Intercenter reproducibility of binary typing for Staphylococcus aureus.

The reproducibility of the binary typing (BT) protocol developed for epidemiological typing of Staphylococcus aureus was analyzed in a biphasic multicenter study. In a Dutch multicenter pilot study, 10 genetically unique isolates of methicillin-resistant S. aureus (MRSA) were characterized by the BT assay as presented by van Leeuwen et al.

A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells.

In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a crucial role in the mechanism of resistance to cytarabine (AraC). The resistant phenotype in vitro is always a result of mutational inactivation of dCK, leading to defects in the metabolic pathways of AraC. Although inactivation of dCK has shown to be one of the major mechanism of resistance to AraC in vitro, limited in vivo data are available.

High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia.

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified.

Complete remission of accelerated phase chronic myeloid leukemia by treatment with leukemia-reactive cytotoxic T lymphocytes.

Relapse of chronic myeloid leukemia (CML) in chronic phase after allogeneic stem cell transplantation (SCT) can be successfully treated by donor lymphocyte infusion (DLI). However, relapse of accelerated phase CML, blast crisis, or acute leukemia after allogeneic SCT are resistant to DLI in the majority of cases. In vitro-selected and expanded leukemia-reactive T-cell lines may be more effective in inducing an antileukemic response in vivo.

Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification.

With the ongoing progress in human genome projects, many genes are discovered whose function and/or expression pattern are not known. Most of these genes are expressed in relatively low abundance compared to housekeeping genes such as elongation factor-1alpha and beta-actin. Gene expression is studied by Northern blot assays or by semiquantitative PCR methods.

Correction of abnormal T-cell receptor repertoire during interferon-alpha therapy in patients with hairy cell leukemia.

Patients with the B-cell malignancy hairy cell leukemia (HCL) exhibit a skewed T-cell repertoire with oligoclonal expression or absence of many members of the T-cell receptor (TCR) BV gene families. To evaluate whether interferon-alpha (IFN-alpha) therapy would not only restore normal hematopoiesis, but also the abnormal T-cell repertoire, we studied T lymphocytes from a cohort of HCL patients treated by IFN-alpha in the past, at initiation, and at several intervals up to 6 years of IFN-alpha treatment. The junctional regions from 22 TCRBV gene families were analyzed after polymerase chain reaction amplification of cDNA (RT-PCR) using family specific primers.

Extramedullary hematopoietic growth factor and cytokine gene expression indicate endocrine regulation of hematopoiesis in patients with acute appendicitis.

To analyze the role of hematopoietic growth factors (HGFs) and other cytokines in the regulation of hematopoiesis in vivo, we investigated HGFs and cytokine gene expression in appendices obtained from patients who underwent surgery for suspected appendicitis. Concomitantly, HGF gene expression was studied in bone marrow (BM) biopsy specimens and plasma HGF levels were measured. G-CSF gene expression was detected in inflamed but not in normal appendices.

Sublocalization of the breakpoints of a t(5;16) in myelodysplasia.

As a first step in characterizing a t(5;16)(q31;p11.2) in a patient with the diagnosis refractory anemia with ring sideroblasts, a cell fusion was carried out between bone marrow cells from the patient and the Chinese hamster cell line A3. Using PCR and FISH analysis on hybrid lines containing the human derivative 16 chromosome, the breakpoints could be mapped between the markers TCF-7 and IL-9 on chromosome 5 and OL-7 and s30A4 on chromosome 16, both regions spanning approximately 1 Mb.

T cells selectively infiltrate bone marrow areas with residual haemopoiesis of patients with acquired aplastic anaemia.

Aplastic anaemia (AA) is characterized by pancytopenia and bone marrow (BM) hypocellularity. In some patients AA may be mediated by T cells. To localize inflammatory cell infiltrates, we carried out a quantitative immunohistochemical analysis of BM biopsies of AA patients.

Absence of mutations in the RET gene in acute myeloid leukemia.

Expression of the tyrosine kinase receptor RET has previously been detected in normal hematopoietic cells, and especially in cells of the myeloid lineage. Furthermore, RET was shown to be differentially expressed in acute myeloid leukemia (AML), a disease characterized by excessive cell growth and aberrant maturation of cells, with the highest levels of expression in leukemias with monocytic differentiation. RET is known to be expressed in cells from the excretory system and from the developing central and peripheral nervous system.

The role of cytokines and hematopoietic growth factors in the autocrine/paracrine regulation of inducible hematopoiesis.

To elucidate the role of hematopoietic growth factors (HGFs) and other cytokines in the autocrine or paracrine regulation of inducible hematopoiesis we studied cytokine gene expression in the bone marrow (BM) of patients after myelosuppressive treatment. Furthermore, we studied the cytokine gene expression profile in healthy individuals before and after bone marrow harvesting for the purpose of bone marrow transplantation. We speculated that the bone marrow harvesting procedure might induce changes in cytokine gene expression.

Mosaicism of the 5q deletion as assessed by interphase FISH is a common phenomenon in MDS and restricted to myeloid cells.

Interstitial deletion of chromosome 5q is a common cytogenetic abnormality observed in MDS. We have used fluorescence in situ hybridization (FISH) to determine accurately the percentage of cytogenetically abnormal peripheral blood cells. YAC and cosmid probes localized to chromosome 5q were hybridized to interphase nuclei from purified polymorphonuclear cells (PMNs) from six MDS patients with chromosome 5 deletions.

Analysis of T-cell clonality in bone marrow of patients with acquired aplastic anaemia.

Acquired aplastic anaemia (AA) represents a state of bone marrow (BM) failure which is characterized by BM hypocellularity and pancytopenia. It has been hypothesized that in some AA patients, bone marrow failure is secondary to the targeted destruction of haemopoietic stem cells by autoreactive T cells. The response of T cells to antigenic stimulation has been shown, in a number of animal models and in autoimmune diseases, to result in the (oligo)clonal expansion of positively reacting T cells.

Haemopoietic growth factor tyrosine kinase receptor expression profiles in normal haemopoiesis.

Expression profiles were generated for the haemopoietic tyrosine kinase receptors (HGF-TKRs or class III TKRs) by PCR on cDNA samples (RT-PCR) using a degenerate primer set. Each profile consisted of primary and secondary, i.e.

Direct display of hematopoietic tyrosine kinase receptor expression profiles in KG1 cells by PCR using degenerate primers.

Hematopoietic tyrosine kinase receptors (HGF-TKRs or class III TKRs) are essential for the growth and differentiation of hematopoietic cells. In this report we present a novel method that generates expression profiles of these receptors. The method was tested and optimized using the myeloblastic/ promyelocytic cell line KG1.

Aplastic anaemia patients with clonal X-chromosome inactivation pattern in haemopoietic cells exhibit polyclonal TCRgamma and IgH gene rearrangements.

Previously, we reported that 13/18 (72%) female patients with aplastic anaemia (AA) exhibited a clonal X-chromosome inactivation (XCI) pattern in all haemopoietic lineages. To study the consequences of a clonal haemopoiesis for the randomness of immunoreceptor rearrangements in lymphocytes we determined clonality of T-cell receptor gamma (TCRgamma) and immunoglobulin heavy chain (IgH) gene rearrangements in purified cell fractions. Peripheral blood granulocytes, monocytes, and B and T lymphocytes from 18 female patients in remission from AA were studied by PCR for randomness of XCI and rearrangement at the IgH and TCRgamma locus.

Persistent clonal excess and skewed T-cell repertoire in T cells from patients with hairy cell leukemia.

Hairy cell leukemia (HCL) is characterized by a severe T-cell-mediated immune deficiency. At the same time, spontaneous T-cell activation is noted when splenic T cells are studied in vivo and in vitro. Therefore, we searched for oligoclonal T-cell populations in the blood and spleens of 25 patients with HCL using a T-cell receptor gamma-polymerase chain reaction (TCR gamma-PCR).

Detection of partial cDNA sequences differentially expressed in patients with myelodysplasia.

The most common chromosomal aberrations in myelodysplastic syndromes (MDS) are complete or partial loss of chromosomes 5 and 7, and trisomy 8. To identify genes important in the pathogenesis of this disease that could be associated with these gross chromosomal defects, we have employed the differential display PCR (DDPCR) procedure developed by Liang and Pardee. This method allows simultaneous comparison of several cDNA sources for the presence of differentially expressed genes.

Apoptin, a protein encoded by chicken anemia virus, induces cell death in various human hematologic malignant cells in vitro.

Apoptin, a small protein encoded by chicken anemia virus (CAV) was expressed in various human hematologic malignant cell lines derived from leukemias and lymphoma. Three of these cell lines contain bcl-2 or BCR-ABL proteins, known to block apoptosis induced by chemotherapeutic compounds. By immunofluorescence and propidium-iodide staining apoptin was shown to induce apoptosis in all analysed cell lines.

In vitro-induced resistance to the deoxycytidine analogues cytarabine (AraC) and 5-aza-2'-deoxycytidine (DAC) in a rat model for acute myeloid leukemia is mediated by mutations in the deoxycytidine kinase (dck) gene.

The deoxycytidine kinase (dck) gene encodes the enzyme responsible for the metabolic activation of the antileukemic drugs cytosine arabinoside (AraC) and 5-aza-2'-deoxycytidine (decitabine, DAC). The dck locus was analyzed at the chromosomal and the molecular level in a model of rat leukemic cell lines, in which AraC and DAC resistance was induced, that was marked by dck deficiency. At the chromosomal level, karyotype analysis of metaphase spreads revealed the presence of an aberrant 2q + chromosome in the AraC-resistant cell line and a (Xq:11q) translocation in its subclone RA/7.

De novo induced mutations in the deoxycytidine kinase (dck) gene in rat leukemic clonal cell lines confer resistance to cytarabine (AraC) and 5-aza-2'-deoxycytidine (DAC).

We have investigated whether cytarabine (AraC) or decitabine (DAC) induce deficiency of deoxycytidine kinase (DCK) through different mutations of the dck gene, related to their distinct interference with DNA replication. Also, it is not known whether mutations of the dck gene are the result of selection of mutants or de novo induction. To address these issues, three subclones of a rat leukemic cell line (RCL/O), sensitive to cytotoxicity mediated by AraC and DAC, were exposed to gradually increasing concentrations (from 0.

Constitutive in vivo cytokine and hematopoietic growth factor gene expression in the bone marrow and peripheral blood of healthy individuals.

We investigated hematopoietic growth factor (HGF) and cytokine gene expression in the bone marrow (BM) and peripheral blood (PB) of healthy individuals as a starting point for delineating the physiologic role of cytokines in steady state hematopoiesis. BM biopsy specimens and PB samples from 7 healthy individuals were analyzed by polymerase chain reaction amplification of reverse-transcribed RNA using gene-specific primer sets. Consistent gene expression in the BM of all 7 individuals was detected for macrophage colony-stimulating factor (CSF), stem cell factor, interleukin-6 (IL-6), IL-7, erythroid-potentiating factor, erythroid-differentiating factor, and insulinlike growth factor 1, all cytokines with reported direct stimulatory effects on in vitro hematopoiesis.

Transfection of wild-type deoxycytidine kinase (dck) cDNA into an AraC- and DAC-resistant rat leukemic cell line of clonal origin fully restores drug sensitivity.

The AraC-resistant rat leukemic cell line RO/1-A has been shown to have a typical deoxycytidine kinase (DCK)-deficient phenotype and cannot metabolize the antileukemic drugs cytarabine (AraC) and decitabine (DAC). To investigate the relative contribution of mutations in the dck gene to the development of in vitro-induced AraC-resistance, a neomycin selectable plasmid construct harboring the wild-type dck coding region was transfected into RO/1-A. Polymerase chain reaction analysis confirmed the presence of vector DNA in the target cells (RO/1-ADCK) that were stably transfected and monitored over a period of 14 weeks.