julilili42/papers huggingface dataset:
Decrease of Malignant Potential of t(8;21) Positive Cells after Stable Expression of RUNX-CBFA2T1-Specific Small Interfering RNA.
2004-11-16

Abstract The reciprocal chromosomal translocation t(8;21) results in the fusion of the DNA-binding domain of the transcription factor RUNX1 (also called AML1 or CBFα) to CBFA2T1 (also called MTG8 or ETO). The chimeric protein RUNX1-CBFA2T1 interferes with hematopoietic gene expression by recruiting histone deacetylases via N-CoR and mSin3. In addition, impairment of the function of transcription factors such as C/EBPα has been described. To further investigate the function of RUNX1-CBFA2T1 in the development of leukemia, the expression of the fusion protein was inhibited by small interfering RNAs (siRNAs). For stable expression of the siRNA in the target cells, a RUNX1-CBFA2T1-specific siRNA-sequence was cloned into a lentivial vector as short-hairpin-RNA (shRNA). Subsequently, the t(8;21) positive Kasumi-1 cell line was infected with an effeciency of &amp;gt;95% as detected by expression of the GFP reporter gene. As a control, an shRNA targeting luciferase mRNA was used. Expression of the anti-RUNX1-CBFA2T1-shRNA in the Kasumi-1 cells led to a marked reduction of mRNA and protein expression of the fusion gene, whereas the expression of the wildtype RUNX1 gene was not affected. The surface expression of the M-CSF-Receptor, which is a known target gene of C/EBPα, was increased in the RUNX1-CBF2T1 depleted cells. Also, Kasumi-1 cells treated with the shRNA displayed a decrease in CD34 surface expression. In parallel, CD34 mRNA expression was reduced to 10%. To analyze, if CD34 downregulation of the Kasumi-1 cells after RUNX1-CBF2T1 depletion correlates with a loss of progenitor status, the clonogenicity of the cells in semisolid medium was investigated. In Kasumi-1 cells treated with the shRNA against RUNX1-CBFA2T1 the number of spontaneous colonies after 14 days of culture was reduced to about 30% in comparison to cells expressing the control shRNA. In conclusion, these experiments show that RUNX1-CBF2T1 expression can be stably suppressed in t(8;21) positive cells by endogenously expressed shRNAs thereby reducing their clonogenic potential.
Highly-Purified Aspergillus Fumigatus Specific T-Cells as a Potential Treatment Option of Invasive Aspergillosis.
2004-11-16

Abstract Invasive aspergillosis is still a life-threatening complication, in particular in patients after allogeneic hematopoietic stem cell transplantation (HSCT). Whereas prolonged neutropenia is a well established risk factor for invasive fungal disease, there is a growing body of evidence that T-cells also play an important role in the immunological response to Aspergillus species. Since invasive aspergillosis often occurs during the phase of postengraftment, which is characterized by impaired cell-mediated immunity, Aspergillus-specific T-cells could be a potential therapeutic target in these patients. We therefore analyzed in a first step the response of T-cells to several potential antigens of Aspergillus fumigatus by means of 3H-thymidine incorporation assay. In order to generate Aspergillus-specific T-cells, the antigens with the highest proliferation indices (EC-SAB and 90 kDa catalase) were used to stimulate 1.0 x 108 mononuclear cells from healthy donors. The activated T-cells were isolated on the following day using the IFN-γ secretion assay (Miltenyi Biotec, Germany) and then expanded for 14 days. Intracellular cytokine analysis of EC-SAB generated cell lines (n=7) revealed a significant IFN-γ secretion by 13.6%±2.3 of CD4+ cells (seven out of seven tested cell lines) and an Aspergillus-specific IL-2 secretion by 6.5%±1.9 of CD4+ cells (three out of three tested cell lines), which supports the TH1 response of the generated cells to Aspergillus antigen. In contrast to EC-SAB generated T-cell populations, all three cell lines which were generated with 90 kDa catalase were not informative. Further analysis showed that restimulation with EC-SAB induced a strong proliferation of EC-SAB generated T-cell populations (all three populations tested), whereas alloreactivity was unaffected. The number of these cells could be expanded within 14 days up to 20fold using OKT-3, IL-2 and feeder cells. Currently, we investigate the impact of these Aspergillus-specific cell populations in the defense to different species of Aspergillus. Our preliminary results suggest that Aspergillus-specific T-cells could be an interesting option in prophylaxis and therapy of invasive aspergillosis in patients undergoing HSCT.
Pediatric Patients Transplanted with Positive Selected Stem Cells from Alternative Donors Have a Low Incidence of CMV Dnaemia.
2004-11-16

Abstract Human cytomegalovirus (HCMV) remains a cause of serious infectious complications after allogeneic transplantation of hematopoietic stem cells, especially in recipients of T cell depleted grafts. Here, we investigated the incidence of CMV-DNAemia in a cohort of 100 pediatric patients with leukemias and nonmalignant diseases, who received megadoses of CD34+ or CD133+ selected (and therefore highly T-cell depleted) grafts from matched unrelated or mismatched related (haploidentical) donors. Graft versus host disease was minimized with this approach (acute GvHD grade III-IV 5%) but T-cell recovery was delayed in most patients. All patients received prophylactic acyclovir. PCR screening for CMV was performed weekly from leukocytes and plasma and additional antiviral treatment was started in the case of positive findings. Cumulative incidence of CM-DNAemia at day 100 was 29%. Seropositive recipients (n=44) had a significantly higher incidence of CMV-DNAemia than seronegative recipients (n=56) (64% vs. 0.05%; p=0.0001). In contrast, the incidence was not influenced by the serostatus of the donors (patients with seropositve donors (n=41): 33%; patients with seronegative donors (n=59): 28%; p=0.6). D+R+ pairs were not superior to D-R+ pairs. CMV related over-all mortality was&amp;lt;10%. Conclusions: the incidence of CMVDNAemia was remarkably low in our transplanted patients, despite profound T-cell depletion. Donor seropositivity had no influence. An explanation for these observations may be, that only purified stem cells were transplanted (representing less than 1% of the total cell number of an unmanipulated graft) and therefore, patients received a minimal number of potentially CMV infected leukocytes.We suppose, that this effect may counterbalance the transiently impaired T-cell function in our patients and supports the use of CD34+ or CD133 selection in order to prevent GvHD.
Determination of Soluble MICA in Serum as a Novel Marker for Tumor Stage and Metastasis.
2004-11-16

Abstract The human NKG2D ligands (NKG2DL) MICA and MICB have been shown to be expressed on tumors of epithelial and hematopoietic origin in vivo. Recently we reported that MICA is shed from the cell surface of tumor cells and is present in sera of tumor patients (J Immunol169:4098 (2002), Blood102:1389 (2003)). Since the strength of an anti-tumor response by NK cells and CD8 T cells is critically depending on NKG2DL expression levels, down-regulation of MICA-expression on tumor cells represents an immune escape mechanism that diminishes anti-tumor reactivity of NKG2D-bearing lymphocytes. However, no data are yet available regarding the correlation of soluble MICA (sMICA) levels with specific tumor entities, aggressiveness of the disease, and hence the potential implementation of sMICA as novel marker in differential diagnosis and prognosis of cancer. In this study, we determined sMICA levels in sera of 512 individuals including 296 patients with various cancers, 154 patients with benign disorders and 62 healthy individuals. Healthy individuals revealed significantly lower sMICA values (median&lt;30pg/mL) than patients with benign diseases (84pg/mL; p=0.005) and cancer patients (161pg/mL; p&lt;0.0001). In addition, sMICA levels differed significantly between cancer patients and patients with benign disorders (p&lt;0.0001) that represent the most relevant control group for differential diagnosis. In cancer patients, while there was no association between sMICA levels and tumor size (p=0.456), cell differentiation (p=0.271), or lymph node involvement (p=0.674), sMICA correlated significantly with cancer stage and metastasis (p=0.015 and p=0.007, respectively). Our data indicate that release of MICA might play a role in late stages of tumor progression by overcoming the confining effect of NK cells and CD8 T cells. Thus, determination of sMICA levels provides valuable information for cancer staging, and sMICA in serum seems to be an indicator for systemic manifestation of malignancy rather than for local tumor extent.
Therapeutic Plasma Levels of OKT3 Muromonab Are Effective as Second-Line and Subsequent Treatment in Recipients of Stem Cell Allografts with Steroid-Resistant Acute Graft-Versus-Host Disease.
2004-11-16

Abstract Acute graft-versus-host disease (aGvHD) remains a major complication in recipients of hematopoietic cell allografts and is associated with poor outcome. Salvage treatment is difficult after failure of steroids. OKT3 muromonab is a monoclonal murine IgG2a antibody directed against the CD3 antigen on T-lymphocytes that has proven effective in transplant settings both of solid organs and bone marrow. Here we report on the results of OKT3 treatment in 43 patients who had received stem-cell or bone marrow allografts for hematologic malignancies and subsequently been diagnosed with steroid-resistant aGvHD. Overall, 48 treatment cycles of OKT3 (5 mg intravenous injection once daily) were administered for acute GvHD of grades II (n = 12), III (n = 27) and IV (n = 9). Mean duration of OKT3 therapy was 9 (range, 1 –16) days. Twenty cycles of OKT3 were administered as second line and 28 as third-plus line (third line, n = 14; fourth line, n = 14) treatment regimens. Side effects were mild to moderate, comprising fever, dyspnea, tachycardia, and vomiting. Thirty of 42 evaluable patients responded to therapy for an overall response rate of 69 %. Complete remissions were seen in 6 (11.9 %) and partial remissions in 24 patients (57.1 %). Response to treatment was 88.8 % for skin GvHD; 53.3 % for gut aGvHD; and 8.7 % for liver involvement. Recurrent or progressive acute GvHD was observed in 23 patients (54.8 %) after a median of 9 (range, 1 – 58) days. Pharmacokinetic studies of monoclonal antibody OKT3 were performed in 17 patients, 16 of whom achieved at least a partial remission. Treatment duration was &amp;gt; 5 days in 16 patients. Adequate plasma levels (&amp;gt; 1000 ng/ml) were observed in 13 patients after a median of 6 (1 – 11) days on treatment. OKT3 concentration became undetectable at a median of merely 4 (3 – 11) days after discontinuation of therapy. Survival at 100 days from transplantation was 58.1 % and at one year, 25.5 % with the most important causes of death being infectious complications (44.7%) and GvHD (33.2%). Patients treated with OKT3 earlier in the course of aGVHD showed a trend to better survival: 35% of patients with 2nd-line therapy were alive at one year from transplantation, compared to 17.4% with 3rd- plus line treatment (p=0.21). There was also a trend to a better one-year survival for patients with grade II (33% of patients alive) when compared to grade III/IV aGvHD (16% of patients alive; p=0.24). Thus, a significant proportion of patients with corticosteroid-resistant acute GvHD showed a response to salvage treatment with OKT3. More effective antimicrobial prophylaxis and treatment are mandatory to overcome considerable treatment-related mortality in those high-risk patients.
PPAR-gamma Agonists Inhibit Toll-Like Receptor Mediated Activation of Dendritic Cells Via the MAP Kinase and NF-kB Pathways.
2004-11-16

Abstract Dendritic cells (DC) play an important role in initiating and maintaining of primary immune responses. However, little is known about the termination of once induced immunological responses. Cyclopentenone prostaglandins (15d-PGJ2, PGD2) are produced during the late phase of inflammation due to upregulation of COX2 and can lead to the resolution of an inflammation by activation of PPAR-gamma dependent and independent pathways. In this study we analyzed the effects of 15d-PGJ2 and the synthetic PPAR-gamma ligand troglitazone (TGZ) on the immunogenicity of monocyte derived DC upon stimulation with toll-like receptor (TLR) ligands. Activation of PPAR-gamma resulted in a reduced activation of DC via the TLR ligands 2, 3, 4 and 7 characterized by downregulation of CD83, adhesion and costimulatory molecules. Moreover, these cells secreted lower levels of cytokines and chemokines involved in T lymphocyte activation and recruitment including IL-12 and RANTES. In contrast to these results, MCP-1 production was increased due to the treatment with PPAR-gamma agonists. To determine the mechanisms by which PPAR-gamma regulates DC function we performed Western blot analyses and found that the inhibitory effects on TLR induced DC activation were mediated via inhibition of the NF-kB and MAP kinase pathways. Incubation of DC during the differentiation from monocytes with 15d-PGJ2 or TGZ resulted in downregulation of TLR ligands induced phosphorylation of ERK1 as well as reduced expression of nuclear localized members of the NF-kB family. No effect on the expression of MyD88, an adaptor molecule involved in the signal transduction of TLR, was observed. Our results demonstrate that inhibition of the MAP kinase and NF-kB pathways is critically involved in the regulation of TLR and PPAR-gamma mediated signaling in DC an represent a novel negative feed-back mechanism involved in the resolution of immunological responses.
Identification of novel heat shock factor‐dependent genes and biochemical pathways in Arabidopsis thaliana
2004-11-17

Summary In order to assess specific functional roles of plant heat shock transcription factors (HSF) we conducted a transcriptome analysis of Arabidopsis thaliana hsfA1a/hsfA1b double knock out mutants and wild‐type plants. We used Affymetrix ATH1 microarrays (representing more than 24 000 genes) and conducted hybridizations for heat‐treated or non‐heat‐treated leaf material of the respective lines. Heat stress had a severe impact on the transcriptome of mutant and wild‐type plants. Approximately 11% of all monitored genes of the wild type showed a significant effect upon heat stress treatment. The difference in heat stress‐induced gene expression between mutant and wild type revealed a number of HsfA1a/1b‐regulated genes. Besides several heat shock protein and other stress‐related genes, we found HSFA‐1a/1b‐regulated genes for other functions including protein biosynthesis and processing, signalling, metabolism and transport. By screening the profiling data for genes in biochemical pathways in which known HSF targets were involved, we discovered that at each step in the pathway leading to osmolytes, the expression of genes is regulated by heat stress and in several cases by HSF. Our results document that in the immediate early phase of the heat shock response HSF‐dependent gene expression is not limited to known stress genes, which are involved in protection from proteotoxic effects. HsfA1a and HsfA1b‐regulated gene expression also affects other pathways and mechanisms dealing with a broader range of physiological adaptations to stress.
Host and non‐host pathogens elicit different jasmonate/ethylene responses in Arabidopsis
2004-11-17

Summary Arabidopsis does not support the growth and asexual reproduction of the barley pathogen, Blumeria graminis f. sp. hordei Bgh ). A majority of germlings fail to penetrate the epidermal cell wall and papillae. To gain additional insight into this interaction, we determined whether the salicylic acid (SA) or jasmonate (JA)/ethylene (ET) defence pathways played a role in blocking barley powdery mildew infections. Only the eds1 mutant and NahG transgenics supported a modest increase in penetration success by the barley powdery mildew. We also compared the global gene expression patterns of Arabidopsis inoculated with the non‐host barley powdery mildew to those inoculated with a virulent, host powdery mildew, Erysiphe cichoracearum. Genes repressed by inoculations with non‐host and host powdery mildews relative to non‐inoculated control plants accounted for two‐thirds of the differentially expressed genes. A majority of these genes encoded components of photosynthesis and general metabolism. Consistent with this observation, Arabidopsis growth was inhibited following inoculation with Bgh , suggesting a shift in resource allocation from growth to defence. A number of defence‐associated genes were induced during both interactions. These genes likely are components of basal defence responses, which do not effectively block host powdery mildew infections. In addition, genes encoding defensins, anti‐microbial peptides whose expression is under the control of the JA/ET signalling pathway, were induced exclusively by non‐host pathogens. Ectopic activation of JA/ET signalling protected Arabidopsis against two biotrophic host pathogens. Taken together, these data suggest that biotrophic host pathogens must either suppress or fail to elicit the JA/ET signal transduction pathway.
Essential role of the V‐ATPase in male gametophyte development
2004-11-17

Summary Intracellular pH homeostasis is a prerequisite for biological processes and requires the action of proton pumps. The vacuolar H + ‐ATPase (V‐ATPase) is involved in regulating pH in endomembrane compartments of all eukaryotic cells. In plants, there is an additional endomembrane proton pump, H + ‐pyrophosphatase (H + ‐PPase). However, the relative roles of the two types of pumps in endomembrane acidification and energization of secondary active transport are unclear. Here, we show that a strong T‐DNA insertion allele of VHA‐A , the single copy gene encoding the catalytic subunit of the Arabidopsis V‐ATPase, causes complete male and partial female gametophytic lethality. Severe changes in the morphology of Golgi stacks and Golgi‐derived vesicles in male gametophytes are the first visible symptoms of cell degeneration leading to a failure to develop mature pollen. Similar effects on Golgi morphology were observed in pollen tubes when growth was blocked by Concanamycin A, a specific V‐ATPase inhibitor. Taken together, our results suggests that V‐ATPase function is essential for Golgi organization and development of the male gametophyte.
Arabidopsis vacuolar H+‐ATPase subunit E isoform 1 is required for Golgi organization and vacuole function in embryogenesis
2004-11-17

Summary Vacuolar H + ‐ATPases play an important role in maintaining the pH of endomembrane compartments in eukaryotic cells. The functional relevance of this homeostasis for multicellular development has not been studied in plants. Here, we analyze the biological consequences resulting from the lack of subunit E isoform 1 (VHA‐E1) encoded by the Arabidopsis TUFF gene. tuff mutant embryos are lethal, displaying variably enlarged cells with multiple nuclei, large vacuoles containing inclusions, abnormal organization of Golgi stacks, and cell wall defects. Rescue of embryo lethality by cell cycle‐regulated expression of VHA‐E1 results in abnormal seedlings with non‐functional meristems and defective cell differentiation. VHA‐E1 is the predominant isoform in embryogenesis whereas VHA‐E3 is expressed mainly in the endosperm and surrounding maternal tissues during seed development, and VHA‐E2 is pollen‐specific. VHA‐E1 protein accumulates at endomembrane compartments including vacuoles and endosomes, but appears absent from the plasma membrane. Our results suggest an essential role for VHA‐E1 in maintaining a functional secretory system during somatic development but not in the haploid gametophytes.
The chromatin remodelling complex FACT associates with actively transcribed regions of the Arabidopsis genome
2004-11-17

Summary The packaging of the genomic DNA into chromatin in the cell nucleus requires machineries that facilitate DNA‐dependent processes such as transcription in the presence of repressive chromatin structures. Using co‐immunoprecipitation we have identified in Arabidopsis thaliana cells the FAcilitates Chromatin Transcription (FACT) complex, consisting of the 120‐kDa Spt16 and the 71‐kDa SSRP1 proteins. Indirect immunofluorecence analyses revealed that both FACT subunits co‐localize to nuclei of the majority of cell types in embryos, shoots and roots, whereas FACT is not present in terminally differentiated cells such as mature trichoblasts or cells of the root cap. In the nucleus, Spt16 and SSRP1 are found in the cytologically defined euchromatin of interphase cells independent of the status of DNA replication, but the proteins are not associated with heterochromatic chromocentres and condensed mitotic chromosomes. FACT can be detected by chromatin immunoprecipitation over the entire transcribed region (5′‐UTR, coding sequence, 3′‐UTR) of actively transcribed genes, whereas it does not occur at transcriptionally inactive heterochromatic regions and intergenic regions. FACT localizes to inducible genes only after induction of transcription, and the association of the complex with the genes correlates with the level of transcription. Collectively, these results indicate that FACT assists transcription elongation through plant chromatin.
Chemotherapy‐induced cell death in primary cerebellar granule neurons but not in astrocytes: in vitro paradigm of differential neurotoxicity
2004-11-18

Abstract The nervous system is frequently the site of symptomatic toxicity of antineoplastic agents. However, there is limited information about the differential vulnerability of neurons, astrocytes and glioma cells. We have analyzed the effects of four chemotherapeutic drugs (lomustine, cisplatin, topotecan and vincristine) on primary cerebellar granule neurons and astrocytes derived from rats. All drugs led to cell death in cerebellar granule neurons in a concentration‐dependent manner. Comparison of the EC 50 values for cerebellar neurons and astrocytes with the median EC 50 values of 12 malignant glioma cell lines demonstrated a large therapeutic range for lomustin and cisplatin. Further, this comparison revealed a 100‐fold higher sensitivity of cerebellar neurons towards vincristine and 10‐fold higher sensitivity towards topotecan compared with glioma cells. Astrocytes were generally resistant to vincristine. In cerebellar granule neurons, vincristine and to a lesser extent topotecan induced caspase 3 and caspase 9 cleavage, and enhanced caspase activity and Akt‐dependent expression of phosphorylated BAD. zVAD‐fmk, a caspase inhibitor and brain‐derived neurotrophic factor (BDNF), but not MK‐801, a non‐competitive NMDA receptor antagonist, significantly reduced vincristine‐ or topotecan‐induced cell death.