bluuebunny/crossref_metadata_2025 huggingface dataset:
Rimcazole - A Sigma Receptor Antagonist - As a Potential Novel Therapy for Acute Myeloid Leukemia and Myeloma.
Authors: Upadhye Yogesh , Watt Stephen , Spruce Barbara A. , Bowen David T.
Published: April 2020

The cure rates for most subtypes of acute myeloid leukaemia (AML) have been static for a decade or more. Risk directed therapy is paying dividends for younger patients with AML characterised by balanced translocations, but therapeutic success is more limited for the majority of patients. Although several new therapeutic options are available to prolong survival in myeloma, cure is rarely attainable. Thus novel therapeutic approaches are required for both diseases. Sigma receptors are widely expressed in the cells of the nervous system, and are aberrantly increased in malignant tumour cells, including hematological malignancies. Sigma receptor antagonists have previously been tested in humans as candidiate antipsychotics and also in diagnostic tumour imaging. We have previously shown that sigma receptor antagonists induce apoptotic cell death in a range of cancer cell lines, while sparing normal cells (Spruce et all 2004).

To investigate the potential for sigma receptor ligands in the treatment of hematological malignancies, we studied the effect of rimcazole, a sigma receptor antagonist, in 3 established and well characterized cells lines derived from malignant haemopoetic cells - HL60, K562 and OPM-2. Rimcazole induced dose-dependent cell death in all 3 haemopoetic cell lines.

Figure Figure

Rimcazole induced cell death was shown to be apoptotic, as assessed by FACS analysis using Annexin V and Propidium Iodide staining as markers of apoptosis. Rimcazole also inhibited colony formation by HL60 and K562 cell lines in a standard methycellulose assay in a dose-dependent manner.

Malignant hematological cells are known to have increased NF kappaB activity. Suppression of NF kappaB activity by treatment with a TAT-tagged IkappaB superepressor peptide resulted in attenuation of rimcazole-induced cell death.

Figure Figure

This suggests an NF kappaB-dependent pathway of rimcazole action. We also measured the I kappaB levels in the 3 cell lines after treating them with rimcazole. Decreased concentration of IkappaB was detected by Western Blot method in the samples treated with Rimcazole. Untreated cells were used as control.

We further evaluated in vitro response of primary AML cells stored in the tissue bank from 3 patients diagnosed with AML. The primary AML cells also showed dose dependent death on treatment with Rimcazole. The drug concentrations at which the optimum effect was observed was similar for the 3 primary AML cells as well as established cell lines.

Evaluation of sigma receptor antagonists in other primary leukaemic and myeloma cells is currently in progress.
Stromal Cell IL-8 Production Is Increased in MM and May Impact Both Tumor Cells and the Microenvironment.
Authors: Kline Michael , Donovan Kathleen , Wellik Linda , Lust Christopher , Jin Wendy , Moon-Tasson Laurie , Kumar Shaji , Witzig Thomas E. , Rajkumar S. Vincent , Lust John A.
Published: April 2020

Background: Multiple myeloma (MM) is an incurable hematological malignancy characterized by the expansion of a plasma cell clone that localizes to the bone marrow. Stromal cells residing in the bone marrow respond to signals from MM cells and other cell types by producing cytokines and other proteins that stimulate tumor cell growth, survival, adhesion, migration, and drug resistance. We have examined the proteins produced by stromal cells in response to stimulation by bone marrow from patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and MM. Furthermore, we have begun analyzing the properties of one of these proteins, the pro-angiogenic chemokine IL-8, in MM.

Methods: Bone marrow aspirates from patients with MGUS (n=3), SMM (n=7), and MM (n=6) were cultured for 48 hours, and the culture supernatants were incubated with stromal cells for an additional 48 hours. Protein levels were analyzed using antibody array and ELISA. Microvessel density (MVD) was determined as a measure of angiogenesis in patient bone marrow samples using CD34 staining. Flow cytometry analysis of MM cell lines and patient bone marrow samples was performed using monoclonal antibodies against IL-8 receptors CXCR1 and CXCR2.

Results and Conclusion: We observed a significant increase in stromal cell IL-8 production stimulated by bone marrow cells from patients with active myeloma and a subset of SMM patients (16.67 ± 9.82 ng/ml) in comparison to bone marrow of patients with MGUS and all other SMM patients (0.55 ± 0.17 ng/ml; P=0.0004). Use of an IL-1 inhibitor and recombinant IL-1β demonstrated that IL-8 production was dependent upon IL-1β signaling. Increased BM microvessel density correlated with stimulation of stromal cell IL-8 production (P=0.0005). Furthermore, the majority of MM cell lines (7/9) and MM patient plasma cells were found to express IL-8 receptors CXCR1 and CXCR2. In addition to its function as a pro-angiogenic factor, IL-8 may directly influence MM cells through its CXCR1 and CXCR2 receptors. We conclude that stromal cell IL-8 production parallels MM disease activity, is IL-1β induced, correlates with bone marrow angiogenesis, and may influence MM disease via impact upon both the microenvironment and tumor cells.
Tumor Evasion of the Immune System: Inhibiting p38 MAP Kinase Signaling Restores the Function of Dendritic Cells.
Authors: Yi Qing , Wang Siqing , Yang Jing , Wang Jianfei , Wezeman Michele , Kwak Larry W.
Published: April 2020

Dendritic cells (DCs) from cancer patients are functionally defective, however, molecular mechanisms underlying are still poorly understood. In this study, we used the murine 5TGM1 myeloma model to examine the effect and mechanism of tumor-derived factors on the differentiation and function of DCs. Myeloma cells (5TGM1) or tumor culture conditioning medium (TCCM) were shown to inhibit differentiation and function of BM-derived DCs (BMDCs), evidenced by the downregulated expression of DC-related surface molecules, decreased IL-12 but increased IL-10 secretion, and compromised capacity of the cells to activate allospecific T cells in vitro. Similar results were obtained with other murine myeloma cells MOPC-315 and MPC-11. Moreover, TCCM-treated BMDCs were inferior to normal BMDCs at priming tumor-specific humoral and cellular immune responses in vivo (in the 5TGM1 mouse model). Neutralizing antibodies against IL-6, IL-10, and TGF-β partially abrogated the effects. TCCM treatment activated p38 mitogen-activated protein kinase (MAPK) and JNK but inhibited extracellular signal-related kinase (ERK). Inhibiting p38 MAPK by three different specific inhibitors was found to restore the phenotype, cytokine secretion, and function of TCCM-treated BMDCs. Vaccinating mice with BMDCs obtained from cultures in which both TCCM and p38 inhibitor were added was as efficacious as normal BMDCs at inducing tumor-specific antibody, type-1 (IFN-γ) T-cell, and CTL responses. Thus, our results suggest that tumor-induced p38 MAPK activation and ERK inhibition in DCs may be a new mechanism for tumor evasion, and regulating these signaling pathways in vivo or during DC differentiation may provide new strategies for generating potent DC vaccines for immunotherapy of multiple myeloma and other tumors.
Decitabine Low-Dose Schedule (100 mg/m2/Course) in Myelodysplastic Syndrome (MDS). Comparison of 3 Different Dose Schedules.
Authors: Kantarjian Hagop , O’Brien Susan , Giles Francis , Ravandi-Kashani Farhad , Faderl Stefan , Garcia-Manero Guillermo , Davis Jan , Issa Jean-Pierre
Published: April 2020

Background Decitabine (DAC), a hypomethylating agent, has shown activity in MDS. DAC 150 mg/m2 by continuous infusion has been associated with CR rates of 10% to 20%. We investigated optimizing the dose schedule of DAC in MDS.

Study Group and Methods Patients (pts) with IPSS intermediate 1–2 & high risk MDS were randomized to one of 3 schedules of DAC: 1) 20 mg/m2 IV over 1 hour daily x 5; 2) 10 mg/m2 IV over 1 hour daily x 10; or 3)10 mg/m2 subcutaneously (SQ) BID x 5. A total of 95 pts are to be treated; Bayesian randomization is implemented based on CR rates. Courses were given every 28 days. Delays to allow counts recovery were permitted every 3 courses, or if myelosuppression without disease, or severe myelosuppression complications. Pts were allowed to receive erythropoietin 40,000 units weekly for anemia, or GCSF if needed. Response criteria for CR & PR were as for AML (PR requiring also ↓ blasts by >50%). Clinical benefit (CB) referred to one or more of: platelets î by ≥ 50% and >30 x 109/L, or granulocytes increase by ≥ 100% and to >109/L, or hemoglobin î by ≥ 2 g/dl or transfusion independence, or splenomegaly ↓ by 50% or more, or monocytes ↓ by 50% or more (pretreatment >5 x 109/L).

Results 92 pts have been treated; median age 65 (31–90) yrs; 66% >60 yrs old. IPSS: intermediate-1 25%; intermediate-2 38%; high 19%; CMML 17% Cytogenetic abnormalities 57%; secondary MDS 17%; marrow blasts > 10% in 31%. 27 pts had prior erythropoietin; 17 had prior GCSF; 22 had other prior therapies. Presently, 89 pts have received 1 course. Results: 32 CR (36%); 7 PR (8%); 13 marrow CR + CB (15%); 16 CB (18%); overall response 68/89=76%. Median courses to CR 3 (range 1 to 6). Median follow-up of 9 months; 48 pts continue on DAC. Compared with a 114 pts with MDS who received intensive chemotherapy (2000–2004), CR rate was lower with DAC (36% vs. 45%), overall response rate was favorable; 6-week mortality was lower with DAC (1% vs. 21%); and estimated survival favorable (p = 0.00007). CR rates by schedule: 5 days IV 24/58 (41%); 5 days SQ 4/14 (28%); 10 day IV 4/17 (24%). There was more myelosuppression with 10 day IV. After 55 patients were randomized, the 5 day IV arm was determined statistically superior, therefore, remaining patients were not randomized, but were treated with 5 days IV therapy.

Conclusions DAC has significant anti-MDS activity; 2) optimal schedule: 20 mg/m2 IV over 1 hour daily x 5; 3) timely repeated courses needed for optimal response.
High Circulating CD34 Cells, Dacrocytes, Clonal Hematopoiesis, and JAK 2 Mutation Differentiate Secondary Myelofibrosis Associated with Pulmonary Hypertension from Myelofibrosis with Myeloid Metaplasia.
Authors: Popat Uday , Liu Enli , Guan Yongli , Prchal Josef T.
Published: April 2020

Myelofibrosis with myeloid metaplasia (MMM) is a clonal myeloproliferative disorder, characterized by splenomegaly and myelofibrosis. Like all myeloproliferative disorders, it is due to an acquired somatic mutation of a hematopoietic progenitor resulting in clonal erythrocytes, platelets, and granulocytes. A single acquired point mutation of JAK2 1849G>T (V617F), a tyrosine kinase with a key role in signal transduction from growth factor receptors, occurs in 50%–97% of patients with MMM, essential thrombocythemia (ET) and polycythemia vera (PV). We studied 25 myelofibrosis patients with myeloid metaplasia and myelofibrosis complicating PV or ET. These were compared to 19 patients with secondary myelofibrosis of equivalent severity associated with pulmonary hypertension (PH) treated by epoprostenol. In these two groups we compared peripheral blood CD34 counts, clonality of granulocytes and platelets in peripheral blood, mutational status of JAK 2 kinase gene, and morphology of peripheral blood and bone marrow. Heterozygosity for JAK 2 1849G>T somatic mutation was seen in 17 of 23 (74%) patients with MMM including 10 of 15 patients (67%) with idiopathic myelofibrosis and 7 of 8 patients (88%) with myelofibrosis complicating PV or ET, but was absent in all 19 patients with myelofibrosis associated with PH (P<0.0001, Fisher exact test 2 tailed). The number of peripheral blood CD 34 cells was significantly higher in patients with MMM compared to patients with secondary myelofibrosis associated with PH; 1.1% (95% CI, 0.58%–1.64%) in patients with MMM versus 0.08% (95% CI, 0.04%–0.12%) in patients with PH, or 0.05% (95% CI, 0.03%–0.06%) in normal healthy controls (P<0.01). Further, patients with PH lacked dacrocytes, erythroid precursors, or myeloid precursors (leukoerythroblastic picture) on examination of the blood smear. Clonal hematopoiesis was found only in those women with marrow fibrosis due to MMM, PV and ET and not in patients with secondary marrow fibrosis (P < 0.0001).

We conclude that a high circulating CD34 count, clonal platelets and granulocytes, presence of dacrocytes, and JAK2 1849G>T (V617F) mutation of clonal progenitors are the intrinsic features present in patients with myelofibrosis due to myeloproliferative disorders and that these features are not due to the abnormal marrow architecture that is seen in secondary myelofibrosis.
Analysis of JAK2V617F Mutation, PRV-1 Overexpression and Endogenous Erythroid Colony (EEC) Formation Show Different Coexpression Patterns among Ph-Negative Chronic Myeloproliferative Disorders.
Authors: Bellosillo Beatriz , Besses Carles , Florensa Lourdes , Longaron Raquel , Navarro Gemma , Gimeno Eva , Sole Francesc , Vila Rosa M. , Serrano Sergi
Published: April 2020

Introduction. Recently, the JAK2V617F mutation has been reported in the majority of PV patients as well as in a variable percentage of ET and IMF patients. Some authors have reported a high correlation of JAK2 mutation with PRV-1 overexpression and the formation of EECs. In the current study we have analyzed the pattern of positivity of these three biomarkers in a cohort of Ph-negative MPD patients.

Patients and methods. 103 Ph-negative MPD patients (58 ET, 37 PV and 8 IMF) from a single institution were studied. Patients were diagnosed according to the PVSG and Barosi criteria. EEC formation was determined at diagnosis. At the time when PRV-1 and JAK2 mutation were analyzed 29/103 patients were receiving platelet-lowering therapy ± ASA, 26/103 patients only received ASA and 48/103 received no specific treatment. PRV-1 expression was quantified by real-time reverse transcriptase (RT)-PCR in RNA from granulocytes. Analysis of JAK2V617F was performed by direct sequencing using granulocyte RNA. Results. JAK2 mutation was observed in 21/58 ET (36.2%), 30/37 PV (81.1%) and 5/8 IMF (62.5%). PRV-1 was overexpressed in 25/58 ET (43.1%), 35/37 PV (94.6%) and 6/8 IMF (75%) and EECs formation was seen in 31/58 ET (53.4%), 34/37 PV (91.9%) and 6/8 IMF (75%).

All markers were simultaneously positive (group A) in 43/103 patients (41.7%), concurrently negative (group B) in 25/103 patients (24.3%) and both positive and negative markers (group C) were observed in 35/103 patients (34%). In group A, 65% were PV patients, 26% were ET patients and 9 % were IMF patients. In group B, 8% were PV patients, 84% were ET patients and 8 % were IMF patients. In group C, 20% were PV patients, 74% were ET patients and 6 % were IMF patients.

Regarding diagnosis, 76% of PV and 50% of IMF patients belonged to group A, whereas the majority of ET patients (45%) pertained to group C (table). When comparing ET and PV, a significant difference was observed (p<0.001) concerning group distribution.

Conclusion. These results show that, although a good correlation has been observed for the simultaneous expression of these three biomarkers, differences in the pattern of positivity, specially in ET, indicate that not all Ph-negative patients share the same pathogenetic mechanisms and point to other coexisting genetic abnormalities.

Diagnosis ET PV IMF All markers positive (group A) 11 (19%) 28 (76%) 4 (50%) All markers negative (group B) 21 (36%) 2 (5%) 2 (25%) Positive and negative markers 26 (45%) 7 (19%) 2 (25%) Total 58 37 8
Inactivation of the PRDM1/BLIMP1 Gene in Non-GC-Type Diffuse Large B-Cell Lymphoma.
Authors: Pasqualucci Laura , Compagno Mara , Houldsworth Jane , Monti Stefano , Grunn Adina , Shipp Margaret A. , Dalla-Favera Riccardo
Published: April 2020

PRDI-BF1/Blimp1 is a zinc finger transcriptional repressor expressed in post-germinal center (GC) B cells and required for terminal B cell differentiation.

The PRDM1/BLIMP 1 locus lies on chromosomal band 6q21–q22.1, a region frequently deleted in B-cell non-Hodgkin’s lymphomas suggesting the existence of one or more tumor suppressor loci (Gaidano et al, Blood80:1781, 1992). To test whether genetic alterations affecting the BLIMP1 gene may be involved in DLBCL pathogenesis, we performed PCR amplification and direct sequencing of the BLIMP1 coding sequences in 136 cases, including 20 cell lines and 116 primary biopsies. Gene expression profiling analysis using the Affymetrix Gene Chip system was used in 93 cases to classify them as germinal center B-cell like (GCB, N=38), activated B-cell like (ABC, N=35) and type III (N=20). Nonsense mutations were found in 7 of 136 cases. Four of these mutations generated premature stop codons, predicting severely truncated proteins of 61 to 244 aminoacids; in three cases, a single bp substitution affecting the exon 2 splice donor site led to insertion of 101 nucleotides from intron 3 and a premature stop codon. One primary tumor case carried a mutation within the intron 3 splice acceptor site, and one cell line displayed a gene rearrangement in one allele with deletion of the second allele. Strikingly, 6 of the 7 nonsense mutations identified segregated with the ABC phenotype (the remaining case was not profiled), suggesting that these alterations may be common and specific in this subtype of DLBCL (6/35, 17%). In addition, missense mutations generating aminoacid substitutions were found in 8 additional cases, and complete lack of Blimp-1 protein expression was found in most ABC-type DLBCL cases. These observations suggest that inactivation of the BLIMP1 gene may occur also by other mechanisms, including the generation of dominant negative mutants, chromosomal deletion or epigenetic silencing, in a large fraction of DLBCL. Functional studies are currently being performed to corroborate these data. Overall, these results suggest that BLIMP1 may act as a tumor suppressor gene, whose loss or inactivation may contribute to lymphomagenesis by blocking post-GC differentiation of B cells toward plasma cells.
Defining Candidate Oncogenes Leading to Increased Susceptibility to Leukemia in eed Mutant Mice.
Authors: Sauvageau Martin , Sauvageau Guy
Published: April 2020

We previously showed that loss of function of the chromatin regulator polycomb group gene (PcG) eed increases susceptibility to lymphoid tumour development (Sauvageau, M. et al. Blood 104 (11); 213). The human eed locus is in a region of chromosome 11 (11q14.2–22.3) which is associated with recurrent deletions in several reported cases of lymphoproliferative diseases. The persistant expression of the wild-type allele of eed is maintained in lymphomas developing in eed+/− animals. This suggests a haploinsufficient function for this gene in preventing tumorigenesis. We recently exploited an insertional mutagenesis screen to identify new cooperative leukemia-inducing genes in eed mutant mice. 325 retroviral insertion sites (RIS) representing 189 different gene loci have been identified by inverse-PCR from a total of 51 lymphomas (25 eed−/− and 26 wild-type). This screen revealed 18 gene loci known to be cancer related and 21 new common insertion sites (CIS) such as sh2d3c (n=4 tumours), fgfr3 (n=15 tumours) and myo5b (n=7 tumours). More than 97% of these CIS were common to both eed mutant and wild-type animals. We next assessed the expression levels of all the genes found in a 100 Kb region of genomic DNA surrounding the CIS identified in our screen in eed mutant and wild-type tumours as well as in the corresponding non-leukemic tissues. Almost 17% of the 48 genes analyzed, including fgfr2, fgfr3, ttc16 and adam15, are aberrantly expressed in non-transformed eed mutant cells when compared to wild-type controls (levels 5–100+ fold). Importantly, several of these genes are either aberrantly expressed in wild-type and eed mutant tumours or represent true CIS thus indicating their possible contribution to early tumour development in eed mutant mice. Moreover, our results do not incriminate eed as a modifier of gene expression once cells are fully transformed, since the oncogenic complement is similar in eed mutant and wild-type leukemias. Collectively, our results link the haploinsufficient role of eed to the appropriate regulation of genes involved in leukemia development.
Fine and Ultrafine Particles Differentially Affect the PFA-100-Times and Raise Proinflammatory Cytokines in Human Blood.
Authors: Forestier Marc , Buechi Linda , Hermann Corinna , Hartung Thomas , Beer Juerg H.
Published: April 2020

INTRODUCTION: Peaks of ambient air pollution with increased concentrations of particulate matter (i.e. PM2.5: Fine particles of < 2.5 μm diameter and UFP: Ultrafine particles of <0.1 μm diameter) easily reach levels of 100’000 particles/cm3 and are associated with increased cardiovascular morbidity and mortality. Especially the UFP-fraction rapidly crosses cellular membranes by non-phagocytotic mechanisms and is translocated into the circulation. The absorbed microparticles may affect the coagulation directly or indirectly via monocyte/macrophage activation. The data on prothrombotic and inflammatory effects are controversial, depending on the type, size and charge of particles and the experimental models chosen.

HYPOTHESIS: We therefore hypothesized that typically sized particles differentially affect human platelets and coagulation either directly or indirectly by the proinflammatory response.

METHODS AND RESULTS: Three microparticle sources were mixed with citrated human blood at concentrations of 5 and 50 μg/ml for one hour:

a) PM2.5 polystyrene beads (PS) of 0.66 μm diameter. b) UFP ultrafine carbon black (ufCB, 14 nm, Printex), and c) residual oil fly ash (ROFA, with contents of Al, Zu, Cu-ions, and residual polyaromatic hydrocarbons).

We calculated that >800 μg of microparticles may be absorbed by the human lung daily and thus a dose of appr. 5 μg/ml blood could be achieved easily.

Platelet aggregation with ADP, collagen, arachidonate and ristocetin at different concentrations was not affected by any of the three types of microparticles and neither was PT, aPTT, fibrinogen, DDimer or soluble P-selectin. Direct microscopical observation did not show platelet aggregates, nor rosetting of platelets with leucocytes, nor mixed platelet aggregates with microparticles. PFA-100 CADP, however, was prolonged in the presence of PS beads from 85 ± 3 sec to 101 ± 6 (n = 3, p < 0.04); ROFA, on the other hand, shortened it by more than 20 sec to 78 ± 2 vs 110 ± 6 sec (n = 3, p < 0.05) and ufCB/UFP did not affect it. The standardized endogenous thrombin potential as measured by the thrombinoscope did not exhibit significant changes under the conditions chosen.

Preliminary data of a highly standardized assay of cytokine release after 24 hours of stimulation with low dose LPS (1μg/ml blood) indicates a 2 - 3 fold increase of interferon, interleukine-1 and TNFa, but not of interleukine-8.

CONCLUSION: Our data demonstrate a direct shortening effect on shear induced platelet aggregation (PFA-100) by ROFA and a prolongation by positively charged PS beads. None of the three particle types showed a direct effect in any of the other coagulation assays evaluated. The strong proinflammatory cytokine response to all three microparticle preparations is likely responsible for the procoagulant / prothrombotic effects which have been observed in experimental mouse and hamster models.
Inactivation of p38 MAPK Extends Self-Renewal Capacity of Haematopoietic Stem Cells.
Authors: Ito Keisuke , Hirao Atsushi , Arai Fumio , Matsuoka Sahoko , Takubo Keiyo , Ikeda Yasuo , Suda Toshio
Published: April 2020

Haematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime. Appropriate control of HSC self-renewal is critical for maintenance of haematopoietic homeostasis. Here we show that activation of p38 MAPK limits lifespan of HSCs in response to increasing levels of reactive oxygen species (ROS) in vivo. Although normal quiescent HSCs maintain a low level of oxidative stress, an increase in ROS was observed in HSCs after transplantation as well as in aged mice. In vitro treatment with BSO (Buthionine sulfoximine), which depletes intra-cellular glutathion, increased ROS (H2O2) level in immature hematopoietic cell population, c-kit+Sca1+Lin- (KSL) cells, in a dose-dependent manner. Low dose concentration of BSO suppressed reconstitution capacity of HSCs, whereas higher concentration did not affect progenitors. These data indicate that HSCs are much more sensitive to increased ROS than progenitors and are consistent with our previous results from Atm−/− mice in which ROS level is elevated in vivo. Here we focused on MAPKs for the stem cell dysfunction since it has been shown that several MAPKs are activated in response to ROS. We evaluated effects of MAPK inhibitors for p38, JNK or ERK in incubation of KSL cell with BSO. p38 inhibitor (SB203580), neither JNK nor ERK inhibitor, restored reconstitution capacity of HSCs after transplantation, suggesting that activation of p38 may contributes to defect of stem cell function in vivo. To address the question, we evaluated p38 activation in Atm−/− BM cells by immunohistochemistry. Surprisingly, p38 protein was phosphorylated only in KSL cells, but not other more differentiated cell populations, despite that the ROS levels were comparable among the cell population of mice. In response to activation of p38, p16INK4a was up-regulated only in KSL cells. The data indicates a possibility that stem cell-specific p38 activation negatively regulates self-renewal of HSCs. We then investigated a role of p38 activation on self-renewal of HSCs in vivo. When p38 inhibitor was intraperitoneally administered both before and after BMT, the level of repopulation achieved was comparable to that of the wild-type. Furthermore, Atm−/− mice that received long-term p38 inhibitor treatment did not show either anemia, a decrease in progenitor colony-forming capacity, or reduced frequencies of stem cell subsets. These data demonstrate that the activation of p38 present in HSCs promotes the exhaustion of stem cell pool in response to elevation of ROS. It has been proposed that aging is driven in part by a gradual depletion of stem cell functional capacity. There are evidences that inappropriate production of oxidants is connected to aging and life span. We propose a possibility that p38 activation in response to ROS plays a critical role for limit of stem cell capacity.
Activation of the Classical Pathway of Complement by Resting and Shear Stress-Stimulated Human Endothelial Cells.
Authors: Yin Wei , Weksler Babette , Varon David , Savion Naphtali , Ghebrehiwet Berhane , Peerschke Ellinor
Published: April 2020

Complement activation is associated with a variety of inflammatory conditions including atherosclerosis, but the mechanism of complement activation in these settings is poorly understood. Endothelial cells (EC) play an important role in vascular pathology and express a variety of complement receptors, including gC1qR/p33, recognizing the globular domain of the complement component C1q. In preliminary studies, purified recombinant gC1qR/p33 was found to support C1q-dependent C4 activation in vitro, comprising 19.5% ± 8.3% (mean ± S.D., n=5) of that produced by aggregated IgG. In contrast, a truncated form of gC1qR/p33, lacking the C1q binding domain, failed to support C4 activation. Additional studies were performed with immortalized bone marrow microvascular EC to investigate classical complement pathway activation and deposition. EC were exposed to anticoagulated (0.32 % sodium citrate) human plasma, diluted (1/10) in 0.01 M HEPES buffered modified Tyrode’s solution, pH 7.5, containing 2 mM Mg Cl2 and 1 mM CaCl2, for 60 min, 37°C. A solid phase ELISA approach was used to detect EC-associated C1q and C4 activation (C4d). Statistically significant deposition of C4d (0.72 ± 0.3. OD units (ODU), n=4)(p=0.04) and C1q (0.57 ± 0.19. ODU, n=4) (p=0.002) was observed on EC that had been immobilized on poly-L-lysine coated microtiter wells. Consistent with classical complement pathway activation, C4d deposition remained at baseline (0.23 ± 0.13, ODU, n=4) in the presence of 10 mM EDTA, but C1q deposition was unaffected. Moreover, no significant C1q or C4d deposition occurred when endothelial cells were exposed to C1q depleted serum. Similar studies were performed using EC grown to confluence on Type I collagen to examine the effect of shear stress (12 dynes/cm2 for 1 hour in a cone-and-plate shearing device), simulating flow conditions in coronary arteries, on classical complement pathway activation and deposition. Compared to static conditions, shear stress resulted in an approximately 50% increase in C1q and C4d deposition on EC. This was accompanied by an approximately 2-fold increase in EC binding of a monoclonal antibody, 60.11, recognizing the N-terminal C1q binding domain of gC1qR/p33. Taken together, these data present evidence for a potential paradigm shift, illustrating immune complex independent classical complement pathway activation by gC1qR/p33, and deposition of activated classical complement components on EC. The generation and deposition of active complement components on EC is likely to contribute directly to vascular inflammation and atherosclerotic changes.
Response Adapted Therapy: Using FDG-PET after Chemotherapy in Order To Restrict Indication for Radiotherapy in Children and Adolescents with Early Stages of Hodgkin’s Lymphoma (HL).
Authors: Mauz-Koerholz Christine G. , Kluge Regine , Sorge Ina , Merziger Barbara , Krausse Antje , Hasenclever Dirk , Kamprad Friedrich H. , Amthauer H. , Franzius Christiane , Gruenwald F. , Sabri Osama , Hirsch Wolfgang , Korholz Dieter
Published: April 2020

Introduction: Radiotherapy is an important risk factor for the development of secondary malignancies in children with Hodgkin’s lymphoma. The GPOH-HD-study group tries to restrict the indication for radiotherapy after two cycles of intensive OPPA (OEPA) chemotherapy. In the GPOH-HD 95 trial 113/394 (29%) of patients with early stage HL were in complete remission (CR) after chemotherapy based on CT/MRI (=CRI) criteria. They were not irradiated and achieved an excellent 97% EFS rate at 5 yrs (Dörffel, 2003).

Objective: Our next study will drop radiotherapy also in patients with residual findings according to CRI criteria if FDG-PET converts to negative after chemotherapy as ascertained by real-time central review of all images. Here we report pilot data on the conversion rate.

Results: During the GPOH-HD 2002 Pilot study 41 patients with early stage HL received both a staging PET at diagnosis and a restaging PET after chemotherapy. Images were centrally reviewed in Leipzig. 7 patients were in CR based on both PET and CRI criteria, one patient with CRI-based CR had still a positive PET result; in a further patient PET was not completely evaluable. In 17/32 patients with residual masses PET was completely negative after chemotherapy. In one patient PET was completely negative except for one initially involved site, where it was not informative due to a local artefact; however, CRI showed local CR in this region. In summary, 27/41 (66%) patients would qualify for no radiotherapy within a PET-based response adaptation strategy in contrast to only 9/41 (22%) patients when CRI-based. Interestingly, in 13/17 patients with negative PET, a residual mass within the mediastinum was found, indicating that a PET-based strategy will prevent many patients from mediastinal irradiation.

Conclusion: A FDG-PET-based response evaluation has the prospect to avoid radiotherapy in about 60 – 70% of all patients with early stage HL. Our next trial is designed to demonstrate that this does not lead to an unacceptable increase of the relapse rate.
Incidence of Pulmonary Hypertension in Haemoglobinopathic Patients without Left Ventricular Disfunction.
Authors: Derchi Giorgio , Formisano Francesco , Lamagna Martina , Galanello Renzo , Bina Patrizio , Cappellini Maria Domenica , Fasulo Maria Rosaria , Prossomariti Luciano , Cinque Patrizia , Maggio Aurelio , Renda Disma , Cianciulli Paolo , Cogliandro Tiziana , Piga Antonio , Donato Guido , Forni Gian Luca
Published: April 2020

Incidence of Pulmonary Hypertension (PH) has been described in haemoglobinopathic patients, ranging from 10% to 50%, depending on age, therapy, coexisting left ventricular dysfunction (LVD) and classification of the disease. To determine whether PH can occurs in haemoglobinopathic patients in absence of overt LVD, we studied 1121 pts from 7 Thalassemia Centers in Italy(485 Thalassemia Major (TM), 458 thalassemia intermedia (TI), 178 Sickle Cell or Sickle Thalassemia Disease (SCD),49% males, mean age 30 yrs, range 13–56 yrs). In all pts echocardiographic (Echo) defined ejection fraction (EF%) was > 45% and clinically overt LVD was excluded. A cross-sectional Echo study was performed in order to detect PH that was prospectively defined as mild with tricuspid regurgitant jet velocity (TRV)of at least 2.5 m per second and defined as severe with a TRV> 3.5 m per second. Severe PH was observed in 9 pts, 3 with TM, in 6 with TI and in 1 patients with SCD. Mild PH occurs in 46 patients with TM, in 57 patients with TI and 11 pts with SCD. In the remaining patients, PH was in the normal range (TM 89,9%, TI 86,2% and SCD 93,2% of pts, respectively). In the whole group of hemoglobinopathic pts the incidence of PH (mild plus severe) range from 6.5 % to 13.8% of pts. PH (mild to severe) particularly affects pts with TI (13,8% of pts).

Incidence of PH in TM, TI and SCD Tricuspid Regurgitant Jet Velocity Thal Major 485 pts Thal Int 458 pts SCD 178 pts % pts TRV<2.5 m/sec 436 (89.9%) 395 (86.2%) 166 (93.2%) 89% 2,5<TRV<3.5 m/sec 46 (9.4%) 57 (12.5%) 11 (6.2%) 10.1% TRV>3.5 m/sec 3 (0.6%) 6 (1.3%) 1 (0.9%) 0.9%

Our data support a significant presence of PH in haemoglobinopathic pts (6.2 to 13,8%) even though well treated and without LVD. Considering the pathophisiology of haemoglobinopathies a specific consideration to PH in these diseases is required in order to establish a preventive and therapeutic approach.
Endogenous Platelet PF4 Promotes In Vivo Activated Protein C (APC) Generation and Survival after Lethal Lipopolysaccharide Challenge in Mice: A Potential Physiologic Role for PF4.
Authors: Kowalska M. Anna , Mahmud Shawn A. , Poncz Mortimer , Slungaard Arne
Published: April 2020

Pharmacologic infusion of activated protein C (APC) improves survival in human sepsis and genetically induced deficiencies in APC generation increase mortality in a murine lipopolysaccharide (LPS) model of inflammation/sepsis. We have shown that the platelet α-granule-specific chemokine, platelet factor 4 (PF4), accelerates thrombin·thrombomodulin complex APC generation in vitro and in vivo when recombinant PF4 is pharmacologically co-infused in a primate thrombin infusion model system. Hypothesizing a potential physiologic role for PF4 in stimulating APC formation in vivo, we tested whether variations in the level of endogenous platelet PF4 content affect APC generation and also influence survival in the murine LPS challenge model. To stimulate APC formation and platelet PF4 release in mice, we injected murine thrombin (80U/kg IV). Ten min after thrombin injection into wildtype (WT) C57BL/6J mice, plasma APC levels, assessed by enzyme capture amidolytic assay, increased ~10-fold over basal levels. Injection of concurrent murine PF4 (7.5 mg/kg IV) further increased APC levels 2-2.6-fold. We then studied APC generation in mice that were either completely deficient in PF4 (PF4−/−) or transgenic mice that had 6-fold normal levels of human PF4 (hPF4+), both of which had been crossbred onto the same C57BL/6J background >10 times. Plasma APC generation by thrombin was 74 ± 6% in the PF4−/− mice (n = 7) compared to WT littermates (n = 15, p<0.1) and increased to 178 ± 49% in hPF4+ mice (n= 22, p< 0.003 compared to WT (n= 36)). Heterozygous protein C deficient (PC+/−) mice had 65 ± 21% of WT APC generation (n= 5, p= 0.05 compared to WT (n=7)), but APC generation was restored to 96 ± 6% level in hPF4+/PC+/− mice (n= 6, p= 0.88 compared to WT (n=12)). Only PF4−/− and PC+/− mice died during this thrombin injection trial with mortality rates of 30% and 58%, respectively. A lethal LPS challenge model (25 mg/kg IP) was used to test the susceptibility of mouse strains with varying PF4 levels. Significant numbers of WT and PF4−/− mice died before the end of the first day [(18 of 44 (40% mortality) and 6 of 21 (28% mortality), respectively], while none of the 19 hPF4+ animals died (p< 0.001). However, after the first day, mortality did not differ significantly between the groups and final mortality for the three groups was ~70%. In summary, these studies of mice with varying platelet PF4 suggest that endogenous PF4 plays a physiologic role to stimulate APC production. Moreover, mice with high platelet PF4 content appear to be protected from both thrombin- and LPS-provoked deaths. This suggests that PF4 plays a physiologic role to enhance APC generation, that patients with high PF4 levels may be intrinsically protected from sepsis and supports the possibility that PF4 infusions in patients with sepsis may be a viable therapeutic strategy.
The CBFb-SMMHC Oncoprotein Inhibits Binding of the Runx1 Runt Domain to DNA.
Authors: Bushweller John H. , Lukasik Stephen M. , Speck Nancy A.
Published: April 2020

Core-binding factors (CBFs) are heterodimeric transcriptional factors consisting of a DNA-binding Runx1 (CBFα) subunit and a CBFβ subunit. Cbfβ allosterically increases the affinity of Runx1 for DNA ~2.5 fold. CBF subunits are encoded by four genes in mammals. RUNX1 (AML1), RUNX2, and RUNX3 encode for CBFα subunits, and CBFB encodes the CBFβ subunit. Homozygous disruption of either the Runx1 or the Cbfb genes in mice results in essentially identical phenotypes: midgestation embryonic lethality accompanied by extensive hemorrhaging and a profound block at the fetal liver stage of hematopoiesis. In humans, chromosomal rearrangements that disrupt the Runx1 and CBFB genes are associated with a significant percentage of leukemias. CBFβ is disrupted in acute myeloid leukemia by inv(16)(p13;q22), t(16;16), and del(16)(q22). These translocations result in the production of novel fusion proteins containing most of the CBFβ protein fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (SMMHC) encoded by the MYH11 gene. A knock-in of the CBFB-MYH11 allele in mice resulted in embryonic lethality with a profound block in hematopoietic development, the same phenotype observed for the Runx1 and Cbfb knockouts. We recently demonstrated that the CBFβ-SMMHC fusion protein binds to the DNA binding Runt domain from Runx1 with both higher affinity and altered stoichiometry relative to native CBFβ. We also provided NMR-based evidence for multiple sites of contact between Runx1 and CBFβ-SMMHC, proving the role of the SMMHC sequence in creating this altered affinity. Here we demonstrate that CBFβ-SMMHC inhibits DNA binding of the Runx1 Runt domain by ~6-fold for the CD4 dual-site silencer element. Cross-saturation NMR mapping on the Runt domain in complex with CBFβ-SMMHC reveals that the SMMHC portion of the oncoprotein makes contacts with β-strands 1 and 2 in the Runt domain. We propose that the inhibition of DNA-binding and increased affinity combine to mediate the dysregulation of Runx-regulated genes caused by CBFβ-SMMHC. These results also clearly suggest that targeting of the CBFβ-SMMHC protein for drug development may well be a viable approach for the treatment of the associated leukemia.
Reduction of Core Binding Factor beta (CBFβ) Dosage Blocks T Cell Development.
Authors: Guo Yalin , Talebian Laleh , Maillard Ivan , Speck Caroline L. , Pear Warren S. , Rothenberg Ellen V. , Speck Nancy A.
Published: April 2020

Core binding factors (CBFs) are heterodimers consisting of a DNA binding subunit (Runx1, Runx2, or Runx3) and a non-DNA binding CBFβ subunit. CBFβ increases the affinity of the Runx subunits for DNA. Embryos deficient for Runx1 or CBFβ die at midgestation with a complete failure of definitive hematopoiesis due to a block in hematopoietic stem cell (HSC) emergence. To examine the role of core binding factors at later stages of hematopoiesis, we generated a hypomorphic Cbfb allele (Cbfbrss), that when carried over a Cbfb null allele (Cbfbrss/−) results in a 3-4 fold reduction in CBFβ protein levels. Although HSCs emerge in Cbfbrss/− animals, fetal T cell development is severely impaired. Here we examined the T cell developmental block in more detail by culturing fetal liver cells from Cbfbrss/− animals on OP9 stromal cells that express the Notch ligand Delta-like-1 (DL1) (Schmitt and Zúñiga-Pflücker, Immunity17: 749, 2002). Fetal livers (E14.5) from Cbfbrss/− animals contained normal numbers of both c-kit+Sca-1+lin- cells and c-kit+IL7r+ lymphoid progenitors. Lin- fetal liver progenitors cultured on OP9-DL1 cells in the presence of IL-7 and Flt3L displayed a growth disadvantage relative to wild type cells, and a block at the double negative 1 (DN1, CD44+ CD25−) stage of T cell development. The T cell defect could be rescued by retroviral transduction of the CBFβ heterodimerization domain into lin- fetal liver cells, but not by a G61A/N104A mutant that cannot bind the Runx subunits. Genes whose expression was decreased in DN1 cells purified from the OP9-DL1 cultures included CD3 and the early T cell transcription factors GATA3 and TCF. Although expression of several Notch pathway genes (Notch1, Hes-1/5, Deltex-1) was mildly decreased, Notch signals were clearly transduced, suggesting that Notch signaling was intact. These data demonstrate that reduced CBFβ levels impair the differentiation of stem cells/progenitors into T cells at the earliest stage of T cell development. This in vitro model will be useful for characterizing the molecular circuitry involving CBFβ in T cell development, and for identifying CBFβ protein partners.
Transcriptome Analysis of Murine Myeloid Development.
Authors: Yuan Wenlin , Ley Timothy J.
Published: April 2020

To characterize the transcriptional program that governs myeloid differentiation, we performed microarray analyses of cells derived from a G-CSF-dependent in vitro myeloid differentiation system. Using a high speed cell sorter, we purified CD34+/Lin− hematopoietic progenitors from wild type C57Bl/6 murine bone marrow cells cultured for 3 days in stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL3), and FLT3 ligand. These enriched progenitors were then treated for 7 days with G-CSF and SCF, which induces a coordinate wave of myeloid maturation. Linearly amplified cRNAs were prepared from cells harvested on each day of differentiation (d0-d7), labeled, and hybridized with Affymetrix Mouse 430v2.0 arrays. Comparing the percentage changes within two biological replicate sets by statistical analysis, we found that 51.5% of expressed probesets (15,693) were differentially regulated between day 0 and day 7. Among them, 10,627 probesets (34.9%) displayed expression levels higher than 500 (the mean expression level of all probesets was normalized to a value of 1500 before analysis) on at least one day. To avoid the high variability associated with probesets displaying low hybridization signals, we focused on these 10,627 probesets. The data showed the expected expression patterns for several genes that are known to be regulated during myeloid differentiation, including C/EBPa, Pu.1, c-Myb, CDP, c-Jun, M-CSF, GATA-1, AML1, CBFb, NE, MPO, MMP9, CD11b, lysozyme, and several others. In addition, the expression profiles were very consistent with the array data obtained using highly enriched human bone marrow-derived cells at early, mid, and late stages of myeloid development (Theilgaard-Monch et al., Blood105: 1785, 2005). However, our data also includes expression patterns from earlier stages of myeloid differentiation, which may be important to identify genes that are specifically expressed in committed myeloid progenitors. We organized the expressed genes into several categories, including transcription factors, cell cycle regulators, apoptosis genes, receptor and ligand in signal transduction pathways, etc. Some genes relevant for the pathogenesis of acute myelogenous leukemia (AML) also showed dynamic changes during normal myeloid differentiation, including (but not limited to) AML1, Flt3, HoxA9, Npm1, Ncoa2, Crebbp, Etv6, Meis1, Bcl2, Mcl-1, P53, Junb, Myc, and others. This dataset will be invaluable for evaluating the molecular events that underlie normal myeloid differentiation, and for defining the normal expression patterns of genes that are disregulated during the development of myeloid malignancies.
The Role of Alemtuzumab Compared to Antitymocyte Globulin in Non-Myeloablative Stem Cell Transplantation; Especially for the Prevention of GVHD and Immune Recovery.
Authors: Park In Hae , Cheong June-Won , Lee Jee Young , Kim Jin Seok , Chun Hyun Ju , Kim Yuri , Park Sun Young , Lee Jin Koo , Min Yoo-Hong
Published: April 2020

Alemtuzumab is an anti-CD52 antibody, and has been used for lymphoid malignancies or as a member of non-myeloablative conditioning regimen in allogeneic transplantation. Especially, in non-myeloablative stem cell transplantation (NST), it has been reported that alemtuzumab is effective for graft-versus-host disease (GVHD), but the immune reconstruction after transplantation is delayed. In this study, we comparatively evaluated the efficacy of alemtuzumab for non-myeloablative conditioning, GVHD prophylaxis, and immune recovery in NST for hematologic diseases. We have compared the results in 28 recipients of a sibling or unrelated NST enrolled. The recipients were divided into 2 groups according to the use of alemtuzumab. In group A (n=21), the conditioning regimen was a combination of fludarabine, cyclophosphamide (or busulfan) and antithymocyte globulin (ATG), and group B (n=7) received fludarabine, cyclophosphamide (or busulfan), and alemtuzumab instead of ATG. GVHD prophylaxis was by cyclosporin A or FK506 plus methotrexate. There were no significant differences in the graft engraftment and period of granulocyte colony-stimulating factor infusion. Patients receiving alemtuzumab had a significantly lower incidence of acute GVHD (stage 2 or more) (14.3% versus 38.1%, P=0.03) and chronic GVHD (14.3% versus 52.4%, P=0.005). The relapse rate after transplantation was 28.6% (6 patients) in group A and 14.3 (1 patients) in group B (P=0.04). Flow cytometric analysis of peripheral mononuclear cells for evaluation of immune recovery showed that T-cell and NK-cell recovery were delayed in both groups. However, T-cell and NK-cell recovery after transplantation occurred earlier in patients received alemtuzumab. No significant differences were observed in disease-free or overall survival between two groups. In conclusion, alemtuzumab can be recommended for immune suppression in NST, with successful control over acute/chronic GVHD and inducing relatively earlier immune recovery after transplantation.