bluuebunny/crossref_metadata_2025 huggingface dataset:
A Validated Gene Expression Signature of High Risk Multiple Myeloma Is Defined by Deregulated Expression of Genes Mapping to Chromosome 1.
Authors: Shaughnessy John D. , Zhan Fenghuang , Burington Bart , Huang Yongsheng , Hanamura Ichiro , Stewart James P. , Hollmig Klaus , Pineda-Roman Mauricio , Tricot Guido , van Rhee Frits , Zangari Maurizio , Crowley John , Barlogie Bart
Published: April 2020

In an effort to better define the molecular mechanisms of aggressiveness in multiple myeloma (MM), we performed microarray analysis on tumor cells from 532 newly diagnosed patients treated on two separate protocols. We hypothesized that expression extremes of a subset of genes linked to poor survival might point to important regions of the genome that when amplified or deleted may lead to altered expression of resident genes and hence disease progression. Using log rank tests of expression quartiles, 70 genes, 30% mapping to chromosome 1 (P <.001) were linked to early disease-related death. Importantly, the majority of up-regulated genes mapped to 1q and down-regulated genes mapped to 1p. This high-risk signature, seen in 13% of patients, was linked to poor outcome in both a training (HR 5.16, P <.0001) and test cohort (HR 4.96, P <.0001) and was an independent predictor of outcome in multivariate analysis (P <.0001) that included the ISS. Using a combination of high-resolution aCGH and microarray profiling, we recently identified 47 minimal common regions (MCRs) of genomic gain across the myeloma genome and 207 genes in these MCRs whose expression increases with copy number changes (Carrasco et al., Cancer Cell 2006 9:313–325). When the expression of these copy number-sensitive genes was compared between the high and low risk diseases defined by the 70 gene model, we found that only genes mapping to MCRs at 1q21, 1q22 and 1q43–q44 were significantly over expressed in high-risk disease. These data support prior studies indicating that gains of 1q and amplification of 1q21 and loss of 1p define a distinct subset of disease with poor survival. Novel treatment modalities are justified in these high-risk patients who have not yet benefited from advances in MM therapy.
Control of Meiotic and Mitoic Progression by the F-Box Protein NIPA In Vivo.
Authors: Illert Anna L. , Kawaguchi Hiroyuki , Quintanilla-Fend Letitia , Bassermann Florian , von Klitzing Christine , Seipel Petra , Hiwatari Misuteru , Morris Stephan W. , Peschel Christian , Justus Duyster
Published: April 2020

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein (FBP) that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry.

Using a conditional knockout strategy we inactivated the gene encoding the FBP NIPA to determined the consequences of NIPA deletion in vivo. The targeting construct was designed to flank exon 1 and 2 of the NIPA gene with loxP-sites. Deletion of this region was obtained by crossing the floxed mice to a cre-transgenic mouse strain expressing cre ubiquitously. NIPA deficiency did not affect the viability of NIPA −/− animals. Mating of heterozygotes yielded NIPA +/+, NIPA +/− and NIPA −/− offspring approximately at the expected Mendelian ratio. Although copulatory behavior was normal and vaginal plugs were produced, NIPA-deficient animals have a fertility defect. 100% of the tested NIPA −/− males and 60% of NIPA −/− females never produced progeny with young fertile wild-type mice. Interestingly, histological evaluation showed progressive testis atrophy in NIPA −/− males. Further analyses indicate a block in germ cell differentiation at the stage of meiotic prophase, no spermatides or spermatozoa were observed in NIPA-deficient animals. High levels of nuclear cyclin B1, a previously reported NIPA substrate, were present in NIPA-deficient germ cells. Furthermore, inactivation of NIPA leads to premature mitotic entry and subsequent mitotic catastrophe and TUNEL positive apoptosis in germ cells. Long-term studies of NIPA deficient mice may display a potential role of NIPA in tumor development.

Since we found the most striking phenotype in high proliferating germ cells our results strongly confirm the cell cycle regulatory function of NIPA.
gp55 from Friend Erythroleukemia Virus Induces Oligomerization and Cell Surface Localization of a Truncated Form of the STK Receptor Tyrosine Kinase.
Authors: He Shihan , Ni Shuang , Paulson Robert F. , Correll Pamela H.
Published: April 2020

Friend disease is an acute erythroleukemia induced by various strains of Friend virus and provides an excellent model to study the multistage progression of carcinogenesis. In the first stage of the disease, the virus infects erythroid progenitor cells and a viral glycoprotein, gp55, interacts with both the EpoR and a truncated form of the stem cell derived tyrosine kinase (STK), Sf-stk. This results in Epo independent expansion of erythroid progenitor cells. Companion with proviral integration, inactivation of p53 and activation of ets gene family members (Spi-1/PU1, Fli-1) in the late stage of disease will cause leukemic transformation. The majority of gp55 is retained on the ER membrane, with only 5% transported to the cell surface. Previous data has demonstrated that the activation of EpoR by gp55 through the dualtropic and transmembrane binding domain on the cell surface is critical for the early stage Friend disease. However, the mechanism by which gp55 leads to Sf-stk activation remains largely unknown. Consistent with previous reports, we find that gp55 immunoprecipitates with Sf-stk under nonreducing conditions and that coexpression of Sf-stk with gp55 results in enhanced tyrosine phosphorylation of Sf-stk. Moreover, we find that Sf-stk forms homo-oligomers when coexpressed with gp55 and, by introducing point mutations in the four cysteine residues in the gp55 ecotropic domain, the cysteine 338 mediates homo-oligomer formation of Sf-stk. Furthermore, mutation of cysteine 338 to alanine inhibits the ability of gp55 to induce Epo independent polyclonal expansion of erythroid precursor cells. We also demonstrate that, in the absence of gp55, Sf-stk is primarily located in the ER, but coexpression of gp55 induces cell surface localization of the truncated receptor. Further, we show that by adding a 6aa ER retention signal to the C-terminal of Sf-stk (Sf-stk-ER), Sf-stk-ER cannot be processed to cell membrane. However, gp55 retains the ability to bind and induce the tyrosine phosphorylation of Sf-stk-ER, and Grb2 is recruited to both Sf-stk and Sf-stk-ER when gp55 is present. Despite active signaling, our preliminary work failed to detect Epo independent colonies from BM cells transfected with Sf-stk-ER followed by Friend virus infection, indicating that a productive interaction with EpoR at the surface may be required for the differentiation of infected progenitors in vitro. Taken together, our data supports a model in which gp55 interaction with Sf-stk through cysteine 338 in the ecotropic domain of gp55 leads to oligomerization of the receptor, resulting in enhanced autophosphorylation and recruitment of Grb2, as well as relocation of active Sf-stk to the cell surface.
The C-Terminal Region of the G-CSF Receptor Is Required for Induction of Neutrophil Elastase Expression in Myeloid Cells: Implication for Understanding CSF3R Mutations in Acute Myeloid Leukemia Evolving from Severe Congenital Neutropenia.
Authors: Dong Fan , Qiu Yaling , Friedman Alan D. , Liu Qingquan
Published: April 2020

Severe congenital neutropenia (SCN) is characterized by early onset of bacterial infections and maturation arrest of myeloid cells at early stages of differentiation in the bone marrow. Point mutations in ELA2 encoding neutrophil elastase (NE) have been identified in 60% to 80% of patients with SCN. SCN patients are predisposed to acute myeloid leukemia (AML), which occurs in approximately 15 % of cases. With rare exceptions, leukemic cells from these patients carry mutations in CSF3R encoding the G-CSF receptor, leading to C-terminal truncation of the receptor. Notably, the nonsense mutations in CSF3R are present only in SCN/AML patients, particularly those with ELA2 mutations, but not in other types of neutropenias and de novo AML. The mechanism for the exclusive presence of the nonsense CSF3R mutations in SCN/AML is unknown. In myeloid 32D cells transfected with the wild type (WT) G-CSF receptor (32D/WT), G-CSF treatment induced the expression of NE. However, NE expression was not upregulated by G-CSF in 32D cells expressing the truncated G-CSF receptor d715, derived from an SCN patient. It has been shown that myeloid cells from patients with SCN/AML express both the wild type and the truncated G-CSF receptors. Indeed, the d715 mutant acted in a dominant negative manner to suppress NE upregulation by the WT G-CSF receptor. In luciferase reporter assays, the WT G-CSF receptor, but not the d715 mutant, activated a 1.8-kb fragment of the mouse Ela2 promoter. Significantly, forced expression of an SCN-associated NE mutant G185R caused premature apoptosis of differentiating 32D/WT cells in response to G-CSF with no significant effect on IL-3-stimulated survival. To address whether the d715 mutant may abolish the proapoptotic effect of the G185R mutant via suppressing its expression, we transfected 32D/WT and 32D/d715 cells with an expression construct in which the expression of the G185R mutant was driven by the 1.8-kb fragment of the Ela2 promoter. G-CSF treatment induced the expression of the G185R mutant and subsequent apoptosis in 32D/WT cells. In 32D/d715 cells, however, the expression of the G185R mutant was not induced by G-CSF and accordingly its proapoptotic activity was not evident. We propose that acquisition of the nonsense mutations in CSF3R may represent a mechanism utilized by the myeloid cells harboring the ELA2 mutations to evade the proapoptotic effect of the NE mutants. However, expression of the truncated G-CSF receptors has other biological consequences: they transduce strong proliferative signals but are defective in inducing granulocytic differentiation, which may initiate the leukemogenic process.
Erythropoietin Fusion Proteins Comprised of Identical Head-to-Tail Repeats with Enhanced Biological Activity.
Authors: Jeong Jee-Yeong , Chen Changmin , Davis Kerry L. , Breidbach Andreas , Catlin Don H. , Sytkowski Arthur J.
Published: April 2020

Recombinant human erythropoietin (EPO, epoetin) is used widely for treatment of chronic anemia due to renal failure, cancer, and other causes. However, considerably high and frequent doses of EPO are required to maintain therapeutic effectiveness, since it has a relatively short in vivo half-life. Thus, alternatives with higher efficacy and/or longer half-life are being developed. We have shown previously that EPO-dimers, either produced by chemical cross-linking of monomeric EPO or expressed as a recombinant fusion protein from COS cells, exhibit enhanced biological properties in vitro and in vivo (Sytkowski, et.al. Proc. Natl. Acad. Sci. USA 95, 1184; Sytkowski, et.al. J. Biol. Chem. 274, 24773). We now report increased activities of EPO-dimer fusion protein and EPO-trimer fusion protein comprised of identical head-to-tail repeats and a 15 or 20-amino acid linker (for dimer), or 17-amino acid linkers (for trimer) produced from stably transfected CHO cells. EPO-fusion proteins were expressed under a CMV promoter with a signal peptide present on the first monomer coding sequence. The EPO-dimer fusion protein was connected with either three or four repeats of Gly-Gly-Gly-Gly-Ser as a 15 or 20-amino acid linker sequence, respectively. The expression levels of EPO-dimer fusion protein from cloned CHO cells to supernatant of protein-free medium ranged from 4 to 40 mg/L determined by EPO-ELISA, and from 2.0×105 to 4.5×106 IU/L determined by in vitro bioassay. We selected clones producing EPO-dimer fusion protein with the greatest extent of glycosylation, as indicated by SDS-PAGE and isoelectric focusing. Subcutaneous injection of mice with three doses of EPO-dimer fusion protein resulted in percent increases in mean hematocrit of 32.6% (300 IU/kg) or 18.2% (100 IU/kg), while equivalent unit doses of EPO-monomer increased mean hematocrit by 12.5% (300 IU/kg) or 6.4% (100 IU/kg). Moreover, a single dose of EPO-dimer fusion protein (100 IU/kg) increased their mean hematocrit by 4.3% within 7 days, while an equivalent unit dose of EPO-monomer had no effect. Importantly, three doses of EPO-trimer fusion protein increased their mean hematocrit by 8.83% per IU injected, which was much greater than that observed with EPO-monomer (0.69%) or EPO-dimer fusion protein (1.81%). The results show that EPO-fusion proteins exhibit biological activities superior to those of EPO-monomer, suggesting important therapeutic advantages.
Transgene Insertion into the Proximity of the c-myb Gene Disrupts Erythroid-Megakaryocytic Lineage Bifurcation.
Authors: Mukai Harumi Yamamoto , Motohashi Hozumi , Nagasawa Toshiro , Yamamoto Masayuki
Published: April 2020

The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival and differentiation of hematopoietic cells. We established three lines of erythropoietin receptor transgenic mice and found that one of them exhibited anemia, thrombocythemia and splenomegaly. These abnormalities were independent of the function of the transgenic erythropoietin receptor, and were observed exclusively in mice harboring the transgene homozygously, suggesting transgenic disruption of a certain gene. The transgene was inserted 77-kb upstream of the c-myb gene and c-Myb expression was markedly decreased in megakaryocyte-erythrocyte lineage-restricted progenitors (MEPs) of the homozygous mutant mice. In the bone marrows and spleens of the mutant mice, megakaryocytes were increased, but erythroid progenitors were decreased. These abnormalities were reproducible in vitro in a co-culture assay of MEPs with OP9 cells, but eliminated by the retroviral expression of c-Myb in MEPs. The erythroid-megakaryocytic abnormalities were reconstituted in mice in vivo through the transplantation of mutant mouse bone marrow cells. To better understand the transcriptional program that accompanies the decline of c-myb gene expression, we performed DNA microarray analysis of MEPs. We identified 74 genes that are upregulated and 36 genes are downregulated in the homozygous mutant mice. Of these genes, expression levels 10 genes are actually changed in bone marrow cells of the homozygous mutant mice, and harbor c-Myb recognition elements in the upstream region. These results thus demonstrate that the transgene insertion into the c-myb gene far upstream regulatory region affects the gene expression at the stage of MEPs, leading to an imbalance between erythroid and megakaryocytic cells, and suggest that c-Myb is an essential regulator of the erythroid-megakaryocytic lineage bifurcation. Elucidation of transcription network based on the c-myb gene in erythroid and megakaryocytic lineages will take a new turn of lineage specific regulation.

Figure Figure
Gfi1-N382S Mutants from Human Severe Congenital Neutropenia Patients Function through a Transcriptional Dominant Negative Mechanism.
Authors: Zarebski Adrian P. , Baktula Avinash M. , Basu Sudeep , Trent John O. , Grimes H. Leighton
Published: April 2020

The Growth factor independence 1 (Gfi1) zinc finger transcriptional repressor is a T cell leukemia oncoprotein that also plays a crucial role in granulopoiesis in both mice and humans. A single point mutation in the amino terminal SNAG repressor domain (P2A) is able to ablate both Gfi1 transcriptional repression activity and linked oncogenic activity in T lymphoctyes. Mice deleted for Gfi1 are lymphopenic, but also lack mature neutrophils. Gfi1−/− mice display a profound block to myeloid differentiation and abnormal promyelocytes accumulate in the blood. Humans with Severe Congenital Neutropenia (SCN) with heterozygous mutations in Gfi1 have similar abnormal promyelocytes. We introduced the SCN patient Gfi1N382S DNA-binding-deficient mutation into murine Gfi1 and overexpressed it in primary murine bone marrow cells. While expression of the wild type Gfi1 resulted almost exclusively in mature granulocyte differentiation, forced expression of the N382S mutant resulted almost exclusively in monocytic differentiation. Flow cytometric analysis revealed a population of N382S-expressing cells with markers of both monocytes and neutrophils resembling the atypical Gfi1−/− promyelocytes. To determine if mutation of the N382 residue is uniquely able to block Gfi1 function, we constructed a virtual model of Gfi1 zinc fingers 3, 4 and 5 interacting with DNA. The model revealed several possible protein-DNA interactions. In order to validate the model we mutated those residues to alanine and performed EMSA with in vitro transcribed/translated proteins. The same alanine substitution mutants were expressed in primary murine bone marrow and tested for their ability to control myelopoiesis. Lack of DNA binding in EMSA tightly correlated with impaired granulopoesis in our in vitro model, suggesting the necessity of intact DNA binding for proper Gfi1 function. These data suggested that the non-DNA binding mutants were able to inhibit repression by wild type endogenous Gfi1, perhaps through the sequestration of limiting corepressor proteins. The Gfi1P2A mutant is unable to repress transcription. We therefore tested the effect of Gfi1P2A expression on myelopoiesis and found that it blocked granulopoiesis equivalently to Gfi1N382S. To rigorously determine whether the titration of limiting corepressors was the cause of N382S neutropenia, we constructed a compound mutant containing both N382S and P2A and expressed it in primary murine bone marrow cells. Expression of Gfi1P2A-N382S had little effect on myelopoiesis. We conclude that SCN patients with heterozygous Gfi1 mutations have blocked granulopoiesis because the non-DNA binding mutant protein competes with the wild type allele for titratable associated cofactors.
Identification of Oxidative Post-Translational Modifications on Plasma Albumin in Patients with Pulmonary Hypertension of Sickle Cell Anemia.
Authors: Klings Elizabeth S. , Odhiambo Adam , Perlman David , Huang Hua , Costello Catherine , Farber Harrison W. , McComb Mark , Steinberg Martin H.
Published: April 2020

Pulmonary hypertension (PH) in sickle cell anemia (SCA) is characterized by decreased nitric oxide bioavailability; which may, in part, be related to increased oxidative stress. It is possible that via protein post-translational modifications, oxidants are able to affect both protein structure and function. We hypothesized that, in patients with SCA and PH, oxidative post-translational modifications occur on plasma proteins and are important in disease pathogenesis. To examine this hypothesis, we chose to determine if oxidative post-translational modifications occur in a large-abundance protein, albumin, as a reflection of the presence of more widespread protein oxidative damage. Plasma was obtained from subjects with:SCA and PH (n=5);SCA steady-state without PH (n=4);Pulmonary Arterial Hypertension (PAH) (n=4);no evidence of cardiopulmonary disease (n=4).

Platelet-poor plasma was separated into albumin-enriched and albumin-depleted fractions. The albumin-enriched fraction was subjected to proteolytic digestion by trypsin and studied by matrix assisted laser desorption ionization (MALDI) mass spectroscopy (MS) and liquid chromatography (LC)-MS/MS tandem mass spectrometry. Proteomics analyses were performed on all samples and post-translational modifications characterized by MS/MS. Results were confirmed by Western and dot-blot analysis using commercially available antibodies. In the albumin fraction, we identified peaks of differential post-translational modification on albumin peptide 146–159 only in PAH and SCA and PH samples; this PTM was identified to be a malondialdehyde adduct. The presence of a malondialdehyde adduct on albumin was confirmed by LC-MS/MS and Western analysis. We have identified a malondialdehyde adduct in plasma albumin that appears to be a common link between PH related to SCA and PAH. This finding supports the notion that oxidative stress modulates the pathogenesis of PH of SCA and suggests that this and other post-translational modifications may be important biomarkers of disease.
New Approach for Finding Novel Antisickling Agents at the NIH NHLBI Sickle Cell Disease Reference Laboratory.
Authors: Asakura Toshio , Yang Jisheng , Chen Qiukan , Evans Greg , Abdulmalik Osheiza
Published: April 2020

Although numerous antisickling agents that chemically bind with sickle hemoglobin (Hb S) and inhibit sickling of sickle erythrocytes (SS cells) in vitro were reported and some of them were brought to clinical trials, none were satisfactory for clinical use. The major problem with this type of drugs is the difficulty of delivering a sufficient amount of antisickling drug to intracellular Hb S because:chemicals that bind with the side chains of hemoglobin also bind with the side chains of other proteins in the gastrointestinal tract, blood and red blood cells (RBCs) prior to binding with intracellular Hb S; andthe concentration of Hb S in SS cells is very high (5 mM of tetrameric Hb S, 20 mM of Hb subunits, or a total amount of approximately 500 grams of Hb S in an adult patient), and the amount of chemical that reaches SS cells upon oral administration cannot modify a significant portion of Hb S to inhibit sickling of SS cells.

For these reasons, drugs that chemically bind with Hb S including those that form Schiff base adducts with Hb S seem to have no clinical value. We found that certain aromatic aldehydes that bind with Hb S allosterically do not have these problems. Since these compounds combine with Hb S physically like a key-and-lock relationship, a low concentration (such as less than 2 mM) of such compound in the blood can bind with a large portion of intracellular Hb S, shift the oxygen equilibrium curve toward the left and inhibit sickling of SS cells (Brit. J. Hematol.128:552–561, 2005). This type of allosteric binding between aromatic aldehydes and Hb is analogous to the binding between 2,3-diphosphoglycerate and Hb. Quite interestingly, our investigations indicate that NIPRISAN (NICOSAN), an herbal medicine that shows a strong antisickling effect both in vitro and in vivo (in transgenic sickle mice) (Brit. J. Haematol.118:337–343, 2002; Brit. J. Haematol.122:1001–1008 2003), contains various aromatic aldehydes (unpublished data). Allosteric drugs designed to interact with specific proteins in the body may be useful as a new type of therapeutic modality for the treatment of sickle cell disease and other diseases. [Researchers with candidate drugs that may have beneficial effects for SCD are welcome to contact the SCD Reference Laboratory for free in vitro evaluation of their agents (for more information, please see our website at www.tatcom.com/sickle-cell).
Placental Growth Factor Prevents Erythroid Suppression by Inflammatory Mediators.
Authors: Means Robert T. , Dallalio Gail , Law Erin R.
Published: April 2020

Although sickle cell anemia is the result of an abnormality in the beta globin chain of hemoglobin, its clinical manifestations cannot be explained solely on that basis. Inflammatory mechanisms contribute to the clinical syndromes associated with sickle cell disease. Placental growth factor (PlGF) is a member of the vascular endothelial growth factor family and is associated with inflammation and with pathologic angiogenesis. Perelman and colleagues have reported that PlGF is released from marrow erythroid cells (Blood2003;102:1506–13). These and other investigators have reported that serum PlGF concentration distinguishes patients with sickle cell disease from healthy controls, although results vary depending upon disease activity at the time of study (Blood2003;1514–24 and Brit J Haematol2006;134:116–9). In studies we reported previously, we measured PlGF and other cytokines in the marrow of stable homozygous sickle cell (SS) patients and controls. Plasma PlGF did not differ between homozygous sickle cell SS patients and controls, but bone marrow PlGF concentrations were significantly higher in SS patients. This was not a phenomenon general to cytokines, and did not reflect increased erythroid activity but rather represented an increase per erythropoietic unit (J Invest Med2005;53:S305–6). In this report, we address the consequences of exposure to PlGF for erythropoiesis in the context of inflammation. We observed that CFU–E from SS patients were less sensitive to inhibition by recombinant human gamma interferon (rhgIFN) than those from healthy controls, with this effect being most pronounced at lower gIFN concentrations. The potential contribution of PlGF to this process was evaluated. At PlGF concentrations 10 – 1000 pg/mL, no inhibition or enhancement of CFU-E colony formation was observed, and there were no differences between the responses of progenitors from SS patients or from normal volunteers to PlGF. However, PlGF 100 pg/mL reversed the inhibitory effects of rhgIFN on CFU-E colony formation (p = 0.04). This effect was most apparent at lower rhgIFN concentrations. Using the HCD57 erythroleukemia cell line as a model, it was found that PlGF produced significant attenuation of Fas ligand induction by rhgIFN. Both HCD57 and CD36+ human marrow mononuclear cells were shown to express Flt-1, a receptor for PlGF. Neutralizing antibody against Flt-1 attenuated the gIFN-protective effect of rhPlGF on CFU-E colony formation. In conclusion, increased PlGF concentrations in the marrow of stable SS patients may contribute to protecting erythroid progenitors from cytokine-induced apoptosis, and may be a mechanism by which erythropoiesis in sickle cell disease is preserved despite concurrent inflammation.
Platelets from Patients with Sickle Cell Disease and Pulmonary Arterial Hypertension Are Hypersensitive to Agonist-Induced Activation.
Authors: Villagra Jose D. , Nichols James T. , Gladwin Mark T. , Kato Gregory J.
Published: April 2020

Pulmonary arterial hypertension (PAH) in sickle cell disease (SCD) is associated with chronic hemolysis and nitric oxide (NO) consumption and characterized by abnormal vascular tone, vascular proliferation and thrombosis. In previous studies, we demonstrated that increased platelet activation is linked to the severity of PAH in SCD. In addition, we reported that patients with SCD and secondary PAH have decreased platelet activation when given sildenafil, a finding that highlights the role of nitric oxide inhibition by hemolysis in this pathological mechanism. Since the vascular homeostasis is altered by the release of hemoglobin in plasma, we hypothesized that platelets of patients with PAH may show an enhanced degree of reactivity to platelet agonists released during hemolysis.

Whole-blood samples were used to determine the in vivo baseline and stimulated platelet activation from SCD patients with PAH, without PAH during steady state (not in pain crisis) and healthy controls. PAH was defined by a tricuspid regurgitant jet velocity ≥ 2.5 m/sec by Doppler echocardiography. Upon withdrawal, citrated venous whole blood was mixed gently with either phosphate-buffered saline (PBS) or escalating doses (1 – 10 μM final concentration) of adenosine diphosphate (ADP), a platelet agonist present in red blood cells. Platelet activation was measured by the flow cytometry detection of activated fibrinogen receptors (activated GPIIb/IIIa) and surface expression of P-selectin. Patients with SCD and PAH reached high degrees of activation with significantly lower concentrations of ADP (EC50 0.88 μM, 95% confidence interval 0.64 to 1.23, n=5) compared to controls (1.3 μM, 1.00 to 1.64, p < 0.01, n=8), and particularly to patients without PAH (1.7μM, 1.31 to 2.27, n=5)(Figure below, left panel). Similar results were observed in parallel experiments with thrombin-receptor activating peptide, another strong platelet agonist.

In separate experiments on platelets from healthy subjects, the addition of cell-free plasma hemoglobin induced platelet activation in dose-dependent fashion (p<0.05), supporting intravascular hemolysis as a mechanism of platelet activation in SCD (Figure below, right panel). Furthermore, cell-free hemoglobin blocked the ex vivo platelet inhibitory effects of an exogenous NO donor, MAHMANONOate (p<0.05), consistent with NO scavenging by cell-free hemoglobin.

These findings suggest that platelets from patients with SCD and PAH are unusually sensitive to activation. Our data also suggests that most of the variability in platelet hyperreactivity observed by other groups in SCD is primarily associated with PAH. Our data support a role for intravascular hemolysis and NO scavenging by cell-free hemoglobin in platelet hyperreactivity. Additional research is needed to define this mechanistic pathway in more detail.

Figure Figure
CCR5 and RANTES/CCL5 Gene Polymorphisms Affect the Risk of EBV Reactivations after Allogeneic HSCT.
Authors: Bogunia-Kubik Katarzyna , Kuzyk Anna , Jaskula Emilia , Lange Andrzej
Published: April 2020

Epstein-Barr virus (EBV) reactivation is a serious complication affecting the recipients of allogeneic hematopoietic stem cell transplants (HSCT). Chemokines and their receptors play a major role in the inflammatory and immune responses that mediate allograft outcome. CC-chemokine receptor-5 (CCR5) is known to be involved in perpetuation of HIV infection (as a co-receptor for HIV entry) and the clinical course of this infection is favored by the presence of the 32-nucleotide deletion within the CCR5 gene (CCR5Δ32 polymorphism). The CCR5 deletion mutation (CCR5Δ32) results in a non-functional chemokine receptor. In the present study the CCR5Δ32 polymorphism and two single nucleotide substitutions (−28 C/G; −403 G/A) within, one of the CCR5 ligands, the CCL5/RANTES gene were analyzed in 75 HSCT recipients, 75 donors and related with EBV load. The control group constituted 99 healthy individuals. DNA was extracted from peripheral blood taken into EDTA using silica membranes. Viral load were assessed 2-3 moths after transplantation in blood cells employing a real-time PCR technique. The detection threshold for viral reactivation equaled 10 EBV-DNA copies/105 peripheral blood cells. EBV reactivation was detected in 26 patients. The CCR5Δ32 and RANTES (−28 C/G; −403 G/A) polymorphisms were analyzed by PCR and PCR-RFLP technique, respectively. Distributions of CCR5 and RANTES −28 genotypes were similar in patients, donors and healthy individuals. RANTES −403 AA homozygosity was more frequent in donors than controls (0.61 vs 0.47, p=0.036). The higher number of EBV copies was detected in patients lacking CCR5Δ32 deletion (0.92 vs 0.71, p=0.031). No significant correlation between EBV reactivation and RANTES −28 C/G polymorphism. However, patients transplanted with donors homozygous for RANTES −403 AA (genotype associated with an increased expression of the RANTES gene) more frequently presented with EBV reactivation (0.85 vs 0.49, p=0.002). In conclusion, the presence of the CCR5 deletion-mutation in the recipient and RANTES-403 G in the donor of HSC appeared to lower the susceptibility for EBV reactivation.
In Vivo Permanent Marking of Cells with RAG1 Activation History Demonstrates Only a Minor Contribution of the Lymphoid Pathway to Dendritic Cell Development.
Authors: Chong Yong , Mizuno Shin-ichi , Shigematsu Hirokazu , Arinobu Yojiro , Iino Tadafumi , Iwasaki Hiromi , Akashi Koichi
Published: April 2020

Dendritic cells (DCs) have been shown to arise from both myeloid and lymphoid pathways, by evaluating the in vivo reconstituting potential of myeloid- and lymphoid-committed progenitor populations. It has been suggested that these “myeloid” or “lymphoid” DCs may constitute independent DC compartments with different functions. However, evaluation of DC development after conventional adoptive transfer experiments may not correctly represent normal DC development including lineage contribution toward the formation of the DC pool. In order to accurately evaluate the distribution and the function of DCs originated from each lineage, it is ideal to mark their origin in vivo in steady-state hematopoiesis. By crossing RAG1-Cre knockin with yellow fluorescence protein (YFP)-floxed reporter lines, we developed a mouse line in which cells with a history of RAG activation should be permanently marked with YFP as a result of lymphoid commitment. The YFP expression started at the common lymphoid progenitor (CLP) stage (~35%), and the percentages of YFP+ cells progressively increased as they differentiate into mature lymphocytes. More than 99.5% of mature T and B cells were marked by YFP, while <0.5% of granulocyte/monocyte progenitors (GMPs) or granulocytes were YFP+, indicating that this system efficiently and exclusively marks cells of lymphoid origin. We found that only 4.1, 4.6, and 9.3% of DCs were YFP+ in the spleen, the lymph nodes, and the thymus, respectively. This is the formal evidence that the vast majority of DCs in steady-state hematopoiesis originate from stages that have committed to the myeloid lineage, even in the thymus. We then profiled the gene expression of the YFP+ and YFP- DCs at a genome wide level by DNA microarray analysis. Unexpectedly, the gene expression profiles of myeloid and lymphoid-derived DCs are virtually identical. These results collectively suggest that myeloid and lymphoid DCs might use a common developmental program that can be activated even after myeloid or lymphoid commitment. Thus, the DC developmental program is unique, which should be independent of either myeloid or lymphoid commitment program.
Mouse Model for Shwachman-Diamond Syndrome with the R126T Disease Mutation Leads to Severe Growth and Developmental Deficiencies with Impairment of Hematopoiesis.
Authors: Zhang Siyi , Zhong Jian , Gan Olga I. , Dick John E. , Rommens Johanna M.
Published: April 2020

Shwachman-Diamond syndrome (SDS) is a bone marrow failure syndrome characterized by exocrine pancreatic insufficiency and skeletal abnormalities, as well as hematological dysfunction. SDS is caused by mutations in SBDS, a highly conserved gene that has been suggested to be involved in RNA metabolism and/or ribosome biogenesis. It is essential, based on our mouse knock-out model studies and the absence of observed patients with the combination of two null alleles. We have generated an allele with the disease mutation, R126T, in the murine ortholog in order to develop new models of SDS. This mutation has been interpreted to be hypomorphic in nature, as it occurs in combination with the common null disease allele in two patients. Interestingly, these patients also have severe hematological disease phenotypes.

SbdsR126T/+mice develop normally and show no disease phenotypes, in accordance with the recessive inheritance of SDS. However, the SbdsR126T/R126T and SbdsR126T/- mice did not survive birth, and exhibited marked size reduction. The growth difference became apparent in embryos during the mid-fetal period, at E12.5–14.5; there were also noted disturbances of major organs including the skeleton, brain and lung. Comparable deficiencies were noted overall, but the SbdsR126T/- embryos were consistently more severely affected than were SbdsR126T/R126T embryos. Examination of hematopoietic progenitors from the fetal livers of SbdsR126T/R126T and SbdsR126T/- mutant mice also showed marked reductions in BFU-E, CFU-G, CFU-M, CFU-GM and CFU-GEMM numbers, as revealed by standard colony formation assays, when compared to wild type and heterozygote littermates.

In additional studies, primary fibroblast cultures from E16.5 day mutant embryos were found to exhibit slower growth and an extended G1 phase of the cell cycle compared to fibroblasts from wild type embryos. Further, total protein synthesis was measured by incorporation of radio-labeled amino acids and found to be notably reduced in the mutant fibroblasts. The small size, organ deficiencies and early death of the mutant mice together with the cellular deficiencies, which indicate severe autologous growth problems, emphasize the severe consequences of loss of Sbds. These animal disease models provide new avenues for investigation to elucidate basic functions of SBDS and the pathobiology of SDS.
Hematopoietic Specific GATA-1-Improved Cre Mouse for Erythroid-Specific Gene Modification.
Authors: Yoon Donghoon , Kim Bumjun , Kwon Myunghi , Prchal Josef T.
Published: April 2020

Animal models of erythropoiesis related genes have been limited by the fact that some of these genes have non-erythroid expression and other functions in addition to erythropoiesis and thus their knock-out may be embryonic lethal. Tissue specific knock-out or knock-in mice models employing GATA-1-Cre and other constructs showed that these promoters are also active in non-hematopoietic tissues, i.e. GATA-1 has activity in early embryonic development and in neuronal tissue. Suzuki et al (Blood, 2002, 100; 2279) isolated the GATA-1 locus hematopoietic regulatory domain (GATA-1-HRD) and demonstrated that the expression of a transgene under its control is limited to the hematopoietic tissue. We generated a transgenic mouse expressing an improved Cre (iCre) under GATA-1-HRD promoter control. This mouse was crossbred with ROSA 26 mouse and the progeny was examined for tissue specificity of iCre expression using beta-galactosidase staining. Brain, spleen, kidney, heart, thymus, liver, lung and ovary were examined for whole organ LacZ staining. All tested organs were negative except kidney and spleen where some positivity was observed. Subsequently, we prepared tissue sections from kidney, spleen and bone marrow and stained with LacZ and anti-beta-galactosidase antibody. Only the bone marrow EpoR expressing cells were positive; the kidney and the spleen cells were negative. Although Suzuki et al previously showed expression of the GATA-1-HRD driven erythropoietin receptor in spleen using RT-PCR, we were not able to find iCre expression in the splenic cells using these approaches. We demonstrate that our transgenic mouse (GATA-1-HRD-iCre) showed a restricted iCre expression in hematopoietic tissue that differs from previous studies of other hematopoiesis specific cre mouse. We conclude that this mouse model should be useful in studies of function of erythroid specific genes.
A Non-Invasive Evaluation of Hemoglobin with a New Optical Sensor.
Authors: Berrebi Alain , Amir Orna , Weinstein Aharon , Monashkin Emmanuel , Holland Paul
Published: April 2020

Background: The performance of the NBM-100, a new non-invasive Hematocrit (Hct) measurement device, was tested in primary and secondary medical care environments. The device utilizes a finger sensor with Occlusion Red/Near-Infrared Spectroscopy (O-RNIRS) technology to non-invasively measure hemoglobin and Hct levels (Figure). A non-invasive Hct measurement has many potential advantages including the prevention of pain and potential transmission of infectious diseases, and the reduced need for trained technicians.

Methods: In order to investigate the use of the device in both primary and secondary medical environments, clinical trials were conducted in a blood donation center (USA), an oncology clinic (USA) and a hematology clinic (Israel). The studies were carried out on a group of 304 subjects (155 Female, 149 Male), ages 18–96 (average age 54.9).

After signing a written consent, all study volunteers were tested non-invasively with the NBM-100 device, and invasively, using a venous sample evaluated on a Cell-Dyn (Abbott) blood analyzer. At the blood donation center the NBM-100 measurements were performed before donation, and the venous blood sample was taken after donation. To neutralize the decrease in Hct occurring during blood donation, as described in published clinical trials, a value of 2.2% was added to the reference venous values obtained from the blood donation center.

Results: The operating staff at all centers found the NBM-100 easy to use and appreciated by subjects. The venous Hct measurements ranged from 15% to 51%. The mean NBM-100 Hct was 36%, the same as the mean venous result. The mean absolute error (MAE) was 2.66% and the relative absolute error (RAE) was 7.9%. The standard deviation of the difference between the venous reference and noninvasive Hct readings was found to be 3.34% and the correlation between the two devices was r=0.9. The bias between the two methods was a negligible 0.40%.

Conclusions: This study supports the use of the NBM-100 device as a useful, safe and accurate non-invasive method for measuring a wide range of Hct in both a hospital (e.g. surgery and blood transfusions) and ambulatory setting (e.g. blood banks). Good agreement was found between the non-invasive NBM-100 and the invasive Cell-Dyn Hct measurements. The non-invasive system was found to be environmentally - and user-friendly by the operating staff. These findings support the high clinical utility of a commercial application of the NBM-100 device.
Altered Immune Response in Severe Malaria Anemia in Children.
Authors: Gordeuk Victor R. , Kasvosve Ishmael , van Dijk Janneke , Weiss Guenter , Debebe Zufan , Nekhai Sergei , Kuddo Thea , Thuma Philip E.
Published: April 2020

We prospectively assessed immune markers in children <6 years with severe malarial anemia (hemoglobin <5.0 g/dL; n = 72) and uncomplicated malaria (n = 69) who presented to Macha Mission Hospital in Zambia’s Southern Province. We also studied 70 children <6 years who presented to well child clinics in Harare, Zimbabwe as controls. Compared to controls, children with uncomplicated malaria had significantly higher temperatures and parasite counts, lower hemoglobin and platelet concentrations, higher plasma levels of interferon-gamma, tumor necrosis factor alpha, and interleukin 10 and lower levels of monocyte inhibitory factor (MIF). Compared to uncomplicated malaria, severe malaria anemia was associated with younger age, longer duration of fever and lower temperature on admission. Reticulocyte index and serum concentrations of bilirubin and LDH did not differ between the malaria groups, suggesting that unusually severe extra- or intra-medullary hemolysis did not explain the severe anemia. Higher white blood cell and platelet counts in the severe malaria group suggested that pan-suppression of the marrow was also not the primary cause. Of originally selected measures of inflammation, plasma levels of TNF-alpha and MIF did not differ between the malaria groups, but concentrations of both interferon-gamma and interleukin-10 were significantly lower in the severe anemia group (P <0.006). Additional testing revealed levels of interleukin-1alpha, interleukin-6, and IP-10 to be lower and levels of sFAS to be higher in the children with severe anemia versus uncomplicated malaria (P <0.0005). In a logistic regression model, severe malarial anemia was associated with younger age (P = 0.010), prior treatment with sulfadoxine/pyrimethamine or traditional medicine (P <0.32), lower levels of Interleukin-10 (P = 0.025) and higher levels of sFAS (P = 0.003) and TNFa (P = 0.013). Our results are consistent with a multifactorial cause of severe malarial anemia, possibly including infection with resistant plasmodia, over-expression of TNF-alpha in conjunction with under-expression of IL-10, and increased apoptosis.
SNP Associations with Event Free Survival in Myeloma from Two Phase III Clinical Trials Using the Bank On A Cure Chip.
Authors: Van Ness Brian G. , Crowley John C. , Ramos Christine , Grindle Suzanne M. , Hoering Antje , Haessler Jeff , Jacobus Susanna , Durie Brian G. , Katz Michael , Rajkumar Vincent S. , Barlogie Bart , Johnson David C. , Morgan Gareth J.
Published: April 2020

While there are certain common clinical features in myeloma, the disease shows significant heterogeneity with regard to disease progression, and responses to therapy, affecting both survival and toxicities. Heritable variations in a wide variety of genes and pathways affecting cellular functions and drug responses likely impact patient outcomes. In the Bank On A Cure (BOAC) program we have developed a custom chip that assesses 3,404 SNPs representing variations in cellular functions and pathways that may be involved in myeloma progression and response. The chip has gone through rigorous quality controls checks for high call rates, accuracy, and reproducibility that will be presented. Using the BOAC chip, we have conducted studies to look for SNPs that may identify biologic variations that are associated with good or poor response across a variety of treatments. In this study we looked for SNPs that may distinguish short term and long term survivors in two phase III clinical trials: ECOG E9486 and intergroup trial S9321. E9487 patients were treated with VBMCP followed by randomization to no further treatment, IFN-alpha, or cylcophosphamide; and, although there was variation in survival, no significant differences in survival were noted among the 3 arms of the trial. Patients included in this SNP study from S9321 received VAD induction followed by randomization to VBMCP or high dose melphalan + TBI. SNP profiles were obtained for patients with less than 1 year EFS (n=20 in E9487; n=50 in S9321) and patients showing greater than 3 years EFS (n=32 in E9486; n=41 in S9321). Statistical approaches were performed to identify single and groups of SNPs that best discriminated the survival groups. Previous studies have suggested genetic variations in drug metabolism genes, p-glycoprotein transport, and DNA repair genes may influence survival outcomes. Our results show significant survival associations of genetic variations in genes within these functional categories (eg. GST, XRCC, ABCB, and CYP genes). Although genetic variations were found that were uniquely associated with each clinical trial, several of these genetic variations show survival associations that increase in significance when the two trials were examined as a conglomerate data set. Grouping genetic variations through common pathway approaches using gene set enrichment analysis, as well as clustering or partitioning algorithms, further improve the value of the SNPs as potential prognostic markers of survival outcomes. These results and statistical approaches will be presented, and represent steps toward identifying patient variations in biologic mechanisms important in predicting therapeutic outcomes.