bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic Graft Versus Host Disease (cGVHD) Following Unrelated Donor Hematopoietic Stem Cell Transplantation (HSCT): Higher Response Rate in Recipients of Unrelated Donor (URD) Umbilical Cord Blood (UCB).
Authors: Arora Mukta , Nagaraj Sriharsha , Wagner John E. , Barker Juliet N. , Brunstein Claudio G. , Burns Linda J. , MacMillan Margaret L. , Miller Jeffrey S. , Weisdorf Daniel J.
Published: April 2020

The nature of chronic graft versus host disease (cGVHD) after umbilical cord blood (UCB) transplant has not been well characterized. We analyzed clinical presentation and response to treatment in 170 patients with cGVHD (123 following transplant from an unrelated donor (URD) and 47 following transplant from UCB). URD transplant recipients were significantly younger (median age 25 (0.5–57) versus 39 (0.2–61) years, p = 0.002; and were less likely to receive a transplant from an HLA mismatched donor (33% versus 90%, p<0.0001). Seven% and 43% of patients underwent a transplant using a non myeloablative conditioning in the URD and UCB groups, respectively (p< 0.001). Progressive onset of cGVHD was seen in 27% and 19% of patients in URD and UCB cohorts (p =0.15) and platelet count of < 100,000/μl was seen in 36% and 15% of patients (p =0.014), respectively. The median follow up was 5 (0.14–10.8) and 1.5 (0.02–8.8) years in the 2 groups, respectively. A higher rate of response (CR+PR) to treatment was seen in UCB group across all time periods. Rates of response were 48% versus 74% at 2 months (p =0.005); 49% versus 78% at 6 months (p =0.001) and 51% versus 72% at 1 year (p =0.03) in the URD and UCB groups, respectively. Overall survival at 2 years was 59% (50–67%) versus 71% (53–83%) at 2 years and at 5 years was 54% (44–63%) versus 64% (42–79%) (p=0.17). In multivariate analysis, progressive onset of CGVHD (RR=2.3, 95% CI 1.4–3.8−2.3,p=0.002), platelet count of <100,000/μl (RR=3.5, 95% CI 1.7–7.3,p=0.0009), liver involvement (RR=2.5, 95% CI 1.4–4.6,p=0.002), and no CR or PR at 2 months (RR=3.5, 95% CI 2.1–5.9,p<0.0001) were associated with significantly increased mortality. These data suggests that among patients undergoing transplant from unrelated donor or cord blood, despite similar clinical presentation, higher rates of response to treatment were seen in UCB recipients. Early identification of high risk groups (progressive onset, less than PR at 2 months, platelet count < 100,000/μl and liver involvement should facilitate timely assignment of intensified therapy.

Response to Treatment Response 2 mo 2 mo 6 mo 6 mo 1 year 1year URD UCB URD UCB URD UCB CR+PR 49 (48) 29 (74) 54 (49) 32 (78) 53 (51) 23 (72) SD+Prog 53 (52) 10 (26) 56 (51) 9 (22) 52 (49) 9 (28)
Obesity Independently Predicts Event Free Survival (EFS) in Adults with BCR-ABL-Negative Acute Lymphoblastic Leukemia (ALL). A Retrospective Analysis of Two GIMEMA Studies.
Authors: Butturini Anna , Vignetti Marco , Gubbiotti Stefania , Meloni Giovanna , Recchia Monica , Di Raimondo Francesco , Morra Enrica , Palmieri Salvatore , Fabbiano Francesco , Liso Vincenzo , Pizzolo Giovanni , Marmont Filippo , Cantore Nicola , Cascavilla Nicola , Luppi Mario , Camera Andrea , Ronco F. , Sora F. , Peta Antonio , Tabilio Antonio , Foa Roberto , Mandelli Franco
Published: April 2020

Obesity predicted decreased EFS in pediatric ALL (ASH 2004, Abstract#992). Here we report the effect of obesity, defined as body mass index >30, in 906 adults enrolled in two GIMEMA trials. The first study (1996–2001) included 45 obese (9.5%) and 426 non-obese patients. Obese patients were older, received lower total doses of vincristine (VCR) and daunomicin (DNR) and had more hepatic and pulmonary toxicity during induction than not-obese patients; the incidence of toxic deaths and complete remissions (CR) were similar. By multivariate analysis, obesity, high WBC, age >30y and presence of BCR-ABL at diagnosis independently predicted EFS. Hazard ratios (HR) for any events were respectively 1.506 (1.059–2.142, p=0.0228), 1.002 (1.001–1.003, p=0.0022)), 1.015 (1.006–1.025, p=0.0014) and 1.8 (1.4–2.4, p<0.0001). The effect of obesity on EFS was present across all levels of WBC, but was observed prevalently in BCR-ABL-negative patients. 3y-EFS in obese vs non-obese patients was 22.9% vs 39.5% (p=0.0064) in BCR-ABL negative patients and not significantly different (14% vs 22%, p=0.77 n.s.) in BCR-ABL positive patients. These results were verified in the following study started in 2000, which included 50 obese (11%) and 385 non-obese patients. In this study, obese patients received 28% and 21% less than the planned doses of VCR and DNR respectively; also they were more likely than non-obese patients to be >30 y of age and BCR-ABL-positive. The incidence of toxicity, toxic deaths and CR were similar in obese and non-obese patients. In the BCR-ABL negative patients, obesity tended to predict EFS. 2y-EFS was 26.6% in the obese and 49.4% in the non-obese patients (p=0.067). By multivariate analysis HR for any events of obesity, WBC>50 and age>30 were respectively 1.53 (0.92–2.58, p=0.107), 1.64 (1.12–2.39, p=0.012) and 1.74 (1.19–2.54, p=0.004). In conclusion, obesity seems associated with a poorer EFS in patients with BCR-ABL negative ALL; this might be caused by the lower doses of chemotherapy obese patients receive. Studies aimed to clarify chemotherapy pharmacokinetics, toxicity and efficacy in obese patients are warranted.
Scoring Adherence in Hemophilia Care and Comparing It to Quality of Life (QofL).
Authors: duTreil Sue , Rice Janet , Leissinger Cindy A.
Published: April 2020

Non-adherence to home based treatment even 10% of the time in hemophilia care may portend a poor outcome and long-range quality of life issues, despite a still enormous financial cost. This study was a rigorous effort to quantify adherence and then compare it to QofL. 79 subjects with hemophilia (48 >18-years and 31<18-years) participated in the study and completed the SF-36 or Child Health Questionnaire (validated QofL forms). Subjects were either using on-demand (OD) treatment regimens or on high intensity (HI) treatment strategies i.e. prophylaxis or immune tolerance therapy. Physician’s specific treatment order for dose and frequency of clotting factor concentrate (CFC) were collected. Factor infusion logs kept by patients were reviewed to determine how factor use matched treatment recommendations. Infusion logs were maintained by 47 of 79 subjects (59%). The amount of CFC used was entered into a database along with the date of the infusion and the reason for the infusion. Due to vial size variability doses that were within 80% to 150% of the prescribed dose for children and 80% to 120% for adults were considered adherent. A scoring system was developed for both OD and HI users based on documented usage patterns. A given subject’s adherence score could range from 0 (representing total non-adherence) to 100% (representing perfect adherence). A subject’s adherence percentile was then ranked accordingly: 0% to 33% = Low Adherence, 34% to 66% = Moderate Adherence, 67% to 100% = High Adherence. Chi Square was used to determine the relationship between adherence and treatment regimen and a two-way analysis of variance was used to compare QofL in subjects with treatment logs to adherence and treatment regimen. Subjects on HI had lower adherence patterns than those on OD (p=. 018). Significant findings related to QofL included that body pain in children was higher with OD compared with HI (p=. 036), however this was not demonstrated in adults. Adults with low adherence patterns had higher mean scores in mental health then high (p=. 021) and moderate (p=. 068) adhering adults. Quantifying adherence and attempting to understand the relationship between adherence and QofL is an important component in the management of hemophilia.
Activation of C/EBPα in Hematopoietic Stem Cells Leads to Growth Suppression and Impairment of Megakaryocyte Differentiation.
Authors: Fukuchi Yumi , Shibata Fumi , Ito Miyuki , Goto-Koshino Yuko , Sotomaru Yusuke , Ito Mamoru , Kitamura Toshio , Nakajima Hideaki
Published: April 2020

C/EBPα is a critical regulator of early myeloid differentiation. Resent study showed that the loss of C/EBPα led to an augmentation of competitive repopulating activity of hematopoietic stem cells (HSCs). Our previous study using the transgenic (Tg) mice expressing an inducible form of C/EBPα (C/EBPα-ER) also showed that in vivo activation of C/EBPα by 4-hydroxy tamoxifen (4-HT) led to remarkable decrease of HSC population (Abstract #671, Blood104, 193a, 2004). In order to investigate further the role of C/EBPα in HSC homeostasis, we purified CD34− c-Kithi Sca-1hi lineage− cells (CD34−KSLs) from the C/EBPα-ER Tg mice and examined the effect of C/EBPα activation on their growth, self-renewal, and differentiation potential. Clonal proliferation assay showed that the growth of about 95 % of CD34−KSLs was suppressed in the presence of 4-HT. Single cell colony assay showed that the percentage of CFU-GEMM and CFU-MK remarkably decreased from 80 % to 2.1 % and from 2.1 % to 0 %, respectively by 4-HT treatment. In contrast, percentage of CFU-GEM increased from 0 % to 74 %, indicating that differentiation potential to megakaryocytes (MKs) was selectively lost by C/EBPα activation. To gain more insights into the cell fate determination at clonal level, we performed paired daughter cell colony assay. Sorted CD34−KSLs were clonally deposited into 96-well plates and allowed to divide once to generate daughter cell pairs, which were then separated by micromanipulation, and transplanted into control or 4-HT containing methylcellulose. 26 daughter cell pairs out of assessable 41 colony pairs lost MK differentiation capacity in the presence of 4-HT. In contrast, remaining 15 pairs took the same fate of differentiation in control and 4-HT. Surprisingly, while most of the CD34−KSLs stopped their growth upon C/EBPα activation, approximately 5 % of the cells proliferated even in the presence of 4-HT. These cells grew from a single cell to generate more than 45 cells and sustained multipotentiality to generate GEMM-colonies even after 8-days of culture. To further examine the effect of C/EBPα activation on self-renewal potential of HSCs, clonally sorted CD34−KSLs were allowed to divide once in 4-HT- or control-media, and the fate of each daughter cell was traced in methylcellulose. The result showed that GEMM-colony pairs were generated at similar frequency in control and 4-HT culture, indicating that the self-renewal capacity of CD34−KSLs was not affected by C/EBPα. These results indicated that activation of C/EBPα caused growth suppression and impaired differentiation to MKs in the majority of CD34−KSLs. However, small fraction does not respond to C/EBPα activation, indicating CD34−KSLs could be classified into two subpopulations by their sensitivity to C/EBPα.
Roles of MOZ in Hematopoiesis.
Authors: Katsumoto Takuo , Aikawa Yukiko , Ochiya Takahiro , Kitabayashi Issay
Published: April 2020

The AML1-CBFβ transcription factor complex is the most frequent target of specific chromosome translocations in acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene, which encodes a MYST-type histone acetyltransferase (HAT), is also involved in leukemia-associated translocations such as t(8;16), t(8;22) and inv(8), which are associated with acute myeloid leukemia with M4/5 subtypes. We previously found that MOZ functions as a potent coactivator for AML1. To investigate roles of MOZ in normal hematopoiesis, we generated MOZ-deficient mice using gene-targeting method. MOZ homozygous mutant is embryonic lethal and it died between days 14 and 15 of gestation. In fetal liver of MOZ-deficient E14.5 embryos, the total cell numbers and the colony-forming cells (CFCs) in a methylcellulose medium were remarkably reduced when compared with wild-type littermates. Flow cytometry analysis indicated that hematopoietic stem cells (HSCs) and progenitors of both myeloid and lymphoid lineages were severely reduced in MOZ-deficient embryos. Especially, the levels of c-kit expression were strongly reduced in lineage-negative cells. Differentiation arrest of erythroid progenitors at a terminal stage and increase in the numbers of Mac-1 and Gr-1 positive cells suggest that MOZ also plays roles in cell differentiation of erythroid, monocytic and granulocytic lineages. In E12.5 MOZ deficient fetal liver cells, expression profile analysis revealed decreases in expressions of thrombopoietin receptor c-mpl, Wnt related ligand dkk2 and HoxA9 and increase in HoxA5 expression. To further determine roles of MOZ in HSCs functions and their progenitors differentiation ability, competitive reconstitution assays were performed. Ly5.2+ fetal liver cells from wild-type, heterozygous or homozygous mutant embryos together with Ly5.1+ competitor fetal liver cells were transplanted into γ-irradiated Ly5.1+/Ly5.2+ recipient mouse. Ly5.2+ wild-type cells were observed in recipient mice after transplantation. However, cells derived from MOZ homozygous mutant embryos were not detected in peripheral blood, bone marrow, spleen and thymus. Reduced population of cells derived from heterozygous mutant embryos were observed. These data suggest that MOZ is required for lymphoid and myeloid development and for self-renewal of HSCs.
Human Conventional and Plasmacytoid Dendritic Cells Can Originate from Both Lymphoid and Myeloid Progenitors in a New Humanized Mouse System.
Authors: Ishikawa Fumihiko , Iino Tadafumi , Niiro Hiroaki , Yoshida Shuro , Miyamoto Toshihiro , Shigematsu Hirokazu , Shultz Leonard D. , Harada Mine , Akashi Koichi
Published: April 2020

Dendritic cells play a key role in host defense by presenting exogenous antigens to T cells. Two dendritic cell subsets, conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs), express distinct repertoire of Toll-like-receptors and recognize different antigens. We previously reported that murine cDCs and pDCs differentiate via either the myeloid or the lymphoid pathway (Shigematsu et al. Immunity ). It is, however, still unclear whether human cDCs and pDCs develop from myeloid, lymphoid or both lineages. In order to analyze the in vivo differentiation of human dendritic cells, we employed the newly-developed xenotrasplant assay system which utilizes newborn NOD-scid/IL2rgnull mice (Ishikawa et al., Blood, in press). Transplantation of 104 Lin-CD34+CD38- hematopoietic stem cells into sublethally irradiated newborn NOD-scid/IL2rgnull mice resulted in generation of all hematopoietic and lymphoid components for a long-term via physiological intermediates such as common myeloid progenitors (CMP) and common lymphoid progenitors (CLP). We found that in this system, dendritic cell subcomponents such as hCD11c+hIL3Ralow cDCs and hCD11c-hIL3Rahigh pDCs, efficiently developed in recipients’ bone marrow, spleen and peripheral blood. To elucidate the origin of human mDCs and pDCs, we purified CMP or CLP from the cord blood, and transplanted these cells into sublethally irradiated newborn NOD-scid/IL2rgnull mice via facial vein. At 4-6 weeks post-transplantation, CMP gave rise only to myeloid cells such as erythroid cells, platelets and granulocytes, while CLP exclusively generated T, B and NK cells. Interestingly, in either mouse group injected with CMP or CLP, cDCs and pDCs were easily detected in the spleen and in the bone marrow. Phenotypic and RT-PCR analyses of purified CMP- or CLP-derived DCs revealed that DCs possessed similar phenotypic characteristics, and transcription profiles in TLR families, BDCA antigens and costimulation molecules, irrespective of their lineage origin. Thus, human cDCs and pDCs develop through both myeloid and lymphoid pathways as in case of mouse hematopoiesis. Further characterization of DCs of different lineage origin is currently performed by microarray analyses in order to find genes specifically expressed in each DC subset.
p15Ink4b Maintains Normal Levels of Granulocyte-Macrophage Progenitors In Vivo.
Authors: Rosu-Myles Michael , Wolff Linda
Published: April 2020

Maintaining a balance between the proliferation and differentiation of hematopoietic stem and progenitor cells is crucial for normal maintenance of the hematopoietic system. The INK4 family of cyclin-dependent kinase inhibitors traditionally function in regulating cell proliferation by blocking G1/S phase transition during cell cycle. Loss of expression of one INK4 family member, p15INK4B, has been associated with acute myeloid leukemia (AML) and myelodysplasia and mice that lack p15Ink4b are more prone to developing retrovirus-induced AML. These data suggest an important role for p15INK4B in myeloid differentiation. Here we examined the hematopoietic progenitor pool in p15Ink4b deficient (Ink4b−/−) mice to determine its potential role in regulating hematopoietic precursors. In myeloid progenitor colony assays, bone marrow (BM) from Ink4b−/− mice were found to contain a 1.4 fold greater number of progenitors committed to the formation of granulocytes and macrophages (CFU-GM). This in vitro data was supported by flow cytometric analysis which determined that Ink4b−/− BM contained a 3-fold greater proportion of granulocyte-macrophage progenitors (GMP) (11+/−1.2% vs 34+/−9%) concomitant with a 2-fold decrease in common myeloid progenitors (CMP) (56+/−2 % vs 34+/−7 %). GMP isolated from Ink4b−/− BM also demonstrated a 3-fold greater propensity to form CFU-GM. Despite these differences, the proportion of cycling GMP or CMP in Ink4b−/− and wt mice were identical as determined by propidium iodide and Hoecsht dye DNA stains and in vivo BrdU incorporation. However, in response to specific cytokines, Ink4b−/− CMP showed an increased potential for GMP differentiation and a decreased capacity to form megakaryocyte-erythroid progenitors (MEP). Our work demonstrates that p15Ink4b functions in maintaining normal levels of CFU-GM and may regulate CMP differentiation in response to specific factors.
CD97 Is Differentially Expressed on Murine Hematopoietic Stem Cells and Progenitor Cells.
Authors: van Pel Melissa , Hagoort Henny , Willemze Roel , Hamann Jorg , Fibbe Willem E.
Published: April 2020

CD97 is a member of the EGF-TM7 family of class II seven-span transmembrane receptors and is broadly expressed on hematopoietic cells including lymphocytes, granulocytes, and monocytes. We have recently demonstrated that CD97 is involved in IL-8-induced hematopoietic stem cell (HSC) mobilization (Blood (2003) 102:455a). To determine a possible role of HSC in this process, we studied the expression of CD97 on HSC. Murine HSC are characterized as c-KitPOSThy-1LOLinNEG/LOSca-1POS and isolation of these cells involves a multistep process consisting of lineage depletion and positive selection.

We first studied expression levels on murine bone marrow (BM) cells using a CD97 specific monoclonal antibody (clone 1B2) and FACS analysis. Based on CD97 expression levels, BM cells were then sorted into different fractions and further characterized in colony-forming assays, CAFC assays and in an in-vivo transplantation model.

FACS analysis of BM cells showed three major populations i.e. CD97HI, CD97INT and CD97NEG cells (71.5%, 24.4% and 4.4% of total BM cells respectively). Analysis of CFU-GM colony forming capacity of these BM subsets revealed that the majority of colony-forming cells were present in the CD97NEG (7142 ± 5057 CFU-GM) population compared to CD97HI (178 ± 170CFU-GM) and CD97INT (3047 ± 2902 CFU-GM per 106 BM cells). Analysis of CAFC frequencies of CD97HI, CD97INT and CD97NEG BM cells showed that CAFC-day 35 activity resided in the CD97INT BM population (3.0 ± 1.2 vs. 0.9 ± 0.1 CAFC-day 35 per 105 cells for CD97INT and total BM respectively), whereas no CAFC-day 35 activity was found in CD97HI and CD97NEG BM fractions. In addition, FACS analysis revealed that the majority (82.6 %) of c-kitPOSthy-1LOLinNEG/LO cells were present in the CD97INT BM population. To investigate the in-vivo repopulating ability of the different CD97 BM subsets, lethally irradiated (9.5 Gy) BALB/c recipient mice (n=10 per group) were reconstituted with 105 syngeneic BALB/c total BM or with CD97 sorted BM cells. Repopulating capacity entirely resided in the CD97INT subset (repopulation rate 90% versus 0% in the CD97HI and CD97NEG subset).

These data indicate that 1) CD97 is differentially expressed on HPC and HSC and that 2) CD97 expression can be used to separate colony forming cells from repopulating hematopoietic stem cells.
Human Mesenchymal Stem Cells Enhances the Growth of Acute Myeloid Leukemia Cells In Vivo.
Authors: Cen Jian-nong , Chen Zi-xing , Qian Jun , Li Zhen-jiang , Qiu Qiao-cheng
Published: April 2020

Human mesenchymal stem cells (MSC) are adhering cells capable of self-renew, proliferation and multilineage differentiation. MSC have the capacity to differentiate into osteoblasts, chondroblasts, myocytes and adipocytes when exposed to appropriate in vivo or in vitro stimuli. MSC descendants are involved in the formation of stroma maintaining the hemopoiesis. In this study, we investigated whether in vivo coinjection of hMSCs could enhance acute myeloid leukemia cells growth. Acute myeloid leukemia line (SHI-1) cells were grown in IMDM with 10%FCS. The hMSCs in adult BM were isolated and cultured in 60%DMEM (low glucose) and 40% MCDB-201 medium, 5%FCS, ITS+1. The hMSCs were immunotyped by using FACS. The differentiation ability in vitro towards osteoblasts and adipocytes was examined. BABL/c nude mice, aged 5 to 6 weeks, were used to explore the hMSCs’ function in vivo. SHI-1cells and hMSCs were prepared either as single-cell type suspensions (3×106 cells in 100ul PBS) or a mixture of cells (3×106 SHI-1 cells plus 4×105 hMSCs in 100ul PBS ). Subcutaneous injection was performed in alar area. Injection of hMSCs alone was used as control. Mice were examined 3 times a week and tumor growth was evaluated by measuring the length and width of tumor mass. After 30 days, animals were sacrificed and tumor masses were weighed. Our experiments showed that the hMSCs have the potential of proliferation and the capacity to differentiate into osteoblasts and adipocytes in vitro. The immunophenotype of MSCs is CD34−,CD45−,CD105+,CD19−,CD13+,CD14−.10 days after injection with a mix of cells small tumor mass occurred in mice while no tumor mass could be observed in mice injected SHI-1 cells alone. Upon day 20, small tumor mass started to emerge in mice injected with SHI-1 cells alone. By the end of the experiment, mice were sacrificed and tumors were recovered. The weight of tumor masses from the mouse injected with mixed cells of SHI-1 and hMSCs was 0.42±0.18 g and from the mouse injected SHI-1 cells alone was 0.11±0.089 g, (P<0.01). No tumor masses could be found in mice injected with hMSCs alone. From this animal model, we proposed that human mesenchymal stem cells favors the growth of acute myeloid leukemia cells in vivo.
Physiological Gradients of Serum Albumin Regulate SDF-1/CXCR4 Dependent Migration.
Authors: Ryser Martin F. , Roesler Joachim , Malech Harry L. , Roesen-Wolff Angela , Brenner Sebastian
Published: April 2020

Hematopoietic stem cell (HSC) egress to the circulation and homing to the bone marrow (BM) are regulated by interactions between CXCR4 and SDF-1. Serum albumin is the major protein component of plasma. Concentration gradients of albumin between plasma and interstitial fluid account for the colloid osmotic pressure, which is a central regulator of the intravasal blood volume. SDF-1/CXCR4 dependent migration of mobilized peripheral blood CD34+ hematopoietic stem cells (PBSC) was studied in transwell migration assays. We compared the effect of RPMI diluted Heparin-plasma (50% plasma, 50% RPMI) versus undiluted plasma as a migration medium. To our surprise the use of undiluted plasma in the upper chamber containing the cells and diluted plasma in the lower chamber containing 100ng/ml SDF-1, resulted in a 3 fold increase of migrating PBSCs compared to experiments without a gradient (undiluted versus undiluted plasma or diluted versus diluted plasma) while diluted plasma in the upper chamber and undiluted in the lower chamber inhibited the SDF-1 dependent migration 3,5 fold. To further characterize this observation we removed high molecular weight proteins (>50kd) by filtration of the plasma. The filtrate was used to dilute plasma to obtain samples with reduced protein contents. Transwell experiments showed that negative gradients of plasma proteins (high concentration in the upper chamber vs low concentration in the lower chamber) stimulate (up to 2 fold) while positive gradients inhibit SDF-1/CXCR4 dependent migration of PBSC (up to 3 fold). Migration experiments were repeated with RPMI, supplemented with varying concentrations of serum albumin as migration medium. Negative gradients of albumin stimulated migration while positive gradients were inhibiting. Interestingly, a gradient of 4% albumin in the upper chamber and 1% albumin in the lower chamber enabled migration in the absence of a SDF-1 gradient (100ng/ml SDF-1 in both upper and lower chamber). Albumin gradients did not stimulate migration in the absence of SDF-1. The absence of serum albumin in both chambers abolished the SDF-1/CXCR4 dependent migration of PBSCs. Our results show that gradients of serum albumin strongly influence the SDF-1/CXCR4 dependent migration of PBSCs. Negative gradients of albumin between blood and bone marrow might be supportive for the homing of PBSC to the stem cell niche, which suggests a new function of the multitask protein serum albumin.
Human Mesenchymal Stem Cells as Precursors of Functional Fibroblastic Reticular Cells: Implications for the Physiopathology of Secondary Lymphoid Organs.
Authors: Tarte Karin , Ame-Thomas Patricia , Maby Hélène , Caulet-Maugendre Sylvie , Jean Rachel , Monvoisin Céline , Lamy Thierry , Fest Thierry
Published: April 2020

Several subsets of stromal cells are found among secondary lymphoid organs where they play a key role in the initiation and maintenance of immune response. In particular, fibroblastic reticular cells (FRC) of the paracortex secrete extracellular matrix (ECM) components that constitute a dense network of conduits allowing antigens carried within the subcapsular afferent lymph to reach the lumen of the medullary high endothelial venules. FRC produce also several chemokines that recruit T, B, and dendritic cells from blood and favour their reciprocal interactions. In addition, follicular dendritic cells (FDC) are located exclusively into germinal centers and allow normal B-cell selection through a complex set of survival signals, including BCR-mediated signal, chemokines and adhesion molecules. FRC and FDC networks are phenotypically and probably functionally altered during development of follicular lymphomas and diffuse large B cell lymphomas, the two most frequent Non-Hodgkin Lymphomas. FRC and FDC are supposed to be of mesenchymal origin even if no conclusive work has been conducted to date in human. We have obtained 15 tonsil-derived stromal cell lines, that displayed all the morphologic, phenotypic, and functional characteristics of FRC, including synthesis of inflammatory (CXCL10, CXCL9, CCL5) and lymph-node specific (CCL19, CCL21) chemokines, and secretion of ECM organized in a reticular meshwork after long-term culture in the presence of TNF-α and lymphotoxin-α1β2 (LT). These cells induced tonsil leukocyte migration and adhesion in vitro. Tonsil-derived stromal cells expressed LTβR, TNFR, and CD40 but were negative for FDC specific markers, such as CD21 or CXCL13, even following in vitro stimulation by TNF-α, LT, and trimeric CD40L. Interestingly, such TNF and LT-dependent FRC differentiation could also be induced in adult bone marrow-derived mesenchymal stem cells (MSC). In addition, MSC-like cells able to differentiate along osteogenic, adipogenic, and chondrogenic lineages at the clonal level were found in normal tonsils. These data shed new lights on our current understanding of lymph node stromal cell origin and strongly suggest that MSC are the precursors of FRC in secondary lymphoid organs, and perhaps in bone marrow in case of FL involvement where ectopic lymph node-like stromal cells are detected in close association with tumor cells. In conclusion, MSC and their progeny trigger differential immune effects, depending on cytokine context, localization and cell contact with immune cells. These properties are probably modified during lymphomas where the contact between malignant B cells and stromal cells is crucial for tumor development.
Increased Red Cell Adhesion Is Associated with Overt Stroke in Sickle Cell Disease.
Authors: Rennie Kimberly M. , Scott J. Paul , Panepinto Julie A. , Hillery Cheryl A.
Published: April 2020

The major cause of morbidity and mortality in sickle cell disease (SCD) is tissue ischemia and infarction due to vascular obstruction. One of the most devastating vascular complications of SCD is clinical stroke caused by occlusion of the large cerebral arteries in the Circle of Willis or its branches. Overt stroke occurs with obvious speech or motor deficits in approximately 10% of children with SCD. Additional complications due to stroke in SCD include an increased risk of secondary disabilities such as mental retardation and learning disabilities, with the most severe forms of neurocognitive impairments associated with large vessel disease (overt stroke). When compared to normal RBCs, sickle red blood cells (SS-RBCs) have markedly increased adhesion to the vascular endothelium and several subendothelial matrix proteins. The increased sickle RBC adhesion likely plays a significant role in the pathogenesis of vascular obstruction. We have previously shown that SS-RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In addition, we have observed that the level of adhesion of RBC adhesion varies from patient to patient (i.e., there are some patients whose RBCs tend to consistently be “stickier” or “less sticky” than other patients). In this study, we proposed that patients who develop large vessel (Circle of Willis and major branches) central nervous system (CNS) disease (e.g., clinical stroke) have higher baseline levels of RBC adhesion. Thus, we examined the level of sickle RBC adhesion to immobilized TSP under flow conditions from patients with SCD with and without stroke. We recruited 87 male and female patients over the age 3 yrs with HbSS disease from the Wisconsin Sickle Cell Disease Comprehensive Center. Of these, 8 patients later developed an overt stroke following their participation in this study. None of the patients were transfused prior to studying RBC adhesion. After obtaining informed consent, blood samples were collected into citrate from patients with HbSS disease. Washed RBCs were perfused through flow chambers previously coated with TSP (2 μg/cm2) at a wall shear stress of 1 dyne/cm2 that mimics the forces in post-capillary venules and optimizes sickle RBC adhesion. After rinsing, adherent RBCs per unit area were counted in four random fields in each of two duplicate wells by direct microscopic visualization. We found that patients with SCD who developed an overt stroke (n=8) had a higher level of RBC adhesion (1436 ±101 RBCs/mm2) compared to 79 unselected patients with SCD who had a mean RBC adhesion of 1004 ± 53 RBCs/mm2 (p=0.016, student t-Test). Thus, RBCs derived from the subpopulation of patients with SCD who developed an overt stroke associated with large vessel disease have an abnormally enhanced adhesive phenotype. These data suggest that enhanced levels of RBC adhesion may play a role in the development of large vessel disease in SCD.
Long Term Follow-Up of Pediatric Sickle Cell Disease Patients with Conditional Velocities on Transcranial Doppler (TCD).
Authors: Coïc Lena , Verlhac Suzanne , Lesprit Emmanuelle , Fleurence Emmanuelle , Bernaudin Francoise
Published: April 2020

Abnormal TCD defined as high mean maximum velocities > 200 cm/sec are highly predictive of stroke risk and justify long term transfusion program. Outcome and risk factors of conditional TCD defined as velocities 170–200 cm/sec remains to be described. Patients and methods Since 1992, 371 pediatric SCD patients (303 SS, 44 SC, 18 Sß+, 6 Sß0) were systematically explored once a year by TCD. The newborn screened cohort (n=174) had the first TCD exploration between 12 and 18 months of age. TCD was performed with a real-time imaging unit, using a 2 MHz sector transducer with color Doppler capabilities. Biological data were assessed at baseline, after the age of 1.5 years and remotely of transfusion or VOC. We report the characteristics and the outcome in patients (n=43) with an history of conditional TCD defined by mean maximum velocities ranging between 170 and 200 cm/s in the ACM, the ACA or the ICA.

Results: The mean follow-up of TCD monitoring was 5,5 years (0 – 11,8 y). All patients with an history of conditional doppler were SS/Sb0 (n=43). Mean (SD) age of patients at the time of their first conditional TCD was 4.3 years (2.2) whereas in our series the mean age at abnormal TCD (> 200 cm/sec) occurrence was 6.6 years (3.2). Comparison of basal parameters showed highly significant differences between patients with conditional TCD and those with normal TCD: Hb 7g4 vs 8g5 (p<0.001), MCV 82.8 vs 79 (p=0.047). We also had found such differences between patients with normal and those with abnormal TCD (Hb and MCV p< 0.001). Two patients were lost of follow-up. Two patients died during a trip to Africa. Conditional TCD became abnormal in 11/43 patients and justified transfusion program. Mean (SD) conversion delay was 1.8 (2.0) years (range 0.5–7y). No stroke occurred. 16 patients required a treatment intensification for other indications (frequent VOC/ACS, splenic sequestrations): 6 were transplanted and 10 received HU or TP. Significant risk factors (Pearson) of conversion to abnormal were the age at time of conditional TCD occurrence < 3 y (p<0.001), baseline Hb < 7g/dl (p=0.02) and MCV > 80 (p=0.04). MRI/MRA was performed in 31/43 patients and showed ischemic lesions in 5 of them at the mean (SD) age of 7.1 y (1.8) (range 4.5–8.9): no significant difference was observed in the occurrence of lesions between the 2 groups. Conclusions This study confirms the importance of age as predictive factor of conditional to abnormal TCD conversion with a risk of 64% when first conditional TCD occured before the age of 3 years. TCD has to be frequently controled during the 5 first years of life.
Excellent Results in Children and Adolescents with Isolated Mature B-Acute Lymphoblastic Leukemia (B-ALL) (Burkitt): Report from the French-American-British (FAB) International LMB Study FAB/LMB96.
Authors: Goldman Stan , Gerrard Mary , Sposto Richard , Davenport Ginny , Auperin Anne , Pinkerton Ross , Michon Jean , Weston Claire , Perkins Sherrie L. , Raphael Martine , McCarthy Keith , Patte Catherine , Cairo Mitchell S.
Published: April 2020

The prognosis for Burkitt leukemia has improved steadily over the past thirty years with short intensive therapy utilizing high dose methotrexate and fractionated cyclophosphamide (Cairo et al Br J Haem, 2003, Patte et al Blood, 2001, Reiter et al Blood, 2005). The presence of CNS involvement at diagnosis with B-ALL is, however, associated with a poorer survival (3yr EFS 60%) as demonstrated previously in 235 children and adolescents with B-ALL (≥25% L3 cells) with or without CNS involvement randomized to standard intensity FAB/LMB therapy vs adjusted (decreased) intensity and maintenance therapy demonstrating an advantage to standard intensity therapy (Cairo et al ASH, 2004). We now analyze the results in 110 children and adolescents with B-ALL and no evidence of CNS involvement at diagnosis treated on FAB/LMB96 (4–6 mo TX). 110 patients, 18% 0–4, 47% 5–9, 29% 10–14 and 6% 15–20yrs of age, 77% male; 109/110 Burkitt histology (one Burkitt-like); 89% ≥2 LDH upper limits, 90% ≥ 70% L3 BM involvement. 44% were treated in COG, 38% SFOP and 18% in UKCCSG. The 4yr EFS and OS was 88±3.1% and 89±3.0%, respectively. Patients randomized to standard vs adjusted (decreased) intensity had a 4 yr EFS of 94±3.8% vs 88±4.8%, respectively (p=0.26). Overall, readjusted for randomized intensity of therapy, the 4 yr EFS was 91±3.1%. Response to reduction therapy with COP (cyclophosphamide, vincristine, prednisone) was assessed at day 7. Patients with a complete response (M1) vs incomplete response (20–99% reduction of BM L3 cells) had a similar outcome; (4yr EFS) CR vs IR (93±5 vs 88±2.9%) (p=0.51). Patients with a higher percentage of L3 blasts, (≥70% vs 25–69% bone marrow involvement), had an improved 4 yr EFS. 4 yr EFS was 90±3.0 vs 70±15% (p<0.06) and 4 yr OS 91±3.0 vs 70±15% (p<0.03). Elevated LDH ≥ 2NL at diagnosis had no impact on prognosis. Furthermore, there was a dismal outcome for patients who relapsed (10% 2 yr PFS). In summary, children and adolescents with isolated B-ALL have an excellent (>90% 4 yr EFS) prognosis with 6 months of standard intensity FAB/LMB therapy. Mature B-acute lymphoblastic leukemia (Burkitt) (B-ALL) should now be considered one of the most curable forms of childhood ALL. Furthermore, as therapy reduction has been unsuccessful in this patient group with B-ALL, future strategies should consider whether substitution of targeted immunotherapy might allow for chemotherapy intensity reduction (with reduced morbidity) in this population. The outcome is poor for the few patients who relapse and new therapeutic strategies are required for this small subgroup.
Tinzaparine in the Management of Painful Vaso-Occlusive Crisis of Sickle Cell Anaemia.
Authors: Qari Mohamad H. , Mousa Shaker , Alsaigh Mohamad A. , Zografos Panagiotis , Aljaouni Suad K. , Fatani Huda , Alalfi Adnan , Alsayes Fatin M. , Beirouti Basim T. , Alamin Mohamad A. , Gadi Abdulilah
Published: April 2020

Introduction

Several components of the haemostatic system are altered in sickle cell anaemia(SCA). The sum of these alterations is manifested as a state of hypercoagulability. Many therapeutic approaches including anticoagulants has been tried in managing the acute painful crisis of SCA along with the analgesics.

Aim

This trial aimed at assessing the efficacy and safety of low molecular weight heparin Tinzaparine (innohep®) as an adjuvant therapy in the management of acute painful vaso-occlusive crisis.

Material and Methods

This two arm pilot, randomised multi-centre controlled prospective trial has incorporated 253 patients admitted through emergency in acute painful crisis, with no other complications of SCA. Subjects were consecutively randomized to the study or control group. In the study group 127 patients (mean age 22,83 years) received tinzaparine 175 i.u, s.c once daily along with analgesics, while in the control group 126 patients (mean age 21,64 years) received placebo and the same analgesics. Subjects were followed for improvement using the numerical pain scale (NMS), assessing the number of days in which the patient experienced the severest degree of pain on the NMS, the hospitalization period, the duration of painful crisis, and the rate at which the pain intensity declined over time. The maximal treatment period was seven days. The complications encountered are treated and estimated in both groups. The results were analysed using “SSPS-V10” statistical tests

Results

The results displayed in the table.1 showed statistically significant reduction in the duration of morbidity in the study group. The drop in the pain intensity is sharper, and the complications experienced in the study group were two events of minor bleeding treated conservatively.

Patient Control No. of days with severest pain score on the NMS (Days) 1,28 1,74 Duration of painful crisis(Days) 2,57 4,35 Total duration of hospitalization(Days) 7.08 12,06

Conclusion

The results indicate a favourable outcome in the study group compared to the control group. The use of tinzaparine in the acute painful crisis of SCA as an adjuvant to the strong analgesics is recommended due to its proven efficacy and safety in shortening the period of the agonizing pain at presentation, the period of painful crisis and the hospital stay.
Early Predictors of Fetal Hemoglobin Response to Hydroxyurea in Sickle Cell Disease.
Authors: Partovi Kristine , Martyr Sabrina , McGowan Vicki , Machado Roberto , Taylor James , Kato Gregory , Gladwin Mark , Little Jane
Published: April 2020

We investigated the kinetics of hematologic change in patients with sickle cell disease (SCD, HbSS, n=6) or SC disease (HbSC, n=1) who had been newly started on hydroxyurea (HU), with the intention of identifying early correlates to fetal hemoglobin (HbF) responsiveness. We found that HbF increased in all patients on HU, and that the half-maximal degree of HbF response could be estimated by 2 months, in patients’ whose MCVs had risen ≥ 10% above baseline.

All 7 patients were treated with HU and followed closely for 6 months or more, until hematologic stability. Hematologic stability was apparent by ≥ 5 months. White blood cell count (WBC), absolute neutrophil count (ANC), reticulocyte (retic) count, % HbF, and mean corpuscular volume (MCV) were examined at bi-weekly intervals. Baseline values (1 or 2 values averaged) were compared with mean values obtained during weeks 2 to 8 (3 or 4 values averaged). As expected, by 2 months WBC and ANC had fallen 30 +/− 8% and 26 +/− 8%, respectively. Change in total hemoglobin (5.8 +/−6.7%), total platelet count (less 11 +/− 10.8%), and LDH (5.3 +/− 8.7%) was not consistent during this two month interval. By eight weeks after initiation of HU, retic counts had dropped in all six SS patients, from 15 to 52% less than baseline while MCV rose 9–21% above baseline; in general, rise in MCV preceded the rise in HbF. Overall, by the time of hematologic stability, all patients had increased their percent HbF, at between 3–8.5-fold relative to baseline; baseline percent HbF of total hemoglobin (Hgb) ranged from 0.7 to 8.3% and, after stabilization, from 5.2% to 24.9%. Maximal percentage of Hgb that was accounted for by HbF at stabilization was arbitrarily set at 100; at 8 weeks, all patients had achieved ≥ 42% of their maximal HbF level, mean 55 +/− 9.4% of maximum HbF. Two additional patients in whom extensive lab data were available, but who were suspected to be non-compliant or sub-therapeutically treated, had a >10% rise in MCV that was temporally associated with an inflection upward for HbF. Patient 8 had mean bi-weekly MCVs of 94, 91, 93, and, after a family conference, 102 (p=.003); Concurrent HbF was 7, 6, 6 and then 10 (p=.046). Patient 9 had mean bi-weekly MCVs on low-dose HU of 97, 96, and, after dose adjustment, 109(p=.003); HbF was 2%, 3%, and then 5 (p=.0094).

We speculate that, in many patients, an increase in MCV above baseline of ≥ 10% is a marker of adequate HU dosing, and that HbF levels at that time approximate half-maximal response. A larger series will be necessary to confirm this relationship; a predictive model, correlating MCV and HbF responsiveness, could be used to determine sufficiency of, and compliance to, HU therapy, and to early identify patients who are at high-risk from SCD (e.g. with pulmonary hypertension) whose HbF responsiveness may not be adequate from HU alone.

Figure Figure
Inactivation of the Suv39h1 Histone Methyltransferase Accelerates Myc-Driven B-Cell Lymphomagenesis and Compromises Drug-Inducible Cellular Senescence.
Authors: Lee Soyoung , Tabor Vedrana , Loddenkemper Christoph , Peters Antoine H.F.M. , Stein Harald , Dorken Bernd , Jenuwein Thomas , Schmitt Clemens A.
Published: April 2020

Activation of the myc oncogene is a frequent finding in human lymphomas. Acute induction of Myc in primary cells initiates massive apoptosis via the ARF/p53 pathway. In turn, apoptotic defects strongly collaborate with Myc in tumor formation. Previous work in a transgenic mouse model demonstrated that DNA damaging drugs can force apoptotically-compromised Myc-lymphomas to enter a terminal cell-cycle arrest, termed cellular senescence, which prolonged overall survival of the tumor-bearing animals. Recently, we identified the histone H3 lysine 9 (H3K9) methyltransferase Suv39h1 as a critical mediator of cellular senescence that acts as an early barrier against Ras-initiated lymphomagenesis, raising the question whether Myc-driven formation of lymphomas and their treatment sensitivity may depend on Suv39h1 function.

Eμ-myc transgenic mice were intercrossed to mice harboring targeted deletions in the Suv39h1 locus. Myc transgenic mice with no additional defined genetic lesions - hereafter referred to as controls - developed B-cell lymphomas with a median onset of more than 100 days, while mice lacking one or both Suv39h1 alleles produced lymphomas significantly earlier, i.e. at a median age of less than 60 days. Importantly, lymphomas arising in female myc transgenic Suv39h1+/− mice invariably lost expression of the X-linked Suv39h1 gene. TUNEL staining in situ and short-term cytotoxicity assays in response to the DNA damaging compound adriamycin in vitro expectedly demonstrated no overt difference in spontaneous or drug-inducible apoptosis in control versus Suv39h1-deficient Myc-lymphomas. However, when the fractions of Myc-driven lymphoma cells in cycle where assessed by Ki67 staining in situ, absence of Suv39h1 allowed Myc to keep nearly all cells in cycle, while a substantial fraction of non-apoptotic control lymphoma cells apparently had exited the cycle. Accordingly, adriamycin-treatment in vitro produced a strong senescent phenotype in control cells in the presence of Bcl2, while no senescence response could be provoked in Suv39h1-deficient cells.

Our findings provide novel insights into Myc-related deregulation of cellular growth control and senescence as a tumor suppressor mechanism, and have important ramifications for the therapeutic utilization of DNA-damage effector programs such as apoptosis and cellular senescence. Whether Suv39h1 inactivation impacts on the long-term outcome of cancer therapy in vivo is currently under investigation and will be reported at the meeting.