bluuebunny/crossref_metadata_2025 huggingface dataset:
CALM-AF10 Positive T-ALLs Show a Pattern of Expression Similar to MLL-Translocated Acute Leukemias.
Authors: Delabesse Eric , Dik Wim A. , Brahim Wajih , Braun Charlene , Asnafi Vahid , Van Dongen Jacques J.M. , Langerak Anton W. , Macintyre Elizabeth A.
Published: April 2020

The t(10;11) translocation is recurrent in T-ALL and AML. The AF10 gene on chromosome 10 is rearranged either with MLL or CALM located on chromosome 11. CALM-AF10 fusion gene is found in T-ALLs in immature (IM) and TCRγδ-expressing (TCRGD+) T-ALLs. We compared 6 CALM-AF10+ T-ALLs cases (4 IM, 2 TCRGD+) to 17 CALM-AF10 negative T-ALLs cases (14 IM, 3 TCRGD+) using Affymetrix U133A microarrays.

44 genes were significantly overexpressed in CALM-AF10+ T-ALLs, the most significant being HOXA9, a homeobox gene overexpressed in MLL-translocated acute leukemias (MLL-t AL), BMI1, a polycomb family member whose function in regulation of HOX genes expression is opposite to Trithorax genes (whose MLL belongs), SOX4, a frequent insertion site in retroviral-induced leukemogenesis, SFRS6 and COMMD3 (p≤0.001). Only two other HOX genes, HOXA5 and HOXA10, were significantly increased. 89 genes were significantly underexpressed in CALM-AF10+ T-ALLs, the most significant being GGH, ARL6IP4, NBS1, OGFR and TUBB (p≤0.001).

An independent analysis of the expression of HOXA5, HOXA9, HOXA10 and BMI1 genes was done by quantitative RT-PCR in 10 CALM-AF10+ T-ALLs and 27 CALM-AF10 negative T-ALLs. These were compared to 19 MLL-translocated acute leukemias (2 MLL-AF10, 5 MLL-AF4, 3 MLL-AF6, 5 MLL-AF9, 3 MLL-ELL and 1 MLL-ENL), since HOXA9 overexpression had been previously associated with MLL-t AL.

HOXA5, HOXA9 and HOXA10 expression were higher in CALM-AF10+ T-ALLs than in CALM-AF10 negative T-ALLs (p<0.001), confirming the microarray results. HOXA5 and HOXA9 expressions in CALM-AF10+ T-ALLs were similar to those detected in MLL-t AL and lower for HOXA10 in CALM-AF10+ T-ALLs as compared to the values of MLL-t AL (p=0.008). BMI1 expression in CALM-AF10+ T-ALLs was significantly higher than in CALM-AF10 negative T-ALLs and MLL-t AL (p<0.001).

Additionally, MEIS1 expression was determined as this gene was associated in MLL-t AL with the overexpression of HOXA9. As for BMI1, MEIS1 expression was significantly higher in CALM-AF10+ T-ALLs compared to CALM-AF10 negative T-ALLs and MLL-t AL (p<0.001 and p=0.019, respectively).

In summary, we demonstrated here the association between CALM-AF10 in T-ALLs and overexpression of HOXA5, HOXA9, HOXA10, BMI1 and MEIS1 genes. Overexpression of BMI1 is restricted to CALM-AF10+ T-ALLs. Although no obvious similarities are apparent between MLL and CALM proteins, the activation of HOXA and MEIS1 genes represent a highly recurrent pattern of expression in CALM-AF10+ T-ALLs and MLL-t AL. Consequently, the leukemias resulting in the activation of HOXA9 (MLL-t AL, CALM-AF10+ AL, NUP98-HOXA9 AML) should be seen as an independent group of acute leukemias and may benefit from common therapeutic protocols.
Identification of 41 Novel Promoter-Associated CpG Islands Methylated in Leukemias.
Authors: Jelinek Jaroslav , Mannari Rajan , Issa Jean-Pierre
Published: April 2020

DNA methylation within promoter-associated CpG islands is a well-recognized mechanism of gene silencing and plays an important role in the development of malignancies. CpG dinucleotides in human DNA are methylated at 5′-cytosine with the exception of areas with dense concentration of CpGs (CpG islands) located in gene promoter regions. In cancer cells, methylation of CpG islands in promoter regions of tumor suppressor genes is a frequent epigenetic change with a gene-silencing effect analogous to inactivating mutations. Methylation profiling can identify biologically and clinically distinct tumor subgroups by mapping the methylation status of multiple genes, and reports in AML and ALL suggest associations between methylation and poor prognosis. Identification of methylated CpG islands can shed new light on the biology of leukemia. We used Methylated CpG Island Amplification coupled with Representative Difference Analysis (MCA-RDA) as a genome-wide screen for promoter-associated CpG islands methylated in leukemic and/or myeloproliferative cell lines and primary malignant cells, but unmethylated in blood cells from normal controls. We identified 51 unique promoter-associated CpG islands in 321 sequenced clones recovered by MCA-RDA. Forty-one CpG islands belonged to known genes, and 10 to annotated mRNAs. Of the genes with known function, 8 are involved in signaling, 7 in transcription, 3 in dephosphorylation, 2 in oxido-reductive processes, 2 in NO synthesis, 2 in adhesion, 2 in solute transport, and 2 in DNA replication. Seven out of the 51 genes were previously reported as methylated in cancer or leukemia (CDH13, HLA-B, HLA-C, PGR, SCGB3A1, SLC26A4, TERT), thus validating the MCA-RDA approach. Of the 41 new hypermethylated CpG islands recovered, 20 corresponded to genes of known function. Published data infer an association with cancer for 10 of these genes (CTDSPL, ECGF1, EDG4, FOXD2, NOR1, NOS3, OLIG2, SLC16A1, TLE1, WNT5B), and no reports were found for the other 10 genes (CNR1, FADS, FBXW3, FGD1, NPM2, P518, PDE4DIP, SNCB, TCEA3, VENTX2). To further validate our findings we are assessing the methylation status of these genes by bisulfite pyrosequencing. Analyses of the bone marrow samples from AML, ALL, CML and MDS patients are ongoing. Our preliminary data confirm methylation of H-cadherin precursor (CDH13), progesterone receptor (PGR) in AML and ALL and cannabinoid receptor 1 (CNR1) in ALL (Table). In conclusion, MCA-RDA identified methylation of 41 new and 10 previously reported promoter-associated CpG islands in leukemia. Functional studies of these may shed new light on the biology of leukemias, and these genes may be useful for methylation profiling of leukemias for prognosis and response to treatment.

Promoter CpG Island Methylation

Gene AML ALL Methylation levels over 10% for CDH13 and PGR and over 25% for CNR1 were scored as positive. CDH13 5/23 22% 12/19 63% PGR A Isoform 11/22 50% 12/18 67% PGR B Isoform 17/24 71% 5/13 38% CNR1 0/24 0% 3/18 17%
Inhibition of Retinoic Acid Receptor Signaling by SKI in Acute Myeloid Leukemia.
Authors: Ritter Markus , Samuelsson Magnus , Hartmann Oliver , Burchert Andreas , Kim Theo D. , Thiede Christian , Hayman Micheal J. , Ueki Nobuhide , Eilers Martin , Neubauer Andreas
Published: April 2020

Objectives: Acute myeloid leukemia (AML) with monosomy 7 or deletion of the long arm of chromosome 7 (-7/7q-) is a leukemia with a poor outcome. Microarray analysis resulted in a gene expression profile specific for this subset of AML and showed SKI, a nuclear Co-repressor protein, being strongly upregulated. SKI is described to be a transforming gene, which inhibits TGF-beta-, SMAD- and retinoic acid (RA)-signaling.

Materials, methods and patients: Gene expression profiling was performed using cDNA microarrays (detecting 4608 genes). Gene expression pattern of 20 AML (9 pts. with -7/7q- and 11 pts. with normal karyotype) patients was compared to 23 healthy bone marrow donors. Expression data of interestings genes were confirmed by real-time PCR in samples of 111 AML patients. In cell culture experiments HL60 and U937 leukemic cell lines were used to investigate the influence of the identified oncogene Ski on differentiating properties. In a reporter gene assay retinoic acid response element (RARE) was transfected in QT6 cells. U937 were retrovirally transfected with Ski wild type and Ski mutant.

Results: Microarray analysis in AML (-7/7q-), revealed SKI being upregulated in AML. The group of patients with a high SKI expression level had a poorer overall survival when compared to the low expressers (p = 0.0279). Real-time PCR analysis comparing 111 AML samples to healthy CD34+ haematopoietic stem cells revealed, that SKI levels were highest in AML with -7/7q-, moderately elevated in most other karyotypes except AML with translocations involving retinoic acid receptor alpha (RARalpha). This suggests that Ski might interfere with RARalpha function in leukemia. Using reporter gene assays we could show that expression of wild-type, but not mutated SKI blocked RA induced differentiation. SKI was down regulated in HL60 leukemia cell line when differentiation was induced by ATRA and or valproic acid.

Conclusion: Upregulation of SKI has prognostic significance in AML. Inhibition of RA signaling may be more often implicated in pathogenesis of AML than anticipated so far. The Co-repressor of histone deacetylases (HDACs) Ski might therefore become a molecular target for the induction of differentiation in the treatment of AML.
Treosulfan-Based Myeloablative Conditioning Prior to Allogeneic Hematopoietic Cell Transplantation Is Associated with Reduced Toxicity without Increased Risk of Relapse.
Authors: Giebel Sebastian , Wojnar Jerzy , Krawczyk-Kulis Malgorzata , Wylezol Iwona , Markiewicz Miroslaw , Kruzel Tomasz , Holowiecki Jerzy
Published: April 2020

The goal of this study was to evaluate toxicity and efficacy of allogeneic hematopoietic cell transplantation (alloHCT) with Treosulfan (alkylyting agent, soluble Busulfan-derivative)-based conditioning as a preparative regimen. The outcome of 27 patients (CML-15, AML-9, ALL-1, SAA-2) given Treosulfan 3x14 g/m2 + Fludarabine 5x30 mg/m2 (n=15) or cyclofosfamide (CTX) 120 mg/kg (n=12) was compared with that of 146 patients treated with Busulfan 16 mg/kg + CTX 120 mg/kg between 2000–2004. In case of unrelated donor (URD)-HCT patients were additionally given anti-thymocyte globulin. GVHD prophylaxis consisted of cyclosporin A and short-course Methotrexate. The indications for alloHSCT were comparable for both subgroups. The patients age was 35(14–56)y and 30(14–55)y, the proportion of URD-HCT was 55% and 45%, peripheral blood was used as a source of stem cells in 33% and 11% of cases, and the proportion of patients with advanced disease (AML and ALL >CR1; CML > CP1) equaled 26% and 19%, respectively.

The cumulative incidence of non-relapse mortality at one year was 8% for the Treosulfan (EBV-LPD n=1, cerebral hemorrhage n=1) and 32% for the Busulfan group (p=0.11). Grade II-IV neutropenic infections occurred in 5% and 13% (p=NS) and grade II-IV mucositis in 9% and 61% of patients (p<0.001), respectively. Other serious adverse events were infrequent. Both subgroups did not differ in terms of time to neutrophil and platelet recovery, as well as for need of RBC and platelet transfusions. Median hospital stay since the date of alloHCT was shorter after Treosulfan- compared to Busulfan-based conditioning (31 (21–55) d. vs. 40.5 (21–153) d., p<0.001).

The overall survival and disease-free survival at one year equaled 92% vs. 67% (p=0.11), and 92% vs. 66% (p=0.09) for the Treosulfan group and the Busulfan group, respectively. None of the patients experienced hematologic relapse among Treosulfan- compared to 3% among Busulfan-treated alloHCT recipients. However, in two CML patients given Treosulfan-based regimen, the immunosuppression taper and interferon or imatinib therapy was neccessary to establish complete chimera. We conclude that Treosulfan + Fludarabine (or CTX) +/− ATG conditioning regimen is characterized by reduced toxicity resulting in shorter hospital stay and low transplat-related mortality. The pattern of hematopoietic recovery is similar to that of Busulfan + CTX indicating myeloablative character of the Treosulfan-based regimen. The risk of relapse is low and comparable for both kinds of treatment.
NOD-Rag1nullPrf1null Mice Support Long-Term Engraftment of Human Hematopoietic Stem Cells.
Authors: Minamiguchi Hitoshi , Livingston Anne G. , Wingard John R. , Shultz Leonard D. , Ogawa Makio
Published: April 2020

NOD-scid mice have been widely used as recipients in the xenograft assay for human hematopoietic stem cells (HSCs). One major problem with the strain is the low level of engraftment except when large numbers of cells are injected. This is probably caused by the presence of residual natural killer (NK) cell cytotoxic activity. NOD-scid/beta 2 microglobulin (B2m)null mice have been reported to have reduced NK cell cytotoxic activity and support higher levels of human cell engraftment. However, use of this strain of mice is limited by their difficulty in breeding and short life span caused by early development of lymphomas, which is accelerated by irradiation. Another immune-incompetent mouse model, NOD-recombination activating gene (Rag1)null mice allow longer observation of human cell engraftment than NOD-scid mice and are easier to breed. Genetic crossing of perforin (Prf) structure gene-targeted mutation onto NOD-Rag1null strain results in absence of NK cell cytotoxic function. In these mice, NK cells are not capable of killing target cells because of the absence of Prf, the major mediator of cytotoxic activity. We have tested the use of NOD-Rag1nullPrf1null mice as recipients of long-term xenograft assay for human HSCs by adopting Yoder’s method of conditioning newborn mice with minor modifications. Pregnant NOD-Rag1nullPrf1null dams were treated with 22.5mg/kg busulfan in 20% dimethylsulfoxide in Hank’s BSS on day 17.5 and 18.5 pc via subcutaneous injection. On the day of delivery, mononuclear cells (MNCs) were isolated from human cord blood (n=3) by density gradient centrifugation and T cell-depleted MNCs were separated by using mouse anti-human CD3, CD4, and CD8 antibodies and sheep anti-mouse IgG immunomagnetic beads to prevent preferential T cell engraftment. The busulfan-exposed pups were transplanted with 4–5 million T cell-depleted MNCs via the facial vein. At 6 months post-transplantation, human cells were detected in the bone marrow of 4 out of 10 transplanted mice. The levels of human CD45+ cells in the bone marrow of engrafted mice were 79.9, 69.8, 60.5, and 7.4%, and those in the peripheral blood were 6.3, 5.8, 4.1, and 1.3%. Multilineage engraftment was confirmed by phenotypic analysis. Next, we tested the hypothesis that human cord blood HSCs have dye efflux activity by injecting T cell-depleted Rhodamine 123 (Rho)− or + cells into conditioned newborn NOD-Rag1nullPrf1null mice. Six-month engraftment was found only with the Rho− cells. Thus, conditioned newborn NOD-Rag1nullPrf1null mice provide an excellent model for assaying long-term engrafting human HSCs.
Recombinant Tpo Stimulates Maturation of Megakaryocytes Derived from Adult Peripheral Blood- but Not Cord Blood-CD34+ Cells.
Authors: Pastos Karen M. , Slayton William B. , Rimsza Lisa M. , Sola Martha C.
Published: April 2020

Umbilical cord blood (CB) is a valuable source of stem cells for transplantation. However, platelet engraftment is slow, taking approximately 70 days for CB transplants versus 20 days for mobilized adult peripheral blood (PB) transplants. This time is not significantly shortened by the administration of recombinant thrombopoietin (rTpo). The cause for the delay in platelet engraftment following CB transplant is unknown. We hypothesized that developmental differences in size and ploidy of neonatal versus adult megakaryocytes (MKs) contribute to this delay. To mimic these two types of transplant in vitro, we compared CB to PB CD34+ cells cultured in adult bone marrow stromal-conditioned media (CM) or unconditioned media (UCM) for 14 days. Increasing doses of rTpo were added to the CM, and the resulting MK maturation was compared with that of UCM with maximal rTpo concentration. MK number and ploidy were determined by flow cytometry using CD41-FITC and propidium iodide, respectively. Increased ploidy levels were expressed as percentage of MKs with a ploidy ≥ 8N. Results represent an average of three independent experiments.

Figure Figure

As seen in the figure, PB-derived MKs reached highest ploidy levels in the presence of UCM + 100 ng/ml rTpo. When cultured in CM, they exhibited lower ploidy levels, regardless of Tpo concentration. In contrast, CB-derived MKs exhibited higher ploidy levels in response to CM with either 0 or 0.1 ng/ml (physiologic concentration) of rTpo, as compared to higher rTpo concentrations or UCM + 100 ng/ml rTpo. MK numbers increased in response to rTpo in a dose-response manner, regardless of the source of the MKs (data not shown). These results indicate that intrinsic differences between CB- and PB-derived megakaryocytes exist, and that maturation is regulated differently in neonatal versus adult MKs. While Tpo is a potent stimulator of MK maturation in PB-derived MKs, it appears to inhibit this process in CB-derived MKs. These differences may be relevant to understanding the delayed platelet engraftment following CB transplants.
Homing Efficiency in Two Models of Submyeloablative Conditioning and Correlation with Long-Term Engraftment.
Authors: Meyers Stephen E. , Meyers Justin L. , Goebel W. Scott
Published: April 2020

Submyeloblative conditioning regimens for bone marrow transplantation (BMT) are desirable to reduce host toxicity in many settings. We previously demonstrated that hosts conditioned with 160 cGy or with two sequential doses of the antimetabolite 5-fluorouracil (5-FU) displayed ~50% and ~35% donor chimerism 6 months after transplantation of 20 x 106 fresh congenic bone marrow (BM) donor cells, respectively. Donor chimerism in 160 cGy- or 5-FU-conditioned mice was proportional to the reduction in host long-term repopulating ability (LTRA) caused by the conditioning regimen (~80–90% vs. ~55%, respectively). In this study, we sought to determine if donor marrow homing efficiency also correlated with long-term engraftment in these models of submyeloablative conditioning. We hypothesized that sequential doses of the cell cycle-specific agent 5-FU (5-FU x 2) may damage the marrow microenvironment less than low-dose radiation, resulting in more efficient homing in 5-FU x 2-conditioned mice. C57Bl6/J (Bl/6) hosts were conditioned with either 5-FU x 2 administered on days -5 and -1 (150 mg/kg IP) or with 160 cGy given the day of transplantation (day 0). Bl/6 (CD 45.2+) hosts were transplanted with 20 x 106 PKH-26 stained Bl/6 or with Boy J (CD 45.1+) whole BM cells via tail vein injection. The percentage of homed cells 20 hours after BMT in 5-FU x 2-conditioned mice was 3.53 % ± 3.1% (n = 13 from 3 independent experiments), while the percentage of homed cells in the 160 cGy group was 8.67 % ± 2.1% (n = 12; p = 0.0375). The fraction of donor cells which homed to the spleen or remained in the peripheral blood after 20 hours was similar between conditioning regimens. This difference in homing efficiency led us to consider other possible explanations as to why BM cells home less efficiently in 5-FU x 2-conditioned hosts than in low-dose irradiated mice. We hypothesized that 5-FU x 2 may have a more injurious effect on the marrow microenvironment than previously thought. Examination of marrow stromal cell potential using a colony-forming unit-fibroblast (CFU-F) assay revealed that marrow from 5-FU x 2-treated mice (n = 3) contained 1.1 ± 0.4 colonies/marrow femur equivalent (FE), whereas marrow from 160 cGy-treated mice (n = 3) contained 33.8 ± 13.4 colonies/FE. In comparison, marrow from untreated mice (n = 3) averaged 95.6 ± 27.5 colonies/FE. These data suggest that homing efficiency correlates with donor chimerism in these two models of submyeloablative conditioning, and establish at least two potential mechanisms (a lesser reduction of host LTRA, and decreased donor cell homing efficiency) for the lower level of long-term donor chimerism observed in 5-FU x 2- compared to 160 cGy-conditioned hosts. Furthermore, 5-FU x 2 conditioning appears to disrupt the marrow microenvironment to a greater degree than low-dose radiation. Current efforts are aimed at exploring the molecular interactions of homed donor cells with conditioned marrow.
Donor CD8+ T Cells Facilitate Induction of Chimerism and Tolerance without GVHD in Autoimmune NOD Mice Conditioned with Anti-CD3 mAb.
Authors: Liang Yaming , Huang Tammy , Zhang Chunyan , Todorov Ivan , Atkinson Mark , Kandeel Fouad , Forman Stephen , Zeng Defu
Published: April 2020

Prevention of autoimmune diabetes and induction of islet transplantation tolerance in NOD mice can be reached by induction of mixed chimerism via bone marrow transplantation (BMT), but this procedure requires total body irradiation (TBI)-conditioning of the recipients. The toxicity of radiation and potential for graft versus host disease (GVHD) prevents its clinical application. Donor CD8+ T cells play a critical role in facilitation of engraftment, but also contribute to induction of GVHD in TBI-conditioned recipients. Here, we showed that high doses of donor CD8+ T cells in combination with bone marrow (BM) cells induced mixed chimerism without GVHD in NOD recipients conditioned with anti-CD3 mAb. The prevention of GVHD in those recipients was associated with low-level production of inflammatory cytokines (i.e. TNF-α), high-level production of anti-inflammatory cytokines (i.e. IL-4 and IL-10), and confining of the donor CD8+ T cell expansion to lymphohematopoietic tissues. The chimeric NOD recipients showed donor specific tolerance and reversal of insulitis. These results demonstrate that donor CD8+ T cell-mediated facilitation of engraftment can be separated from GVHD in non-irradiated recipients. This regimen may have potential application in the treatment of autoimmune disorders as well as induction of transplantation tolerance.
GATA-1, NF-E2 and EKLF/SP1 Binding Elements Cooperate To Specify Patterns of Histone Hyper-Acetylation at β-Globin LCR HS Core Elements.
Authors: Ackley Catherine J. , Lowrey Christopher H.
Published: April 2020

The active elements of the β-globin LCR are necessary for high-level expression of the linked globin genes. These active elements are contained within regions of unique, erythroid-specific chromatin structure characterized by the formation of positioned nucleosomal arrays, areas of approximately 150–200 bp which are highly accessible for protein-DNA interactions (DNase I HSs) and hyperacetylation of histones H3 and H4. At a DNA level, each of the LCR HS cores contain NF-E2, EKLF/Sp1 and tandem, inverted GATA binding elements. Our lab has shown that all of these transcription factor-binding sites are necessary for HS formation. We have previously proposed a model of HS formation in which Sp1 and/or EKLF first bind to the core regions and alter local chromatin structure allowing GATA-1 and NF-E2 to bind and stabilize the HS core structure. Additional factors could then bind to the core and flanking regions. An important aspect of HS core formation that our experiments have not previously addressed is the mechanism of histone acetylation. Based on our previous findings and the fact that GATA-1 and NF-E2 can both interact with histone acetyltransferases (HATs), we hypothesized that histone acetylation at the HS requires the binding of EKLF/Sp1 followed by GATA-1 and/or NF-E2 and therefor should be a late event in HS formation. To test this hypothesis, we have generated a series of 11 artificial HS core elements based on the structure of human HS4. These plasmid-based constructs contain different combinations of the six binding sites in their normal arrangement and spacing: (EKLF/Sp1)-(NF-E2)-(EKLF/SP1)-(GATA-1 x 2)-(EKLF/Sp1). These constructs were stably transfected into MEL cells and assayed for their ability to mediate histone H3 and H4 acetylation as determined by the ChIP assay. The endogenous murine β-globin LCR HS3 served as a positive internal control for histone acetylation. Previous results from our lab showed that all six factor-binding sites within the HS core are required for HS formation. However, when assayed for histone acetylation, the NF-E2 and the tandem inverted GATA sites are able to independently direct H3 and H4 acetylation to between 10 and 50% of that seen at the native HS site. Combination of the NF-E2 and GATA-1 sites results in 89% of normal H4 acetylation. Full HH4 acetylation (98% of the wild-type HS) is only seen with all six factor binding sites. These results indicate that NF-E2 and GATA-1 direct most of the acetylation of LCR HS core histones but that EKLF binding is also likely to contribute. They also indicate that extensive histone acetylation can occur without HS formation. Finally, in contrast to our model of HS formation, GATA-1 and NF-E2 appear able to direct the acetylation of HS core histones without the presence of binding sites for Sp1 or EKLF.
Endogenous Elevations of Short Chain Fatty Acids Up-Regulate Embryonic Globin Gene Expression during Primitive Erythropoiesis.
Authors: Sterner Lauren , Miyazaki Toru , Swift Larry , Dean Ann , Little Jane
Published: April 2020

We examined the effects of short chain fatty acids (SCFAs) on globin gene expression during development. We studied globin gene expression in transgenic mice that have endogenous elevations in the SCFA propionate due to a knockout (KO) of the gene for propionyl CoA carboxylase subunit A (PCCA, Miyazaki et al. JBC, 2001 Sep 21;276(38):35995–9). Serum propionate levels measured by gas chromatography were 2.5 to 3.6 mgms/ml in 2 adult PCCA KO mice and were undetectable in 2 wild type (wt) or heterozygous control adult mice. Embryonic PCCA KO offspring had propionate levels of 2.3 and 5.0 μgms/100 mgms of fetal liver, at day 16.5 (E16.5), while wt or heterozygotes at E14.5 had levels <1 μgm/100 mgms. Analysis of expression from alpha (α), beta major (βmaj), embryonic beta-type epsilon-y (εy), embryonic beta-type beta H1 (βH1) and embryonic alpha-type zeta (ζ) globin genes plus 18S ribosomal RNA as a control was undertaken using real-time PCR with gene-specific primers and taqman probes. cDNA was reverse-transcribed from the mRNA of yolk sac (YS) and fetal liver of PCCA KO and wt progeny of more than one litter from timed pregnancies. Individual PCCA embryos at E10 (n=10), E12 (n=9), and E14 (n=7) were analyzed for globin gene expression, normalized to18S expression and were compared to age-matched wt embryos (n>=4 for each time point). As expected, embryonic alpha- and beta-type globin gene expression (ζ and βH1 plus εy) predominated in E 10 YS, and definitive globin gene expression, α and βmaj, predominated in E12 or E14 fetal liver. Expression from embryonic alpha-type globin was calculated as normalized ζ/(ζ+α) and from embryonic beta-type globins as normalized (βH1+εy)/(βH1+εy+βmaj), see table. Embryonic globin gene expression was statistically significantly increased in PCCA KO E12 YS at 1.3 fold relative to wt ζ and in PCCA KO E14 YS at 1.8 fold and 2.1 fold relative to wt ζ or βH1 and εy respectively (p<.05). No increase in embryonic globin mRNA was seen in adult PCCA KO animals. We conclude that elevations of SCFAs during normal murine development causes a persistence of both embryonic alpha-type and embryonic beta-type globin gene expression during primitive, but not definitive, erythropoiesis, suggesting that SCFAs cannot reactivate silenced murine embryonic globin genes in the absence of erythroid stress.

Embryonic Globin Gene Expression in Mice with Endogenous Elevations of SCFAs

% Expression PCCA KO wild type p value, t test E10 ζ Yolk Sac 53+/− 2 nd E10 βH1 & ε y Yolk Sac 99 +/− 0.3 nd E12 ζ Yolk Sac 32 +/− 3 25 +/− 1 p < .05 E12 βH1 & ε y Yolk Sac 77 +/− 6 74 +/− 3 ns E14 ζ Yolk Sac 7 +/− 1.5 4 +/− 1.4 p < .05 E14 βH1 & ε y Yolk Sac 13 +/− 6 6 +/− 0.5 p < .05 E12 ζ Fetal Liver 11 +/− 4 9 +/− 2 ns E12 βH1 & ε y Fetal Liver 13 +/− 5 13+/− 3 ns E14 ζ Fetal Liver 1 +/− 0.4 0.7 +/− 0.2 ns E14 βH1 & εy Fetal Liver 6 +/− 1.8 4 +/− 1 ns
Cooperative Silencing of Chicken ρ- and βH- Globin Gene Expression by Chicken β-Globin Regulatory Elements: Involvement of GATA-1, NF-E2, and RNA Polymerase II.
Authors: Wang Jin , Liu Hui , Epner Elliot
Published: April 2020

The chicken β-globin locus represents a well characterized, model system where the relationship between chromatin structure, transcription and DNA replication can be studied. The locus contains several regulatory elements including an intergenic enhancer as well as upstream regulatory elements that may function either alone or in combination with the intergenic enhancer as an LCR. The availability of the recombination proficient chicken B cell line DT40 has allowed the introduction of mutations into the endogenous chicken β-globin locus and phenotypic analysis after microcell mediated chromosome transfer into human erythroleukemia (K562) cells. Using this system, we have introduced deletions in the chicken β-globin intergenic enhancer as well as 5′ HS 1,2, and 3. A chicken chromosome marked by insertion of a selectable marker gene into the upstream folate receptor gene locus or 5′ HS1 followed by Cre recombinase mediated removal of the selectable marker gene served as control “wild type” chromosomes. Expression of the embryonic ρ and fetal βH chicken globin genes were repressed by the intergenic enhancer, 5′ HS1, or 5′HS2. No ρ or βH globin gene expression was detected in K562 cells containing control chicken chromosomes, while ρ and βH mRNA were activated when the intergenic enhancer, 5′ HS1, or 5′HS2 were deleted. Transcriptional activation of the ρ and βH genes correlated with hyperacetylation of histones H3 and H4, loss of methylated K9 histone H3, and binding of RNA polymerase II to the gene promoters. The status of methylated CpG dinucleotides at the promoters did not correlate with the transcriptional status of the genes. The phenotype of the 5′ HS1, and HS2 deletions were surprisingly similar to that of the intergenic enhancer, which suggested these elements may function either in the same pathway or complex. Chromatin immunoprecipitation (ChIP) experiments that assayed RNA polmerase II (pol II), GATA-1 and NF-E2 p45/ p18 binding at regulatory elements and gene promoters in targeted cell lines supported this hypothesis and suggested a potential role for 5′HS3 in gene activation. However, targeted deletion of 5′ HS3, unlike the other chicken β-globin regulatory elements, showed no transcriptional phenotype. Our results demonstrate the intergenic enhancer, 5′HS1, and 5′ HS2 function through a common silencing mechanism involving pol II, GATA-1, and NF-E2/P18.
Long-Range Enhancement of β-Globin Gene Expression Is Dependent on Gt Motifs Residing in the HS3 Core Element of the Locus Control Region.
Authors: Navas Patrick A. , Stergachis Andrew B. , Sheffer Hadar H. , Ye Xin , Stafford Mary , Stamatoyannopoulos George
Published: April 2020

Normal expression of the human β-globin genes is dependent on a powerful regulatory element residing upstream of the globin gene cluster referred as the locus control region (LCR), physically characterized by five DNAseI-hypersensitive sites (HS1 to HS5). Of particular interest is HS3, which is characterized by a 225 bp core element region containing seven GT motifs (GT1 to GT7) that alternate with four GATA-1 binding sites. The GT motifs are bound by a family of Kruppel-like factors and are important for proper expression of many housekeeping and tissue-specific genes. We previously demonstrated in transgenic mice carrying a 248 kb human β-globin yeast artificial chromosome in which the GT6 motif (GT6m β-YAC) was mutated resulted in a decrease in ε- and γ-globin gene expression during embryonic erythropoiesis and a decrease in γ-globin expression during definitive erythropoiesis, thus, providing evidence that a single transcriptional motif distantly located can have profound effects on gene expression. We have used the same β-YAC transgenic mouse model system to analyze the function of the remaining six GT motifs of the HS3 core element. Currently we have produced transgenic mice carrying either a mutation of GT1/2 (GT1 and GT2 motifs overlap) or GT3. Total RNA was isolated from yolk sac, liver and blood samples of transgenic F2 embryos at 12- and 14-day postconception and adult blood, and subjected to RNAse protection analysis. The GT1/2m β-YAC transgenic mice exhibited normal ε- and γ-globin gene expression during embryonic erythropoiesis, but during definitive erythropoiesis γ-globin gene expression was reduced 4 to 5 fold in day 12- and day 14- fetal livers. In the two GT1/2m β-YAC transgenic lines produced, β-globin gene expression was reduced and levels varied from 12.3% ± 2.9% to 63.2% ± 5.8% of endogenouse murine α-globin indicating that β-globin expression is strongly influenced by the position of integration of the transgene. In contrast, the GT3m β-YAC transgenic mice the mutation to GT3 had no effect on globin gene expression during development. Our results suggest that multiple GT motifs within the same highly complex regulatory element contribute differently to the enhancement of the downstream genes.
Keratinocyte Growth Factor (KGF) Is Required for Post-Natal Thymic Regeneration.
Authors: Alpdogan Onder , Hubbard Vanessa M. , Muriglan Stephanie J. , Kochman Adam A. , Eng Jeffrey M. , Zakrzewski Johannes L. , Goldberg Gabrielle L. , Smith Odette M. , Waldman Elisha , van den Brink Marcel R.M.
Published: April 2020

KGF is a fibroblast growth factor family member (FGF-7) that mediates epithelial cell proliferation and differentiation in a variety of tissues, including thymic epithelial cells. Recent studies have demonstrated that KGF administration (before conditioning) to the recipients of allogeneic bone marrow transplantation (BMT) can enhance T cell and thymic reconstitution. Therefore, we studied the role of KGF on T cell development under normal and stress conditions (such as irradiation) by using KGF (−/−) mice in experimental murine models. Phenotypic analysis of KGF (−/−) mice at varying ages demonstrated that the bone marrow, thymus and lymph node cellularity and the cell distribution among KGF (−/−), wild type (WT) and KGF (+/−) mice (6–11 weeks of age) were similar. However, splenic cellularity and splenic T cell numbers were slightly lower than WT and littermate controls. KGF (−/−) mice are more vulnerable to sublethal irradiation (450–600 cGy) and a more than six fold decrease was found in thymic cellularity when analyzed on day 28 after irradiation, whereas there was no delay in the erythroid, myeloid and platelet recovery after irradiation.

We used bone marrow transplantation models to assess the relative contribution of KGF produced by thymocytes or thymic epithelial cells during thymic regeneration after irradiation. T cell reconstitution was impaired in syngeneic or allogeneic KGF (−/−) BMT recipients in comparison to littermate controls but there was no difference in the distribution of thymocyte subsets or splenic T cell content. The recipients of KGF (−/−) bone marrow had decreased thymic and splenic cellularity after allogeneic BMT. These data demonstrate that both donor and host derived KGF play a role in thymic regeneration. Finally, KGF administration to young and old mice (10 month old) enhanced thymopoiesis when analyzed 28 days after KGF administration. Pre-BMT KGF administration to the recipients of syngeneic and allogeneic BMT also resulted in a 2–6 fold increase in the thymic cellularity compared to the control group.

We conclude that KGF produced by both thymocytes and epithelial cells is not required for normal and post-natal thymic development, but plays a role in post-natal thymic regeneration after irradiation. These data support the potential use of KGF to protect or restore thymic damage after chemo/radiotherapy.
Petreatment with Rituximab Reduces the Incidence of Chronic Graft-Versus-Host Disease after Allogeneic Stem Cell Transplantation in Patients with B Cell Lymphoma.
Authors: Bornhaeuser Martin , Schwerdtfeger Rainer , Kroschinsky Frank , Hanel Mathias , Treptow Uli , Theuser Catrin , Ehninger Gerhard
Published: April 2020

The use of anti-B cell antibodies before allogeneic hematopoietic stem cell transplantation (HSCT) induces a depletion of recipient B cells. This leads to a reduction of the number of potential antigen presenting cells and thereby might hamper the induction phase of graft-versus-host disease (GvHD). The humanized anti-CD20 antibody Rituximab has been used with increasing frequency to treat patients with B cell Non-Hodgkin’s Lymphoma. Only recently, several investigators have reported on the efficacy of Rituximab for the treatment of chronic GvHD. We therefore hypothesized, that patients who received Rituximab before being scheduled for allogeneic HSCT may have a decreased risk of GvHD.

A retrospective analysis in 72 patients after allogeneic hematopoietic stem cell transplantation for indolent (n=18), aggressive (n=45) B cell lymphoma or mantle cell lymphoma (n=9) was performed at two centres. Whereas 28 patients had received chemotherapy only before allogeneic HSCT, 44 patients had received 4–8 (median 6) cycles of 375 mg/m2 Rituximab before the start of conditioning therapy. Patient characteristics’ were comparable except for a higher proportion of unrelated donors (73% vs. 54%) and a higher median age (48 vs. 42 years) in the cohort with Rituximab pre-treatment. In addition the proportion of patients having received a previous autologous transplantation was higher (63% vs. 46%) and therefore more recipients received dose reduced conditioning before allogeneic tranplantation (70% vs. 50%) in the Rituximab group.

With a median follow-up of 24 months for all patients, no significant differences in overall survival, progression-free survival and treatment-related mortality could be detected between both groups. Although the proportion of transplants from unrelated donors was lower in the No-Rituximab group, the rate of acute GvHD grade I–IV was higher (88% vs. 63%, p=0.03). The incidence of grade II–IV and grade III–IV GvHD was not significantly different between both cohorts. Chronic GvHD occurred more often in patients who had not received Rituximab before allogeneic transplantation (52% vs. 23%, p=0.02).

Although the retrospective nature of our study does not allow to draw definitive conclusions, the rate of chronic GvHD seems to be lower when anti-CD20 treatment is performed before allogeneic HSCT. Prospective studies incorporating Rituximab for the prophylaxis and therapy of GvHD are warranted.
Steroid Refractory GVHD of the Gut Can Be Treated Effectively with Daclizumab and Sirolimus.
Authors: Weisser Martin , Goede Valentin , Schiel Xaver , Kolb Hans-Jochem
Published: April 2020

Steroid refractory GVHD is a major cause of morbidity and mortality after allogeneic transplantation. The response rates of grade III–IV GvHD to second line therapy are 20–30%, while long term survival is less than 10% (Arai et al. 2002). The monoclonal antibody Daclizumab targets the α-chain of the IL-2 receptor (CD25) , an early activation marker of TH-cells. We adminstered Daclizumab in combination with Sirolimus, an immunosuppressive agent that inhibits the signal transduction of IL-2 as second line therapy for servere acute GvHD of the gut. Thirty five patients with acute GvHD grade III and IV were evaluated. Thirteen patients had GvHD of the gut exclusively. Twenty-two patients suffered from GvHD of the gut and liver. Diagnosis of GvHD of the gut was performed by total colonoscopy including biopsy. Liver GvHD was diagnosed clinically. All patients had received and not responded to steroids (300 mg/d) for at least five days. Daclizumab (1 mg/kg) was given on days 1, 4, 8, 15, 21 and 28. Sirolimus was given orally. Sirolimus blood levels were targeted at 10–15 ng/ml. The underlying diseases were AML (18), ALL (9), CML (2), NHL and MMY (6). Twenty patients had an unrelated donor, 9 had a matched sibling donor and 6 had an haploidentical sibling donor. The stem cell source was bone marrow (6), peripheral stem cells (21) and both (6). In two patients GvHD developed after donor lymphocyte infusion. No adverse reactions related to infusion of the antibody were observed. Adverse side effects of Sirolimus included cytopenia and mucositis. These were especially observed with high Sirolimus blood levels. Viral reactivations occurred in 20 patients (8 x HHV6, 8 x EBV, 2 x CMV, 1 x Adenovirus and 1 x HSV). In four patients FACScan analysis of peripheral blood was performed to measure the CD25 expression on TH-cells. CD25/CD4 positivity ranged from 57–100% (median 69%) before the start of therapy. After the first antibody infusion CD25 was not detectable on TH-cells. Overall response to therapy was 48% (17/36) with 37% complete responders (13/35). Overall survival was significantly higher in those patients responding to Daclizumab/Sirolimus therapy (p<0.00001). In addition those patients that suffered from GvHD of the gut without liver affection had a significantly better prognosis. The 2 year overall survival after transplantation of patients with isolated GvHD of the gut was 45% versus 5% of the patients with gut plus liver GvHD (p=0.0001). Overall 74% of the patients (26/35) died. Five patients died of relapsing acute leukaemia, 8 of GvHD and 13 of infective complications. Taken together this analysis demonstrates that 1) GvHD of the gut can be treated effectively with Daclizumab and Sirolimus, 2) isolated GvHD of the gut has a signifcantly higher overall survival than GvHD of the gut and liver, 3) combination of Daclizumab and Sirolimus provides potent immunosuppression and patients should be monitored cautiously for infectious complications and relapse.
Improved Immune Reconstitution with Low Incidence of Severe Graft-Versus-Host Disease after Transplantation of CD34+ or CD133+ Enriched Stem Cells with Add-Back of Ten Million T-Cells Per Kg.
Authors: Greil Johann , Lang Peter , Bader Peter , Eyrich Matthias , Schlegel Paul G. , Schumm Michael , Feuchtinger Tobias , Niethammer Dietrich
Published: April 2020

Unrelated donors are commonly used for hematopoietic stem cell transplants, but graft-versus-host disease (GVHD) is a major problem. We investigated whether transplantation of CD34+ or CD133+ enriched stem cells with add-back of ten million T-cells per kg from unrelated donors would prevent acute GVHD in pediatric patients in combination with pharmacologic immunosuppression. Eighteen patients (1 CML in second chronic phase, 2 MDS, 4 ALL in CR1, 4 ALL in CR2, 1 JMML, 1 AML in CR1, 3 AML in CR2, 1 Wiskott-Aldrich syndrome and 1 NHL in CR2) were transplanted with G-CSF mobilized peripheral blood stem cells (PBSC) from HLA-matched unrelated donors (n = 18). Median of age was 8.9 years (0.5 to 18 years). Conditioning regimens were performed according to national therapy protocol guidelines. On the day of transplant patients received a median of 13.5 (4.5 to 30.0 x 106) CD34+ or CD133+ enriched stem cells and an aliquot of unmanipulated PBSC containing 10 x 106 T-cells per kg. GVHD prophylaxis consisted of cyclosporine A (CSA) and short course methotrexate (MTX) on day +1, +3 and +6. Engraftment was rapid with a median of 19.6 days in sixteen patients. Two patients failed to engraft at first. However, full donor chimerism and stable engraftment was achieved in both patients after cessation of CSA treatment and an additional stem cell boost without any reconditioning. One patient with ALL developed acute GVHD grade III (skin and gut) after cessation of CSA treatment, but responded well to treatment with CSA and steroids. None of the other 17 patients developed acute GVHD > grade I. Thirteen are alive and well with a median follow-up of 578 days (101 to 1095 days). Three patients died of severe infectious complications and two due to relapse (JMML, AML). Compared to a historical control group of patients transplanted with highly purified CD34+ selected cells, the group with add-back of 10 x 106 T-cells per kg showed significantly higher T-cell counts (p=0.008, Wilcoxon Rank sum test) on day 90 after transplantation with a median number of six T-cells/μl in the control group and a median number of 294 T-cells/μl in the study group. We conclude that add-back of 10 x 106 T-cells per kg in combination with CSA and short course MTX improves T-cell recovery and appears to be a safe T-cell dosage regarding acute GVHD in the setting of allogeneic peripheral blood stem cell transplantation from unrelated donors.
Increased Expression of Activating Receptors on NK Cells in the Post-Transplant Period.
Authors: Boyiadzis Michael , Carson Jesse M. , Memon Sarfraz A. , Dean Robert , Castro Kate , Odom Jeanne , Bishop Michael R. , Gress Ronald E. , Hakim Frances T.
Published: April 2020

Following allogeneic and autologous hematopoietic stem cell transplantation (HSCT) natural killer (NK) cells are among the first lymphocyte populations to recover, returning to normal levels within one month after HSCT. We previously reported that levels of IL-15, a cytokine critical to NK cell homeostasis, are elevated during the peri-transplant period (ASH, 2003). Modulation of the relative frequency and intensity of expression of the activating NK cell receptors by IL-15 may contribute to NK-mediated cytotoxicity post transplant. We compared the expression of activating NK receptors in the donor apheresis product and in recipient peripheral blood at one and three months following reduced intensity allogeneic HSCT from HLA-matched siblings. NK cells, divided into CD56bright CD16− and CD56dimCD16+ subsets, were assessed for receptors that trigger cytotoxicity: the natural cytotoxicity receptors (NCR) NKp30 and NKp46, and the C-type lectin receptors NKG2D and NKG2C-CD94. During the first post transplant month the NK cell numbers recovered to normal levels, but the CD56brightCD16− subset increased disproportionately (p = .005). By three months the proportions of the NK cell subsets returned to pre-transplant levels. In both NK subsets, the percentage of cells expressing NCR NKp46 or NKp30 doubled by one month and was sustained at three months. The percentage of cells expressing the C-type lectin receptor CD94 similarly increased by 50% in both NK subsets. The heterodimeric activating partner of CD94, NKG2C, was also increased, but the increase in frequency of cells expressing this receptor was less than those pairing CD94 with its inhibitory NKG2A partner. The homodimeric activating receptor NKG2D increased in the NK CD56brightCD16− subset, but did not significantly change in the CD56dim CD16+ NK population. Consistent with responsiveness to elevated IL-15 during the peri-transplant period, expression of both the IL-2/IL-15 beta and common gamma receptors increased at one and three months. Furthermore, when NK cells from normal donors were cultured with IL-15, we observed both a population shift towards CD56brightCD16− and an up-regulation of the NK cell activating receptors, similar to the changes observed during the early post transplant period. These results suggest that IL-15-dependent changes in NK subset distribution and activating NK receptor repertoire occur during the early post-transplant period. Up-regulation of activating NK cell receptors may contribute to NK cell anti-tumor efficacy in the post transplant period.