bluuebunny/crossref_metadata_2025 huggingface dataset:
Hematopoietic Stem Cells Fail to Trans-Differentiate into Smooth Muscle-Lineage Cells In Vivo.
Authors: Yokota Takafumi , Sato Sanai , Kawakami Yutaka , Nagai Yoshinori , Ma Jian-xing , Tsai Jen-Yue , Oritani Kenji , Tomiyama Yoshiaki , Matsumura Itaru , Kincade Paul W. , Kanakura Yuzuru
Published: April 2020

Since bone marrow has been shown to contain cells capable of differentiating into various lineages, it is attracting considerable attention as a potential source for tissue regeneration. The plasticity of hematopoietic stem cells (HSC), however, remains controversial. While recent studies have suggested that HSC give rise to other tissue types, others argue against HSC plasticity. Because any tissue injury results in extensive recruitment of inflammatory cells, evidence for stem cell plasticity with non-specific green fluorescent protein (GFP) transgenic mice is often confounded by the results of adhesion and/or cell fusion by macrophages. Some reports using non-specific GFP transgenic mice demonstrated that HSC could contribute to the generation of smooth muscle type α-actin (αSMA)-expressing cells in injured arteries or renal gromeruli. We developed a new transgenic αSMA-GFP mouse with GFP coupled to an αSMA regulatory sequence. Bone marrow transplantation was then used to evaluate the potential of marked cells to generate donor type tissues in irradiation chimeras. There was a highly restricted pattern of GFP expression in the transgenics, marking vascular smooth muscle cells, pericytes and mesangial cells in the kidney, but no hematopoietic cells while the marrow contained infrequent cells positive for αSMA-GFP (0.02% of mononuclear cells). When the marrow cells were cultured, stromal cells with high GFP expression were abundantly produced in two weeks. Similarly cultured glomeruli generated many mesangial cells with high GFP expression. These characteristics, however, were not transferable to lethally irradiated (950rad) RAG-2 deficient mice, even when 1 x !07 transgenic marrow cells per recipient were infused via tail vein and the hematopoietic system was largely replaced by recipient cells. At 6 months after transplantation, no GFP+ cells were observed in either bone marrow or renal glomeruli of 20 RAG-2 deficient recipients. Furthermore, cultivation of their marrow or glomerular cells did not produce αSMA-GFP expressing cells. Our findings support earlier studies suggesting that the bone marrow microenvironment is difficult to transplant by standard methods and indicate that HSC are unlikely to give rise to αSMA expressing progeny.
Caspase-3 Has Apoptotic and Non-Apoptotic Functions in Regulating the Fate of Hematopoietic Stem Cells.
Authors: Janzen Viktor , Fleming Heather , Reynolds Griffin , Waring Michael , Scadden David T.
Published: April 2020

The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Much emphasis has been placed on differentiation and proliferation in hematopoiesis, but little is known about control of the stem cell pool by mediators of apoptosis. We are particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitor, p21Cip1. p21 is known to regulate hematopoietic stem cell (HSC) cycling and we demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse HSC. Using mice engineered to be deficient in Caspase-3, we observed no difference from wild-type in blood counts. However, knock-out animals had reduced bone marrow cellularity and an increase in the stem cell enriched lin-kit+Sca-1+ CD34 low (LKS-CD34lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions were minimally different from controls. However, under conditions of stress (gamma irradiation, heat shock or cytokine withdrawal), Caspase-3 deficient LKS cells demonstrated reduced sensitivity to apoptotic cell death as measured by TUNEL or Annexin-V assay. Primitive hematopoietic cells from these animals in vivo also had reduced BrdU incorporation reflecting decreased proliferation and, upon transplantation, demonstrated marked differentiation abnormalities. Caspase-3−/− marrow cells had significantly reduced ability to differentiate into lymphoid and myeloid lineages 12 weeks after transplantation. Similarly, WT bone marrow cells treated prior to transplantation with a selective Caspase-3 inhibitor, z-DEVD.fmk, displayed a significant disadvantage in peripheral blood reconstitution, compared with a mock control. These data indicate that Caspase-3 is involved in the regulation of apoptosis, differentiation and proliferation of HSC. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.
The Membrane Proximal Portion of the Thrombopoietin Receptor (Mpl) Elicits Novel Pivotal Signals for Hematopoietic Stem Cell Self-Renewal.
Authors: Tong Wei , Lodish Harvey F.
Published: April 2020

Thrombopoietin (Tpo) and its receptor, mpl, are important for hematopoietic stem cell (HSC) self-renewal in addition to their roles in megakaryocyte and platelet development. In this study we dissected the signaling pathways that mpl transduces to support megakaryocytopoiesis as well as stem cell production and function using knockin mice bearing a truncated mpl receptor lacking the distal 60 amino acids (delta-60). This 60 amino acid deletion removes three major signaling tyrosines on the mpl cytoplasmic domain, but retains the membrane proximal Box 1 and Box 2 domains that are required for JAK2 activation. We show that delta-60 bone marrow (BM) cells cultured in Tpo generated normal numbers of megakaryocytes but with greatly reduced ploidy. Freshly-isolated delta-60 megakaryocytes showed marked reduction in most, if not all, known Tpo-stimulated signaling pathways, including Stat5, Stat3, Akt, and p42/44MAPK. Using competitive bone marrow transplantation (BMT) to analyze HSC activities and serial BMT to assess HSC self-renewal, we found that delta-60 mice displayed normal short-term (ST-HSC) activities and marginally compromised long-term (LT-HSC) stem cell activities in primary BM transplantation. In addition, delta-60 mice supported HSC self-renewal for at least two serial BMTs. Thus, our results reveal a pivotal role for the membrane proximal region of the mpl receptor in maintaining stem cell activity and self-renewal by eliciting novel intracellular signaling pathways.
In Vitro and In Vivo Induction of Human Hematopoietic Stem Cell Migration by Extracellular UTP.
Authors: Rossi Lara , Manfredini Rossella , Bertolini Francesco , Ferrari Davide , Fogli Miriam , Zini Roberta , Salati Simona , Ferrari Sergio , Di Virgilio Francesco , Baccarani Michele , Lemoli Roberto M.
Published: April 2020

Regulatory mechanisms governing homing and engraftment of hematopoietic stem cells (HSCs) involve a complex interplay between chemokines, cytokines, growth factors and adhesion molecules in the intricate architecture of bone marrow (BM) microenvironment. HSCs express P2Y and P2X receptors for extracellular nucleotides, which activation by ATP and UTP has been recently demonstrated (Lemoli et al. Blood. 2004) to produce potent stimulatory effects on HSCs. Moreover extracellular nucleotides are emerging as key factors of flogosis phenomena and related chemotactic responses of several cell types, such as dendritic cells, monocytes and endothelial cells. In this study we investigated the biologic activity of extracellular ATP and UTP and their capacity to cooperatively promote SDF-1 (stromal cell-derived factor-1)-stimulated cell chemotaxis. Low concentrations of UTP (10uM) significantly improved, in vitro, HSCs migration. Moreover, UTP inhibits CXCR4 down-regulation of migrating CD34+ cells and increased cell adhesion to fibronectin filaments. Furthermore, in vivo competitive repopulation assays showed that preincubation with UTP significantly improved the homing efficiency of human CD34+ HSCs in nonobese diabetic/severe combined immunodeficient mice.

Inhibition assays with Pertussis Toxin from B. Pertussis blocked SDF-1- and UTP-dependent chemotactic responses, suggesting that Gαi proteins may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP-treated CD34+ cells and subsequent in vitro inhibition assays with Toxin B from C. Difficile suggest that RhoGTPase Rac2 and his downstream effectors ROCK1 and ROCK2 are involved in the UTP-promoted, SDF-1-dependent HSCs migration.

Taken together, our data suggest that UTP may physiologically modulate HSC migration and homing to the BM, in concert with the chemotactic peptide SDF-1, via the activation of converging signaling transduction pathways between CXCR4 and P2Y receptors, involving Gαi proteins and RhoGTPases.
Histone H3 Lysine 9 Methylation and HP1γ Are Associated with Transcription Elongation through Mammalian Chromatin.
Authors: Vakoc Christopher R. , Mandat Sean A. , Olenschock Ben A. , Blobel Gerd A.
Published: April 2020

Epigenetic regulation of gene expression plays a fundamental role during tissue specification and cellular memory. Cells that are committed to a given lineage, for example during hematopoiesis, “remember” their phenotype throughout successive rounds of cell division, reflecting alterations in chromatin structure at genes that are permanently activated or silenced. Cellular memory is anchored in specific sets of histone modifications, which together form the basis for the histone code. This is illustrated in the methylation of histone molecules: while methylation of histone H3 on lysines 4, 36, and 79 is linked with gene activation, methylation of H3 on lysines 9 and 27 and histone H4 on lysine 20 is associated with transcriptionally silent heterochromatin and repressed genes within euchromatin. Not surprisingly, dysregulation of histone methylation contributes to human diseases such as leukemias. Here we examined the methylation of histone molecules during gene activation and repression triggered by the hematopoietic transcription factor GATA-1. Surprisingly, we found that during activation by GATA-1 in erythroid cells, the levels of H3K9 di- and tri-methylation increase dramatically at all examined GATA-1-stimulated genes, including alpha- and beta-globin, AHSP, Band 3 and Glycophorin A. In contrast, at all GATA-1-repressed genes examined (GATA-2, c-kit, and c-myc) these marks are rapidly lost. Peaks of H3K9 methylation were observed in the transcribed portion of genes with lower signals at the promoter regions. Heterochromatin Protein 1γ (HP1γ), a protein containing a chromo-domain that recognizes H3K9 methylation, is also present in the transcribed region of all active genes examined. We extended these analyses to include numerous genes in diverse cell types (primary erythroid cells, primary T-lymphoid cells, epithelial cells and fibroblast) and consistently found a tight correlation between H3K9 methylation and gene activity, highlighting the general nature of our findings. Both the presence of HP1γ and H3K9 methylation at active genes are dependent upon transcription elongation by RNA polymerase II. Finally, HP1γ is in a physical complex with the elongating form of RNA polymerase II. Together, our results show that H3K9 methylation and HP1γ not only function in repressive chromatin, but play a novel and unexpected role during transcription activation. These results further elucidate new combinations of histone modifications that distinguish between repressed and actively transcribing chromatin.
Maturation Stage-Specific Regulation of Megakaryocytic Genes by Pointed-Domain Ets Proteins.
Authors: Pang Liyan , Wang Xun , Wang Yuhuan , Blobel Gerd , Poncz Mortimer
Published: April 2020

The pointed-domain Ets transcription factor Fli-1 has a critical role during megakaryocyte-specific gene expression. Previously, we demonstrated that Fli-1 occupies the early megakaryocyte-specific gene αIIb in vivo. Moreover, our work suggested a mechanism for Fli-1 function by showing that Fli-1 facilitates GATA-1/FOG-1 dependent expression of the αIIb gene. However, studies by others with a targeted disruption of the Fli-1 gene in mice showed that while Fli-1 is essential for normal megakaryocyte maturation, αIIb mRNA levels were not significantly reduced in the resulting megakaryocytes, suggesting that a related Ets factor(s) might compensate for the loss of Fli-1. Here we show that the widely expressed pointed domain Ets protein GABPα specifically binds in vitro to Ets elements from two early megakaryocyte-specific genes, αIIb and c-mpl. Chromatin immunoprecipitation (ChIP) experiments using primary murine fetal liver-derived megakaryocytes reveal that GABPα associates with αIIb and c-mpl in vivo. Moreover, GABPα is capable of mediating GATA-1/FOG-1 synergy in the context of αIIb promoter constructs. These results suggest that GABPα contributes to megakaryocyte-restricted gene expression and is capable of at least partially compensating for the loss of Fli-1. However, loss of Fli-1 leads to a pronounced decrease in the expression of the late megakaryocyte-specific gene GPIX, indicating that compensation by GABPα is incomplete. Consistent with this observation, ChIP experiments fail to detect significant levels of GABPα at the regulatory region of GPIX while Fli-1 is readily detected there. Together, these results point to a model in which Fli-1 and GABPα serve overlapping, but distinct roles, during the development of megakaryocytes. GABPα may be important during early megakaryopoiesis, but Fli-1 exerting an essential role during late stages of maturation.
Sequential Analysis of the α- and β-Globin Gene Expressions during Erythropoietic Differentiation from Primate ES Cells.
Authors: Katsutsugu Umeda , Heike Toshio , Shinoda Gen , Arai Masato , Ma Feng , Suemori Hirofumi , Luo Hong Yuan , Chui Devid H.K. , Torii Ryuzo , Shibuya Masabumi , Nakatsuji Norio , Nakahata Tatsutoshi
Published: April 2020

Globin switching of erythrocytes during primate (human and monkey) hematopoietic development has been most thoroughly investigated not only as a model of tissue- and temporally specific transcriptional control but also as a tool for drug discovery against hemoglobinopathies. However, the regulatory mechanisms of globin switching in primates remains to be unresolved in primates mainly due to lack of available model system to date which reproduces the process of hematopoiesis to reflect accurately in vivo development. Recently primate ES cell lines were established, which are expected to serve as an experimental model for tissue growth and development, along with an efficacy and toxicity screening system for new drugs and a cell source for regeneration therapy. Among them, we previously demonstrated that the transition from primitive into definitive erythropoiesis was induced from primate ES cells by coculture with OP9 stromal cells. We also demonstrated that the VEGFR-2high CD34+ cells, emerging onto OP9 stromal layer after initial 6-day differentiation, contain the hemogenic progenitors.

In this study, we quantitatively analyzed temporal pattern of embryonic, fetal, and adult globin expression in the α (ζ to α), and β (ε to γ and γ to β) cluster both at the transcriptional and translational level in erythrocytes induced from primate ES cells by the coculture system. When VEGFR-2high CD34+ cells were harvested and reseeded onto OP9 stromal cells in the presence of EPO, IL-3, SCF, and TPO, two waves of erythropoiesis occurred sequentially thereafter. Immunostaining and real-time reverse transcription-polymerase chain reaction analyses of floating mature erythrocytes revealed that the globin switches occurred in parallel with the erythropoietic transition. However, the temporal patterns of switching were asynchronous in the α- and β-globin genes; sudden fall of ζ-globin expression coincides increase of β-globin expression, while ε-globin expression gradually decreased and were still detected in many erythrocytes. Colony-forming assays demonstrated that the replacement of primitive clonogenic progenitor cells by definitive ones occurred during culture. The decline in embryonic ζ- and ε-globin expressions at the translational level occurred in a single definitive erythroid progenitor. The β-globin expression in individual definitive erythroid progenitors is up-regulated in the presence of OP9 stromal cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of globin switch in human.
GATA-1, but Not GATA-2, Antagonizes PU.1 Mediated Transcriptional Activity at the CBFA2T3 (ETO2, MTG16) Promoter through a Mechanism Dependent on GATA DNA Binding.
Authors: Saunthararajah Yogenthiran , Yang SiJun , Kadkol ShriHari , Baraoidan Marie , Ibanez Vinzon , Mortazavi Fariborz
Published: April 2020

CBFA2T3 (ETO2, MTG16), a target of chromosomal translocation in acute myeloid leukemia, has its highest expression in hematopoietic cells compared to other tissues. This suggests that its expression is regulated by major hematopoietic transcription factors. The proximal promoter from −171 to −65 bp has greater than 90% identity between mouse and human and contains recognition sites for major hematopoietic transcription factors PU.1, GATA-1 and GATA-2. Using chromatin immuno-precipitation and the MPD hematopoietic cell-line, this segment was pulled down with endogenous PU.1, GATA-1 and GATA-2. In luciferase reporter gene assays, PU.1 and GATA-2, but not GATA-1, activated the promoter. As would be expected from these findings, CBFA2T3 levels declined during terminal erythroid differentiation of primary hematopoietic cells. GATA-1, but not GATA-2, antagonized PU.1 mediated activation but this effect of GATA-1 was abrogated by mutation of the GATA DNA binding sites. Both GATA-1 and GATA-2 have been reported to antagonize PU.1 transcriptional activity by antagonizing PU.1 interactions with c-Jun (Zhang et al, Proc Natl Acad Sci USA1999;96:8705–8710); however, the DNA binding dependent mechanism reported here allows GATA-2 and GATA-1 to have contrasting relationships with PU.1 and may be the basis for the co-operation of GATA proteins with PU.1 in some contexts yet antagonism of PU.1 activity in others.
Transcriptome Analysis of Human Hematopoietic Progenitor Cells during In Vitro Erythroid Differentiation.
Authors: Keller Margaret A. , Addya Sankar , Vadigepalli Rajanikanth , Ponte Christine M. , Banini Bubu , Delgrosso Kathleen , Surrey Saul
Published: April 2020

The hematopoietic progenitor cell (HPC) has the potential to differentiate into multiple cell types. Deciphering transcriptional regulatory networks necessary for HPCs to undergo lineage commitment is critical. Toward this goal, we established a serum-free in vitro differentiation system for human HPCs using CD34−positive cells isolated from peripheral blood of normal donors. Cells were cultured in the presence of Epo, SCF and IL-3 for two-weeks, and assessed for total hemoglobin and percent fetal hemoglobin. Adult-derived HPCs express large amounts of Hb A after approximately one week of culture with little expression of Hb F. Transcriptome analysis, using Affymetrix Human Genome U133 Plus 2.0 Arrays on RNA from days 1, 3, 5, 7, 9, 11 and 13 of culture shows more than 1500 differentially-expressed (DE)(> 2-fold up or down) genes, a subset of which were validated independently by real-time RT-PCR. Up-regulated erythroid-specific genes included those for blood group antigens (Kell, Duffy, Rhesus), membrane proteins (ankryin 1, α- and β-spectrin, Band 4.1, Band 4.2, Band 4.9, glycophorin A), receptors (transferrin and erythropoietin receptors), heme biosynthesis enzymes (ALAS, ALAD, UROS, UROD, CPOX, PPOX, FECH), and transcription factors important in globin-gene expression (EKLF, BKLF, NFE2, GATA1) as well as α-, β- and γ-globin mRNAs. Down-regulated genes included those expressed in hematopoietic stem cells (GATA2, CD34) and non-erythroid hematopoietic lineages (MHC class I and II genes, CD33, CD53). The DE genes were clustered into a limited number of expression patterns, suggesting coordinate regulation of genes within each cluster. The DE gene set was examined in silico for co-regulation using transcriptional regulatory network analysis employing PAINT v3.2 software (Vadigepalli R et al., OMICS.2003; 7(3):235–52). GATA binding sites were represented on 149 of the DE gene promoters. Several transcriptional regulatory elements (TRE) were statistically significantly enriched in the DE gene set when compared to the array gene set. These include TRE for AP-1, CREB, GATA-1 and Oct-1. Co-regulation of genes in the different gene clusters will be analyzed by PAINT and findings validated by examination of TRE:transcription factor interactions. These studies should help define timing of transcription factor occupancy in relation to epigenetic changes occurring prior to transcriptome alteration during differentiation of human erythroid cells.
Pulmonary Mortality after High-Dose Oral Busulfan and Autologous Stem Cell Transplant (ASCT) in Patients with Non-Hodgkin Lymphoma (NHL).
Authors: Kalaycio Matt , Pohlman Brad , Rybicki Lisa , Sobecks Ronald , Kuczkowski Elizabeth , Andresen Steven , Brown Stacey , Bolwell Brian
Published: April 2020

Many high-dose chemotherapy preparative regimens include agents known to cause pulmonary toxicity such as BCNU. Chemotherapy induced pulmonary toxicity may be fatal. From 1/1/93 to 12/31/04 we treated 533 NHL patients with oral busulfan 14mg/kg, VP-16 60 mg/kg, and cyclophosphamide 120 mg/kg followed by ASCT. Busulfan levels were not measured. We have followed these patients for toxicity, survival, and cause of death (COD). Of these 533 patients, 214 have died, and 53 have died without relapse (15%). Most patients (n=329; 62%) had intermediate grade NHL by the IWF system. The median age was 50 years (range, 16–77) and 43% had a history of smoking. Radiotherapy was delivered to 27% and less than 3 chemotherapy regimens were administered to 75% of patients before ASCT. Sixteen patients (3%) died of non-relapse pulmonary complications. The median time to pulmonary mortality was 5 months (range, 2.5 – 21.4 months). We routinely screen patients with pulmonary function tests including DLCO before ASCT and most patients (n=490; 92%) were screened at a median of 45 days after ASCT (range, 7–90 days). For patients for whom data was available, there was no significant decrease in the median pre-ASCT and the post-ASCT DLCO in the either the whole cohort of patients or the patients dying of pulmonary complications. However, a lower baseline DLCO before ASCT predicted for pulmonary mortality after ASCT in univariable and multivariable analysis. Patients with a baseline DLCO <94% predicted, had an increased risk of pulmonary mortality compared to patients with a baseline DLCO ≥94% predicted (p=0.016). Other significant risk factors included older age, lower baseline FEV1, prior radiotherapy, and longer time from diagnosis to ASCT. A history of smoking and disease status at the time of ASCT did not predict for pulmonary mortality. Because patients died of pulmonary complications at other institutions, we could neither determine treatment, nor confirm chemotherapy induced pulmonary toxicity. However, the early onset of pulmonary mortality after ASCT suggests the possibility of a toxic insult. Busulfan is a known pulmonary toxin, but the dose in this series of patients is relatively low and we have not seen similar pulmonary complications in 95 patients (median age 52 years) with myeloma treated with the combination of busulfan 16 mg/kg and cyclophosphamide 120mg/kg. Nonetheless, caution is warranted for older patients being treated with high-dose oral busulfan.
Does Highly Active Anti-Retroviral Therapy (HAART) Delay Myeloid Engraftment after Autologous Stem Cell Transplant (ASCT) in HIV Associated Lymphoma (HIV+Ly) Patients?.
Authors: Diez-Martin Jose L. , Carrion Rafael , Anguita Javier , Balsalobre Pascual , Miralles Pilar , Berenguer Juan , Buno Ismael , Gomez-Pineda Alfonso , Conde Eulogio , Varela Rosario , Ribera Jose M. , Espigado Ildefonso , Escoda Lourdes , Loscertales Javier , Serrano David
Published: April 2020

HAART has improved the outcome of AIDS patients (pts). Thus high-dose chemotherapy followed by ASCT has been used for the treatment of HIV+Ly pts, similarly to HIV-neg-Ly pts. In a recent report we found delayed engraftment after ASCT in HIV+Ly compared with HIV-neg Ly pts (Exp.Hem33;487–494,2005). To test the role played by HAART on engrafment we conducted this study, comparing engraftment results on HIV+Ly pts that received HAART with those in wich HAART was withdrawn after ASCT. Patients and Methods: From June 2000 a total of 19 HIV+Ly pts (17 male) received an ASCT in a multicenter cooperative study; 5 were Hodgkin disease (HD) and 14 non-Hodgkin high-grade malignant lymphoma (NHL). Median age was 43 years (range, 31–61); adjusted IPI (aIPI) was: 0–1 in ten pts and 2–3 in nine more pts; advanced Ann Arbor stage (III–IV) was present in 14 pts.Ten pts were treated with HAART during ASCT (HAART+) and in nine pts HAART was withdrawn due to gastrointestinal toxicity (HAART neg). Both groups were similar for the most relevant clinical characteristics (p value Mann-Whitney test or chi-square test > 0.05): HD/NHL, Ann Arbor stage, aIPI, status at ASCT (CR-1/more than CR-1), age, number of lines of chemotherapy before ASCT, number of cycles of mobilization to obtain an adequate amount of CD34+ cells, number of CD34+ cells infused, day of start of GCSF and duration of GCSF-treatment. Results: Median time to reach PMN>0.5 x 109/L was 12 days (9–17) for HAART neg group and 18 days (9–33) for HAART+ pts (p=0.069). Platelet engraftment (>20x109/L) was achieved after a median of 20 days (11–28) and 26 days (11–455) respectively (p=0.25). Nevertheless, a multivariate regression analysis using as covariates HAART, GCSF (starting day) and amount of CD34+ cells infused, was performed. Accordingly no statisticaly significant results were found to relate with myeloid engraftment. Conclusions: Despite the HAART+ group shows longer interval to reach PMN and platelets engraftment following ASCT, we can not definitively conclude that HAART plays a negative influence on engraftment in this clinical setting. In order to clarify these results further recruitment of patients is needed.
Serial Analysis of Whole Blood Cell and CD3+ T-Lymphocyte Chimerism Following Allogeneic Stem Cell Transplantation with Alemtuzumab Containing Reduced-Intensity-Conditioning (RIC).
Authors: Tauro S. , Lovell R. , Begum G. , McMullan D. J. , Griffiths M. J. , Clark F. J. , Cook M. A. , Mahendra P. , Milligan D. W. , Craddock C. F.
Published: April 2020

Serial analysis of haemopoietic chimerism can be used to predict outcome after allogeneic SCT using a RIC regimen and guide post-transplant intervention. Although alemtuzumab is increasingly used as a component of RIC regimens, there have been few studies of its impact on chimerism status post-transplant. We have therefore measured chimerism following allogeneic SCT in whole blood/marrow nucleated cells (WB) and magnetically selected CD3+ T-lymphocytes in 64 patients with lymphoid (8 high-grade NHL, 18 low-grade NHL, 5 myeloma, 3 mantle cell lymphoma, 9 Hodgkin’s lymphoma) or myeloid malignancy (14 AML, 3 MDS, 1 myelofibrosis, 3 CML) conditioned with alemtuzumab and either fludarabine with melphalan (n=46), BEAM (carmustine, etoposide, cytarabine and melphalan) (n=16) or fludarabine/busulphan (n=2). Forty-seven patients received a transplant from an HLA compatible sibling donor and 17 from a matched unrelated donor. All patients achieved neutrophil and platelet engraftment. Donor chimerism was quantified within the first year post-allograft as well as following donor lymphocyte infusions (DLI) by FISH or PCR-based analysis of polymorphic microsatellite regions. 85% of patients demonstrated full donor chimerism (FDC, defined as ≥ 95% cells of donor origin) in WB within the first 90 days post-transplant. By contrast FDC was only present in the CD3+ compartment of 45% of patients. The proportion of patients with WB FDC declined to 64% by 12 months post transplant whilst the proportion of patients with CD3+ FDC remained constant. Thirteen patients received DLI using escalating CD3+ doses for management of mixed chimerism (MC) including 6 with evidence of disease relapse. Following DLI, 7 patients achieved FDC in WB and CD3+ compartments and 4 failed to switch to FDC. Seven patients developed acute GVHD post-DLI. Acquisition of FDC in the CD3+ compartment within 90 days post-transplant correlated with the presence of acute GVHD (p=0.003). Sixteen patients relapsed of whom 13 exhibited MC in WB or CD3+ compartments. Three patients relapsed despite the presence of FDC in WB and CD3+ cells. There was a trend towards improved disease free survival in patients who achieved FDC in WB within 90 days of transplantation compared to patients with MC (median 30 months v 11 months respectively). These data define a different pattern of WB and CD3+chimerism after alemtuzumab regimens compared with T-replete RIC regimens and confirm a correlation between chimerism status and outcome post-transplant.
Secondary Long-Term Prophylaxis in Severe Patients with von Willebrand’s Disease: An Italian Cohort Study.
Authors: Federici Augusto B. , Gianniello Francesca , Canciani Maria T. , Mannucci Pier M.
Published: April 2020

Background and Objectives. Patients with severe forms of von Willebrand’s disease (VWD) may have frequent episodes of mucocutaneous bleeding and also of hemarthrosis or hematomas. However, little retrospective or prospective data on secondary long term prophylaxis in VWD are available. Aim of this study was to evaluate the efficacy and safety of fixed regimens of prophylaxis with factor VIII/VWF concentrates in our cohort of VWD patients with recurrent joint and gastrointestinal (GI) bleeds. Design and Methods. This is a cohort study on 452 VWD patients regularly followed up at our Center for at least three years. 89/452 cases (20%) were treated with FVIII/VWF concentrates during the last two years because of one or more bleedings and 11/89 (12%) were included in a long term prophylaxis program because of frequent recurrence of bleeds at the same sites. All patients were characterized by a bleeding severity score derived from a detailed history of 11 symptoms. Since concentrates available in Italy are still labelled in FVIII IU, patients were given 40 FVIII IU/Kg of high- (Alphanate, Fanhdi) or intermediate-purity (Haemate-P) concentrates, two times a week (joint bleeds) or every other day (GI bleeds) to maintain FVIII/VWF levels higher than baseline during prophylaxis. Effectiveness of prophylaxis was based on resolution/reduction of bleeding as well as on numbers of transfused packed red blood cells (PRBC) and days of hospitalization. Safety was measured by monitoring side effects and FVIII levels before and after every injection during the first three weeks of prophylaxis. Results. All the 11 patients were severe, as shown by high bleeding scores (> 20) and VWF:RCo baseline levels <10 U/dL. Prophylaxis was started because of GI bleeds in 7 patients with VWD type 3 (n=1), 2A (n=4), 2M (n=1) and 1 (n=1) and for joint bleeds only in VWD type 3 (n= 4). Prophylaxis could stop bleeding in 8 patients and largely reduced hospitalization for PRBC transfusions in the remaining 3. When prophylaxis was compared with previous on demand regimen in all 11 cases, the annual total FVIII IU (x 1000) of concentrate (a) as well as number of PRBC used (b) and days of hospitalization (c) were significantly reduced (mean ± SD, with * p < 0.01): a * = 239±207 versus 385±247; b* = 9±11 versus 3±4; c* = 18±13 versus 4±3. As far as safety, FVIII levels were always <180 U/dL in all VWD and no side effects, including thrombosis, were observed. Interpretations and Conclusions. Secondary long-term prophylaxis by high-and intermediate purity FVIII/VWF concentrates is effective and safe in severe forms of VWD. Cost-effectiveness of these prophylaxis regimens versus on demand therapy should be further investigated in large prospective studies.
Antigen Levels of Thrombospondin-1 and ADAMTS13 Are Reduced in Parallel in TTP.
Authors: Liu Fang , Feys Hendrik B. , Dong Ningzheng , Bai Xia , Vanhoorelbeke Karen , Hans Deckmyn , Ruan Changgeng
Published: April 2020

Background Thrombospondin-1 (TSP-1) is considered to control the von Willebrand factor (VWF) multimer size by reducing interchain disulfide bridges, whereas ADAMTS13 is the major VWF cleaving protease. However the exact contribution of each of these to VWF size control is not known. In this study we investigate the relationship between ADAMTS13 antigen levels and TSP-1 levels in diseased plasmas.

Methods Antigen levels of TSP-1 and ADAMTS13 were determined in 21 plasma samples derived from 11 patients with TTP, and compared to normal control, 20 ITP samples and 20 SLE samples. TSP-1 antigen was measured by a commercial kit (Cytimmune, Rockville, MD), which can detect both bound and free TSP-1 in plasma. ADAMTS13 antigen was measured in a newly developed sandwich ELISA using 3 monoclonal antibodies to ADAMTS13. All donors were Chinese.

Results The average TSP-1 antigen level in normal plasma was 18.34±7.24 μ g/ml (mean ± SD, n=30) and the mean ADAMTS13 level in Chinese normals (CN) was 601±145 mU/ml (n=26) compared to the level set at 1 U/ml in pooled (n=20) normal human plasma from Caucasian origin. In TTP plasma, before plasma exchange (pre-PE), 5.79±2.35 μg/ml TSP-1 was detected (n=11, p<0.01 versus CN), which upon PE increased to 9.06±6.52 μ g/ml (n=10, p>0.05 versus pre-PE). The mean antigen level of ADAMTS13 was 98.7±82.8 mU/ml (n=11, p<0.01, versus CN) and 449±232 mU/ml (n=10, p<0.05 versus pre-PE), before and after PE, respectively. Furthermore TSP-1 antigen levels and ADAMTS13 titres in these patients plasma correlated linearly with an R=0.7 (n=21). To determine whether this significant decrease in both TSP-1 and ADAMTS13 antigen levels is restricted to TTP, plasma from ITP and SLE patients was analyzed next, however no significant decrease of TSP-1 was found. In contrast, the average antigen level of ADAMTS13 was raised to 830±229 mU/ml (n=20, p<0.01 versus CN) and 721±192 mU/ml (n=20, p<0.05 versus CN), for ITP and SLE plasmas, respectively.

Conclusion Both ADAMTS13 and TSP-1 antigen levels are significantly reduced in TTP plasma and hence the combined effect may be responsible for the presence of ultra-long VWF multimers in TTP plasma. Interestingly ADAMTS13 antigen level appears to be increased in SLE/or ITP parients, in contrast to previous reports that showed reduced activity, whereas no changes in TSP-1-levels were found in those patients.
Pharmacokinetics and Dose Linearity of von Willebrand Factor /Factor VIII Concentrate (Humate-P®) in Elective Surgery in von Willebrand Disease Patients.
Authors: DiPaola J. , Gill J. , Bernstein J. , Leissinger C. , Valentino L. , Friedman C. , Knaub S.
Published: April 2020

The VWF/FVIII concentrate Humate-P® (ZLB Behring) was studied in a prospective, uncontrolled, open-label clinical trial in VWD patients undergoing elective surgery. An interim analysis was performed after 24 patients (18 female, 6 male) with the following types of VWD had been included into the study: type 1 (n=11), type 2A (n=2), type 2B (n=4), type 2M (n=2) and type 3 (n=5). All patients underwent the pre-operative PK assessment to individualize the loading and maintenance doses of Humate-P® and 18 patients had completed surgery. The primary objective of the study - to demonstrate safety and hemostatic efficacy - was achieved in 100% of the patients. All patients received at least two infusions: a single infusion of 60 IU VWF:RCo per kg b.w. for a pharmacokinetic (PK) assessment prior to surgery and a PK guided loading dose before start of surgery. In addition, all patients received more infusions for further treatment after surgery. PK results: VWF:RCo: The overall median IVR was 2.1 [% per IU/kg] (range 1.1–4.2). The overall median response was 75.3% (range 37.0–169.4). Type 3 VWD patients had a slightly lower median response of 73.4% (range 63.7–96.1). Median value of Cmax was 142 IU/dL (range 84–330). Median effective half-life was 6.7 h (range 0.2–74.9), median clearance 3.6 [mL/kg/h] (range 1.0–16.6). FVIII:C: The median IVR was 2.8 [% per IU/kg] (range 1.4–4.9). Type 3 VWD patients had a median IVR of 3.4 [% per IU/kg] (range 1.7–4.9). The median response was 96.4% (range 63.0–164.8). Type 3 VWD patients had a median response of 104.5% (range 63.0–155.3). Dose-linearity: Graphical presentation of the relation between IVR and dose administered demonstrated the dose linearity of the VWF concentrate within a wide dose range.

IVR of VWF:RCo versus the amount of VWF:RCo administered (all doses) IVR of VWF:RCo versus the amount of VWF:RCo administered (all doses)

Statistical modeling using all doses (loading dose and maintenance infusions) supported this finding. Using the log-transformed IVR plotted against the dose administered the slope was 0.0004 (95% confidence interval: −0.0045 to 0.0053. Conclusion: The PK results are comparable to PK data from other clinical studies with Humate-P®/Haemate P® and given the interindividual variability underline the importance of pharmacokinetic studies prior to surgical interventions. Dose linearity of VWF:RCo with Humate-P® has been shown over a wide range of dosages (10–135 IU/kg b.w.). These results increase our confidence in the treatment of VWD patients with Humate-P®.
Tacrolimus/Methotrexate (MTX) Versus Cyclosprorine/MTX for Graft-Versus-Host Disease (GVHD) Prophylaxis in Patients with Severe Aplastic Anemia (SAA) Who Received Marrow Transplantation from Unrelated Donors: Results of a Matched Pair Analysis.
Authors: Kojima Seiji , Yabe Hiromasa , Kato Koji , Kigasawa Hisato , Sakamaki Hisashi , Tsuchida Masahiro , Kato Shunichi , Morishima Yasuo , Kodera Yoshihisa
Published: April 2020

Acute and chronic GVHD contribute to much of the morbidity and mortality associated with unrelated bone marrow transplantation (UBMT). Effective prevention of these complications is crucial for the success of UBMT. Cyclosporine(CSA) was introduced for the prophylaxis of GVHD and has been shown to be effective; similar findings have been shown for tacrolimus. However, there are no comparative studies between tacrolimus/MTX and CSA/MTX in patients with SAA who undergo UBMT. We compared tacrolimus/MTX with CSA/MTX in patients with SAA who received UBMT using a matched-pair analysis. Patients with acquired SAA who received UBMT between July 1997 and December 2002 through the Japan Marrow Donor Program were eligible for analysis. Recipients of UBMT were divided into two cohorts for comparison: those who received tacrolimus/MTX and those who received CSA/MTX. Because our previous studies identified that recipient age and conditioning regimen are the most important variables associated with treatment failure, we selected pairs of patients treated with either tacrolimus/MTX or CSA/MTX by matching these variables. We were able to identify 47 pairs that could be matched exactly for the recipient age and conditioning regimens. There was an imbalance of HLA-A, -B, and -DRB1 antigen mismatches, with 18 mismatched pairs in the tacrolimus group and 7 in the CSA group (p=0.005). Other variables were comparable between the two groups. Engraftment took place in 46 patients (98%) in the tacrolimus group and 43 patients (92%) in the CSA group. The median time to neutrophil recovery was the same in the two groups (tacrolimus group: 18.0 days, CSA group: 17.5 days). The cumulative incidence of grade 2 to 4 acute GVHD was 28.9% in the tacrolimus group and 32.6% in the CSA group (p=0.56). The estimate of chronic GVHD occurrence was 13.3% for the tacrolimus group and 36.0% for the CSA group (p=0.10). 8 patients died of transplantation related toxicities in the tacrolimus group and 22 patients in the CSA group (p=0.002). There was a higher frequency of deaths from thrombomicroangiopathy in the CSA group than in the tacrolimus group (6 vs 0: p=0.03). The 4-year survival rate was 82% for patients in the tacrolimus group and 52% for patients in the CSA group (p=0.001). Although a disadvantage existed in terms of HLA disparity in the tacrolimus group, there was no difference in the incidence of acute and chronic GVHD between the two groups. The present study shows the superiority of tacrolimus/MTX over CSA/MTX in overall survival because of lower incidence of regimen-related toxic deaths.
Revised Response Criteria for Malignant Lymphomas From the Members of the International Harmonization Project (IHP) of the Competence Network Malignant Lymphoma, Represented by:.
Authors: Cheson Bruce D. , Pfistner Beate , Juweid Malik E. , Specht Lena , Rosen Steven T. , Gascoyne Randy , Stroobants Sigrid , Diehl Volker
Published: April 2020

Standardized response criteria are essential for interpretation of clinical data, comparisons among clinical trials, development of new therapies, and approval of agents by regulatory agencies. In 1999, an International Working Group (IWG) developed recommendations for response assessment for non-Hodgkin’s lymphomas (NHL) that were adopted internationally by study groups and regulatory agencies and, subsequently, by clinical trials groups for Hodgkin lymphoma (HL) as well (Cheson et al, J Clin Oncol, 17:1244, 1999). Since their publication, several observations compelled a reassessment and, ultimately, revision of those guidelines, e.g., the availability of FDG-PET scans, new insights into lymphoma pathology and biology, the failure of the IWG guidelines to include HL and extranodal NHL, and features of the original guidelines that were found to be unclear as they were implemented into clinical trials. Most notable of these was the interpretation of the response category of Complete Remission unconfirmed (CRu). In the context of the IHP, a group of international lymphoma investigators with expertise in medical hematology/oncology, radiation oncology, nuclear medicine and imaging, pathology, biostatistics, and pediatrics were convened to revise the IWG guidelines. Committees focused on Response Criteria, Pathology/Biology, Endpoints, and Clinical Features, and discussions were initiated to undertake a major revision of the IWG guidelines. The important modifications that will be presented included, but were not limited to, integration of PET according to recent data (Juweid et al, J Clin Oncol, 23:4652, 2005), to facilitate the distinction between persistent tumor and scar/fibrosis, virtually eliminate the designation of CRu, and improve prediction of outcome. Guidelines were provided for the specific indications where this test can be currently recommended. Other proposals involved the role of flow cytometry and assessment of minimal residual disease. Response criteria for extranodal sites were also incorporated. Adoption of these revised guidelines by study groups will further improve the conduct and interpretation of clinical trials leading to more effective therapies for patients with lymphomas.
Acute GVHD May Play a Role in Preventing Relapse Following Reduced Intensity Allografts for Acute Myeloid Leukemia.
Authors: Andritsos Leslie , Augustin Kristan , Vij Ravi , DiPersio John F. , Devine Steven M.
Published: April 2020

The role of reduced intensity allografting in the treatment of patients (pts) with acute myeloid leukemia (AML) and the role that graft versus host disease (GVHD) plays in preventing relapse in this setting is unclear. While more pts may survive the initial period following allografting in comparison to those receiving full intensity transplants, the risk of relapse may be greater, particularly in those not in remission at the time of transplantation. Here we report the results of allogeneic transplants following a reduced intensity conditioning (RIC) regimen given to 158 consecutive pts with AML. All were all conditioned with an identical regimen containing Cyclophosphamide at 120mg/kg iv over 2 days (days −3 and −2) followed by a single dose of total body irradiation at 550cGy on day −1. HLA-identical (HLA-ID) siblings donated cytokine mobilized peripheral blood (PB) to 68 recipients and unrelated donor (UD) bone marrow (BM) was used for 90 patients. GVHD prophylaxis consisted of single agent cyclosporine (CSP) for recipients of HLA-ID allografts and CSP, methotrexate, and methylprednisolone in recipients of UD BM. The median age of recipients was 44 years (range 6–70), with 74 males and 84 females. 43 pts were in CR1, 47 in CR2 or 3, and 68 had relapsed or refractory disease. The majority of UD BM recipients were serologically identical with their donors at 6/6 antigens. There were no graft failures in either group.The median follow-up in survivors was long at 1903 days (range 938–2757). Overall survival (OS) was evaluated in relation to the development of any grade of acute GVHD. There were no significant differences in the incidence of grade 2–4 (30+/− 6%) or grade 3–4 (7+/−3%) acute GVHD in the HLA-ID PB or UD BM groups. Since OS was not significantly different between CR1 and CR2 pts, the results were pooled. The Kaplan-Meier (KM) estimate of 3 year OS in UD BM pts in CR was 45+/−9% in those with no acute GVHD compared to only 25+/−10% with any grade of acute GVHD (p=0.24). In recipients of HLA-ID PB who were in CR, 3 yr OS was 39+/−12% without acute GVHD and interestingly, 54+/−10% with acute GVHD. UD BM recipients not in remission at the time of transplant had dismal outcomes with or without acute GVHD, with less than 5% OS. No recipients of HLA-ID PB with active disease at the time of transplant remained alive if they did not develop acute GVHD versus 19+/−10% if they did (p=0.17).These data obtained in a large group of homogeneously treated pts suggest that acute GVHD may play an important role in preventing relapse after RIC and HLA-ID sibling PB transplantation. Following UD BM, acute GVHD appears to adversely affect OS. RIC should not be proposed in the unrelated donor setting if pts with AML are not in remission. Future efforts aimed at harnessing graft versus leukemia effects in the HLA-ID setting following RIC may lead to improvements in OS and are clearly warranted.