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Major histocompatibility complex class I polypeptide-related sequence A (MICA) is a highly polymorphic ligand of the activating NKG2D receptor on natural killer (NK) cells, γδ-T cells, and NKT cells. MICA incompatibilities have been associated with an increased graft-versus-host disease (GVHD) incidence, and the MICA-129 (met/val) dimorphism has been shown to influence NKG2D signaling in unrelated hematopoietic stem cell transplantation (uHSCT). We investigated the effect of MICA matching on survival after uHSCT. We sequenced 2172 patients and their respective donors for MICA. All patients and donors were high-resolution HLA-typed and matched for 10/10 (n = 1379), 9/10 (n = 636), or 8/10 (n = 157) HLA alleles. Within each HLA match group, cases matched and mismatched for MICA and MICA-129 were analyzed for the end points overall survival (OS), disease-free survival (DFS), nonrelapse mortality (NRM), relapse-incidence (RI), and GVHD. Mismatches at the MICA locus as well as MICA-129 increased with the number of HLA mismatches (MICA mismatched 10/10, 9.2% [n = 127]; 9/10, 22.3% [n = 142]; 8/10, 38.2% [n = 60]; MICA-129 mismatched 10/10, 3.9% [n = 54]; 9/10, 10.2% [n = 65]; 8/10, 17.2% [n = 27]). Adverse OS was observed in the 10/10 match group if MICA-129 was mismatched (10/10, hazard ratio [HR], 1.77; confidence interval [CI], 1.22-2.57; P = .003). MICA-129 mismatches correlated with a significantly worse outcome for DFS in the 10/10 HLA match group (HR, 1.77; CI, 1.26-2.50; P = .001). Higher rates of aGVHD were seen in MICA-129 mismatched cases. Our results indicate that MICA-129 matching is relevant in uHSCT. Prospective typing of patients and donors in unrelated donor search may identify mismatches for MICA-129, and compatible donor selection may improve outcome for this small but high-risk subgroup.

Erythropoietin (EPO), the key hormone in red blood cell renewal, is mainly produced in the adult kidney. Anemia and hypoxia substantially enhance EPO expression to increase erythropoiesis. Investigations of the cellular physiology of renal EPO production have been hampered by the lack of an adequate human cell line. In the present study, we present the human kidney cell line REPC (for renal Epo–producing cells), established from an explanted human kidney exhibiting EPO gene expression and release of the EPO protein in an oxygen-dependent manner. Hypoxic induction of EPO mRNA showed the typical transient increase and peak in expression after 36 hours under continuous conditions of hypoxia. Bioactive EPO protein accumulated in the culture supernatant. The induction of EPO gene expression in REPCs critically depended on the activation of hypoxia-inducible transcription factors (HIFs). SiRNA treatment revealed that the expression of EPO was largely dependent on the activation of the transcription factor complex HIF-2. In addition, hepatic nuclear factor 4α was shown to be critically involved in hypoxia-induced renal EPO expression. Using the human kidney cell line REPC, we provide for the first time a powerful tool with which to study the cellular and molecular regulation of renal EPO production.

Members of the Toll/interleukin-1 receptor (TIR) family are of importance for host defense and inflammation. Here we report that the TIR-family member interleukin-33R (IL-33R) cross-activates the receptor tyrosine kinase c-Kit in human and murine mast cells. The IL-33R–induced activation of signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase B (PKB), and Jun NH2-terminal kinase 1 (JNK1) depends on c-Kit and is required to elicit optimal effector functions. Costimulation with the c-Kit ligand stem cell factor (SCF) is necessary for IL-33–induced cytokine production in primary mast cells. The structural basis for this cross-activation is the complex formation between c-Kit, IL-33R, and IL-1R accessory protein (IL-1RAcP). We found that c-Kit and IL-1RAcP interact constitutively and that IL-33R joins this complex upon ligand binding. Our findings support a model in which signals from seemingly disparate receptors are integrated for full cellular responses.

Two human neuroblastoma (NB) cell lines, SH-SY5Y and Kelly, were found to express the gene for erythropoietin (EPO) in an oxygen (O2)-dependent manner. However, NB cells had maximal production of EPO with lower partial pressure of O2 values than the well-characterized hepatoma cell line HepG2. This maximal EPO expression was preceded by accumulation of the O2-sensitive α subunit of the heterodimeric transcription-factor complex hypoxia-inducible factor 1 (HIF-1). Western blot analysis revealed that the amount of the β subunit of HIF-1, identical to aryl hydrocarbon receptor nuclear translocator 1 (ARNT1), and the homolog ARNT2 increased in nuclear extracts from SH-SY5Y cells exposed to anoxia. In neuronal cells, ARNT1 and ARNT2 can form a heterodimer with HIF-1α, generating a functional HIF-1 complex. Using the hypoxia response element of the human EPO enhancer, we conducted electrophoretic mobility shift assays that showed accumulation and binding of HIF-1 complexes containing both ARNT1 and ARNT2 in NB cells. In addition to the HIF-1 complex, hepatocyte nuclear factor 4α (HNF4α) was found to be indispensable for hypoxia-induced EPO gene expression in hepatoma cells. Western blot analysis and polymerase chain reaction assessment showed that NB cells express neither HNF4α nor the splicing variant HNF4α7 and thus express EPO in an HNF4α-independent manner. Together, SH-SY5Y and Kelly cells may provide a new in vitro model for studying the mechanism of tissue-specific, hypoxia-inducible EPO gene expression.

Introduction:A common genetic variant within the TCRA-TCRD locus has been recently identified as a predictive factor of thymic function and T cell repertoire diversity (Clave et al., 2018). Specifically it was shown in a mouse model that transplantation of rs2204985 AA human hematopoietic stem cells (HSC) into immunodeficient mice led to lower thymocyte counts and poorer TCR diversity. T cell mediated pathways are known to play a significant role in immunological processes affecting HSCT outcome like GvL, GvH and infection. Aim of this study was to investigate the potential impact of donor rs2204985 genotype on patient's outcome after unrelated HSCT.

Mycobacterium avium-intracellulare(MAI) is an opportunistic pathogen commonly found in acquired immunodeficiency syndrome patients, whose immune systems are severely compromised. However, normal responses to this bacterium are apparently sufficient to prevent disseminated infection because disease is rarely found unless an immunocompromised state is present. Because interleukin-6 (IL-6) is an inflammatory cytokine with a multitude of activities, we investigated the potential of MAI to induce IL-6 from normal human leukocytes. Peripheral blood mononuclear cells were fractionated into monocytes (Mo), large granular lymphocytes (LGL), and T cells and stimulated with bacteria. Culture supernatants were collected and assayed for IL-6 activity by bioassay. Mo and LGL, but not T cells, were found to release IL-6 within 12 hours of stimulation, with optimal production occurring by 2 days of culture. Production of IL-6 from human leukocyte subsets was confirmed by Northern blot analysis and by neutralization of biologic function of the culture supernatants with specific antisera. Taken together, these results indicate that production of IL-6 is a key response of Mo and LGL to MAI. The role of IL-6 in MAI infection, therefore, needs to be further investigated.

Mycobacterium avium-intracellulare (MAI) is an opportunistic pathogen commonly found in acquired immunodeficiency syndrome patients, whose immune systems are severely compromised. However, normal responses to this bacterium are apparently sufficient to prevent disseminated infection because disease is rarely found unless an immunocompromised state is present. Because interleukin-6 (IL-6) is an inflammatory cytokine with a multitude of activities, we investigated the potential of MAI to induce IL-6 from normal human leukocytes. Peripheral blood mononuclear cells were fractionated into monocytes (Mo), large granular lymphocytes (LGL), and T cells and stimulated with bacteria. Culture supernatants were collected and assayed for IL-6 activity by bioassay. Mo and LGL, but not T cells, were found to release IL-6 within 12 hours of stimulation, with optimal production occurring by 2 days of culture. Production of IL-6 from human leukocyte subsets was confirmed by Northern blot analysis and by neutralization of biologic function of the culture supernatants with specific antisera. Taken together, these results indicate that production of IL-6 is a key response of Mo and LGL to MAI. The role of IL-6 in MAI infection, therefore, needs to be further investigated.

Based on animal experiments, a switch of the erythropoietin (EPO) production site from the liver in the fetus to the kidneys in the adult has been postulated. To study the switch in humans, we have quantitated EPO mRNA expression in liver, kidney, spleen, and bone marrow of human fetuses and neonates by means of a competitive polymerase chain reaction (PCR). Tissue samples from 66 routine postmortem examinations were obtained. EPO mRNA was expressed in 97% of the tissue specimen derived from the liver (n = 66) and in 93% of those from the kidneys (17 weeks of gestation until 18 months after birth; n = 59). For the first time the EPO gene was found expressed in vivo in human spleen (96% of 64 samples) and in fetal and neonatal bone marrow (81% of 21 samples). EPO mRNA expression in the kidneys increased significantly beyond 30 weeks of gestation (P < .05). Although there was a slight decrease in EPO mRNA content per g liver tissue towards birth, the liver accounted for about 80% of the total body EPO mRNA. The contribution of the spleen and bone marrow were minor compared with liver and kidneys. Our results indicate that in humans the liver is the primary site of EPO gene expression not only in fetal, but also in neonatal life. A significant increase of renal EPO mRNA expression after 30 weeks of gestation might indicate the beginning switch. © 1998 by The American Society of Hematology.

Hillengass: Novartis: Research Funding; Sanofi: Research Funding; BMS: Honoraria; Celgene: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Goldschmidt:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Durie:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

Myelodysplastic syndromes (MDS) are mainly characterized by dyserythropoiesis resulting in anemia. So far, this pathological hallmark is incompletely understood. Notch signalling has recently been linked to impaired erythropoiesis and megakaryopoiesis of CD34+ progenitor cells, however its role in MDS is unclear. On the other hand, in MDS demethylating therapy results in decreased transfusion dependency, indicating aberrant methylation of key erythroid genes. Therefore, we have analyzed Notch pathway elements and its association with the key erythroid factors GATA1 and BCLxl and examined the methylation of CpGs flanking cis-regulatory elements (including N-box suppressor binding site for HES1 and GATA box) of the GATA1 erythroid promoter in differentiating CD34+ cells selected from MDS patients. We have generated an in-vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ bone marrow cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO. Cell harvest was at days 0, 4, 7 and 11. RNA-expression of GATA1, BCLxl, DLK1, Notch1, HES1 and HERP2 was measured by real time RT-PCR (qPCR). GPI was used as a housekeeping gene. DNA methylation at 7 CpGs of the GATA1 gene promoter was quantitatively analyzed by Pyrosequencing (Pyro Mark ID, Biotage, Uppsala, Sweden) of bisulfite treated genomic DNA at any specific time point. In normal erythropoietic cells, RNA expression of GATA1 and of BCLxl was steadily up regulated, particularly during late erythropoietic differentiation. In contrast, during MDS erythropoiesis a loss of typical up regulation of GATA1 (day 11: 2.08 vs. 0.11; p=0.001) and BCLxl (day 11: 7.46 vs. 0.16; p=0.0005) was observed. Notch ligand DLK1 showed increased expression during erythropoiesis particularly in high risk MDS as compared to normal controls (days 4–11: 0.38 vs. 0.03; p=0.02). Furthermore, expression of HES1 was increasing during the course of normal erythropoietic differentiation but not in lineage specific cells from MDS patients (day 11: 0.01 vs. 0.006; p=0.1). No hypomethylation of CpGs flanking repressor HES1 binding site within the 5′-GATA region was detected in MDS erythropoiesis. Interestingly, decremental GATA1 promotor methylation values were seen during normal erythropoiesis matching GATA1 RNA up regulation in contrast to MDS erythropoiesis (day 11: 26% vs. 58%; p=0.00004). Our data show that the critical erythropoietic transcription factor GATA1 as well as the antiapoptotic molecule BCLxl fail to be up regulated during MDS erythropoiesis. The higher residual 5′-GATA1 methylation values in MDS erythropoiesis but decremental loss thereof in normal erythropoiesis suggests a gene dose effect for GATA1 during erythropoiesis being finely tuned by CpG methylation. Its dysregulation may contribute to the ineffective erythropoiesis observed in MDS. However, a transcriptional activation of the Notch pathway leading to increased expression of the GATA1 repressor HES1 and a hypomethylation of its binding site could not be detected in MDS.

Notch signals have recently been shown to inhibit erythroid and megakaryocytic differentiation of hematopoietic progenitor cells. In myelodysplastic syndrome (MDS) its role in dyserythropoiesis has not been fully elucidated. Therefore we asked whether dysregulation of Notch pathway elements might be associated with impaired GATA1 and BCLxl expression and ineffective erythropoiesis being a hallmark of MDS hematopoiesis. We have generated an in-vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ bone marrow cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO and TPO. Cell harvest was at days 0, 4, 7 and 11. Expression of GATA1, BCLxl, DLK1, Notch1, HES1 and HERP2 was measured by real time RT-PCR (qPCR). RNA expression of GATA1 and of BCLxl was steadily upregulated, particularly during late normal erythropoiesis. During normal megakaryopoiesis expression of both genes was up to 50 times lower as compared to normal erythropoiesis. In contrast, during MDS erythropoiesis a loss of typical late upregulation of GATA1 and BCLxl was observed. DLK1 expression during erythropoiesis showed increased expression particularly in high risk MDS vs. normal controls. Expression of HES1 was increasing during the course of normal erythropoietic and megakaryopoietic differentiation but not in lineage specific cells from MDS patients. In conclusion our data show that the central erythropoietic transcription factor GATA1 and the associated antiapoptotic molecule BCLxl are markedly downregulated during MDS erythropoiesis which may contribute to the ineffective erythropoiesis seen in this disease. Increased DLK1 expression in differentiated stem cells from high risk MDS patients was seen. However, an upregulation of the Notch pathway leading to increased expression of the GATA1 repressor HES1 could not be detected.

Recent findings suggest that in myelodysplastic syndrome (MDS) several key regulatory genes are affected by aberrant promotor methylation. To explore the molecular basis of this impairment we have generated an in vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Cell harvest was at days 0, 4, 7 and 11. Promotor methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite treated genomic DNA for each lineage at each time point. In addition, expression of DNMT1 (maintenance DNA methyltransferase), DNMT3a and DNMT3b (both de novo DNA methyltransferase) was analyzed by real time RT-PCR and correlated with gene promotor methylation at any time point. DNMT1 expression was increased during erythropoiesis in both, normal controls and MDS patients. On the other hand, expression of de novo DNMTs was elevated during thrombopoiesis at all time points. During erythropoiesis hypermethylation of p73, hMLH1 and RARb was associated with elevated DNMT1, hypermethylation of p15, p16, p73 and survivin was positively associated with increasing DNMT3 expression. Interestingly, DNMT1 was only elevated in low risk MDS, but not further increased in high risk MDS patients. Surprisingly, MDS specific survivin promotor methylation was inverse correlated with DNTM1 and DNMT3a expression. However, a negative correlation of DNMT3a with survivin expression was found in low risk MDS but not in high risk MDS. In summary our data indicate that all mammalian DNMT isoforms may be involved in the aberrant DNA-methylation phenotype in MDS. Elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in low risk MDS. DNMT3a and 3b were elevated during megakaryopoiesis and their expression was inversely correlated with MDS disease risk (IPSS). We conclude that the knowledge about distinct expression patterns of DNMT isoforms in hematopoiesis may be of help for further strategies to implicate DNMT-inhibitors in the treatment of patients with MDS.

Survivin has recently been shown to critically regulate erythroid versus megakaryocytic differentiation in mouse hematopoietic stem cells. Its role in human hematopoiesis has not been fully elucidated yet. To answer the question whether dysregulation of expression of survivin and its protein stabilisator HSP90 contributes to the ineffective erythropoiesis in myelodysplastic syndrome (MDS), we have generated an in vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Cell harvest was at days 0, 4, 7 and 11. Expression analysis of survivin splicing variants (wt, 2a, d3ex) and of HSP90 was done by real time RT-PCR (qPCR) for each lineage at each time point. Furthermore, epigenetic analysis of key regulatory genes by methylation specific PCR and qPCR to detect DNMT1 (maintenance methylation) and DNMT3a and 3b (de novo methylation) expression was performed for each of the experimental conditions. RNA expression of all survivin isoforms and of HSP90 was continously upregulated during normal erythropoiesis. During late megakaryopoiesis of normal hematopoietic stem cells a significant downregulation was seen with elevated expression values for survivin wt and survivin 2b during early thrombopoiesis. In contrast, during MDS erythropoiesis a significant downregulation of expression of all survivin isoforms and HSP90 during late erythropoiesis was observed. Lower expression for survivin wt, survivin 2b and HSP90 was seen during early MDS megakaryopoiesis. Promotor methylation analyisis revealed MDS specific aberrant methylation for survivin, particularly during MDS high risk erythropoiesis (p=0.02). However, expression of DNMT1 was only elevated during erythropoiesis and in low risk MDS. A significant inverse correlation between RNA expression of DNMT enzymes and survivin splicing variants could not be detected. In conclusion our data support the essential role for survivin in regulation of erythropoietic versus megakaryopoietic differentiation of hematopoietic precursor cells. The dysregulation of survivin expression during MDS erythropoiesis may therefore account for the ineffective erythropoiesis known in MDS. Furthermore, downregulation of survivin (protein) stabilisator HSP90 might additionally contribute to this effect. Although an inverse correlation with overexpression of DNMTs and downregulation of survivin splicing variants was not seen, the aberrant survivin promoter methylation during MDS erythropoiesis indicates that epigenetic dysregulation results in low survivin expression.

Rationale:No published randomized trials have compared low molecular weight heparin (LMWH) to unfractionated heparin (UFH) for thromboprophylaxis in medical-surgical ICU patients.