Molecular-defined clonal evolution in patients with chronic myeloid leukemia independent of the BCR-ABL status.

To study clonal evolution in chronic myeloid leukemia (CML), we searched for BCR-ABL-independent gene mutations in both Philadelphia chromosome (Ph)-negative and Ph-positive clones in 29 chronic-phase CML patients by targeted deep sequencing of 25 genes frequently mutated in myeloid disorders. Ph-negative clones were analyzed in 14 patients who developed clonal cytogenetic abnormalities in Ph-negative cells during treatment with tyrosine kinase inhibitors (TKI). Mutations were detected in 6/14 patients (43%) affecting the genes DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1 and ZRSR2. In two patients, the mutations were also found in corresponding Ph-positive diagnostic samples. To further investigate Ph-positive clones, 15 randomly selected CML patients at diagnosis were analyzed. Somatic mutations additional to BCR-ABL were found in 5/15 patients (33%) affecting ASXL1, DNMT3A, RUNX1 and TET2. Analysis of individual hematopoietic colonies at diagnosis revealed that most mutations were part of the Ph-positive clone. In contrast, deep sequencing of subsequent samples during TKI treatment revealed one DNMT3A mutation in Ph-negative cells that was also present in Ph-positive cells at diagnosis, implying that the mutation preceded the BCR-ABL rearrangement. In summary, BCR-ABL-independent gene mutations were frequently found in Ph-negative and Ph-positive clones of CML patients and may be considered as important cofactors in the clonal evolution of CML.

Simulations of slip flow on nanobubble-laden surfaces.

On microstructured hydrophobic surfaces, geometrical patterns may lead to the appearance of a superhydrophobic state, where gas bubbles at the surface can have a strong impact on the fluid flow along such surfaces. In particular, they can strongly influence a detected slip at the surface. We present two-phase lattice Boltzmann simulations of a flow over structured surfaces with attached gas bubbles and demonstrate how the detected slip depends on the pattern geometry, the bulk pressure, or the shear rate. Since a large slip leads to reduced friction, our results give assistance in the optimization of microchannel flows for large throughput.

Methylprednisolone therapy in deceased donors reduces inflammation in the donor liver and improves outcome after liver transplantation: a prospective randomized controlled trial.

OBJECTIVE: To investigate potential beneficial effects of donor treatment with methylprednisolone on organ function and outcome after liver transplantation. SUMMARY BACKGROUND DATA: It is proven experimentally and clinically that the brain death of the donor leads to increased levels of inflammatory cytokines and is followed by an intensified ischemia/reperfusion injury after organ transplantation. In experiments, donor treatment with steroids successfully diminished these effects and led to better organ function after transplantation. METHODS: To investigate whether methylprednisolone treatment of the deceased donor is applicable to attenuate brain death-associated damage in clinical liver transplantation we conducted a prospective randomized treatment-versus-control study in 100 deceased donors. Donor treatment (n = 50) consisted of 250 mg methylprednisolone at the time of consent for organ donation and a subsequent infusion of 100 mg/h until recovery of organs. A liver biopsy was taken immediately after laparotomy and blood samples were obtained after brain death diagnosis and before organ recovery. Cytokines were assessed by real-time reverse transcriptase-polymerase chain reaction. Soluble serum cytokines were measured by cytometric bead array system. RESULTS: After methylprednisolone treatment, steroid plasma levels were significantly higher (P < 0.05), and a significant decrease in soluble interleukins, monocyte chemotactic protein-1, interleukin-2, interleukin-6, tumor necrosis factor-alpha, and inducible protein-10 was observed. Methylprednisolone treatment resulted in a significant downregulation of intercellular adhesion molecule-1, tumor necrosis factor-alpha, major histocompatibility complex class II, Fas-ligand, inducible protein-10, and CD68 intragraft mRNA expression. Significantly ameliorated ischemia/reperfusion injury in the posttransplant course was accompanied by a decreased incidence of acute rejection. CONCLUSIONS: Our present study verifies the protective effect of methylprednisolone treatment in deceased donor liver transplantation, suggesting it as a potential therapeutical approach.

Rapid screening and sensitive detection of NPM1 (nucleophosmin) exon 12 mutations in acute myeloid leukaemia.

Nucleophosmin mutations of exon 12 (NPM1 mutations) represent the most frequent molecular aberration that can be found in patients with acute myeloid leukaemia (AML) and can be detected in about 35% of AML patients. NPM1 mutations are characterised by four basepair insertions within the region corresponding to the C-terminus of the protein leading to its translocation out of the nucleus. Until now, more than 40 different subsets of mutations have been identified and about 90% of NPM1 mutations are represented by subtype A and B (78% versus 12%, respectively). So far, standard screening of NPM1 mutations using conventional polymerase chain reaction (PCR) followed by capillary electrophoresis is rather time consuming. We established a new method for rapid screening of NPM1 mutations using the fluorescence resonance energy transfer (FRET) principle. Furthermore, based on individual NPM1 mutations type A and B, we designed mutation specific primers to perform a highly sensitive PCR assay that can be applied for the detection of minimal residual disease (MRD). In summary, we demonstrate new methodological approaches for rapid screening of NPM1 mutations as well as for MRD analyses based on the most frequent NPM1 mutations.

A long distance-PCR derived FISH probe detects a deletion between p15 and p16 in CML and T-ALL patients.

The tumor suppressor genes p15INK4B and p16INK4A, located in the chromosomal region 9p21, are frequently inactivated by homo- or hemizygous deletions, point mutation or promotor methylation in various types of cancer. No commercial probe is yet available that allows the detection of such deletions by FISH. Long distance (LD)-PCR was successfully used to generate a FISH probe, that covers a sequence stretch of 11.68 kb, located between the tumor suppressor genes p15 and p16. The LD-PCR amplicon was cloned and biotinylated by DOP-PCR (degenerated oligonucleotide primed-PCR) or nick translation. The FISH probe was hybridized on different samples of 16 patients with leukemia (3 T-ALL, 13 CML) and normal controls. Loss of at least one FISH-signal was found in 2/3 (67%) of the T-ALL- and 2/13 (15%) of the CML-cases. The new FISH probe presented here was proven to be advantageous for the detection of deletions in chromosomal region 9p21, especially between p15 and p16.

[The effect of erythropoietin (rh-EPO) om in vitro proliferation of granulocyte/monocyte-determined stem cells in bone marrow and peripheral blood]. / Der Einfluss einer rh-EPO-Therapie auf die In-vitro-Proliferation granulozytär-monozytär determinierter Stammzellen aus Knochenmark und peripherem Blut.

AIM: The aim of the investigation was to determine the effect of recombinant human erythropoietin (rh-EPO) therapy on in-vitro proliferation of granulocyte/monocyte(gm)-determined stem cells in bone marrow and peripheral blood. PATIENTS AND METHODS: The study comprised 28 dialysis patients (15 female, 13 male, aged between 22 and 78 years, dialysis sessions 3 times weekly 4 to 5 hours, mean duration time of dialysis treatment 4 to 99 months). Stem cell parameters were estimated before and after reaching the target hematocrit and the absolute number of leukocytes in peripheral blood was measured. RESULTS: No alteration in total leukocyte number (PMN, monocytes, lymphocytes) of peripheral blood was found. Dialysis patients had a diminished proliferation capacity of colony forming unit-granulocyte/monocyte (CFU-GM) in bone marrow and an increased one in peripheral blood. The CFU-GM proliferation capacity in bone marrow improved during rh-EPO (34.3 +/- 5.8 x 10(5)/ml vs 120.5 +/- 37.8 x 10(5)/ml), whereas no changes in peripheral blood could be observed (46.7 +/- 7.15 x 10(5)/ml vs 37.6 +/- 12.05 x 10(5)/ml).

Correlation between granulocyte/macrophage-colony-forming units and CD34+ cells in apheresis products from patients treated with different chemotherapy regimens and granulocyte-colony-stimulating factor to mobilize peripheral blood progenitor cells.

We examined the efficiency of disease-specific "standard" chemotherapies epirubicin, cyclophosphamide (EC); cyclophosphamide, vincristine, doxorubicin, etoposide, prednisolone (CHOEP); epirubicin, ifosfamide (EPI/IFOS) for peripheral blood progenitor cell (PBPC) mobilization in comparison to well-characterized mobilization protocols, i.e. etoposide, ifosfamide, cisplatin, epirubicin (VIPE) and dexamethasone, carmustine, etoposide, cytarabine, melphalan (DexaBEAM). Twenty-seven patients with various malignancies underwent 75 apheresis procedures for PBPC collection. Median cell yields from all 75 aphereses were 1.18 x 10(5) mononuclear cells/kg [range (0.28-3.7) x 10)8)], 1.4 x 10(5) granulocyte/macrophage-colony-forming units (CFU-GM)/kg [range (0.2-11) x 10(5)] and 3.3 x 10(6) CD34+cells/kg [range (0.35-17.7) x 10(6). CD34+/ CD90+ cells could be mobilized by all mobilization regimens used. The difference observed in the mobilization of CD34+ cells was only of low significance when the mobilization regimens were compared, whereas the mobilizations of MNC and CFU-GM were significantly different between the groups. Breast cancer patients treated with the VIPE regimen (including pretreated women) had a significantly higher CFU-GM rate than patients treated with EC (P=0.0005). Mobilized CD34+ PBPC were correlated with CFU-GM in all apheresis products. The linear correlation coefficients differed for the various mobilization groups: DexaBEAM (r=0.9, P < 0.0001), VIPE (r=0.68, P=0.0024), CHOEP (r=0.52, P=0.022), EPI/ IFOS (r=0.34, P=0.11) and EC (r=0.23, P=0.2). We conclude that clonogenic assays can provide additional information about the autotransplant quality, particularly when alternative or new mobilization regimens are being investigated.

Sequence homologies among the three yolk polypeptide (Yp) genes in Drosophila melanogaster.

To identify candidates for cis-acting sequences that regulate the sex-, stage-, and cell-specific expression of three coordinately regulated yolk polypeptide genes (Yp) in Drosophila melanogaster, we have mapped the Yp3 transcript, sequenced a 4278 bp DNA fragment containing the Yp3 gene, compared Yp3 region sequences to corresponding parts of Yp1 and Yp2, and compared the predicted amino acid sequence of YP3 to YP1 and YP2. The results showed that the Yps are largely homologous in translated regions, especially in the 3' half of the genes. Untranscribed flanking regions had little homology. A conserved inverted repeat (the H-box) has homology both to vertebrate steroid hormone receptor binding sites and to the ecdysone control region of Drosophila's hsp23. These results identify sequences to mutate in order to define elements that regulate Yp gene expression and govern YP polypeptide function.

Sex- and cell-specific regulation of yolk polypeptide genes introduced into Drosophila by P-element-mediated gene transfer.

To find whether cis-acting regulatory sequences necessary for sex- and cell-specific expression of two yolk polypeptide genes (Yps) reside near the structural genes themselves, we introduced a 5.0-kilobase genomic DNA segment containing a 3' truncated Yp1 and a complete Yp2 into five different autosomal locations by P-element-mediated gene transfer. Transcripts from the introduced Yp1 were not found in male flies but appeared on a normal developmental schedule in adult females, accumulating in their body walls and ovarian follicles but not in guts or malpighian tubules. Protein from the introduced Yp2 allele was present in female hemolymph and vitellogenic ovaries but was lacking from male hemolymph. We conclude that sequences necessary for the correct stage-, cell-, and sex-specific expression of the Yp1 and Yp2 genes are included in this genomic fragment. These results combined with published work suggest that two tissue-specific, cis-acting, bidirectional, positive regulatory elements placed on either side of a centrally located HindIII site govern expression of both Yp genes--one element specific for fat body and the other specific for ovarian follicle cells.