Runx1 repression by histone deacetylation is critical for Setbp1-induced mouse myeloid leukemia development.

Abnormal activation of SETBP1 through overexpression or missense mutations is highly recurrent in various myeloid malignancies; however, it is unclear whether such activation alone is able to induce leukemia development. Here we show that Setbp1 overexpression in mouse bone marrow progenitors through retroviral transduction is capable of initiating leukemia development in irradiated recipient mice. Before leukemic transformation, Setbp1 overexpression significantly enhances the self-renewal of hematopoietic stem cells (HSCs) and expands granulocyte macrophage progenitors (GMPs). Interestingly, Setbp1 overexpression also causes transcriptional repression of critical hematopoiesis regulator gene Runx1 and this effect is crucial for Setbp1-induced transformation. Runx1 repression is induced by Setbp1-mediated recruitment of a nucleosome remodeling deacetylase (NuRD) complex to Runx1 promoters and can be reversed by treatment with histone deacetylase (HDAC) inhibitors Entinostat and Vorinostat. Moreover, treatment with these inhibitors caused efficient differentiation of Setbp1 activation-induced leukemia cells in vitro, and significantly extended the survival of mice transplanted with such leukemias, suggesting that HDAC inhibition could be an effective strategy for treating myeloid malignancies with SETBP1 activation.

Inflammatory cytokines in relation to adrenal response following total hip replacement.

Our objective was to investigate the initiation and course of pro- and anti-inflammatory cytokines in early inflammatory response and to elucidate the cytokine system in relation to the adrenal response caused by stress. Seven blood samples were collected, pre- and postoperatively (0-72 h) after total hip replacement (THR) due to osteoarthritis. The following cytokines were measured using Cytometric Bead Array: interleukin-1beta (IL-1beta), IL-6, tumour necrosis factor-alpha, IL-8, IL-12 and IL-10 (B&D). Thirteen patients took part in the study (67 +/- 9 years). C-reactive protein increased from <6 to over 200 mg/l on the second post-op day. The concentration of IL-6 increased 10-fold just 3 h post-op (4-47 pg/ml) and reached its maximum value 6 h post-op (77 pg/ml; Wilcoxon test P < 0.01) Repeated measurements were also significant (Friedman P < 0.05). The concentration of IL-8 doubled the day of surgery but did not reach a significant level (Friedman test =0.069). None of the other cytokines showed any significant changes. The diurnal cortisol rhythm was interrupted after the surgery and there was a significant correlation between the cortisol secretion and IL-6 response. This study demonstrates an isolated elevation in IL-6 levels with only a minor elevation in IL-8 following THR. This pro-inflammatory response seemed to decline without activation of anti-inflammatory cytokines (IL-10), but cortisol seemed to play a complicated role in halting the acute inflammatory response.

Increased expression of interleukin-13 but not interleukin-4 in CD4+ cells from patients with the hyper-IgE syndrome.

Hyper IgE syndrome (HIES) is a rare immunodeficiency disorder characterized mainly by high levels of polyclonal IgE in serum and recurrent staphylococcal abscesses of the skin and lungs. The raised IgE levels have led researchers to study the synthesis of cytokines that regulate switching of immunoglobulin production towards IgE such as interleukin-4 (IL-4), IL-12 and interferon-gamma (IFN)-gamma. However, the role of IL-13 in the disease pathogenesis has not been investigated extensively. In this study, we investigated intracellular expression of IL-4 and IL-13 in mononuclear cells and CD4+ cells isolated from patients with HIES and healthy controls. Cells were stained intracellularly with antibodies directed against IL-4 and IL-13 and analysed by flow cytometry before and after activation with PMA and calcium ionophore. The mean proportion of resting or activated IL-4 and IL-13 expressing mononuclear cells were comparable in the two groups as well as the proportion of IL-4 expressing CD4+ cells. In contrast, the mean proportion of IL-13 expressing CD4+ cells was increased significantly in patients with HIES in both the resting and the activated state compared to healthy controls. We conclude that increased expression of IL-13 in CD4+ cells from patients with HIES could account, at least partly, for raised IgE levels in those individuals.

[Mesenchymal stem cells. A review.].

The bone marrow contains various types of stem cells. Among them are hematopoietic stem cells, which are the precursors of all blood cells, and mesenchymal stem cells. Mesenchymal stem cells have recently received a lot of attention in biological research because of their capability to self renewal, to expand and transdifferentiate into many different cell types; bone cells, adipocytes, chondrocytes, tendocytes, neural cells and stromal cells of the bone marrow. Mesenchymal stem cells can be cultured in vitro although their differentiation potential is not yet fully understood. Several experiments have been conducted in animal models where mesenchymal stem cells have been transplanted in order to enhance hematopoiesis or to facilitate the repair of mesenchymal tissue. Similar experiments are being conducted in humans. Mesenchymal stem cells are believed to be able to enhance hematopoietic stem cells transplantation by rebuilding the bone marrow microenvironment which is damaged after radiation- and/or chemotherapy. Mesenchymal stem cells are promising as vehicles for gene transfer and therapy. It may prove possible to tranduce them with a gene coding for a defective protein i.e. collagen I in osteogenesis imperfecta. The cells could then be expanded ex vivo and transplanted to the patients where they home to the bone marrow, differentiate and produce the intact protein. Future medicine will probably involve mesenchymal stem cells in various treatment settings.

Immunoglobulin-secreting cells in cord blood: effects of Epstein-Barr virus and interleukin-4.

The contribution of cord blood B lymphocytes to the immune response has been under considerable investigation. Cord blood B cells produce almost no antibodies except of the immunoglobulin (Ig)M isotype, indicating immaturity of the cells or the environment they reside in. The aim of this study was to investigate the number of circulating IgA-, IgM-, IgG-, and IgE-producing cells in cord blood in comparison to adult peripheral blood using the ELISPOT method. Moreover, we studied the effect of transformation with the Epstein-Barr virus (EBV) on the proportion of cells producing different isotypes with or without interleukin (IL)-4. Cord blood had IgM-producing cells circulating predominantly, but also some IgA- and IgG-producing cells, whereas adult peripheral blood contained high amounts of circulating IgA-producing cells and some IgM- and IgG-producing cells. No circulating IgE-producing cells were found in either group. Transformation by EBV caused significant expansion of IgA-, IgM-, and IgG-producing cells in adult peripheral blood, but almost only of IgM-producing cells in cord blood. A low but detectable expansion of IgA- and IgG-producing cells was found. Cells producing IgE were still not found, even after EBV transformation. However, EBV transformation in the presence of IL-4 increased the numbers of IgE-producing cells significantly both in cord blood and adult peripheral blood. These findings indicate that cord blood contains some circulating IgA- and IgG-producing cells that are expanded to some extent after EBV infection. They also indicate that cord blood B cells have a similar capacity for IgE production to adult peripheral blood B cells when appropriately stimulated.

Hereditary cystatin C amyloid angiopathy: monitoring the presence of the Leu-68-->Gln cystatin C variant in cerebrospinal fluids and monocyte cultures by MS.

Hereditary cystatin C amyloid angiopathy (HCCAA) is an autosomal dominant condition in which the patients suffer at an early age from repeated cerebral haemorrhages. The development of HCCAA is directly linked to a Leu-68-->Gln (L68Q) mutation in the cystatin C protein sequence. The concentration of cystatin C in cerebrospinal fluid (CSF) of HCCAA patients is markedly diminished and cultivated monocytes from affected individuals accumulate cystatin C. The goal of this work was to characterize cystatin C isolated from CSF and monocyte cultures originating from healthy persons and HCCAA patients with respect to the L68Q mutation. Cystatin C was isolated by carboxymethylpapain affinity chromatography. Proteins from CSF and monocyte cultures that bound specifically to the carboxymethylated papain column were resolved by reverse-phase HPLC chromatography and tryptic peptides were subsequently analysed by matrix-assisted laser desorption ionization MS. No evidence for mutated cystatin C protein was found in CSF samples from healthy subjects or HCCAA patients, but approx. 60% of the protein was found to be hydroxylated on Pro-3. No evidence was found for secretion of mutated cystatin C from HCCAA monocytes. However, we obtained evidence for the presence of mutated cystatin C in HCCAA monocytes. These results support the conclusion that the mutated cystatin C is retained in association with the monocytes and not secreted. An increased intracellular concentration would presumably promote the aggregation and denaturation of the mutated cystatin C, leading to the formation of amyloid fibrils and cell death.

[Hematopoietic stem cell grafts.].

INTRODUCTION: The number of stem cell transplantations have greatly increased in the treatment of primary bone marrow disorders and as a rescue therapy following highdose chemoradiofherapy for various malignancies. Hematopoietic stem cells can be mobilized from bone marrow by hematopoietic growth factors enabling their sampling from peripheral blood. Peripheral blood stem cell transplantation is gradually replacing autologous bone marrow transplantation and is increasingly used in allogeneic settings. Transplantation using umbilical cord blood derived stem cells are also well described. OBJECTIVE: To standardize the in vitro methods necessary for the evaluation of stem cell grafts using umbilical cord blood mononuclear cells. MATERIAL AND METHODS: Mononuclear cells (MNC) were isolated from 57 umbilical cord blood samples. The proportion of hematopoietic stem cells (CD34+) was determined by flow cytometry. The number of clonogenic cells (BFU-E, CFU-GM) was determined by culturing MNC in methylcellulose and agar. The number of clonogenic cells was compared before and after freezing in liquid nitrogen. RESULTS: The mean volume of collected cord blood was 43.8 ml and the number of MNC's was 102.7x10" cells of which 0.93% were CD34+. The number of CFU-GM was 23 8/105 MNC and BFU-E 506/105 MNC. After freezing and thawing the MNC, the viability was 94.9% and the number of clonogenic cells was slightly decreased when compared to prefreezing values, the difference being not statistically significant. The purity of CD34+ cells after selection with magnetic beads was over 95%. CONCLUSION: In vitro methods, neccessary for evaluation of hematopoietic stem cell grafts, have been standardized using umbilical cord blood derived stem cells.