bluuebunny/crossref_metadata_2025 huggingface dataset:
Dexamethasone Induces Apoptosis “Ex Vivo” in Chronic Lymphocytic Leukemia Cells with Either Unmutated IgVH Genes or High ZAP-70 Expression.
Authors: Muntanola Ana , Crespo Marta , Gine Eva , Villamor Neus , Montserrat Emili , Bosch Francesc
Published: April 2020

Patients with chronic lymphocytic leukemia (CLL) and unmutated IgVH genes or high ZAP-70 expression have poorer prognosis than those with mutated IgVH genes or low ZAP-70 levels. This is in part related to the resistance of unmutated and ZAP-70 positive cases to treatment agents that induce apoptosis in a p53-dependent manner. It has been suggested that corticosteroids are active in CLL through p53-independent pathways. The aim of this study was to analyze the “ex vivo” response to dexamethasone in CLL cells according to the IgVH mutational status and ZAP-70 expression. Frozen lymphocytes from 60 patients with CLL were analyzed for ZAP-70 expression and IgVH mutational status (n=44). Cells were cultured and treated using fludarabine (5μgr/ml), mitoxantrone (0.5μgr/ml), FCM (fludarabine 1μgr/ml, maphosphamide 1μgr/ml and mitoxantrone 0.5 μgr/ml), and dexamethasone (5.2 μg/mL). Cell viability and apoptosis were determined by annexinV/PI staining and FACscan analysis at three different time points and conditions (0h and 24h without treatment, and 24h with treatment). The expression of glucocorticoid receptor (GR) isoforms alpha, beta and gamma was analyzed by Quantitative RT-PCR in 20 cases. Dexamethasone-induced apoptosis was significantly higher in patients with unmutated IgVH and/or high ZAP-70 expression (≥20%) than in those with mutated IgVH and/or low ZAP-70 expression (median cell viability 65% vs. 81%, respectively; p<0.001). In contrast, mitoxantrone induced higher cell toxicity in patients with IgVH mutations or low ZAP-70 expression (p=0.009). No differences in cell viability were found according to ZAP-70 expression or IgVH mutational status after “ex vivo” treatment with fludarabine or FCM (p=0.649 and p=0.055, respectively). Expression of different GR isoforms was similar in corticosteroid-responders and non-responders. In conclusion, in this study CLL cells with unmutated IgVH genes or high ZAP-70 expression showed a higher cell mortality after “ex vivo” exposure to dexamethasone than those with mutated IgVH genes or low ZAP-70 expression, with no relationship with the expression of the different GR isoforms. These data give conceptual support to trials aimed at determining the role of dexamethasone in the treatment of patients with CLL and poor prognostic features or resistant to fludarabine.

Figure Figure
A Randomized Trial of Valacyclovir Versus Valganciclovir To Prevent CMV Reactivation in Patients with CLL Receiving Alemtuzumab.
Authors: O’Brien Susan , Ravandi-Kashani Farhad , Wierda William G. , Giles Francis , Thomas Deborah , Huang Xuelin , Riehl Todd D. , Tarrand Jeffrey J. , Brandi O’Neal , Kantarjian Hagop M. , Keating Michael J.
Published: April 2020

Alemtuzumab is an effective treatment for patients with CLL, but is an immunosuppressive antibody that depletes normal T-cells as well as B-cells. Prophylaxis for HSV and PCP is standard with this agent. About 25% of patients will experience CMV reactivation, manifested by fever and antigenemia. We conducted a randomized trial wherein patients being treated with an Alemtuzumab-containing regimen received either prophylaxis with valacyclovir 500 mg po daily, or valganciclovir 450 mg po b.i.d. CMV antigen was monitored every 2 weeks for 12 weeks. The study design planned to enroll 128 patients, but stopping rules for early termination were met. Forty-six patients were entered, six were inevaluable. Median age was 58 years (range 25–83); median number of prior therapies was 2 (0–10). Diagnoses included CLL (29), T-PLL (3), HCL (1), ATLL (1), marginal zone leukemia (1), LGL leukemia (2), ALL (1), T-cell lymphoma (2). Of patients with active CLL (some received treatment for MRD), Rai stage was I–II = 13, III–IV = 10. Patients received varying Alemtuzumab-containing regimens including single agent (5), or combined with: Rituximab (2), Pentostatin (6), FCR (23), or Hyper-CVAD (4). Seven of 20 patients enrolled on the valacyclovir arm experienced symptomatic CMV reactivation with the number of CMV antigen containing cells ranging from 19–651. None of the 20 patients randomized to valganciclovir experienced symptomatic CMV reactivation (p=0.004). One patient receiving valganciclovir had 6 CMV antigen + cells at 2 weeks but was asymptomatic and continued on prophylaxis; repeat testing at 4 weeks showed 0 Ag + cells. Given the heterogeneity of regimens and the frequency of myelosuppression expected, it is uncertain as to whether valganciclovir increased the incidence of myelosuppression. In conclusion, this agent was highly effective for prophylaxis of CMV in patients receiving Alemtuzumab. Further studies may address whether the use of this antibiotic with single-agent Alemtuzumab increases the incidence of myelosuppression and whether a lower dose of valganciclovir (450 mg daily) would be as effective in prophylaxis.
A Non-Internalizing Anti-CD40 Antibody, CHIR-12.12, Blocks CD40L-Induced Cytokine Production and Mediates Greater ADCC Than Rituximab in Primary CLL Cells.
Authors: Tong Xia , Aukerman Sharon Lea , Lin Karen , Aziz Natasha , Goldbeck Cheryl , Georgakis Georgios V. , Younes Anas , Weng Wen-Kai , O’Brien Susan , Wierda William , Jallal Bahija , Luqman Mohammad
Published: April 2020

CD40 is expressed on chronic lymphocytic leukemia (CLL) cells, and CD40 activation leads to signaling critical for cell survival and proliferation. We have previously described a novel, fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, generated in XenoMouse® mice (Abgenix, Inc.), and have demonstrated that it inhibits normal human B cell proliferation and survival and mediates potent antibody-dependent cellular cytotoxicity (ADCC) against primary CLL and non-Hodgkin’s lymphoma cells. In this study, we examined the ability of CHIR-12.12 to modulate cytokine production by primary CLL cells and compared the ADCC activity of CHIR-12.12 with rituximab against primary CLL cells. Primary CLL cells stimulated with CD40L produced a variety of cytokines, including IL-10, TNF-α , IL-8, GM-CSF, IL-6, MCP-1, and MIP-1β. Addition of CHIR-12.12 to primary CLL cells inhibited CD40L-mediated production of these cytokines. Cytokine production by primary CLL cells cultured with CHIR-12.12 alone in the absence of CD40L did not exceed levels produced by CLL cells cultured in medium. These data suggest that CHIR-12.12 is a potent antagonist for CD40L-mediated cytokine production by primary CLL cells and shows no agonistic activity by itself. We next compared the relative ADCC activity of CHIR-12.12 and rituximab against ex vivo primary CLL cells from 8 patients. CHIR-12.12 exhibited greater ADCC than rituximab against CLL cells from all patients. The average percent of maximum lysis by CHIR-12.12 and rituximab were 49 ± 16% and 31 ± 14%, respectively. CHIR-12.12 was greater than 10-fold more potent than rituximab, as measured by ED50 values (14.1 pM versus 155.5 pM, respectively). Quantitative CD20 and CD40 density on CLL cells and the degree of antibody internalization were investigated as potential reasons for the difference in ADCC activity. The greater ADCC potency and efficacy of CHIR-12.12 was not dependent on a higher density of cell surface CD40 molecules, as there were 1.3 to 14-fold higher numbers of CD20 than CD40 molecules on the cell surface. Antibody internalization studies using primary CLL cells conducted by flow cytometry and confocal microscopy show that upon binding to CD40 at 37°C, CHIR-12.12 remains uniformly distributed on the cell surface, even after 3 hours. In contrast, after binding at 37°C, rituximab is redistributed into caps and internalized. These data suggest that the potent ADCC activity of CHIR-12.12 may be partly related to its ability to remain on the surface of target cells uniformly, allowing optimal interaction with effector cells. Taken together, these results suggest that CHIR-12.12 may be effective at mediating potent ADCC against CLL cells in vivo. CHIR-12.12 is currently in Phase I trials for B-cell malignancies.
Treatment of Refractory Autoimmune Hemolytic Anemia in B-CLL with Alemtuzumab (Humanized CD52 MAb).
Authors: Lundin Jeanette , Celsing F. , Karlsson C. , Osterborg A.
Published: April 2020

Alemtuzumab is a humanized monoclonal antibody that targets the CD52 antigen. CD52 is abundantly expressed on leukemic B-CLL cells (approximately 4 x 105 binding sites/B-CLL cell) and to a lesser degree on normal B and T cells, which may result in pronounced immunosuppression. Treatment with alemtuzumab has achieved an ORR of 33% in patients with fludarabine refractory B-CLL (Keating et al. J Clin Oncol.2002;20:205–213) and >80% in patients with symptomatic but previously untreated B-CLL (Lundin et al. Blood.2002;100:768–773). Progressive B-CLL is frequently complicated by autoimmune complications such as autoimmune hemolytic anemia (AIHA), which is estimated to occur in 10% to 20% of patients. We report here on 5 patients with advanced B-CLL and 1 with hairy cell leukemia (earlier diagnosed as B-CLL), all of whom developed severe AIHA resistant to conventional therapy (eg, corticosteroids, rituximab, chemotherapy, etc.) that received alemtuzumab as salvage treatment for AIHA. The median age of patients was 66 years (range, 59–77 years). At the start of alemtuzumab therapy, all patients had disease classified as Rai stage III or IV, were heavily pretreated with chemotherapy, and had severe transfusion dependent DAT-positive AIHA, which was resistant to conventional AIHA therapy, with a median Hb of 7.8 g/dL. The alemtuzumab dose was quickly escalated to 30 mg SC tiw (n = 3) or IV (n = 3), and continued for a median treatment period of 4 weeks (range, 3–8 weeks). Of 6 patients who received alemtuzumab, 5 responded with a >2 g/dL rise in Hb concentration, which eliminated the need for transfusion after a median of 3 weeks (range, 2–8 weeks). At the end of therapy, the median Hb concentration in the responding patients was 122 g/dL (range, 100–125 g/dL). Alemtuzumab was well tolerated with only minor “first-dose” reactions. However, 1 patient had a CMV reactivation, as indicated by a fever of unknown origin that responded rapidly to foscarnet. All patients were alive at a median follow-up time of 5 months, and none of the responding patients experienced another episode of AIHA. We conclude that alemtuzumab may be an important therapeutic option for patients with severe, refractory CLL-related AIHA, who have not previously responded to conventional treatment.
A Genome-Wide Retroviral Insertional Mutagenesis Screen for Genes Cooperating with Truncated, Oncogenic GATA1s.
Authors: Li Zhe , Klusmann Jan-Henning , Godinho Frank J. , Lee Hee-Won , Reinhardt Dirk , Orkin Stuart H.
Published: April 2020

Somatic mutations in the hematopoietic transcription factor GATA1 are found in megakaryoblasts of Down Syndrome (DS) patients with transient myeloproliferative disorder (TMD, or transient leukemia or TL) and the related acute megakaryoblastic leukemia (DS-AMKL, or DS- AML M7). These mutations lead to production of a GATA1 variant (GATA1s) lacking its N-terminal domain. Mice carrying GATA1s mutation have normal adult hematopoiesis. However, during embryonic/fetal development, we have identified a transient population of abnormal yolk sac/fetal liver megakaryocytic progenitors in mutant mice. We proposed that these progenitor cells are the target for transformation in DS-AMKL/TMD. GATA1s mice (either during development or as adults) do not develop myeloproliferative disorder or leukemia. To recapitulate human DS TMD in mice, we bred GATA1s mice to mouse DS models (Ts65Dn and Ts1Cje) and generated GATA1s/DS double mutants. The phenotype of GATA1s/DS mice is not different from that of GATA1s mice, suggesting that these mouse DS models (representing ~166 and 112 trisomic genes on human chromosome 21, respectively, including Runx1, Ets2, and Erg) do not accurately recapitulate the effects of trisomy in DS. To search for genes that cooperate with GATA1s in an unbiased fashion, we established a genome-wide retroviral insertional mutagenesis screen. GATA1s mutant fetal liver progenitors proliferate in culture in the presence of thrombopoietin (Tpo) for about 4–5 weeks. We infected mutant fetal progenitors with MSCV retrovirus and selected in vitro in the presence of Tpo for immortalized cell lines. Retroviral integration sites in these cell lines were determined by Splinkerette PCR, and confirmed by genomic PCR. Genes that were affected by retroviral integration were confirmed by real-time PCR for their elevated expression or knock-down. From the genetic screen performed thus far, we identified two common retroviral integration sites, Evi1 and Prdm16 (PR domain containing 16). Interestingly, Evi1 is also overexpressed in M7 leukemias, though its expression in non-DS M7 leukemia is higher than in DS M7 leukemia. By retroviral overexpression, we have confirmed that ectopic expression of Evi1 in GATA1s mutant fetal progenitors further enhanced proliferation. Currently we are testing the in vivo leukemogenic abilities of these cell lines by transplantation. By this approach, we will identify genes that cooperate with GATA1s in cellular transformation and, thereby, gain insights into the mechanism of leukemogenesis in DS-AMKL/TMD.
Dissecting the Molecular Mechanisms of Mutant C/EBPalpha (p30) in Normal and Malignant Human Hematopoiesis.
Authors: Cammenga Jorg , Niebuhr Birte , Horn Stefan , Delwel Ruud , Stocking Carol
Published: April 2020

Acute myelogenous leukemia (AML) is often associated with mutations in transcription factors that are essential for normal hematopoietic development and differentiation. The basic leucine zipper (bZIP) protein C/EBPalpha is mutated in 7–10% of AML. Two different classes of C/EBPalpha mutations are found in AML: 1) mutations that introduce a stop codon between the primary translational initiation site and a downstream ATG, resulting in translation of an N-terminally truncated protein (p30) 2) mutations that disrupt the basic region of the bZIP domain in the C-terminus, abolishing DNA binding. Of note, biallellic mutations are often but not always observed. It has been proposed that p30 is dominant-negative over wt C/EBPalpha (p42), blocking its transcriptional activity. We have previously shown that p30 blocks erythrocytic commitment and inhibits terminal granulocytic differentiation when expressed in human but not murine primary hematopoietic cells. To identify the molecular mechanism by which the p30 inhibits differentiation and to evaluate the role of homo- and heterodimerization between p30 and p42 in leukemogenesis, we created a p30 that carries point mutations in bZIP (p30-L12V) that disrupt dimerization. Expression of p30-L12V had no effect on the differentiation of human CD34+ cells, arguing for the requirement of dimerization. To inhibit potential heterodimerization between p30 and p42 but permit p30 homodimerization, we generated a p30-GZ, in which the leucine zipper was replaced by an artificial dimerization domain. Expression of p30-GZ induced a similar differentiation block as the normal p30, arguing against a dominant-negative function of p30 over p42 mediated by dimerization. In a second approach to understand the mechanism by which p30 inhibits differentiation, we wanted to identify gene targets of p30. For this purpose, p30 was fused to the modified ligand-binding domain of the estrogen receptor (p30-ERtm). Expression of p30-ERtm in CD34+ cells in the presence of tamoxifen showed a phenotype very similar to the one observed after the expression of p30, while in the absence of tamoxifen no effect was observed. We used this system of inducible p30 expression in human CD34+ cells to evaluate gene expression patterns. If p30 is dominant-negative and interferes with the transcriptional activation of p42, reciprocal expression patterns of target genes should be observed for wt C/EBPalpha and p30. Our data show that homodimerization of p30 is necessary and sufficient to block erythrocytic commitment and terminal granulocytic differentiation of human CD34+ cells and to induce known C/EBPalpha target genes. These data argue that the formation of p30/p42 heterodimers is not required for the effect of C/EBPalpha p30, questioning the proposed dominant-negative function.
Reduced Binding of C/EBPα to Myeloid Specific Promoters with Altered Gene Expression in the Presence of PML/RARα.
Authors: Wagner Katharina , Krauter Jürgen , Görlich Kerstin , Drescher Bettina , Johansen Lisa M. , Radomska Hanna S. , Duprez Estelle , Ganser Arnold , Tenen Daniel G.
Published: April 2020

The hallmark of acute promyelocytic leukemia is the reciprocal translocation t(15;17) resulting in the fusion protein PML/RARα . According to the current concept of the pathogenesis of APL, PML/RARα interferes with RARα target gene expression. Until now, the crucial target genes have not been found. The transcription factor C/EBPα is essential for myeloid development and mutations have been found in 10% of AML patients. No mutations of C/EBPα in APL have been described. However, using the U937 cell line with a Zinc-inducible PML/RARα (U937PR9), a decrease of C/EBPα binding to the G-CSF-R promoter in the presence of PML/RARα was detected in EMSA as well as chromatin immunoprecipitation. This finding indicates, that the function of C/EBPα might be impaired in APL. To test whether the effect is limited to the G-CSF-R promoter, C/EBPα binding to other myeloid promoters was analyzed upon Zn-induction of PML/RARα . In those experiments decreased binding of C/EBPα to other promoters of myeloid specific genes, such as neutrophil elastase, was detected. In contrast, no effect of Zn-induction on the binding of Sp3 to the thymidine-kinase promotor was found. Next, the functional importance of the alteration in binding was demonstrated. In the presence of PML/RARα , decreased G-CSF-R RNA expression as well as G-CSF-R surface expression was found in U937PR9 cells. In addition, the RNA expression of elastase was reduced after Zn-induction. To investigate the effect of PML/RARα on C/EBPα function in t(15;17) positive cells, a knockdown of PML/RARα by lentivirally delivered shRNAs in NB4 cells was used. After lentiviral transduction, a marked decrease of PML/RARα RNA and protein was found. Consistent with the results in U937PR9 cells a 5–6 fold increase of elastase RNA expression and a 2 fold increase in G-CSF-R-RNA was observed after PML/RARα knockdown. Taken together, decreased binding of C/EBPα to promoters of myeloid specific genes was found in the presence of PML/RARα . This alteration in binding was associated with a reduction of RNA expression. Decreasing PML/RARα with siRNA had the opposite effects. Currently, the mechanism for this effect is being investigated.
Identification of a MEF Gene Mutation in a Familial Hemophagocytic Lymphohistiocytosis Patient That Decreases MEF Transcriptional Activity.
Authors: Lacorazza H. Daniel , Lopiccolo Serena , Rubenstein Eric , Liu Yan , Nunes Juliana , Hasle Henrik , Fadeel Bengt , Ericson Kim , Henter Jan-Inge , Nimer Stephen D.
Published: April 2020

Familial hemophagocytic lymphohistiocytosis (FHL) is generally an autosomal recessive disease of early childhood characterized by uncontrolled T cell and macrophage activation leading to a fatal accumulation of activated T lymphocytes and histiocytes in different tissues. Immune regulation is severely impaired in these patients (both CTL and NK cells) and defects in perforin gene expression, an important component of the lytic granules, were the first defects identified in these patients. So far, FHL genetics have identified a locus at chromosome 9q22 (not molecularly defined yet), at chromosome 10q21-22 (where mutations in perforin gene have been described in 20% of FHL patients), at chromosome 17q25 (where mutations in the Munc13-4 gene have been found) and most recently at chromosome 6q24 (where mutations in the syntaxin-11 gene have been found). We described previously that MEF-null mice, which lack the ETS-transcription factor also known as ELF-4 (located at Xq26.1), have impaired NK cell development and function, and no NK cell perforin gene expression. We also showed that MEF regulates the human perforin proximal promoter via two ETS binding sites and showed that MEF is bound to the perforin promoter in vivo, using chromatin immunoprecipitation. Subsequently, we initiated a study of MEF gene integrity in male patients that have been studied and lacked perforin gene mutations. In addition to a known polymorphism in exon 9 (503 C→T) present in approximately 30% of unrelated patients and leukemic cell lines, we found a nucleotide change in exon 9 of MEF (408, C→T) in a male FHL patient from Denmark. The mutation found in the son, who was diagnosed with FHL and received a BMT at 12 years of age, was also seen in the patient’s grandmother, mother and one sister (heterozygous). Mutation 408 leads to an amino acid change (Val→Met) in a region located between the Ser/Thr and Pro rich domains in the C-terminal portion of MEF. We generated a MEF-mutant (MEFVal→Met) protein; it has a reduced capacity to activate the perforin gene promoter (only 30–40% of the wild type). MEFVal→Met does not have a dominant negative effect, when co-transfected with wild type protein, which could account for the FHL phenotype found in the male only. We are further examining the biology of this mutation in MEF-null cells to see if it can contribute to FHL pathology. Transcription factor abnormalities, such as the MEF mutation in this family, should be evaluated to help explain the pathogenesis of FHL in different patients.
C/EBP Is Deregulated by PML-RAR in Acute Promyelocytic Leukemia.
Authors: Guibal Florence , Radomska Hanna S. , Johansen Lisa M. , Tenen Daniel G.
Published: April 2020

Acute promyelocytic leukemia (APL) cells are blocked at the promyelocyte stage of myeloid differentiation. The majority of APL cells display the t(15;17) reciprocal chromosomal translocation leading to the expression of the fusion protein promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa). Cells harboring this reciprocal translocation can be induced to differentiate after treatment with all-trans retinoic acid (at-RA) both in vivo and in vitro. During normal hematopoiesis, differentiation is regulated by several key transcription factors. One of them, CCAAT/enhancer binding protein alpha (C/EBPa), controls expression of genes regulating normal myeloid differentiation. Its disruption leads to a block of granulocytic differentiation. We thus hypothesize that C/EBPa could be deregulated in APL and therefore participate in the pathogenesis of APL. Using the U937PR9 cell line, which expresses an inducible PML-RARa, we observed that expression of PML-RARa induced a decrease of both C/EBPa mRNA and protein, leading to decreased C/EBPa DNA binding activity. Using a transient transfection assay with a C/EBPa promoter construct in presence or absence of PML-RARa, we are able to demonstrate that PML-RARa can repress C/EBPa promoter activity. This repression is specific to the fusion protein, as both PML and RARa have no effect upon the C/EBPa promoter. A computer search of the C/EBPa promoter sequence did not exhibit any evident RARE binding site, and therefore we are currently mapping the site(s) responsible for this repression. In conclusion, PML-RARa down regulates C/EBPa expression; this down regulation could participate in the pathogenesis of APL. This hypothesis is also supported by the observation that at-RA treatment of APL cell lines (NB4 and HT93) induces a rapid restoration of both C/EBPa RNA and protein. Thus, a decrease in both C/EBPa expression and activity could contribute to the differentiation block of APL cells by deregulating the normal myeloid differentiation program.
Prevalence of Heparin-Dependent Platelet Antibodies in Children after Cardiopulmonary Bypass Surgery.
Authors: Punzalan Rowena C. , Hanson Sheila J. , Ghanayem Nancy , Curtis Brian R. , Murkowski Kathleen , Havens Peter L. , McFarland Janice G.
Published: April 2020

Heparin is used during cardiopulmonary bypass (CPB) surgery in children. Upon exposure to heparin, heparin-dependent platelet antibodies (HDPA) may form against complexes of platelet factor 4 and heparin on platelet surfaces. Heparin-induced thrombocytopenia (HIT) or heparin-induced thrombocytopenia and thrombosis (HITT) may then ensue. Among adult CPB patients, up to 50% demonstrate HDPA, although only 2% develop HIT and 1% develop HITT. In previous studies done among non-CPB newborns and pediatric intensive care patients, the incidence of HDPA has been zero to minimal.

Objective: To determine prevalence of HDPA among children undergoing CPB who are exposed to unfractionated heparin for >120 hours after CPB.

Methods: We designed a prospective pilot study. All patients ≤ 12 years old who were to receive heparin during CPB were eligible. The presence of HDPA was assessed by heparin-platelet factor 4 enzyme-linked immunosorbent assay (ELISA); positive and equivocal results were confirmed by serotonin release assay. Blood samples were obtained at the time of cessation of heparin or after 10 days on heparin, whichever came first.

Results: Thirty patients were enrolled: 15 were aged 2–19 days (median 6 days); 15 were aged 1.2 to 50 months (median 4.5 months). One patient had borderline positive and 1 had positive results by heparin-platelet factor 4 ELISA; both had negative results by serotonin release assay; neither developed clots. Eighteen patients developed thrombocytopenia, which is common after CPB, including the 2 with equivocal results by ELISA. Six patients, all with negative ELISAs, developed symptomatic thromboses.

Conclusion: There were no HDPA identified among children who received unfractionated heparin for CPB. The specificity of heparin-platelet factor 4 ELISA among children should be assessed.
Absence of P21 Gene Augments Proliferation of Mesenchymal Stem Cells.
Authors: Tsai Anthony Y. , Filice Stephanie C. , Javazon Elizabeth H. , Badillo Andrea T. , Flake Alan W.
Published: April 2020

Bone marrow derived mesenchymal stem cells (MSCs) have the capacity to differentiate into cells of the various mesodermal lineages. Efficiency of engraftment, however, remains a significant obstacle in MSC transplantation. P21 is a G1 checkpoint regulator and cyclin-dependent kinase inhibitor. In its absence, hematopoieitc stem cell (HSC) proliferation increases under normal homeostatic conditions. The aim of the study is to evaluate the effects of p21 deficiency on the proliferative capacity of MSCs. Therefore, MSCs from p21 KO and wildtype mice were maintained in culture in hypoxic and normoxic conditions. Cell counts and respective days in cultures were recorded from which population doubling (PD) times were calculated and compared. 1:1 Competition assays were performed between p21 KO MSCs transduced with DsRed and eGFP transduced WT MSCs as well as between DsRed transduced p21 KO MSCs and non-transduced WT MSCs. Osteogenic, chondrogenic, and adipogenic differentiation assays were performed on both cell populations using standard protocols. Sca-1, CD34, CD45, CD90, MHC Class I and II and other surface antigens were assessed using FACS analysis. In normoxic conditions, the p21 KO MSCs went through 25.68 population doublings in 60 days versus 25.43 population doublings in 103 days for the p21 WT MSCs. Under hypoxic conditions, the KO MSC population doubled 23.3 times in 58 days versus 14.78 times in 60 days for the WT MSCs. All population doubling times were taken from passage 10 cells. When the DsRed transduced KO MSCs (96.6-98.5% DsRed+) was placed in a 1:1 competition assay with eGFP transduced WT MSCs (99.3-99.9% eGFP+) or non-transduced WT MSCs (99.9–100% eGFP−), the normoxic results are as shown in Table 1. Similarly, the competition assay result between DsRed transduced KO MSCs (86.9–94.1% DsRed+) and eGFP transduced WT MSCs (99.7–100% eGFP+) or non-transduced WT MSCs (100% eGFP−) for the hypoxic condition are shown in Table 2. Both the p21 KO and wildtype MSCs populations were able to differentiate into ostenogenic, adipogenic, and chondrogenic lineages. No significant surface marker differences were observed between the 2 populations on FACS analysis. Our results clearly showed that p21 deficient MSCs have increased proliferative ability in vitro compared to normal MSCs. These findings have implications for expansion of MSC populations in vitro , and for the enhancement of competitive capacity of MSCs following in vivo administration.

Table 1 - Normoxic Competition Assay KO MSC (DsRed) WT MSC (eGFP) KO MSC (DsRed) WT MSC (none) results analyzed by FACS Day 6 64.2±3.7% 34.5±4.3% 70.3±0.4% 19.7±0.6% Day 11 84.0±4.5% 13.6±4.6% 92.0±0.8% 6.4±0.7% Table 2 - Hypoxic Competition Assay KO MSC (DsRed) WT MSC (eGFP) KO MSC (DsRed) WT MSC (none) results analyzed by FACS Day 4 70.3±1.7% 30.0±1.7% 83.0±0.8% 18.6±0.9% Day 8 75.7±0.7% 25.8±0.7% 81.3±1.2% 20.6±1.2%
Umbilical Cord Blood Derived CD133+ Cells: Homing Capability in Vasculogenesis and Immunogenicity.
Authors: Fanning L. R. , Finney M. R. , Winter D. G. , Kadereit S. , Banks J. , Greco N. , Kozik M. , Pompili V. , Laughlin M. J.
Published: April 2020

Human CD133+ cells constitute a phenotypically and functionally distinct population of endothelial stem and precursor cells that may play a role in postnatal angiogenesis. CD133+ homing to stimuli, IL-8 production and immunogenic properties are anticipated important characteristics to consider in potential use of allogeneic UCB CD133+ as a therapeutic in arterial ischemia. CD133+ cells were isolated from UCB mononuclear cells (MNC) by magnetic bead selection (AutoMACS, Miltenyi Biotech) according to manufacturer’s protocol and analyzed by flow cytometry. Yields per cord blood unit averaged 1.05 x 106 cells (+/− 0.7 SEM n=30). Surface phenotyping of UCB CD133+ showed co-expression of VEGFR2 (3.5%), CD105 (22.7%) and CXCR4 (8.7%) ligand for SDF-1. Both HLA-DR (57.6%) and HLA-ABC (66.5%) were expressed on CD133+ cells suggesting that CD133+ cells may be capable of presenting immunostimulatory antigens and eliciting an allogeneic reaction. To test this, we performed 96 hour mixed lymphocyte reactions (MLR) using healthy adult peripheral blood MNC as responders stimulated by irradiated (30Gy) CD133+ from UCB (ratio 3:1). Proliferation was measured by 3H-thymidine incorporation. CD133+ and MNC from UCB induced proliferation from allogeneic healthy adult MNC in vitro (46939 +/− 2764 and 49548 +/− 2018 cpm respectively, n=2). MLR studies with CSFE-stained responder cells revealed equivalent rates of lymphocyte cell division comparing selected UCB CD133+ and MNC cells used as stimulators. Comparison studies of responding lymphocyte cytokine production including pro-inflammatory protein assays are ongoing. Initial angiogenic protein assays of CD133+ cells demonstrated elevated levels of IL-8 production as compared to MNC (103+/−380 pg/mL greater in CD133+ than MNC from the same UCB unit) when cultured for 24h in basal media. Transwell migration assays of CD133+ cells to SDF-1 (100ng/mL) demonstrated a 1.8 ± 0.7 fold increase in homing compared to a negative control, coinciding with the CXCR4 expression observed on these cells. In summary, UCB derived CD133+ cells demonstrate homing capability as well as potential for cellular recruitment (IL-8 production) for angiogenesis and cellular therapeutics. CD133+ cells selected from UCB maintain immunostimulatory capacity and initiate proliferation of adult MNC. Further studies of UCB derived CD133+ pro-inflammatory potential; cell recruitment and homing to ischemic signals are warranted.
Gene Transfer of Cytidine Deaminase (CDD) Protects from Cytidine Analogs in an In Vivo Murine Transplant Model.
Authors: Rattmann Ina , Sorg Ursula R. , Brueckner Annette , Opalka Bertram , Flasshove Michael , Moritz Thomas
Published: April 2020

In vitro gene transfer of CDD protects murine and human hematopoietic cells from cytarabine (araC) and gemcitabine induced toxicity. Thus CDD represents a drug resistance gene with considerable clinical potential. We now have examined CDD gene transfer in a bone marrow transplant model with C57Bl/6 mice using the retroviral vectors SF91-CDD-IRES-EGFP and SFβ1-EGFP (control).

Protection from araC was investigated in three experiments. Gene transfer efficiency was analysed by EGFP expression in the peripheral blood after hematopoietic reconstitution. In these experiments 42±4%, 31±1% and 69±2% EGFP+ granulocytes and 9±1%, 10±1% and 36±3% EGFP+ lymphocytes were detected following SF91-CDD-IRES-EGFP transduction (n=5–6 for each experiment). After araC application (500mg/kg; 1xdaily; d1-4; i.p.) substantial protection of granulocytes and thrombocytes was observed. Following the first treatment cycle nadir counts [x103/μl] were 2.9±0.6 vs. 0.7±0.1, 1.1±0.2 vs. 0.5±0.1 and 2.3±0.3 vs. 0.7±0.2 for granulocytes and 510±148 vs. 80±9, 165±48 vs. 70±42 and 728±133 vs. 85±22 for thrombocytes, respectively (SF91-CDD-IRES-EGFP vs. control; all differences significant at p<0.01 for granulocytes and p<0.05 for thrombocytes). Similar protection was noted during repetitive araC treatment cycles. However, no significant enrichment of CDD transduced cells following araC application was seen and lack of selection was confirmed in two additional experiments utilizing low efficiency gene transfer (<5% and <12% EGFP+ granulocytes or lymphocytes throughout the experiment). No major toxicity of CDD-expression in hematopoietic cells was noted and steady levels of gene marked cells were seen for up to six months in primary and at 5 weeks in secondary recipients. Substantial protection of hematopoiesis by CDD overexpression also was observed for gemcitabine. Mice receiving CDD transduced cells and exposed to 250mg/kg gemcitabine (1xdaily i.p., d1-2), showed granulocyte and thrombocyte nadirs [x103/μl] of 2.4±0.6 vs. 1.4±0.2 and 416±62 vs. 277±97 (SF91-CDD-IRES-EGFP vs. control). When a third day of gemcitabine treatment was added, all mice in the control group died within 4 days while animals receiving CDD-transduced bone marrow survived with nadir counts [x103/μl] of 0.8±0.1 granulocytes and 1130±292 thrombocytes [n=3]. Again, no enrichment of transduced cells was observed.

In summary, our data demonstrate efficient protection from araC and gemcitabine hematotoxicity by CDD overexpression. This protective effect was on progenitor rather than stem cells, as no selection of CDD-expressing cells was demonstrated. Thus, a promising approach to utilize CDD clinically may be the combination with a selectable drug resistance gene such as MGMT or MDR-1 to allow enrichment of transduced cells while at the same time protecting against additional drugs.
Cross Talk between IRAK4 and the NADPH Oxidase.
Authors: Catz Sergio D. , Pacquelet Sandrine , Ellis Beverly A. , Johnson Jennifer L.
Published: April 2020

The ability of human neutrophils to combat bacterial and fungal infections depends on their production of reactive oxygen species. The enzymatic complex NADPH oxidase which is responsible for the generation of superoxide anion, is essential for the microbicidal activity of neutrophils, since patients with chronic granulomatous disease, whose NADPH oxidase is inactive, suffer recurrent infections. The exposure of neutrophils to lipopolysaccharide (LPS) amplifies their oxidative response to formylated-peptides (priming). However, the relationship between the signaling downstream of the toll-like receptor 4 after LPS stimulation and the activation of the oxidase has not been elucidated. The phosphorylation of the NADPH oxidase cytosolic factor p47phox is an essential step during the oxidase activation. Here, we test the hypothesis that Interleukin-1 receptor-associated kinase-4 (IRAK4) regulates the NADPH oxidase through phosphorylation of p47phox. We first show that p47phox is a substrate for IRAK4. We showed that IRAK4-phosphorylated p47phox could be subsequently phosphorylated by PKC suggesting that they phosphorylate p47phox at different residues. IRAK4 phosphorylated p47phox to a similar extent as PKC, however, while p47phox was phosphorylated by PKC only in serines, IRAK4 phosphorylated p47phox also in threonines as determined by two-dimensional electrophoresis. Importantly, IRAK4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system (significantly different from the unphosphorylated control, p<0.05). Furthermore, cophosphorylation of p47phox by IRAK4 and PKC significantly potentiated the activity of the NADPH oxidase when compared to PKC phosphorylation alone (p<0.01). Finally, we identified, by mass spectrometry, the residues in p47phox that are targets for IRAK4 phosphorylation. We found that IRAK4 phosphorylates p47phox at a threonine rich domain which possibly constitutes a novel regulatory domain.
The Direct Association of GATA-1 to Rb Is Necessary for Definitive Erythropoiesis.
Authors: Kadri Zahra , Shimizu Ritsuko , Gisselbrecht Sylvie , Ohneda Osamu , Yamamoto Masayuki , Romeo Paul Henri , Chretien Stany
Published: April 2020

GATA-1, the founding member of GATA family, is a master gene for erythropoiesis. It binds to the consensus sequence WGATAR and is involved in the activation of most of the erythroid specific genes. GATA-1 can also repress the transcription of genes coding for proteins involved in the control of cell proliferation. Here we describe a novel function of GATA-1 i.e. the control of the cell proliferation through a direct association to the retinoblastoma protein (pRb). This association required the canonical LXCXE motif localized between amino acids 81 and 85 of GATA-1. This sequence is conserved during evolution, is not found in the other members of the GATA family and is absent in the short form of GATA-1 expressed in the acute megakaryoblastic leukemia of Down syndrome. The inhibition of cellular proliferation induced by GATA-1 over-expression was inefficient in pRb negative cell lines or when pRb was knocked down by specific shRNA and, using a GATA-1 protein mutated on the LXCXE motif (GATA-1Rb-), we showed that (i) the LXCXE motif was necessary for GATA-1/pRb association, (ii) the over-expression of GATA-1Rb- did not inhibit cellular proliferation whereas GATA-1 over-expression did, and (iii) the expression of GATA-1Rb- in a GATA-1 deficient erythroid cell line decreased the number of benzidine positive cells and increased the immature erythroid fraction. Finally, endogenous level expression of GATA-1Rb- with GATA-1 HRD vector in the GATA-1.05 genotype mice resulted in a partial rescue of GATA-1.05 embryos from the early lethality at E11.5, but the embryos then exhibited severe anemia and died by E15.5. Analysis of the E12.5 fetal liver of these GATA-1Rb- rescued mice showed an increased number of immature erythroid cells and a decreased number of mature erythroid cells. These results indicate that the GATA-1/pRB interaction is necessary for erythropoiesis enlighting the mechanism of the autonomous defect of erythropoiesis observed in the Rb−/− mice.
Phenotypic and Genotypic Characteristics of Mastocytosis Differ According to the Age of Disease’s Onset.
Authors: Lanternier Fanny , Cohen-Akenine Annick , Palmerini Fabienne , Feger Frederic , Nasca Laurent , Barete Stephane , Guez David , Casassus Philippe , Bodemer Christine , Arock Michel , Dubreuil Patrice , Hermine Olivier , Lortholary Olivier
Published: April 2020

Mastocytosis is a heterogeneous group of disease with respect to clinical, biological and genotypic features, which may either occur during childhood or at the adult’s age. Adult’s mastocytosis is usually associated with persistent systemic involvement and c-kit 816 mutations, while pediatric’s disease is mostly limited to the skin and often resolves spontaneously during adolescence. No study has attended to compare characteristics of adult’s mastocytosis according to the age of disease’s onset. Among 155 adult patients with histologically proven cutaneous mastocytosis and complete phenotypic and genotypic data available recruited through the French mastocytosis network (AFIRMM), disease started before 15 years in 23 patients (15%) and later than 18 years in 132 patients (85%). We compared phenotypic and genotypic features of patients whose disease started during childhood (Group 1, n = 23) with those of 31 randomly allocated patients whose disease started at adult’s age (Group 2). Genotypic analysis was performed on skin biopsy by direct sequencing of c-kit exons 17 and 8 to 13 in which most of activating mutations are found.

According to the WHO classification, the percentage of systemic disease was similar in both groups (61 vs. 79%) in groups 1 and 2, respectively. Bone pain (57% vs. 23%) and flush (83% vs. 39%) differ between groups (p < 0.01). Bullous lesions were only observed in group 1 (14%) while telangiectasia macularis eruptiva perstans was only observed in group 2 (16%). Major asthenia, syncope, mood disturbances, anaphylactoid reactions, gastrointestinal and respiratory disturbances and blood cell count, serum tryptase and liver enzymes levels did not differ between groups. C-kit 816 mutations was found in 33% and 77% of patients in groups 1 and 2, respectively (p < 0.001). 45% of patients whose disease’s onset occurred before the age of 2 years vs. 7% of those whose disease’s onset occurred later during childhood presented with C-kit 816 mutation (p < 0.001). Other c-kit activating mutations were found in 29% and 3% (p < 0.001) and lack of c-kit activating mutation in 38% and 20% (p < 0.001) in groups 1 and 2, respectively. In conclusion, pathogenesis of mastocytosis significantly differs according to the age of disease’s onset. Our data may have major therapeutic relevance when considering c-kit-targeted therapy.
Extracorporeal Photopheresis Induces the Generation of Regulatory T Cells.
Authors: Strobl Frank , Campbell Kim , Krutsick Amy , Huber Janine , Peritt David
Published: April 2020

Extracorporeal photopheresis (ECP) is an immunomodulatory cell therapy being investigated as a treatment for various immune-mediated inflammatory disorders. Clinically, ECP involves the intravenous reinfusion of autologous, apoptotic peripheral blood leukocytes. The exact mechanism of action behind ECP, however, remains unclear. In animal models, delivery of apoptotic cells has been shown to regulate immune responses through the downregulation of antigen-presenting cell (APC) function, the modulation of cytokines, and the generation of regulatory T cells. We and others, have shown that activation of naive T cells in the presence of APCs that have engulfed ECP-treated apoptotic cells leads to the generation of a T cell population that can suppress syngeneic T cell proliferation and function. In the present study, we demonstrate that the direct interaction of ECP-treated peripheral blood mononuclear cells (PBMCs) with naive human CD4+ T cells in vitro also promotes a T cell phenotype with regulatory activity. Transfer of ECP-derived regulatory T cells to a secondary mixed lymphocyte reaction results in a greater than 50% inhibition of syngeneic responder T cell proliferation and IFNg production at a Treg:responder ratio of 1:10. In addition, transwell assays demonstrate that inhibition of responder T cells by ECP-derived regulatory T cells is contact-dependent. The generation of human regulatory T cells by ECP is dependent on the presence of ECP-treated monocytes and can be reversed by the addition of IL-2. These regulatory T cells also proliferate in response to concanavalin-A, but do not express increased levels of Foxp3 or IL-10, nor do they secrete significant levels of the pro-inflammatory cytokines IL-2, IFNg, IL-4, and TNFa. Additional studies are underway to further characterize the phenotype of these regulatory cells and to determine their suppressive capacity in vivo. It is hoped that a better understanding of the mechanism of action behind ECP will help to define more efficacious ECP treatment regimens.
African American Patients with Multiple Myeloma Have Prolonged Responses after Autologous Stem Cell Transplantation.
Authors: Saraf Santosh , Chen Yi-Hsiang , Dobogai Lisa C. , Saunthararajah Yogen , Peace David , Hoffman Ronald , Mahmud Nadim , Chunduri Sandeep , Rondelli Damiano
Published: April 2020

The incidence of multiple myeloma (MM) in African American (AA) patients is higher than in other racial groups. In this retrospective study we analyzed the outcome of 70 consecutive patients (AA n=38, non-AA n=32) with MM receiving an autologous stem cell transplantat (ASCT) at the University of Illinois at Chicago. No significant differences, including age, gender, stage of disease, Hgb and b2 microglobulin levels, skeletal involvement, plasma cell marrow infiltration, were observed between AA and non-AA patients at diagnosis. Chemotherapy prior to ASCT was also comparable in the two groups. Conditioning regimen with high dose melphalan was used in 84% AA and 72% non-AA, while the remaining patients received BCNU/melphalan. After induction chemotherapy no difference occurred in complete remission (CR) and partial remission (PR) rates in AA (9% and 42%) as compared to non-AA (13% and 33%). Two months after transplantation CR and PR rates were 31% and 25% in AA and 30% and 20% in non-AA. However, at 6 months after ASCT 25%AA vs 60% non-AA patients showed a progression of the disease (p=0.009). At a median follow-up of 26 months (range:1 to 89), 79% AA and 69% non-AA patients are alive. The estimated median Event-Free-Survival in the two groups is 21 vs 12 months (p=0.02), while the estimated median Overall Survival is 64 vs 39 months (p=0.19) (Figure 1). In conclusion, AA and non-AA patients have similar CR and PR rates after ASCT. However, AA patients seem to have more prolonged clinical responses. In a setting where equal access to modern modalities of treatment is available, race does not appear to affect responses to therapy in multiple myeloma.

Figure 1 Figure 1.
Expression of C/EBPε Restores G-CSF-Induced Granulocytic Differentiation of Myeloid Cells Expressing the Carboxy Truncated G-CSF Receptor Associated with AML/SCN.
Authors: Dong Fan , Zhuang Dazhong , Qiu Yaling
Published: April 2020

Mutations in the G-CSF receptor gene causing carboxy terminal truncation of the G-CSF receptor have been associated with the development of acute myelogenous leukemia (AML) in patients with severe congenital neutropenia (SCN). The truncated G-CSF receptors mediate augmented cell proliferation and survival, but are defective in inducing granulocytic differentiation in myeloid cell lines and transgenic mice. Little is known about the mechanism by which the carboxy terminus of the G-CSF receptor regulates granulocytic differentiation. It has been shown that myeloid transcription factors including C/EBPα, C/EBPε and PU.1 are critically involved in granulopoiesis. Of the three transcription factors, expression of C/EBPε was dramatically upregulated upon G-CSF treatment of myeloid 32D cells expressing the wild type G-CSF receptor (32D/WT). C/EBPε upregulation by G-CSF was abolished in 32D cells expressing the G-CSF receptor mutant D715 (32D/D715), which lacked a region of 98 amino acids in the carboxy terminus. Forced expression of C/EBPε in 32D/D715 cells at levels comparable to those in G-CSF-treated 32D/WT cells resulted in markedly attenuated cell proliferation and survival, and rapid terminal granulocytic differentiation in response to G-CSF. These data indicated that the functional defect of the D715 mutant in mediating granulocytic differentiation may arise from its inability to upregulate C/EBPε expression. Notably, forced expression of C/EBPε failed to induce terminal granulocytic differentiation of 32D/D715 cells cultured in IL-3, suggesting that the D715 receptor acted in collaboration with C/EBPε to drive granulocytic differentiation. Of the four cytoplasmic tyrosine (Tyr) residues of the G-CSF receptor, the D715 mutant contains only Tyr 704 that is involved in Stat3 activation. Because Stat3 is an important regulator of granulopoiesis, experiments are under way to address whether Tyr 704 in the D715 mutant is required for granulocytic differentiation driven by C/EBPε.