Medicine JournalFeeds

The carboxyl-terminal region of erythroid-specific 5-aminolevulinate synthase acts as an intrinsic modifier for its catalytic activity and protein stability.

Exp Hematol. 2012 Jan 20;

Authors: Kadirvel S, Furuyama K, Harigae H, Kaneko K, Tamai Y, Ishida Y, Shibahara S

Abstract

Erythroid-specific 5-aminolevulinate synthase (ALAS2) is essential for hemoglobin production, and a loss-of-function mutation of ALAS2 gene causes X-linked sideroblastic anemia (XLSA). Human ALAS2 protein consists of 587 amino acids, and its carboxyl(C)-terminal region of 33 amino acids is conserved in higher eukaryotes, but is not present in prokaryotic ALAS. We explored the role of this C-terminal region in pathogenesis of XLSA. In vitro enzymatic activity was measured using bacterially expressed recombinant proteins. In vivo catalytic activity was evaluated by comparing the accumulation of porphyrins in eukaryotic cells stably expressing each mutant ALAS2 tagged with FLAG, and the half-life of each FLAG-tagged ALAS2 protein was determined by Western blot analysis. Two novel mutations (Val562Ala and Met567Ile) were identified in patients with XLSA. Val562Ala showed the higher catalytic activity in vitro, but a shorter half-life in vivo, compared to those of wild-type ALAS2 (WT). In contrast, the in vitro activity of Met567Ile mutant was about 25% of WT, while its half-life was longer than that of WT. However, in vivo catalytic activity of each mutant was lower than that of WT. Furthermore, the deletion of 33 amino acids at C-terminal end resulted in higher catalytic activity both in vitro and in vivo with the longer half-life compared to WT. In conclusion, the C-terminal region of ALAS2 protein may function as an intrinsic modifier that suppresses the catalytic activity and increases the degradation of its protein, each function of which is enhanced by the Met567Ile mutation and the Val562Ala mutation, respectively.

PMID: 22269113 [PubMed - as supplied by publisher]

Immunomodulatory nonablative conditioning regimen for B-cell lymphoid malignancies.

Exp Hematol. 2012 Jan 20;

Authors: Chinratanalab W, Reddy N, Greer JP, Morgan D, Engelhardt B, Kassim A, Brandt SJ, Jagasia M, Goodman S, Savani BN

Abstract

Twenty six patients with recurrent CD20+ B-cell lymphoid malignancies received fludarabine, cyclophosphamide, and rituximab (FCR) based non-ablative conditioning (NST) followed by either matched related (n=18) or unrelated (n=8) donor allogeneic stem cell transplantation (allo-SCT) between March 2008 and May 2011. The median age of patients at transplantation was 59 years (range 41-64). At diagnosis, 20 (77%) had stage IV disease; 23 (88%) received ≥3, 14 (54%) ≥4 regimens and 4 (15%) had prior autologous-SCT. All patients had either chemosensitive or stable disease and 9 (35%) were in complete remission before transplantation. At the time of analysis, 17 patients were alive with an estimated 2-year overall survival and progression-free-survival of 63%, and non-relapse mortality 25%. Grade II-IV acute graft-verus-host-disease (GVHD) occurred in 8 (31%) and chronic GVHD in 6 (23%) patients (extensive=3). Causes of death include progressive disease 4, acute GVHD 2 (both after receiving donor lymphocyte infusion for mixed chimerism with residual disease), infection 1 and others 2 (substance abuse, leucoencephalopathy). Six patients required re-hospitalization within 100 days of SCT, (average 10 days; range, 3-18). Our data support FCR NST allo-SCT in CD20+ B-cell lymphoid malignancies and it is time to compare this regimen with alternative reduced-intensity conditioning regimen in B-cell malignancies.

PMID: 22269114 [PubMed - as supplied by publisher]

A novel and simple hollow-fiber assay for in vivo evaluation of nonpeptidyl thrombopoietin receptor agonists.

Exp Hematol. 2012 Jan 20;

Authors: Xie CY, Xu YP, Zhao HB, Lou LG

Abstract

Preclinical in vivo assessment of the pharmacologic activity of nonpeptidyl thrombopoietin receptor (TPOR) agonists is very difficult owing to the high species specificity of such agonists. In this study, we have developed a novel and simple in vivo hollow-fiber assay to pre-clinically evaluate TPOR agonists. The 32D-mpl cell line was generated by stable transfection of human TPOR into 32D lymphoblast cells and shown to be a specific model for non-peptide TPOR agonists in vitro. Stably transfected 32D-mpl cells were then sealed in hollow fibers and implanted into nude mice. The cells in hollow fibers specifically responded to TPOR agonists, including thrombopoietin and eltrombopag, a non-peptide small-molecule TPOR agonist, but not to granulocyte colony-stimulating factor or erythropoietin. Oral administration of eltrombopag stimulated 32D-mpl cell proliferation, prevented 32D-mpl cell apoptosis, and stimulated the phosphorylation of cellular signalling transducers and activators of transcription (STAT) in a TPOR- and dose-dependent manner. These results indicate that the hollow-fiber assay is a specific and efficient model for rapidly evaluating the in vivo activity of small-molecule TPOR agonists.

PMID: 22269115 [PubMed - as supplied by publisher]

Lymphoid progenitors in normal mouse lymph nodes develop into NK cells and T cells in vitro and in vivo.

Exp Hematol. 2012 Jan 20;

Authors: Warner K, Luther C, Takei F

Abstract

We have identified a population of normal mouse LN cells, termed LN lymphoid progenitor (LNLP), resembling common lymphoid progenitor (CLP) in the BM. LNLPs lack lineage markers, express CD127, low levels of CD117 (c-Kit) and Sca-1, but lack fms-related tyrosine kinase 3 (Flt3). They efficiently differentiate in vitro into NK cells and T cells, but not mature B cells. LNLPs injected into non-irradiated lymphopenic mice that have no LN develop into mostly splenic T cells with low numbers of NK cells and B cells. When injected into irradiated mice, they generate NK cells and T cells, but not B cells, in the LN. By contrast, BM CLPs develop into mostly B cells with very small numbers of T and NK cells in recipients’ spleen and LN. Thus, LNLPs have NK and T cell potentials but little B cell potential, and they may develop into NK cells within the LN of normal mice, whereas their contribution to the T cell lineage is unknown.

PMID: 22269116 [PubMed - as supplied by publisher]

Consolidative therapy with Stem Cell Transplantation Improves Survival of patients with Mantle Cell Lymphoma following any Induction Regimen.

Exp Hematol. 2012 Jan 20;

Authors: Reddy N, Greer JP, Goodman S, Adetola K, Morgan DS, Chinratanalab W, Brandt S, Englehardt B, Oluwole O, Jagasia MH, Savani BN

Abstract

Intensive induction regimen followed by high dose chemotherapy and autologous stem cell transplantation (auto-SCT) is frequently used to improve outcome in patients with mantle-cell lymphoma (MCL). The comparative impact of conventional vs. intensive induction regimen before transplantation is unknown. Forty eight patients with MCL receiving SCT at our institution between January 2000 and December 2010 were included in this study. At the time of initial presentation, 43 (89.5%) had stage IV disease and 18(37.5%) received more than one chemotherapy regimens prior to transplantation. Forty patients underwent auto-SCT, 7 allo-SCT; one patient had an allo-SCT for relapsed disease after auto-SCT. At the time of this analysis (median follow-up 6 years from diagnosis and 4 years from transplantation), 40 patients (88%) were alive with a 5 year disease free survival of 74.8%. Age, disease stage, number of regimens pre-SCT, pre-SCT disease status, and type of SCT had no impact on long-term outcome. Importantly, there was no difference among the type of induction regimen on outcome in this cohort receiving SCT. Based on our data, we believe that future studies should focus on strategies to prevent disease relapse rather than comparing induction regimens prior to stem cell transplantation.

PMID: 22269117 [PubMed - as supplied by publisher]

The adaptor protein NTAL enhances proximal signaling and potentiates corticosteroid-induced apoptosis in T-ALL.

Exp Hematol. 2012 Jan 20;

Authors: Svojgr K, Kalina T, Kanderova V, Skopcova T, Brdicka T, Zuna J

Abstract

The biology of T-ALL is characterized by functional pre-T-cell receptor (TCR) signaling. NTAL is a non-enzymatic transmembrane adaptor molecule that is involved in the proximal signaling of lymphocytes. In our previous work, we found an association between high NTAL expression in T-cell ALL blasts and a favorable response to initial glucocorticoid treatment. In the present study, we confirm our previous observation in an experimental model. Moreover, the molecular mechanism of the contribution of NTAL to malignant T-ALL blast signaling and to methylprednisolone-induced cell death is analyzed. In the in vitro experiments, we used the T-ALL Jurkat cell line (Jurkat/wt), and derived Jurkat cell line with stable NTAL expression (Jurkat/NTAL+). Cell signaling and cell death after methylprednisolone treatment and after TCR stimulation was analyzed using flow cytometry, western blot and quantitative PCR. Jurkat/NTAL+ cells are significantly more sensitive to both methylprednisolone treatment and TCR-induced stimulation. Moreover, after TCR stimulation, Jurkat/NTAL+ cells show a higher level of intracellular ERK phosphorylation and increased expression of the CD69 activation marker on the cell surface than the Jurkat/wt cells. The ERK inhibitor U0126 almost completely abrogates TCR-induced cell death and, importantly, reverses the sensitizing effect of the NTAL protein on methylprednisolone-induced cell death. In conclusion, NTAL acts as a tumor suppressor that enhances the proximal signaling of leukemic blasts. The key downstream molecule responsible for the biological effect of TCR signaling is ERK. Higher ERK phosphorylation leads to enhanced cell death after TCR stimulation and increases cell sensitivity to methylprednisolone-induced cell death.

PMID: 22269118 [PubMed - as supplied by publisher]

Vitamin-D deficiency, autoimmunity and graft-versus-host-disease risk:Implication for preventive therapy.

Exp Hematol. 2012 Jan 17;

Authors: Benrashid M, Moyers K, Mohty M, Savani BN

Abstract

The majority of patients after allogeneic stem cell transplantation (HSCT) are expected to have vitamin-D deficiency early post-HSCT due to poor nutritional status and limited sun exposure. The importance of vitamin D in the immune system has been well defined over the past several years, as vitamin-D has demonstrated modulatory effects on the immune system through B- and T-lymphocyte, macrophage, monocyte and dendritic cell regulations, which are the effector cells involved in graft-versus-host-disease (GVHD) pathophysiology after HSCT. High dose early replacement of vitamin D might attenuate autoimmune reactions and may decrease the severity of GVHD. In this article, we discuss the hypothetical link between early vitamin-D deficiency and GVHD and its potential therapeutic role in GVHD and long term bone loss after HSCT.

PMID: 22265707 [PubMed - as supplied by publisher]

Abnormal Differentiation of Erythroid Precursors in p45 NF-E2 -/- Mice.

Exp Hematol. 2012 Jan 17;

Authors: Gasiorek JJ, Nouhi Z, Blank V

Abstract

The transcription factor p45 NF-E2 plays major roles in erythroid and megakaryocytic lineages. Here, we investigated the role of p45 NF-E2 in erythoid differentiationin vivo. Absence of p45 NF-E2 in mice leads to a two-fold increase of serum erythropoietin levels. In the bone marrow of these animals, we found a different distribution of precursor populations compared to wild-type mice, suggesting abnormal differentiation. Loss of p45 NF-E2 was also associated with an increase in splenic erythropoiesis, as evidenced by an accumulation of early precursors, namely late basophilic and polychromatic erythroblasts. These observations are consistent with a stress erythropoiesis phenotype and indicate that the spleen is likely compensating for ineffective erythropoiesis in the bone marrow. Analysis of bone marrow samples revealed increased GATA1 levels as well as an increased proportion of erythroid cells arrested at the G1 stage of cell cycle in p45 NF-E2 deficient mice. These results suggest that p45 NF-E2 is required for the differentiation of erythroid precursors.

PMID: 22265708 [PubMed - as supplied by publisher]

Production of large numbers of plasmacytoid dendritic cells with functional activities from CD34+ hematopoietic progenitor cells: use of IL-3.

Exp Hematol. 2012 Jan 10;

Authors: Demoulin S, Roncarati P, Delvenne P, Hubert P

Abstract

Plasmacytoid dendritic cells (pDC), a subset of dendritic cells characterized by a rapid and massive type I interferon secretion through the Toll-like receptor pathway in response to viral infection, play important roles in the pathogenesis of several diseases such as chronic viral infections (HCV, HIV), autoimmunity (psoriasis, SLE) and cancer. As pDC represent a rare cell type in the peripheral blood, the goal of this study was to develop a new method to efficiently generate large numbers of cells from a limited number of CD34+ cord blood progenitors in order to provide a tool to resolve important questions about how pDC mediate tolerance, autoimmunity and cancer. Human CD34+ hematopoietic progenitor cells (HPC) isolated from cord blood were cultured with a combination of Flt3-Ligand (Flt3L), thrombopoietin (TPO) and one of the following cytokine: interleukin-3 (IL-3), interferon-β (IFNβ) or prostaglandin (PG)E2. Cells obtained in the different culture conditions were analyzed for their phenotype and functional characteristics. The addition of IL-3 cooperates with Flt3L and TPO in the induction of pDC from CD34+ HPC. Indeed, Flt3L/TPO alone or supplemented with PGE2 or IFNβ produced smaller amounts of pDC from HPC. Moreover, pDC generated in Flt3L/TPO/IL-3 cultures exhibited morphological, immunohistochemical and functional features of peripheral blood pDC. We showed that IL-3, in association with Flt3L and TPO, provides an advantageous tool for the large-scale generation of pDC. Indeed, this culture condition generated, starting from 2×105 CD34+ cells, up to 2.6×106 pDC presenting features of blood pDC.

PMID: 22245566 [PubMed - as supplied by publisher]

Critical role of CD4+CD25+ regulatory T cells in preventing murine autoantibody-mediated thrombocytopenia.

Exp Hematol. 2012 Jan 9;

Authors: Nishimoto T, Satoh T, Takeuchi T, Ikeda Y, Kuwana M

Abstract

Autoimmune response suppression by regulatory T cells (Tregs) helps to maintain peripheral immune tolerance, and defects in this mechanism are thought to play a role in the pathogenesis of various autoimmune diseases. In patients with immune thrombocytopenia, naturally occurring CD4(+)CD25(+) Tregs are both functionally impaired and reduced in number. This study was undertaken to investigate Tregs’ role in preventing immune thrombocytopenia in mice. Treg-deficient mice were prepared by inoculation of Treg-depleted CD4(+)CD25(-) T cells isolated from BALB/c mice into syngeneic nude mice intravenously. Platelet count, proportion of reticulated platelets, platelet-associated IgG, platelet-associated anti-platelet antibodies, and IgG anti-platelet antibody production in splenocyte cultures were examined by flowcytometry. Of 69 Treg-deficient mice, 25 (36%) spontaneously developed thrombocytopenia that lasted at least 5 weeks. The platelet-associated IgG level and proportion of reticulated platelets were elevated in the thrombocytopenic mice. Platelet eluates and splenocyte culture supernatants prepared from thrombocytopenic mice, but not from non-thrombocytopenic mice, contained IgG antibodies capable of binding to intact platelets. Simultaneous transfer of Tregs completely prevented the onset of thrombocytopenia, but Treg transfer after the onset of thrombocytopenia had no apparent effect. Treatment with IgG anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibody cancelled this Treg-governed suppressive effect. In summary, these results indicate that Tregs play a critical role in preventing murine autoantibody-mediated thrombocytopenia by engaging CTLA-4.

PMID: 22240606 [PubMed - as supplied by publisher]