comparable to that induced by the early acting hematopoietic growth factor FLT3-ligand, whereas the CXCR4 ligand SDF-1 (CXCL12) was unable to expand the number of CD34+ HPC when added to similar cytokine-supplemented liquid cultures. In both, CD34+ cell lines and primary CD34+ progenitor cells, LTD4 induced a rapid phosphorylation of ERK/MAP-kinase, a signaling step related to cell proliferation, and Pyk2, which is connected to downstream signaling pathways resulting in cell migration. Signaling through NF-kappa B, representing an alternative pathway of GPCR signaling associated with cell proliferation, was unaffected. In conclusion, the GPCR cysLT1 activates multiple signal transduction pathways in CD34+ HPC leading to cell migration and proliferation. In contrast to the CXCR4 ligand SDF-1 (CXCL12), the effect of the cysLT1 ligand LTD4 on cell proliferation was more pronounced and even comparable to that induced by early acting hematopoietic growth factors.
The Role of Leukemia Derived B7-H1 in Tumor-T-Cell Interactions in Humans.
2005-11-16

Abstract Objective: Expression of the B7-homologue B7-H1 (PD1-Ligand) has been proposed to enable tumor cells to evade immune surveillance. Recently, B7-H1 on murine leukemia cells was reported to mediate resistance to cytolytic T-cell destruction. In this study we investigated the expression and functional role of the B7-homologue B7-H1 in human leukemia. Patients and Methods: Leukemia cells from 20 patients and 9 human leukemia cell lines were investigated for B7-H1 expression by flow cytometry. Functional relevance of B7-H1 for tumor-immune interactions was assessed by coculture experiments using purified, alloreactive CD4 and CD8 T-cells in the presence of a neutralizing anti-B7-H1 antibody. Results: Significant B7-H1 expression levels on leukemia cells were detected in 13 of 20 patients and in 8 of 9 cell lines. In contrast to various other tumor entities and the data reported from a murine leukemia system we did not observe any significant inhibitory effect of leukemia-derived B7-H1 on CD4 and CD8 cytokine production (IFN-g, IL-2) or expression of T-cell activation markers (ICOS, CD69). In the presence of a neutralizing B7-H1 antibody (mAb 5H1) no significant changes in T cell IFN-g or IL-2 production were observed. Conclusions: Our data demonstrate that leukemia-derived B7-H1 seems to have no direct influence on T-cell activation and cytokine production in humans. Further experiments are warranted to delineate factors and characterize yet unidentified B7-H1 receptor(s) that determine inhibitory and stimulatory functions of B7-H1 in human leukemia.
Reciprocal Interaction of Monocytes and NK Cells by Activation-Induced Expression of Ligands for the Activating Immunoreceptor NKG2D.
2005-11-16

Abstract Reciprocal Interactions of NK cells with Dendritic Cells (DC) can induce activation of NK cells, maturation or lysis of DC and influence subsequent adaptive immune responses. However, little is known about the interaction of peripheral blood monocytes with NK cells, especially regarding involved immunoregulatory surface molecules. Here we report that monocytes express ligands for the activating immunoreceptor NKG2D expressed on NK cells upon treatment with various stimuli. Incubation of monocytes with TNF, GM-CSF, IFN-g and various TLR ligands (LPS, Pam3Cys, R848, PolyI:C) induced surface expression of the NKG2D ligands (NKG2DL) MICA and to a lesser extent MICB, but no relevant changes of ULBP molecules, as determined by FACS analysis. Expression was confirmed by quantitative PCR analysis of NKG2DL mRNA induction. To elucidate the functional consequences of NKG2DL expression on monocytes for NK cell functions we performed coculture assays of monocytes and autologous NK cells. NKG2DL expression on stimulated monocytes lead to a significant induction of IFN-g release into the culture supernatant by NK cells as determined by ELISA. This IFN-g release was blocked by addition of a NKG2D-Ig fusionprotein, but not by isotype control demonstrating that the induction of NK cell IFN-g production was in fact specifically due to NKG2DL expression on monocytes. Since both monocytes and NK cells rapidly migrate to sites of inflammation, and monocytes display a high plasticity regarding their function and maturation which is influenced by IFN-g, our data indicate that NKG2DL expression on monocytes might not only mediate reciprocal activation of NK cells and monocytes but also might influence other components of the innate and adaptive immune system and thereby determine the course of subsequent immune reactions.
BCR-ABL Is Not an Immunodominant Antigen in CML.
2005-11-16

Abstract Chronic myelogenous leukemia (CML) is a myeloproliferative disorder, which originates from pluripotent hematopoietic bone marrow progenitor cells. It is characterized by excessive proliferation of the granulopoiesis in the bone marrow. In approximately 90% of the patients with CML the Philadelphia chromosome (Ph) is the characteristic cytogenetic hallmark. The Ph is a shortened chromosome 22, which arises from a acquired reciprocal translocation between chromosome 9 and 22 (t(9;22) (q34;q11)). This translocation fuses the bcr gene with part of the c-abl gene, which encodes a protein-tyrosine kinase. The chimeric bcr-abl oncogene is translated into a 210-kDa chimeric protein (p210) which exhibits constitutive ABL kinase activity. In the present study, we analysed the involvement of the BCR-ABL protein in the induction of antigen-specific cytotoxic T lymphocytes (CTLs) in order to develop an immunotherapeutic approach targeting this neo-antigen in patients with CML. To accomplish this, we generated dendritic cells (DCs) in vitro and electroporated them with various sources of RNA harbouring the chimeric bcr-abl transcript. These genetically engineered DCs where used as antigen presenting cells (APCs) for the in vitro induction of CTLs. By applying this approach we found that the CTLs induced by DCs transfected with RNA extracted from bcr-abl-positive K-562 cells or CML blasts do not recognize epitopes derived from the chimeric BCR-ABL fusion protein. In contrast, they were able to lyse autologous DCs electroporated with RNA isolated from patients with acute myeloid leukemia (AML), indicating that antigens shared among these malignant cells are involved and recognized by these CTLs. However, we successfully generated BCR-ABL-specific CTLs by DCs electroporated with in vitro transcribed bcr-abl-RNA. In patients with CML in complete cytogenetic remission during IFN-α treatment there was some reactivity against BCR-ABL in IFN-γ ELISPOT assays, which was weaker as compared to proteinase 3 (PR3)- or Prame-directed responses supporting the observations obtained in healthy donors. In summary, BCR-ABL is processed and presented by Ph+ cells but is not the immunodominat antigen that induces CD8+ T lymphocytes in competition with other tumor-associated antigens. Nevertheless, APCs transfected with pure bcr-abl-IVT in vitro are capable of inducing BCR-ABL specific CTLs that efficiently lyse Ph+ target cells.