Blood
0006-4971
Cơ quản chủ quản: Elsevier BV , AMER SOC HEMATOLOGY
Lĩnh vực:
BiochemistryHematologyCell BiologyImmunology
Các bài báo tiêu biểu
Rearrangement of CCND1 (BCL1/PRAD1) 3' untranslated region in mantle- cell lymphomas and t(11q13)-associated leukemias Abstract
Rearrangement and overexpression of CCND1 (BCL1/PRAD1), a member of the cyclin G1 gene family, are consistent features of t(11q13)-bearing B- lymphoid tumors (particularly mantle-cell lymphoma [MCL]). Its deregulation is thought to perturb the G1-S transition of the cell cycle and thereby to contribute to tumor development. As suggested by previously published studies, rearrangement of the 3′ untranslated region (3′ UTR) of CCND1 may contribute to its activation in some lymphoid tumors. To define further the prevalence of such rearrangements, we report here the result of the molecular study of 34 MCL and six t(11q13)-associated leukemias using a set of probes specific to the different parts of the CCND1 transcript. We also sequenced the entire cDNA of the overexpressed CCND1 transcripts in a t(11q13)-associated leukemia. DNA from four of these 40 patients showed rearrangement of the 3′ UTR of CCND1 coexisting with major translocation cluster (MTC) rearrangement. Southern blot and sequence analyses showed that, as a result of these rearrangements, the 3′ AU- rich region containing sequences involved in mRNA stability and in translational control is eliminated. Moreover, the finding that the CCND1 mRNA half-life was greater than 3 hours (normal tissues, 0.5 hours) in three t(11q13)-associated cell lines stresses the importance of posttranscriptional derangement in the activation of CCND1. Finally, we did not observe any mutation in the coding frame of the CCND1 cDNA analyzed.
Tập 83 Số 12 - Trang 3689-3696 - 1994
Detection of Anti-Thrombopoietin Antibodies in Patients with Immune Thrombocytopenia Abstract
Background: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by the presence of autoantibodies against platelet membrane glycoproteins, which cause the autoantibody-mediated destruction of platelets and impaired platelet production. Thrombopoietin (TPO) binds to its receptor on the surface of hematopoietic stem cells and megakaryocytes and induces their maturation and proliferation. Patients with thrombocytopenia due to aplastic anemia have drastically elevated plasma levels of TPO, whereas patients with ITP have normal or slightly elevated plasma levels of TPO despite their low platelet count. Furthermore, based on the existence of a multitude of autoantibody reactivities in ITP, including antibodies against platelets and TPO receptors, the presence of anti-TPO antibodies in patients with ITP may be suspected.
Objective: We developed assay systems to detect plasma anti-TPO antibodies and screen patients with ITP. We examined the clinical characteristics associated with anti-TPO antibodies and their pathogenic roles in patients with ITP.
Methods: Plasma anti-TPO antibodies from 101 patients with ITP and 72 healthy controls were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant human TPO (rhTPO) as an antigen. The specificity of anti-TPO antibody reactivity was confirmed by ELISA competition assay. The presence of anti-TPO antibodies was further examined using immunoprecipitation and immunoblotting using rhTPO. To investigate whether anti-TPO antibodies inhibited functional interactions between TPO and TPO receptors, we examined extracellular signal-regulated kinases (ERKs), downstream signals induced by TPO. The binding of TPO to TPO receptors induced the phosphorylation of ERK in TPO receptor-expressing UT-7/TPO cells.
Results: The level of anti-TPO antibodies measured by ELISA was significantly greater in the samples from patients with ITP than in those from healthy controls (2.91 ± 3.64 units versus 1.45 ± 0.67 units, P < 0.001). Samples were classified as positive or negative for anti-TPO antibody, as determined by immunoprecipitation and immunoblotting. Thus, the ELISA positive-cutoff value was considered to be the mean plus 3.5 standard deviation (SD) of 72 healthy control plasma samples. Plasma anti-TPO antibodies were detected in twenty-four ITP patients (23.8%), but in none of the healthy controls. By ELISA competition assay, anti-TPO antibody reactivity was inhibited dose-dependently by preincubation of patient plasma with rhTPO. In addition, anti-TPO antibody-positive plasma samples inhibited the phosphorylation of ERK in UT-7/TPO cells. In contrast, healthy control plasma had no inhibitory effect. Furthermore, the number of megakaryocytes was decreased relatively in the anti-TPO antibody-positive ITP patients. There was no difference in the TPO levels in plasma between ITP patients with anti-TPO antibodies and patients without anti-TPO antibodies (63.6 ± 79.7 pg/ml versus 45.2 ± 49.3 pg/ml).
Conclusion: Our results have thus demonstrated the presence of anti-TPO autoantibodies in patients with ITP. The ELISA using rhTPO was specific for the detection of anti-TPO antibodies and thus allows their easy and rapid measurement in clinical settings. These findings suggest that functional anti-TPO antibodies cause impaired megakaryocyte proliferation and platelet production in patients with ITP.
Disclosures
Higashihara: Bristol-Myers Squibb: Research Funding; Baxter: Research Funding; Teijin: Research Funding; Pfizer: Research Funding; Astellas: Research Funding; Yakurt: Honoraria; KyowaHakkoKirin: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Eisai: Honoraria; GlaxoSmithKline: Honoraria, Research Funding; Nippon Shinyaku: Research Funding; Shionogi: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria; Takeda: Honoraria; Janssen pharma: Honoraria, Research Funding; Alexion: Honoraria; Dainippon Sumitomo: Research Funding; Taisho Tomiyama: Research Funding.
Tập 124 - Trang 4187 - 2014
Calcitonin receptor-like receptor guides arterial differentiation in zebrafish Abstract The calcitonin receptor-like receptor (crlr) is a major endothelial cell receptor for adrenomedullin, a peptide vasodilator involved in cardiovascular development, homeostasis, and disease. Here, we used the zebrafish (Danio rerio) model to characterize the role of crlr in vascular development. Crlr is expressed within somites from the 4- to the 13-somite stage and by arterial progenitors and axial vessels during zebrafish development. Loss of crlr results in profound alterations in vascular development and angiogenesis, including atrophic trunk dorsal aorta and interruption of anterior aortic bifurcation, delay in intersomitic vessel development, and lack of blood circulation. Remarkably, crlr morphants are characterized by the loss of arterial endothelial cell identity in dorsal aorta, as shown by the lack of expression of the arterial markers ephrin-B2a, DeltaC, and notch5. Down-regulation of crlr affects vascular endothelial growth factor (vegf) expression, whereas vegf overexpression is sufficient to rescue arterial differentiation in crlr morphants. Finally, genetic and biochemical evidences indicate that somitic crlr expression is under the control of sonic hedgehog. These data demonstrate that crlr plays a nonredundant role in arterial differentiation, representing a novel element of the sonic hedgehog–vegf-notch signaling cascade that controls arterial/venous fate.
Tập 111 - Trang 4965-4972 - 2008
BAFF-R Receptor Functions in Transcription Regulation in Genes Critical to Survival and Proliferation in Normal and Neoplastic Human B Lymphocytes Abstract
B-lymphocyte stimulator (BLyS also called BAFF), is a potent cell survival factor expressed in many hematopoietic cells. However, many aspects of BLyS signaling pathway remain unclear. In order to investigate BLyS signaling pathway, we studied the function of BAFF-R (also called BR3), a major BLyS receptor, in normal and neoplastic B cell survival and proliferation. In our initial study, we found that BAFF-R was also present in nucleus, in addition to its presence in the plasma membrane of B cells. A candidate nuclear localization sequence was identified in the BAFF-R protein sequence. We also found BAFF-R mediated transcriptional activity, that could be increased through over expression of a NF-κB family member c-rel. Further study showed that BAFF-R co-localized with, c-rel in the nucleus and bound to the NF-κB binding site on the promoters of NF-κB target genes such as BLyS, CD154, Bcl-xL, Bfl-1/A1 and IL-8. The IκB kinase (IKK) protein complex is critical for regulating NF-κB pathway activation. The IKK complex includes three important subunits: the catalytic subunits IKKα and IKKβ (also known as IKK1 and IKK2) and the regulatory subunit IKKγ (also known as NEMO). In the cytoplasm, activation of the IKK complex induces processing of precursors p105 and p100 into p50 and p52 respectively, resulting in NF-κB subunit dimeric partners that migrate from the cytoplasm into the nucleus. In recent studies, IKKα has also been identified in the cell nucleus, functioning in histone H3 phosphorylation. Although IKKβ was also previously observed in the cell nucleus, its nuclear function has been obscure. Besides functioning as a transcriptional co-factor with c-rel, we also found BAFF-R interacted with IKKβ in the nucleus of normal and neoplastic (lymphoma) B cells, enhancing histone H3 phosphorylation through IKKβ by immuno precipitation experiments and in vitro kinase assays. Inhibition of BAFF-R entry into the nucleus by BAFF-R NLS mutant transfection, decreased the level of phosphorylated histone H3 compared to the controls in NHL-B cells. These findings not only demonstrate a novel nuclear function of IKKβ, but also determine a new mechanism of how BAFF-R promotes survival and proliferation of normal B cells and NHL-B cells. In addition to activating NF-κB pathways in the plasma membrane, BAFF-R also functions as a transcriptional regulator binding to NF-κB targeted gene promoters possibly through a chromatin remodeling mechanism(s).
Tập 112 - Trang 4478 - 2008
The hereditary hemochromatosis protein, HFE, lowers intracellular iron levels independently of transferrin receptor 1 in TRVb cells Abstract Hereditary hemochromatosis (HH) is an autosomal recessive disease that leads to parenchymal iron accumulation. The most common form of HH is caused by a single amino acid substitution in the HH protein, HFE, but the mechanism by which HFE regulates iron homeostasis is not known. In the absence of transferrin (Tf), HFE interacts with transferrin receptor 1 (TfR1) and the 2 proteins co-internalize, and in vitro studies have shown that HFE and Tf compete for TfR1 binding. Using a cell line lacking endogenous transferrin receptors (TRVb cells) transfected with different forms of HFE and TfR1, we demonstrate that even at low concentrations Tf competes effectively with HFE for binding to TfR1 on living cells. Transfection of TRVb cells or the derivative line TRVb1 (which stably expresses human TfR1) with HFE resulted in lower ferritin levels and decreased Fe2+ uptake. These data indicate that HFE can regulate intracellular iron storage independently of its interaction with TfR1. Earlier studies found that in HeLa cells, HFE expression lowers Tf-mediated iron uptake; here we show that HFE lowers non–Tf-bound iron in TRVb cells and add to a growing body of evidence that HFE may play different roles in different cell types.
Tập 105 - Trang 2564-2570 - 2005
Human myeloid leukemia cell lines: a review Abstract
Several human acute myeloid leukemia cell lines were recently established. These lines provide model systems to study the control of differentiation in human myelogenous leukemia and, in a broader framework, the controls of normal myeloid development. The K562 line is composed of undifferentiated blast cells that are rich in glycophorin and may be induced to produce fetal and embryonic hemoglobin in the presence of hemin. The KG-1 cell line is composed predominantly of myeloblasts and promyelocytes. A unique characteristic of the KG-1 cells is their almost complete dependence on colony-stimulating factor for proliferation in soft-gel culture. The HL-60 is a promyelocytic leukemia cell line. In the presence of DMSO, the cells mature into granulocytes. Both the KG-1 and HL-60 cells differentiate into nondividing mononuclear phagocytes when exposed to phorbol esters. Investigations with these cell lines, and selected variants should provide important insights into the cell biology and perhaps therapy of human leukemia.
Tập 56 Số 3 - Trang 344-350 - 1980
An undifferentiated variant derived from the human acute myelogenous leukemia cell line (KG-1) Abstract
A variant subline (KG-1a) of the human acute myelogenous leukemia (AML) cell line (KG-1) has been isolated. The cells retain the same constitutive markers as the parent line, including HLA antigens, isoenzymes, and karyotype. The cells from the subline are morphologically and histochemically undifferentiated blast cells, while the parent cells and several of its clones are at the myeloblast and promyelocyte stages of development. The variant cells do not respond to colony-stimulating factor (CSF), and they do not express the human la antigen, nor a recently characterized AML antigen. The parent KG-1 cells are stimulated to proliferate in the presence of CSF and the cells express the la and AML antigen. Variant AML cell lines, such as KG-1a, will be useful in vitro models for investigating cellular response to CSF and for studying antigen expression in leukemic cells.
Tập 56 Số 2 - Trang 265-273 - 1980
Molecular defect of a phosphoglycerate kinase variant (PGK-Matsue) associated with hemolytic anemia: Leu----Pro substitution caused by T/A- ---C/G transition in exon 3 Abstract
We have identified the mutation in a phosphoglycerate kinase variant (PGK-Matsue) associated with severe enzyme deficiency, congenital nonspherocytic hemolytic anemia, and mental disorders. The mRNA coding for PGK was reverse transcribed and amplified by the polymerase chain reaction. Nucleotide sequencing of the variant cDNA showed a point mutation, a T/A----C/G transition in exon 3 of the variant gene. No other mutation was found in all coding regions of PGK-Matsue. The nucleotide change created an additional NciI cleavage site in the variant gene; thus, the NciI fragment types detected by Southern blot hybridization differ in the variant DNA and normal DNA. The mutation should cause Leu----Pro substitution at the 88th position from the NH2- terminal Ser of PGK. Because the Leu----Pro substitution is expected to induce serious perturbation and instability in the protein structure, the severe enzyme deficiency is mainly caused by more rapid in vivo denaturation and degradation of the variant enzyme.
Tập 77 - Trang 1348-1352 - 1991
AML Cell Vaccines Co-Expressing CD80 and IL-15/IL-15 Receptor Alpha Induce Activation and Cytolytic Activity in Post Remission Autologous Patient PBMC <i>Ex Vivo</i> Abstract
There is a critical need for more effective therapy for acute myelogenous leukemia (AML). Although many patients achieve remission, most relapse with poor outcomes. Even after allogeneic Stem Cell Transplantation (SCT), 30-50% of patients relapse due to the persistence of residual disease.
To address the poor immunogenicity of AML cells and the diminished immune responsiveness of patients, our candidate autologous AML vaccine is lentivirally engineered, in each patient's leukemic cells, to express CD80, IL-15, and IL-15 Receptor alpha (IL-15Rα). In prior studies in a syngeneic 32Dp210 murine AML model, CD80-mediated co-stimulation of T-cells combined with immune activation by the IL-15/IL-15Rα heterodimer showed unprecedented synergy in induction of anti-leukemic cytolytic activity (Shi, Y. et al, 2018). This was observed in both ex vivo co-culture and in vivo where vaccinated leukemic mice had >80% cure rates. No local skin, organ, or systemic toxicity was observed, nor was there evidence of systemic cytokine release of IL-6 or TNFα after SC or IV injection of up to 10 8 transduced irradiated AML cells.
We confirmed the feasibility of producing patient-derived AML vaccines by transduction of 16 independent AML samples with a tri-cistronic lentiviral vector (TLV) that contains human CD80, IL-15 and IL-15Rα. Transduction levels were 11-71% of cells (median 38.6%).
To define the minimum transduction level required for PBMC activation and to assess synergy of co-expressed human CD80, IL-15, and IL-15Rα, allogenic U937 leukemia cells were initially used as stimulators. Transduced U937 (U937-TLV) had high-level surface expression of CD80 and IL-15, secreted IL-15 (7 ng/ml/24 hours from 2 x 10 6 cells/ml) and activated CD3+ T-cells from an AML patient (Fig.1). Mixtures of irradiated U937-TLV with non-transduced U937 were created at fixed ratios (100%, 80%, 40%, 20%, 10%, 5%, 0%) for overnight co-culture with patient PBMC. At 24 hours, the T-cells were analyzed for activation by measurement of the frequency of CD69+ CD4 or CD8 T cells (Fig. 1), normalized to expression of unstimulated PBMC (0%) and the percentage of maximal CD69 expression with 100% U937-TLV (100%). Background levels of activation due to the presence of allogenic U937 were negligible. Co-culture with as little as 10% transduced U937-TLV reliably activated patient T-cells. To assess the synergy of CD80, IL-15 and IL-15Rα expression, parallel experiments were performed with PBMC co-cultured in IL-15 containing supernatants from U937-TLV cells (Fig. 1). The frequencies of activated T-cells were significantly higher after co-culture with U937/U937-TLV cells than after stimulation with IL-15-containing supernatants from similar ratios of U937/U937-TLV, confirming the synergy of CD80 and IL-15/IL-15Rα in the transduced cells.
To better, model the clinical setting, we assessed induction of immune responses of patient T cells to autologous transduced AML. PBMC were stimulated with transduced or non-transduced autologous AML cells vs stimulation with allogeneic U937-TLV, or with anti-CD3/CD28 beads to define maximal stimulation. Negative controls included culture of PBMC alone. All patients had T-cell activation, as measured by induction of CD69, HLA-DR and CD95 (Fas) expression, although there was heterogeneity in the nature of responses, e.g., disparate induction of the markers in individual patients (Fig. 2A and B). Induction of cytotoxic effector pathways was confirmed by detection of CD178 (FasL) and perforin expression (Figure 2C and D). Overall, all patients' PBMC had the capacity to mount T-cell responses of similar magnitude to both allogeneic U937-TLV and autologous vaccine.
These studies establish that autologous AML cells transduced with CD80, IL-15 and IL-15Rα can elicit specific anti-leukemic T-cell responses, even in the face of prior lymphodepleting chemotherapy. A strength of this autologous vaccine strategy is that it is agnostic to which AML proteins are immunogenic for each patient. Although uniformly detected, there was heterogeneity in the induction of activation markers and effector pathways, which may reflect host and/or disease-related differences. The mechanisms underlying differences in the nature of responses in patients will be important to understand and will provide the basis for future immune correlative studies for our Phase 1 vaccine trial in transplant ineligible AML patients.
Figure 1 Figure 1.
Disclosures
Kohn: Lyrik Therapeutics: Membership on an entity's Board of Directors or advisory committees; MyoGene Bio: Membership on an entity's Board of Directors or advisory committees; ImmunoVec: Membership on an entity's Board of Directors or advisory committees; Pluto Immunotherapeutics: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; UC Regents: Patents & Royalties; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Sangamo Biosciences: Membership on an entity's Board of Directors or advisory committees.
Tập 138 - Trang 1706 - 2021
Response to desmopressin is influenced by the genotype and phenotype in type 1 von Willebrand disease (VWD): results from the European Study MCMDM-1VWD Abstract
We have prospectively evaluated the biologic response to desmopressin in 77 patients with type 1 von Willebrand disease (VWD) enrolled within the Molecular and Clinical Markers for the Diagnosis and Management of type 1 VWD project. Complete response to desmopressin was defined as an increase of both ristocetin cofactor activity (VWF:RCo) and factor VIII coagulant activity (FVIII:C) to 50 IU/dL or higher and partial response as VWF:RCo or FVIII:C lower than 50 IU/dL after infusion, but at least 3-fold the basal level. Complete response was observed in 83% of patients; partial in 13%; and no response in 4%. Patients with some abnormality of VWF multimeric pattern had significantly lower basal FVIII:C and VWF, lower VWF:RCo/Ag ratio, and less complete responses to desmopressin than patients with a normal multimeric pattern (P = .002). Patients with mutations at codons 1130 and 1205 in the D′-D3 domain had the greatest relative increase, but shortest FVIII and VWF half-lives after infusion. Most partial and nonresponsive patients had mutations in the A1-A3 domains. Response to desmopressin in these VWD patients seemed to be associated with the location of the causative mutation. The presence of subtle multimeric abnormalities did not hamper potential clinically useful responses, as in typical type 1 VWD.
Tập 111 - Trang 3531-3539 - 2008