Quantitative detection of DNMT3A R882H mutation in acute myeloid leukemia

Background DNMT3A mutations represent one of the most frequent gene alterations detectable in acute myeloid leukemia (AML) with normal karyotype. Although various recurrent somatic mutations of DNMT3A have been described, the most common mutation is located at R882 in the methyltransferase domain of the gene. Because of their prognostic significance and high stability during disease evolution, DNMT3A mutations might represent highly informative biomarkers for prognosis and outcome of disease. Methods We describe an allele-specific PCR with a Blocking reagent for the quantitative detection of DNMT3A R882H mutation providing the possibility to analyze the quantitative amount of mutation during the course of disease. Next, we analyzed 62 follow- up samples from 6 AML patients after therapy and allogeneic stem cell transplantation (alloSCT). Results We developed an ASB-PCR assay for quantitative analysis of R882H DNMT3A mutation. After optimization of blocker concentration, a R882H-positive plasmid was constructed to enhance the accuracy of the sensitivity of quantitative detection. The assay displayed a high efficiency and sensitivity up to 10−3. The reproducibility of assay analyzed using follow-up samples showed the standard deviation less than 3.1 %. This assay displayed a complete concordance with sequencing and endonuclease restriction analysis. We have found persistence of DNMT3A R882H mutations in complete remission (CR) after standard cytoreduction therapy that could be indicating presence of DNMT3A mutation in early pre-leukemic stem cells that resist chemotherapy. The loss of correlation between NPM1 and DNMT3A in CR could be associated with evolution of pre-leukemic and leukemic clones. In patients with CR with complete donor chimerism after alloSCT, we have found no DNMT3A R882H. In relapsed patients, all samples showed an increasing of both NPM1 and DNMT3A mutated alleles. This suggests at least in part the presence of NPM1 and DNMT3A mutations in the same cell clone. Conclusion We developed a rapid and reliable method for quantitative detection of DNMT3A R882H mutations in AML patients. Quantitative detection of DNMT3A R882H mutations at different time points of AML disease enables screening of follow-up samples. This could provide additional information about the role of DNMT3A mutations in development and progression of AML

Successful long-term monotherapy with rituximab in a patient with chronic lymphocytic leukemia of the B-cell-lineage: a case report

<p>Abstract</p> <p>Introduction</p> <p>Treatment of chronic lymphocytic leukemia of the B-cell-lineage is strongly based upon clinical staging because of the heterogeneous clinical course of this disease.</p> <p>Case presentation</p> <p>We describe a 62-year-old patient with newly diagnosed chronic lymphocytic leukemia of the B-cell-lineage who did not respond to several chemotherapy regimens including chlorambucil, fludarabine and cyclophosphamide, developing a marked neutropenia and thrombocytopenia with life-threatening infections. Further chemotherapy appeared not feasible because of bone marrow toxicity. The patient was treated with 600 mg/m²rituximab weekly followed by eight courses of biweekly therapy and then by long-term maintenance therapy, achieving almost complete remission of the symptoms and disease control.</p> <p>Conclusion</p> <p>After resistance to standard chemotherapy with chlorambucil and fludarabine, a patient with chronic lymphocytic leukemia of the B-cell-lineage was successfully treated with rituximab.</p

Perfluorocarbon Particle Size Influences Magnetic Resonance Signal and Immunological Properties of Dendritic Cells

The development of cellular tracking by fluorine (19F) magnetic resonance imaging (MRI) has introduced a number of advantages for following immune cell therapies in vivo. These include improved signal selectivity and a possibility to correlate cells labeled with fluorine-rich particles with conventional anatomic proton (1H) imaging. While the optimization of the cellular labeling method is clearly important, the impact of labeling on cellular dynamics should be kept in mind. We show by 19F MR spectroscopy (MRS) that the efficiency in labeling cells of the murine immune system (dendritic cells) by perfluoro-15-crown-5-ether (PFCE) particles increases with increasing particle size (560>365>245>130 nm). Dendritic cells (DC) are professional antigen presenting cells and with respect to impact of PFCE particles on DC function, we observed that markers of maturation for these cells (CD80, CD86) were also significantly elevated following labeling with larger PFCE particles (560 nm). When labeled with these larger particles that also gave an optimal signal in MRS, DC presented whole antigen more robustly to CD8+ T cells than control cells. Our data suggest that increasing particle size is one important feature for optimizing cell labeling by PFCE particles, but may also present possible pitfalls such as alteration of the immunological status of these cells. Therefore depending on the clinical scenario in which the 19F-labeled cellular vaccines will be applied (cancer, autoimmune disease, transplantation), it will be interesting to monitor the fate of these cells in vivo in the relevant preclinical mouse models

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

T antigen expression by human lymphocytes in relation to E rosette affinity

Active and late rosette-forming cells, separated on the basis of their different affinities for SRBC, were tested for their ability to react with monoclonal antibodies of the OKT series. No significant preferential distribution of T subpopulations defined by these reagents was found in the high affinity E rosette fraction, while in the low affinity T cell subset the major finding was a high number of cells lacking both OKT4 and OKT8 determinants. This seems to be related to methodology, as indicated by experiments in which sequential cycles of rosetting procedures were found to induce loss of reactivity with OKT monoclonal antibodies. The implications of these methodological observations are further discussed. © 1984.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Assessment of the Space Heating and Domestic Hot Water Market in Europe—Open Data and Results

The paper investigates the European space heating (SH) and domestic hot water (DHW) market in order to close knowledge gaps concerning its size. The stimulus for this research arises from incongruences found in SH and DHW market&#8217;s data in spite of over two decades of scientific research. The given investigation has been carried out in the framework of the Hotmaps project (Horizon 2020&#8212;H2020), which aims at designing an open source toolbox to support urban planners, energy agencies, and public authorities in heating and cooling (H&amp;C) planning on country, regional, and local levels. Our research collects and analyzes SH and DHW market data in the European Union (EU), specifically the amount of operative units, installed capacities, energy efficiency coefficients as well as equivalent full-load hours per equipment type and country, with a bottom-up approach. The analysis indicates that SH and DHW account for a significant portion of the total EU energy utilization (more than 20%), amounting to almost 3900 TWh/y. At the same time, the energy consumption provided by district heating (DH) systems exceeds the one of condensing boilers. While DH systems applications are growing throughout the EU, the replacement of elderly, conventional boilers progresses at a slower pace