bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cytoplasmic Tail of GPIbß Modulates Signalling Induced by GPIb/VWF Interactions under High Shear Conditions.
Authors: Martin Kenneth , Feng Shuju , Kroll Michael H. , Kenny Dermot
Published: October 2019

The platelet glycoprotein (GP) Ib/IX/V receptor complex controls crucial steps in haemostasis and thrombosis by mediating adhesion to von Willebrand factor (VWF). Nevertheless, the downstream consequences of VWF binding to GPIb remain unclear. The GPIb/IX/V complex consists of four polypeptides GPIbα, GPIbβ, GPIX and GPV. We have shown that a palmitylated (Pal-) peptide based on the sequence of the GPIbβ cytoplasmic domain between R151 and A161 (Pal-RRLRARARARA abbreviated to RARA) increases the velocity of platelets rolling on VWF, and inhibits VWF-dependent adhesion and aggregation.

The peptide RARA is delivered into platelets and does not cause any reduction in the surface expression of GPIb-IX-V. We examined the effect of RARA and control peptides (Pal-RAAAARARARA and Pal-RRLRADADADA) on GPIb/IX/V-mediated tyrosine kinase signalling induced by VWF/GPIb interactions under shear conditions using a cone and plate viscometer. Whole platelet lysate phosphotyrosine immunoblots were characterised in the presence and absence of RARA and the control peptides before and after shearing at 120 dynes/cm2 for 5 mins. We then further investigated the effect of RARA on PI-3 kinase signalling, the tyrosine kinase Src and calcium influx under the conditions described above.

The platelet permeable peptide RARA alone specifically caused protein tyrosine phosphorylation under resting conditions and caused platelet microaggregate formation with a 35% reduction in single platelets as assessed by flow cytometry. In addition RARA significantly inhibited shear-induced aggregation. When tyrosine phosphorylation was examined in platelets sheared for 5 minutes at 120 dynes/cm2, we observed RARA failed to inhibit tyrosine phosphorylation despite inhibiting shear-induced aggregation.

RARA caused Src kinase activation as determined by site-specific phosphorylation of Y416 on Src. Furthermore the Src inhibitor PP1 inhibited RARA induced tyrosine phosphorylation. RARA caused a Src-dependent increase in intracellular [Ca2+] and inhibited any further increase in intracellular calcium induced by thrombin. Pretreatment of platelets with the PI-3 kinase inhibitor wortmannin demonstrated that tyrosine phosphorylation induced by RARA was not PI-3 kinase dependent.

These results indicate that a platelet permeable peptide corresponding to specific residues of the cytoplasmic tail of GPIbβ directly stimulates Src, and suggest that the mechanism of its inhibitory effect on VWF-mediated platelet adhesion and aggregation involves the tyrosine kinase-mediated desensitisation of a GPIb/IX/V-triggered signalling pathway. This study demonstrates a novel role for the GPIbβ cytoplasmic tail in mediating Src-dependent shear induced signalling through GPIb/VWF.
Activation of G12/13 Pathway Is Essential for the Dense Granule Release in Platelets.
Authors: Jin Jianguo , Kim Soochong , Kunapuli Satya
Published: October 2019

Platelet secretion is an important physiological event in hemostasis. A number of agonists such as thrombin and thromboxane A2 induce platelet secretion. ADP does not cause dense granule release in aspirin-treated platelets, although ADP induce platelet shape change, aggregation and alpha granule release through Gq and Gi pathways. The protease activated receptors 1 and 4, and the thromboxane receptor activate the G12/13 pathways in addition to the Gq pathways. We postulated that the platelet dense granule release reaction depends on both Gq and G12/13, and in the absence of signaling through either G protein abolishes secretion. In other words, co-stimulation of Gq and G12/13 pathways is a major requirement for dense granule release in platelets. We rationalize that because U46619 and thrombin can activate both Gq and G12/13, they cause platelet dense granule release, whereas ADP fails to cause platelet secretion from dense granules because it does not activate G12/13. As a first step towards testing this hypothesis, we supplemented ADP signaling in aspirin-treated platelets with selective activation of G12/13 pathways using YFLLRNP, a partial agonist of PAR-1. YFLLRNP selectively active G12/13 signaling pathway without activating Gq or Gi pathways at low concentrations. YFLLRNP (60 μM) or 2MeSADP (100 nM) failed to cause dense granule release. However, addition of YFLLRNP (60 μM) and 2MeSADP (100 nM) together caused dense granule release. We proceeded to confirm these results in Gq null mice by selectively activating phospholipase C (PLC), a downstream signaling molecule from Gq. In aspirin-treated Gαq knockout mouse platelets 80 μM m-3M3FBS, a direct PLC activator, causes calcium mobilization from intracellular stores and platelet shape change, but does not cause dense granule secretion. We have previously shown that Gq/PLC pathways downstream of ADP are not sufficient for aggregation and require concomitant signaling from Gi for platelet aggregation. Thus, lack of aggregation by PLC activation alone in G?q knockout mouse platelets is consistent with our previous observations. In G?q null mouse platelets, m-3M3FBS (80 μM) or AYPGKF (500 μM) alone failed to cause dense granule release. However, addition of m-3M3FBS (80 μM) and AYPGKF (500 μM) together caused dense granule release. In addition, consistent with our previous findings, co-stimulation of G12/13 pathways and Gi (AYPGKF + 2MeSADP) in G?q knockout mouse platelets caused aggregation, but failed to cause dense granule release. We conclude that supplemental signaling from G12/13 is required for Gq-mediated dense granule release and that ADP fails to cause dense granule release because the platelet P2Y receptors, although activate PLC, do not activate G12/13 pathways.
Effect of Adding Rituximab to Three Cycles of CHOP Plus Invoved-Field Radiotherapy for Limited-Stage Aggressive Diffuse B-Cell Lymphoma (SWOG-0014).
Authors: Miller Thomas P. , Unger Joseph M. , Spier Catherine , Stea Baldassarre , Cantu Emilia , LeBlanc Michael , Fisher Richard I.
Published: October 2019

The 5-year survival rate of patients with limited disease (LD) and aggressive histologies of non-Hodgkin lymphoma (NHL) who have at least one adverse risk factor is about 70% after treatment with three cycles of CHOP followed by involved-field radiotherapy (CHOP(3) plus RT). We tested the effect of adding four infusions of rituximab to CHOP(3) plus RT in 62 evaluable patients. Patients had an aggressive histology of diffuse B-cell NHL (diffuse large cell or Burkitt’s-like) and had LD. LD is defined as stage I with at least one adverse risk factor (age > 60 years, elevated serum LDH, or a performance status of 2) OR non-bulky stage II disease. Adverse risk factors are defined using a stage-modified IPI system and include: non-bulky stage II, age >60 years, elevated serum LDH and a performance status of 2. Patients with bulky stage II disease were excluded as their prognosis is similar to advanced disease, and patients with stage I disease and no risk factors were excluded as their prognosis exceeds 90% survival at 10 years with CHOP(3) plus RT. Rituximab was infused at 375 mg/m2 on days −7, 1, 22, and 43, and CHOP was given at standard doses on days 3, 24, and 45 (CHOP(3) plus R plus RT). Involved-field RT was given as previously described (N Engl J Med1998;339:21–26). With a median follow-up of 2.4 years, the progression-free survival (PFS) and overall survival (OS) measured at 2 years was 94% and 95%, respectively. In order to estimate whether the addition of rituximab adds benefit to CHOP(3) plus RT, and therefore, would be worth testing in a comparative trial, we identified 68 patients from SWOG study 8736 treated with CHOP(3) plus RT using the same criteria to include LD and aggressive diffuse B-cell histologies. These previously reported patients treated with CHOP(3) plus RT (ref above) have a median follow-up of 12.0 years. The PFS and OS for this group measured at 2 years was 85% and 93%, respectively. Four patients have relapsed in the rituximab-treated group and 10 patients had relapsed in the CHOP(3) plus RT alone treated group within the first 2 years. Three patients have died in the rituximab- treated group and five patients had died on the CHOP(3) plus RT alone treated group within the first 2 years (see Table). Short-term toxicity appeared similar. We conclude that the addition of rituximab to standard treatment with CHOP(3) plus RT results in outcome which compares favorably to our historical experience and merits further study.

Effect of Rituximab in Treating Limited Disease

MEASUREMENT CHOP(3) + R + RT (0014) CHOP(3) + RT (8736) Results of SWOG study 0014 compared to SWOG study 8736 No. patients 62 68 2-year PFS 94% 85% 2-year OS 95% 93% No. relapses 2 yrs 4 10 No. deaths 2 yrs 3 5
Red Blood Cells Facilitate Reverse Cholesterol Transport.
Authors: Kuypers Frans A. , Larkin Sandra , Beckstead Jenifer , Oda Michael , Ueda Kazumitsu , Ryan Robert O.
Published: October 2019

Lecithin:cholesterol acyl transferase (LCAT)-dependent conversion of cholesterol (CH) to cholesteryl ester (CE), a key component of the reverse cholesterol transport (RCT) pathway, is essential for cholesterol processing. We hypothesized that red blood cells (RBCs) function in this pathway by facilitating phosphatidylcholine (PC) re-generation from LCAT-derived lysophosphatidylcholine (LPC). Addition of 14C-oleate to fresh RBCs resulted in an ATP-dependent incorporation of radiolabel into PC via the Lands pathway. Prior depletion of red cell LPC content reduced the incorporation of 14C-oleate into PC, which was restored by the addition of LPC. Reconstituted PC/cholesterol/apolipoprotein A-I (apoA-I) high-density lipoprotein (rHDL) was used as substrate for LCAT dependent conversion of CH to CE. Addition of LPC to this reaction mix inhibited CE production. The inhibition was overcome, however, by inclusion of RBCs, suggesting rHDL-generated LPC migration to RBCs. RBCs depleted of LPC increased their ability to generate 14C-PC from 14C-oleate in the presence of rHDL with LCAT, indicating that LCAT-derived LPC can be utilized as a substrate for PC production in RBCs. Radio-labeled CH associated with RBCs was recovered as CE only when rHDL substrate and LCAT were present, indicating RBC-associated CH migration to rHDL. When RBCs containing 14C-PC were incubated with rHDL and LCAT, PC transferred from RBCs to the rHDL particles. The interaction of apoA-I with the membrane lipid transporter ATP-Binding Cassette A1 protein (ABCA1) in cell membranes has been shown to play an essential role in the formation of HDL and facilitates RCT. Using monoclonal antibodies, we were able to show that RBCs contain ABCA7, closely related to ABCA1. Our data show that fluorescently labeled apoA-I binds to RBCs, suggesting that the interaction between ABCA7 and apoA-I may be important for HDL/RBC interaction. Together, our data supports an active role for RBCs, the major cell type in blood, in LCAT-mediated lipoprotein remodeling. Thus, RBCs represent a hitherto underappreciated component of the RCT pathway.
CCAAT Enhancer Binding Protein-α (C/EBPα) Determines Myeloid Versus Erythroid Cell Fate in Multipotential Progenitors.
Authors: Suh Hyung-Chan , Gooya John , Renn Katie , Friedman Alan , Johnson Peter , Keller Jonathan
Published: October 2019

C/EBPα is a bZip transcription factor, which is required for granulocyte development, and loss of C/EBPα function is associated with the development of acute myelogenous leukemia and myelodysplastic syndrome. While the precise mechanisms that regulate cell fate decisions during hematopoietic development are largely unknown, expression of transcription factors (PU.1 and GATA-1) can induce lineage conversion. In this regard, C/EBPα can drive the differentiation of B cells into macrophages, and bi-potential cell lines into granulocytes at the expense of macrophages. C/EBPα can also promote the transdifferentiation of myoblasts into adipocytes. We have recently found that there are increased numbers of erythroid cells in the fetal liver of C/EBPα −/ − mice. Also, C/EBPα is expressed in more primitive progenitor cells than granulocyte/macrophage progenitors (GMP) including hematopoietic stem cells. Therefore, we initiated experiments to evaluate whether C/EBPα has a functional role in regulating cell fate decisions in progenitors more primitive than GMP where it may promote a myeloid versus erythroid cell fate decision. To test this hypothesis, we over-expressed C/EBPα using retroviral vectors in 1) bone marrow cells (BMC) and evaluated their growth and differentiation in vitro, and in vivo when transplanted into mice; 2) purified multipotential progenitors with erythroid and myeloid potential, and erythroid restricted progenitors; and 3) murine erythroid leukemia (MEL) cells. We found that there was a marked decrease in erythroid lineage cells and an increase in myeloid cells in mice transplanted with BMC that over-expressed C/EBPα. We also observed a decrease in erythroid cell growth in vitro with BMC that expressed C/EBPα. Furthermore, when infected with retroviral vectors that express C/EBPα, erythroid restricted progenitors acquired myeloid cell morphology and myeloid specific cell surface markers. In addition, MEL cells that over-express C/EBPα showed increased myeloid gene expression including GM-CSFR, PR3 and myeloid specific esterase, while they showed decreased expression of β-globin and Epo receptor (EpoR) which is required for erythroid cell differentiation and survival. We detected high levels of EpoR in C/EBPα −/ − suggesting an inverse relationship between C/EBPα and EpoR expression. Thus, C/EBPα is a dual function transcription factor that can repress erythroid specific genes while enhancing myeloid lineage gene expression. Consequently, C/EBPα acts as a switch to drive hematopoietic progenitor cells toward myeloid cell development at the expense of erythroid maturation, and can reprogram erythroid cells into myeloid cells. # Funded in part by DHHS #NO1-CO-12400
Characterization of Mammalian Cellular Memory.
Authors: Sawado Tomoyuki , Rincon-Arano Hector , Halow Jessica , Groudine Mark
Published: October 2019

Cellular differentiation is accompanied by the coordinated activation and silencing of specific subsets of genes and associated alterations in chromatin structure. This process, which is under the control of extrinsic and intrinsic cellular signals, results in the establishment of lineage-specific domain structures that must be maintained during dynamic nuclear events, such as cell division. The propagation of specific patterns of gene expression is termed “cellular memory”. Our goal is to identify the elements that are involved in the establishment and propagation of transcription states and to determine the molecular basis of memory establishment in mammals. To this end, K562 cells were transfected with a γ-globin promoter-GFP-metallothionein response element (MRE) cassette (GGM cassettes) and, using a procedure to derive stably transfected cells in the absence of selection, multiple stable transformants harboring a single copy of the cassette were obtained. In most clones, zinc (Zn) induction resulted in transition of the transgene from the silent to the active state, with the maintenance of the active state dependent on continuous exposure to Zn. However, at one genomic site (clone 6177), a high level of GFP expression was maintained even after Zn removal. Characterization of epigenetic marks of this transgene, including CpG methylation, histone modifications and nuclear localization, at various stages of GFP expression may provide insight into the molecular mechanisms of mammalian cellular memory. In initial experiments, Na-bisulfite conversion/sequencing revealed that, prior to the establishment of memory, up to 8% of CpGs in the GFP gene are methylated, and after memory establishment, no CpG methylation is detectable. However, pretreatment of 6177 cells with a DNA methylation inhibitor, 5-dAzaC for 48hs before Zn induction resulted in no change in the memory establishment program, suggestiong that reduction of CpG methylation is not a cause of memory establishment in 6177 cells. We also have examined the relationship between cellular memory and histone modifications. Comparison of the histone modification status of the active GFP gene in cells before and after memory establishment revealed that H4 K20 dimethylation is present in the both the inactive and active GFP gene prior to memory establishment, but is significantly reduced upon memory establishment. Interestingly, this mark is known to be associated with silenced regions of euchromatin and the chromocenter, but not with active genes, in Drosophila. Thus, these results have revealed a unique nature of histone modification at the 6177 site: a marker of silenced chromatin co-exists with active transcription prior to the establishment of cellular memory. This co-existence may be the mechanism by which the establishment of memory of the active state is prevented at stage II. Currently, we are cloning the 6177 integration site to identify cellular memory elements, the function of which will be further analyzed at defined genomic sites.
PU.1 and pRb Bind GATA-1 on DNA and Recruit a Histone H3K9 Methyl Transferase-Containing Complex to Repress the Erythroid Transcription Program.
Authors: Stopka Tomas , Amanatullah Derek F. , Skoultchi Arthur I.
Published: October 2019

Current work indicates that transcriptional repression is at least as important as transcriptional activation in normal development. Inappropriate or untimely transcriptional repression in immature hematopoietic cells is often the basis for a block to differentiation in hematologic malignancies. Activation of PU.1, a myeloid and B-cell specific transcription factor, in erythroid cells plays a key role in Friend virus-induced mouse erythroleukemia (MEL). Previous results from our laboratory showed that PU.1 blocks the erythroid differentiation-promoting activity of GATA-1 by binding directly to GATA-1 on DNA and inhibiting its transcriptional function. PU.1-mediated repression of GATA-1 on transiently transfected GATA-1 target genes is dependent on the corepressor pRb that also binds to PU.1 (Rekhtman et al., Genes & Dev 1999 and Mol Cell Biol 2003). To further investigate the mechanism of PU.1-mediated repression of GATA-1 in chromatin, we examined the occupancy of several GATA-1 target genes by PU.1 and pRb, as well as the state of core histone modifications at these loci in MEL cells by quantitative chromatin immunoprecipitation. These studies included both endogenous GATA-1 target genes and an exogenous GATA-1 target gene (alpha globin) integrated at a specific locus in MEL cells by Recombinase-Mediated Cassette Exchange. We found that GATA-1 sites at both the exogenous, integrated gene as well as at endogenous genes (including the regulatory regions of the alpha globin, beta globin, alas-e, eklf, p45 nf-e2) are occupied by a GATA1 - PU.1 - pRb complex in undifferentiated MEL cells. The presence of all three components of the complex is dependent on intact GATA-1 binding sites in the exogenous, integrated gene. The histone methyltransferase Suv39H1 and the histone H3MeK9 binding protein, HP1alpha, are also present at the repressed loci. During induced differentiation of MEL cells, PU.1, pRb, Suv39H1 and HP1alpha occupancy at these sites declines but GATA-1 continues to be present at its binding sites. The disruption of the repression complex at these loci during differentiation as well as during siRNA-mediated PU.1 knock down is associated with conversion of methylated H3K9 to acetylated H3K9 and significant transcriptional derepression of these GATA-1 target genes. These findings support a model for repression of GATA-1 by PU.1 at endogenous loci through recruitment of the corepressor pRb and associated histone methyltransferase (Suv39H1) and H3MeK9 binding (HP1alpha) activities.
Pharmacogenetics of Outcome in Children with Acute Lymphoblastic Leukemia.
Authors: Rocha Jose Claudio , Cheng Cheng , Liu Wei , Kishi Shinji , Das Soma , Cook Edwin H. , Sandlund John T. , Rubnitz Jeffrey , Ribeiro Raul C. , Pui Ching-Hon , Evans William E. , Relling Mary V.
Published: October 2019

The acquired genetic characteristics of acute lymphoblastic leukemia (ALL) blasts are often used to guide the intensity of therapy, whereas the germline host genetic characteristics of the patient generally have not been considered. Multiple common, functionally important polymorphisms affect genes whose products determine the pharmacokinetics and pharmacodynamics of antileukemic agents. It is not yet known how genetic polymorphisms may interact to affect the outcome of antileukemic therapy. Combining classification and regression tree with failure time analysis, we assessed whether 16 genetic polymorphisms, alone or in combination, predicted relapses in 246 children with ALL, 116 of whom were treated on the lower-risk (LR) and130 on the higher-risk (HR) arms of the St Jude protocol Total XIIIB. Genotyping was performed for the following polymorphic loci: CYP3A4*1B and CYP3A5*3; GSTP1 313A>G, GSTM1 and GSTT1 deletions; MDR1 exon 21 (2677G>T/A) and MDR1 exon 26 (3435C>T); MTHFR 677C>T and MTHFR 1298A>C; NR3C1 1088A>G; SLC19A1 80G>A; TPMT 238G>C, 460G>A and 719A>G; TYMS enhancer repeat; UGT1A1 promoter repeat polymorphism; VDR intron 8 G>A and VDR FokI (start-site) T>C. In all children with available RNA in their diagnostic ALL blasts, gene expression levels of prognostic genotypes were analyzed using the Affymetrix genechip array HG_U95Av2. Among the HR group, the glutathione S-transferase M1 (GSTM1) non-null genotype was associated with the risk of hematological relapse (5-year cumulative incidence, 17.1%±4.5% compared to 5.1%±2.9% for GSTM1 null genotype, p = 0.03), and among the non-null genotypes, the thymidylate synthetase (TYMS) 3/3 genotype was associated with a further increase in hematologic relapse risk (5-year cumulative incidence, 29.2%±9.5% compared to 10.9%±4.7% for TYMS 2/3 or 2/2 genotypes, p = 0.02). Increased expression levels of these two target genes (p < 0.0001 and p = 0.09, respectively) were consistent with resistance to the drugs interacting with these gene products. For central nervous system relapse, among the HR group, the vitamin D receptor (VDR) start site (p = 0.02) and intron 8 genotypes (p = 0.04) predisposed, whereas for LR patients the TYMS 3/3 genotype predisposed (p = 0.04). The genotypes associated with outcome have pharmacologic plausibility: e.g., high GST activity (GSTM1 non-null) could cause anticancer drug resistance; high TYMS activity (TYMS 3/3) would be less inhibited by antifolates. In conclusion, germline polymorphisms influence the outcome of antileukemic therapy, and therefore represent determinants of response that can be used to optimize therapy.
Prognostic Factors and Long-Term Outcome for Patients with Hodgkin’s Lymphoma Who Relapse after an Autologous Stem Cell Transplantation.
Authors: Constans Mireia , Sureda Anna , Arranz Reyes , Caballero Maria Dolores , Lahuerta Juan Jose , Hernandez-Boluda Juan Carlos , Vidal Maria Jesus , Garcia-Laraña Jose , Rifon Jose , Ribera Josep Maria , Fernandez-Abellan Pascual , Moraleda Jose Maria , Bernal Maria Teresa , Mateos Maria Victoria , Martin-Mateos Maria , Cordoba Rafael , Garcia-Conde Javier , Sierra Jorge , Conde Eulogio ,   for the Spanish Cooperative Group GEL/TAMO
Published: October 2019

We have analyzed overall survival (OS-REL), progression-free survival (PFS-REL) and the clinical variables influencing the long-term outcome for patients with Hodgkin′s lymphoma (HL) who relapse after an autologous stem cell transplantation (ASCT). From 639 patients autografted for HL [383 males (60%) and 256 (40%) females, median age of 30 (1–66) years] reported to the GEL/TAMO Cooperative Group between January/1984 and January/2003, 175 patients (27%) relapsed at a median time of 10 (4 – 125) months [median (range)] after ASCT. They were 97 males (56%) and 78 (44%) females with a median age of 28 (10 – 66) [median (range)] years at transplantation. Sixty-three patients were autografted in complete remission, 73 in sensitive relapse and 31 in resistant relapse. One hundred and sixty three patients (94%) received different chemotherapy (CT) protocols as conditioning regimen and 12 patients (6%), CT plus total body irradiation. At relapse, 94 patients (53%) presented with advanced stages (III-IV), 49 patients (28%) with B symptoms and 19 (11%) with bulky disease. Relapse was extranodal in 45% of the patient population. Ten percent of the patients had a poor performance status (ECOG ≥2) and 37 patients (22%) had a hemoglobin (Hb) level < 100 g/l at relapse. Thirty four patients (20%) received no further therapy, 107 (60%) received different CT +/− radiotherapy and the remaining 34 patients (20%) were treated with a second stem cell transplantation (13 patients, an ASCT and 21 an allogeneic transplantation). OS-REL and PFS-REL were of 35% ± 4% and 23% ± 4% at 3 years, respectively. Advanced clinical stage (III-IV) at relapse [relative risk (RR) 4.4, 95% confidence interval (CI) (1.7 – 10.8), p = 0.002] and an interval between ASCT and relapse < 12 months [RR 2.4, 95%CI (1.1 – 5.0), p = 0.03] were independent adverse prognostic factors for PFS-REL. Advanced clinical stage at relapse [RR 4.4, 95%CI (1.4 – 14.4), p = 0.012], extranodal disease [RR 2.4, 95%CI (1.3 – 15.4), p = 0.02] and a Hb level < 100 g/l at relapse [RR 3.4, 95%CI (2.0 – 10.4), p = 0.03] were significant adverse prognostic factors for OS-REL. Although the long-term outcome of HL patients who relapse after ASCT is poor, results are better in patients with late relapses and localized disease. Moreover, a minority of the patients can become long-term progression-free survivors after relapse. The identification and construction of prognostic sub-groups according to the number of risk factors may be useful to develop risk-adapted therapeutic strategies.
Early Response to Induction Is Predictive of Survival in Childhood Philadelphia Chromosome Positive Acute Lymphoblastic Leukaemia: Results of the Medical Research Council ALL 97 Trial.
Authors: Roy Anindita , Bradburn Mike , Moorman Anthony V. , Burrett Julie , Mitchell Chris , Kinsey Sally E. , Vora Ajay J. , Lilleyman John , Eden Osborn , Hann Ian , Saha Vaskar
Published: October 2019

We report the outcome of children with Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL) treated on the national trial for childhood ALL, MRC ALL 97/99, between Jan 1997 and Jun 2002. A diagnosis of Ph+ ALL was made by karyotyping and/or fluorescent in situ hybridisation. Outcome was evaluated according to NCI risk criteria and treatment response. Statistical methods included Kaplan Meier analysis, logrank and stratified logrank tests. Of the 1937 patients treated on the MRC ALL 97/99 protocol, 1836 were successfully screened for Ph chromosome and 42 (2.3%) were positive. This subgroup of patients was heterogeneous at the cytogenetic level with 27 (77%) out of 35 assessable cases having one or more abnormality in addition to the Ph translocation. Four recurrent secondary abnormalities were observed: an extra Ph chromosome, loss of 9p (del 9p), high hyperdiploidy and monosomy 7. The median age at diagnosis and presenting white cell count were 6.8 years and 32.5 × 109/L respectively. 19 (45%) had <25% bone marrow blasts within the first two weeks of treatment and were defined as a Good-response group (GRG), the others were classified as a Poor-response group (PRG). 6/10 (60%) of the patients with an extra Ph chromosome were in the GRG, whereas 8/10 (80%) of those with del 9p belonged to the PRG. 36/42 (86%) achieved complete remission (CR1) at the end of induction of whom 28 underwent bone marrow transplantation (BMT). The median follow up was 41 months (range 21–84 months). The 3-year estimate for event free survival (EFS) (with 95% confidence intervals) was 57% (41–70%). The EFS for the GRG and PRG were 74% (48–88%) and 44% (22–64%) respectively. Significant predictors for survival were presenting white cell count (p=0.02) and early response to treatment (p=0.03), but not age at diagnosis or gender. BMT in CR1 appeared to reduce the risk of a subsequent bone marrow relapse. These results are a significant improvement on previous results, particularly for the PRG. The EFS for the PRG in this report is 44% compared to a 4 yr. EFS of 10% in previously published data from the BFM/AIEOP group. We believe that the early introduction of intensive chemotherapy as in the MRC trial improves early response rates and overall outcome in Ph+ ALL, especially for the PRG. BMT in CR1 is recommended in children with Ph+ ALL, nonetheless newer alternative treatment strategies, such as the use of imatinib mesylate need to be evaluated for children with Ph+ ALL.
Acute on Chronic Pulmonary Hypertension in Patients with Sickle Cell Disease.
Authors: Kato Gregory J. , Martyr Sabrina , Machado Roberto , Sachdev Vandana , Ernst Inez , Hunter Lori , Coles Wynona , Nichols James , Hunter Christian J. , Gladwin Mark T.
Published: October 2019

Secondary pulmonary hypertension (PHT), defined as a tricuspid regurgitant jet velocity (TRV) > 2.4 meters/second at rest, has been identified as an emerging complication in patients with sickle cell disease, with 32% prevalence. Patients with sickle cell pulmonary hypertension tend to have a higher mortality rate than patients with primary PHT at comparable pulmonary artery pressures (PAP). We analyzed prospectively obtained Doppler echocardiography measurements from 11 patients in steady-state and during vaso-occlusive crisis episodes or during exercise to determine whether pulmonary pressures became further elevated during stress. We found that tricuspid regurgitant jet velocity is significantly further elevated during vaso-occlusive crisis (2.61 ± 0.03 vs. 2.81 ± 0.06 m/sec (mean ± SEM), p = 0.01)(Fig. A). We assessed other laboratory parameters and found associated significant decreases in hemoglobin concentration (9.3 ± 0.3 vs. 8.4 ± 0.3 gm/dL, p = 0.02)(Fig. B), and significant elevations in lactate dehydrogenase (381 ± 39 vs. 442 ± 49 IU/L, p = 0.02)(Fig. C). These data are consistent with increased hemolysis during vaso-occlusive crisis, and the association of increased hemolysis with acute exacerbation of PHT in patients with SCD. In three patients at steady state, exercise echocardiograms were performed, showing acute marked rise in TRV during exercise (2.80 ± 0.12 vs. 3.37 ± 0.17 m/sec, p = 0.02)(Fig. D). These data bring to light several important points in the clinical assessment of tricuspid regurgitant jet velocity in patients with sickle cell disease. First, the relatively mild steady state PHT in patients with SCD is associated with previously unappreciated acute increases in PAP that may explain the high mortality rate in this population. This might be responsible for sudden death occurring during vaso-occlusive crisis or physical exertion. Second, this acute rise is associated with markers of increased hemolysis, further implicating hemolysis-associated derangement of nitric oxide homeostasis, endothelial function and PHT. Third, exercise measurements of TRV may be indicated in the clinical evaluation of patients with SCD and dyspnea on exertion, since it may unmask otherwise unappreciated PHT.

Figure Figure
Long Term Treatment with Hydroxyurea Does Not Prevent Development of Renal Dysfunction and Osteodystrophy in Patients with Sickle Cell Disease.
Authors: Voskaridou Ersi , Terpos Evangelos , Margeli Alexandra , Hantzi Eugenia , Stoupa Eleni , Loutradi Aphroditi , Papassotiriou Ioannis , Loukopoulos Dimitris
Published: October 2019

Hydroxyurea (HU) is presently considered as the main treatment for the reduction of sickle-cell crises; however, information regarding the potential of HU to inhibit progressive organ failure is scarce. The gradually failing renal function and renal osteodystrophy are well known complications of sickle cell disease (SCD). The pending question is whether administration of hydroxyurea over very long period of time may delay or prevent the appearance of these abnormalities. To this effect we evaluated the renal function and bone metabolism in 57 patients with HbS/β-thal (31M/26F; median age 32 years, range: 19–67 years) receiving hydroxyurea (usually 1 g/daily) continuously for 1 to 14 years (median 10.2 years). In addition to conventional renal biochemistry we measured the levels of serum and urinary β2-microglobulin (β2M), serum cystatin C (specific and sensitive index of glomerular filtration rate), and urine N-acetyl-b-D-glucosaminidase (NAG; reflecting the distal tubular cells function). The extent of renal osteodystrophy was evaluated by DEXA scans assessing bone mineral density (BMD), and by assaying various markers of (a) osteoclast function [soluble receptor activator of nuclear factor κB ligand (sRANKL), osteoprotegerin (OPG), and tartrate resistant acid phosphatase isoform 5b (TRACP-5b)], (b) bone resorption [C-telopeptide of collagen type I (CTX)], and (c) bone formation [bone-alkaline phosphatase (bALP) and osteocalcin (OC)]. The above parameters were also evaluated in 16 age- and gender-matched controls. Three patients (5.2%) had increased serum creatinine levels; 16 patients (28%) had more than 300 mg/day protein excretion in the urine, and 13 patients (22.8%) had microalbuminuria. Moreover, serum cystatin C was elevated in 16 patients (28%), NAG in 21 (36.8%), serum β2M in 34 (59.6%) and urinary β2M in one patient. In addition, 17 patients (29.8%) had osteoporosis/osteopenia in DEXA scans (comparable to HbS/β-thal patients who did not receive HU). Furthermore, all patients displayed significantly elevated levels of OPG (p=.001), sRANKL (p<.01), sRANKL/OPG ratio (p=.022), and CTX (p=.02), while significant correlations were found between serum cystatin C vs. both serum OPG and β2M levels as well as between cystatin C and the sRANKL/OPG ratio. Not only do these results suggest that HU does not prevent renal dysfunction in this cohort of patients but also highlight the role of RANKL/OPG pathway in the renal-induced bone disease of sickle cell syndromes. Furthermore, NAG, cystatin C and OPG may be useful as early biochemical markers for the assessment of renal impairment in SCD patients.
Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.
Authors: Miyake Noriko , Brun Ann C.M. , Magnusson Mattias , Scadden David T. , Karlsson Stefan
Published: October 2019

Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.
Genetic Evidence of a Novel Blood Differentiation Pathway from Lympho-Myeloid Hematopoietic Stem/Progenitor Cells, Independent of Common Myeloid Progenitors.
Authors: Hultquist Anne , Mansson Robert , Sigvardsson Mikael , Adolfsson Jorgen , Bryder David , Yang Liping , Thoren Lina , Jacobsen Sten E.W.
Published: October 2019

We have recently identified three novel subsets of multipotent hematopoietic stem/progenitor cells (HSCs) in the Lin−Sca-1+c-Kithi (LSK) compartment of adult murine bone marrow based on differential expression of CD34 and the cytokine tyrosine kinase receptor Flt3. Long-term HSCs (LT-HSCs) lack CD34 and Flt3 expression (LSKCD34-flt3-), whereas short-term HSCs (ST-HSCs) are LSKCD34+flt3−. A third LSK population is characterized by co-expression of CD34 and Flt3 (LSKCD34+flt3+) and possess a combined myeloid (granulocyte and monocyte) and lymphoid (B and T cell) differentiation potential, but surprisingly lack megakaryocytic (Mk) and erythroid (E) potential in vitro and in vivo. These findings implicate an alternative road map for blood lineage development distinct from the classical model in which the first lineage commitment step of HSCs is thought to result in a strict separation into myelopoiesis and lymphopoiesis. In the current study we sought genetic evidence in further support of this new model through genetic profiling of these three HSC subpopulations, using affymetrix chips, quantitative (Q)-PCR and single cell PCR. In contrast to the pluripotent LSKCD34−Flt3− LT-HSCs and LSKCD34+Flt3− ST-HSCs, LSKCD34+Flt3+ cells downregulated or turned of genes critically involved in promoting Mk and E lineage development, such as the Epo and Tpo receptors as well as the transcription factor Gata-1. In contrast, the gene for Il-7rα, critically involved in early B and T cell development was upregulated in LSKFlt3+ cells, but absent in LT-HSCs and ST-HSCs. However, in agreement with their sustained ability to produce granulocytes and monocytes, G-CSFR and Pu.1 expression was sustained from LT-HSC through the LSKCD34+flt3+ stage Particularly noteworthy, single cell PCR demonstrated that a fraction of single LSKCD34+Flt3+ cells upregulating Il-7rα gene expression, sustained and co-expressed G-CSFR expression. Thus, genetic profiling at the single cell level provide further and compelling evidence for a novel road map for blood lineage development, independent of the common myeloid progenitor (CMP). Our biological and gene expression data rather supports the existence of a pathway in which HSCs lose their lineage potentials one by one, starting with the Mk and E lineages.
Hematopoietic Cells from Gadd45a, Gadd45b Deficent Mice Exhibit Altered Myelopoiesis and Higher Apoptosis after Cytokine Treatment.
Authors: Gupta Shiv K. , Gupta Mamta , Hoffman Barbara , Liebermann Dan A.
Published: October 2019

Growth arrest and DNA damage, Gadd45 gene family members are rapidly induced by genotoxic agents as well as by apoptosis and differentiation inducing cytokines. Their role in hemetopoiesis, wherein proliferation, differentiation and apoptosis integrate to maintain cellular homeostasis, is not clear. Using bone marrow cells from gadd45a or gadd45b deficient and wild type littermate mice we have investigated the role of Gadd45 proteins in cytokine induced myeloid cell differentiation in vitro. Bone marrow cells obtained from either gadd45a or gadd45b deficient mice displayed compromised cytokines (IL3, GM-CSF, M-CSF or G-CSF) induced myelopoiesis, resulting in a quantitatively decreased population of mature myeloid cells. Immuno-phenotyping with antibodies to cell surface molecules associated with myeloid cell maturation confirmed impaired myeloid cell maturation in Gadd45a or b deficient bone marrow cells treated with the above cytokines. Analysis of apoptosis by annexin-V and PI staining followed by FACS analysis showed a substantially higher apoptosis in Gadd45a−/− as well as gadd45b−/− cells compared to wild type cells after treatment with M-CSF or G-CSF. Gadd45a−/− as well as gadd45b−/− bone marrow cells were found to be less clonogenic in methylcellulose medium. Morphologically compact and round colonies consisting of immature myeloid cells prevailed over dispersed- colonies consisting of mature myeloid cells in gadd45- deficient cells cultured in methyl cellulose containing IL-3. Furthermore, colony re-plating assay showed better self-renewal abilities in gadd45a−/− as well as gadd45b−/− progenitors, compared to wild type progenitor cells. Altered myelopoiesis in gadd45 a or b deficient mice was further confirmed in vivo by intra-peritoneal administration of sodium casienate - a known inducer of inflammatory response and myelopoiesis in mice bone marrow. Sodium casienate failed to enhance myelopoiesis in gadd45a or gadd45b deficient mice bone marrow, while wild type littermate mice showed a rapid induction of myelopoiesis. Simultaneously peritoneal exudates collected from gadd45 deficient mice consisted of 2–3 fold less myeloid cells compared to age matched wild type control mice after sodium casienate treatment. Gadd45a−/− or gadd45b−/− mice showed a slow recovery after myelo-suppressive effect of antimetabolite 5-Fluorouracil, which further confirmed that gadd45 deficiency leads to delayed myelopoiesis in mouse. Mechanistic aspects of Gadd45 deficiency, which results in impaired myelopoiesis are under investigation.
Normal Hematopoietic Stem Cell Functioning in p21−/− Mice.
Authors: Kamminga Leonie M. , van Pelt Kyrjon , Dontje Bert , de Haan Gerald
Published: October 2019

Recently, several studies have suggested that the family of cyclin-dependent kinase inhibitors plays a crucial role in regulating hematopoietic stem and progenitor pool size. However, due to a lack of appropriate transplantation models, competitive repopulation assays have not been performed. In the present study we have backcrossed a p21 null allele from mice with a mixed genetic background to inbred C57BL/6 mice. As expected, mouse embryonic fibroblasts (MEFs) derived from B6p21−/− mice failed to undergo senescence, whereas B6p21+/+ MEFs show a normal senescent phenotype. Moreover, B6p21−/− CFU-GM were more resistant to radiation compared to B6p21+/+. In contrast, homozygous deletion of the p21 allele did not affect the percentage of Lin− Sca-1+ c-kit+ cells in S-phase when measured by 7-AAD staining, and did not result in any alterations of in vitro cobblestone area forming cell activity. In a competitive repopulating assay different ratios of Ly5.2 BM cells from B6p21−/− or B6p21+/+ littermates were competed with 2 x 106 Ly5.1 B6 BM cells. Assuming similar repopulating capacity of both cell populations, expected chimerism was calculated. Surprisingly, observed and expected chimerism were identical, strongly suggesting that B6p21−/− stem cells had completely normal competitive repopulating activity for up to 1 year after transplant. Our data argue against an important role of p21 in maintaining stem cell function during steady-state hematopoiesis.
Human Cytomegalovirus Infection of Primitive Hematopoietic Progenitor Cells: A Model for Latency.
Authors: Goodrum Felicia D. , Jordan Craig T. , Terhune Scott S. , High Kevin P. , Shenk Thomas
Published: October 2019

Human cytomegalovirus (CMV) is beta-herpesvirus that infects the majority of the population worldwide and establishes a lifelong relationship with its host by way of a latent infection. Although, CMV is of little consequence to healthy individuals, reactivation of CMV from latency can cause life-threatening disease in immunocompromised individuals such as bone marrow transplant recipients and AIDS patients. CMV establishes a latent infection in cells of the hematopoietic system. The focus of our work is to elucidate the mechanisms governing a latent infection and reactivation from latency. It is likely that both viral and cellular mechanisms govern the latent program. We have developed a novel in vitro model for CMV latency using primary human hematopoietic progenitors. Using this model to compare CMV infection in CD34+ hematopoietic subpopulations, the outcome of CMV infection was found to depend on the nature of the subpopulation of cells infected. Only primitive CD34+/CD38− progenitor cells harboured an infection with the hallmarks of latency. This subpopulation transiently expressed a unique subset of CMV genes in the absence of viral replication. Importantly, viral replication could be reactivated from this subpopulation. Other primitive (CD34+/c-kit+) and more mature (CD34+/CD38+ and CD34+/c-kit−) subpopulations resulted in an apparently abortive infection. Strikingly, a CD34+ subpopulation expressing a stem cell phenotype supported a productive infection. These results indicate that cellular determinants contribute to the outcome of HCMV infection in hematopoietic cells. Further, we have also demonstrated that viral determinants play a role in establishing a latent infection using our in vitro model. Comparing the clinical Toledo and FIX strains and the laboratory-adapted AD169 strain of CMV, only the clinical strains were able to establish an infection consistent with latency. AD169, by contrast, replicated productively. Genetic sequences unique to clinical strains have been identified and may contribute to its ability to establish a latent infection in vitro. These studies indicate that both viral and cellular determinants contribute to the establishment of CMV latency. Furthermore, these determinants may serve as targets for CMV disease prophylaxis following bone marrow transplantation or in AIDS patients.