bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intergenic Variants of HBS1L-MYB Are Responsible for a Major Quantitative Trait Locus on Chromosome 6q23 Influencing HbF Levels in Adults.
Authors: Thein Swee Lay , Menzel Stephan , Peng Xu , Best Steve , Jiang Jie , Close James , Silver Nicholas , Gerovasilli Ageliki , Ping Chen , Yamaguchi Masao , Wahlberg Karin , Muller Heike , Spector Tim D. , Garner Chad , Matsuda Fumihiko , Farrall Martin , Lathrop Mark 
Published: April 2020

Abstract
Sickle cell disease and β-thalassemia demonstrate a remarkable diversity in phenotypic severity, and the innate ability to produce fetal hemoglobin (HbF, α 2γ 2) is a major ameliorating factor in these β-hemoglobinopathies. HbF and F cells (denoted FC; subset of erythrocytes that contain measurable HbF) are highly heritable quantitative traits; cis-acting variants in the β globin gene locus explain some of the variability but over 50% of the variance is unlinked to the β globin gene locus. We have previously mapped a quantitative trait locus (QTL) controlling FC in an Asian-Indian kindred with β-thalassemia to a 1.5 Mb interval on chromosome 6q23 that encompasses five known protein-coding genes (ALDH8A1, HBS1L, MYB, AHI1 and PDE7B), none of which harbored mutations. From the known genes within this interval, we selected HBS1L, a GTP-binding protein family member, and MYB, a transcription factor central to hematopoiesis, as candidates for detailed study. Two panels of twin-pairs (824 and 1217 individuals, respectively) of North European origin were recruited through the UK Adult Twin Registry. Age and sex, and XmnI-Gγ (−158C/T variant upstream of the Gγ globin gene which is associated with FC levels) show similar associations with FC levels in both panels. Altogether, 121 markers identified by re-sequencing and from public databases were genotyped in the HBS1L-MYB intergenic region. The high-resolution association study in Europeans identified genetic variants within and 5′ to HBS1L that are highly associated with F-cell levels (p=10−75). Additional HBS1L-MYB variants contribute independently to the QTL (p&lt;10−3). Our study provides unequivocal evidence for the mapping of the chromosome 6q HbF / F-cell QTL to a 79 kb region flanked by HBS1L and MYB; the intergenic variants account for 16% of the trait variance. The biological effects of these intergenic variants appear to be complex as they may influence several genes or transcribed sequences in this genomic region. Our study illustrates the power of QTL mapping for positional identification of trans-acting genetic variants influencing fetal hemoglobin levels, which could lead to new diagnostic and therapeutic strategies.
Regulatory T Cells (Tregs) Are Highly Preserved in Multiple Myeloma Patients and Can Dominate Effector Functions.
Authors: Foglietta Myriam , Mariani Sara , Castella Barbara , Coscia Marta , Godio Laura , Zappia Serena , Boccadoro Mario , Massaia Massimo 
Published: April 2020

Abstract
Naturally occurring CD4+CD25+ T regulatory cells (Tregs) are a subpopulation of CD4+ T cells playing a key role in lymphocyte homeostasis and maintenance of tolerance. Tregs play a key role in hampering anti-tumor immune surveillance by regulating immune responses against tumor cells. Depletion of Tregs yields to improved anti-tumor immune responses in both preclinical and clinical settings. Contradictory data have been reported in the frequency and function of Tregs cells in multiple myeloma (MM). The aim of this study was to characterize MM Tregs from the phenotypic, molecular and functional standpoints. We studied both peripheral blood (PB) and bone marrow (BM) samples from patients with symptomatic MM and from healthy donors. CD4+CD25high Tregs were normally represented in both PB and BM of MM patients and expressed a memory and activated phenotype. No differences were observed between PB and BM Tregs of healthy donors and MM patients, based on the expression of CD45R0, HLADR, CD40L and CD69 cell surface antigens. Flow cytometry was also used to assess the intracellular expression of Foxp3, a transcription factor which is crucial for the inhibitory function of Tregs cells. More than 90% of CD4+CD25high T cells were Foxp3+ in the PB and BM of MM patients and healthy donors. CD4+CD25+ T cells were purified from PB of newly diagnosed MM by MACS sorting. These cells were anergic to the stimulation via TCR and as effective as normal donor-derived CD4+CD25+ cells in inhibiting the TCR-mediated proliferation of autologous CD4+CD25- counterparts. To investigate whether clonal restriction had occurred in MM Tregs, we studied TCR diversity of CD4+CD25+ and CD4+CD25- cells by determining the reciprocal usage of BV gene segments (TCRBV repertoire) with a novel multiplex polymerase chain reaction assay recently developed in our laboratory. Our results demonstrate an highly preserved polyclonal TCRBV repertoire, providing the first evidence in cancer patients that TCR diversity of Tregs is not skewed by the long lasting exposure to tumor cells. Based on these data, we propose that inhibitory signals delivered by the highly preserved Tregs subset in MM can easily overwhelm the effector mechanisms of antitumor immunosurveillance which are deeply compromised in MM. Thus, depletion or neutralization of Tregs should be considered in future trials aimed at controlling the disease in MM patients by immune intervention.
CS1 Is Expressed on Myeloma Cells from Early Stage, Late Stage, and Drug-Treated Multiple Myeloma Patients, and Is Selectively Targeted by the HuLuc63 Antibody.
Authors: Szmania Susann , Balasa Balaji , Malaviarachchi Priyangi , Zhan Fenguhuang , Huang Yongsheng , Draksharapu Aparna , Vexler Vladimir , Shaughnessy John , Barlogie Bart , Tricot Guido , Afar Daniel , Frits van Rhee 
Published: April 2020

Abstract
Introduction: One-third of multiple myeloma (MM) patients exhibit high-risk features such as abnormal cytogenetics, high LDH, amplification of CKS1-B or spiked expression of MAF, MAF-B or FGFR3. While not affecting complete response rates, median durations of event-free and overall survival, even with high-dose melphalan-based tandem autotransplants of such patients, do not exceed 24 mo and 36 mo, compared to 60 mo and ≥ 90 mo for the remainder. Monoclonal antibody (mAb)-mediated therapy may target a chemotherapy-resistant myeloma cell pool. CS1 (CD2 subset 1, CRACC, SLAMF7), a member of the CD2 family of cell surface glycoproteins, exhibits high-level expression on primary myeloma cells, indicating that CS1 is a potential target for treatment in MM.
Methods: Gene expression was assessed using an Affymetrix GeneChip array. Protein expression was measured by flow cytometry, and immunohistochemistry (IHC), using HuLuc63, a novel humanized anti-CS1 mAb. HuLuc63-mediated lysis of myeloma cells via antibody dependent cellular cytotoxicity (ADCC) was measured by 51Cr-release.
Results: CS1 mRNA was detected in &gt;95% of CD138+ purified plasma cells from &gt;95% of healthy donors, newly diagnosed myeloma patients, and those with relapsed myeloma (Fig. 1a). CS1 remained highly expressed in patients following VDTPACE treatment, albeit at a reduced level. CS1 expression was also high following bortezomib (Velcade®) treatment, with a subset of patients showing increased expression post-treatment. CS1 protein expression on primary myeloma cells was confirmed by flow cytometry, while IHC analysis of normal tissues revealed anti-CS1 staining primarily on CD138+ tissue plasma cells. Finally, we determined that HuLuc63 could induce killing of myeloma cells using purified allogeneic NK cells (Fig. 1b). Blocking the Fc receptor greatly reduced this activity indicating an ADCC mechanism. Killing of myeloma targets was also observed in autologous systems suggesting that HuLuc63 can overcome KIR-mediated NK cell inhibition of autologous NK cells. In summary, we observed high mRNA and protein expression of CS1 in myeloma from early stage, late stage, and treated patients, and showed enhanced lysis of myeloma cells in vitro with HuLuc63. Our data support the potential clinical utility of CS1-targeted therapy. HuLuc63 will be entering a phase I clinical trial for advanced myeloma patients.
Figure 1a. CS1 mRNA is highly expressed in CD138+ purified plasa cells. Figure 1a. CS1 mRNA is highly expressed in CD138+ purified plasa cells. Figure 1b. CS1 antibody enhances killing of myeloma cells by allo - NK cells. Figure 1b. CS1 antibody enhances killing of myeloma cells by allo - NK cells.
Xenotransplantation of Primary CD34+CD38- Hematopoietic Stem Cells Results in an In Vivo Model of Idiopathic Myelofibrosis.
Authors: Saito Noriyuki , Ishikawa Fumihiko , Shimoda Kazuya , Yoshida Shuro , Saito Yoriko , Kawano Noriaki , Shulz Leonard D. , Akashi Koichi , Harada Mine 
Published: April 2020

Abstract
Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These abnormal cells secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and lead to extramedullary hematopoiesis and the appearance of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate at the level of hematopoietic stem cell (HSC), this has not been demonstrated directly in primary human IMF. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we used the newborn NOD/SCID/IL2rg-null xenotransplantation model. We purified PB CD34+ cells from six IMF patients, transplanted 1–10 x10e4 cells intravenously into newborn NOD/SCID/IL2rg-null recipients and analyzed PB and BM human CD45+ hematopoietic cell chimerism, degree of suppression of murine hematopoiesis, presence of hallmark BM fibrosis and plasma TGF-b1 levels in the recipients at 6 months post-transplantation. Primary IMF PB CD34+ cells from five out of six patients engrafted in twelve out of twelve recipients. BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of murine fibroblasts, the presence of human megakaryocytes and elevated plasma TGF-b1 levels, recapitulating the clinical features of IMF. Three distinct patterns of human hematopoietic reconstitution were observed among the engrafted recipients:
Predominantly malignant myelomonocytic engraftment in the PB and BM (n=4), Reconstitution of both normal human hematopoiesis (with mature B and T cells, myeloid cells and platelets) and malignant myelomonocytic cells (n=6) and Development of acute leukemia (n=2).
Fibrotic change was seen even in the BM of recipients that showed normal human hematopoietic reconstitution, showing that in IMF, there is co-existence of both normal and malignant hematopoietic stem/progenitor cells in the PB CD34+ fraction. Furthermore, when 5–10 x 10e3 sorted PB CD34+CD38– cells from three patients were transplanted into six newborn NOD/SCID/IL2rg-null recipients, reconstitution with human myelomonocytic cells associated with BM fibrosis was demonstrated in all recipients, with compatible level of PB and BM chimerism with those transplanted with PB CD34+ cells. These findings demonstrate that the IMF-initiating cells are contained within the HSC fraction. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and may be useful for development of novel therapeutic agents for IMF.
Mice Lacking the Signaling Molecule, CalDAG-GEFI, Represent a Mouse Model for Leukocyte Adhesion Deficiency Type III.
Authors: Bergmeier Wolfgang , Goerge Tobias , Wang Hong-Wei , Cifuni Stephen M. , Crittenden Jill R. , Graybiel Ann M. , Wagner Denisa D. 
Published: April 2020

Abstract
Defective inside-out activation of β 1, β 2, and β 3 integrins in platelets and leukocytes is a main characteristic of patients with leukocyte adhesion deficiency (LAD)-III syndrome. We have recently shown that CalDAG-GEFI, a member of the CalDAG-GEF/RasGRP family of intracellular signaling molecules that catalyzes the exchange of GTP for GDP bound to Rap1, plays a key role for the activation of α IIbβ 3 in murine platelets. Here we studied the role of CalDAG-GEFI for neutrophil function as well as the activation of β 1 integrins in platelets. Neutrophils from CalDAG-GEFI−/ − mice showed normal surface expression of key adhesion receptors such as L-selectin, PSGL-1, or β 1/β 2 integrins. Calcium flux, degranulation, and oxygen radical formation were similar in wild-type (WT) and mutant cells. In contrast, β 2 integrin-mediated adhesion to fibrinogen was significantly reduced in cells lacking CalDAG-GEFI when compared to controls. In vivo, CalDAG-GEFI-deficient neutrophils showed normal rolling along stimulated venules, while firm adhesion was almost completely inhibited. A similar defect in firm adhesion was observed in WT mice pre-treated with blocking antibodies against β 2 integrins. To determine the role of CalDAG-GEFI in neutrophil emigration, inflammation was induced in the peritoneum or the skin. In both models, neutrophil infiltration was significantly reduced in CalDAG-GEFI−/ − mice when compared to controls. We further demonstrate that CalDAG-GEFI regulates the activation of β 1 integrins in platelets and that CalDAG-GEFI-deficiency leads to a complete inhibition of arterial thrombus formation in mice. Due to its central role in the activation of β 1, β 2, and β 3 integrins, we propose CalDAG-GEFI as a candidate gene defective in LAD-III patients.
The Identification and Characterization of Zebrafish Hematopoietic Stem Cells.
Authors: Ma Dongdong , Lin Hui-feng , Handin Robert Irving 
Published: April 2020

Abstract
Hematopoietic stem cells (HSCs) are defined by their ability to self-renew and maintain hematopoiesis throughout the lifespan of an organism. Because of the optical clarity of their embryos and the ease of genetic manipulation, the zebrafish (Danio rario) is an excellent model for studying hematopoiesis. To date, though, there have been no published studies characterizing zebrafish HSCs. We have engineered transgenic zebrafish strains with green fluorescent protein (GFP+) thrombocytes by driving expression of the GFP reporter with the platelet/thrombocyte-specific CD41 promoter (Lin et. al. Blood. 2005 Dec 1; 106: 3803). We have identified two populations of CD41-GFP+ cells in suspensions of whole kidney marrow by flow cytometry. The majority of the cells strongly express GFP (GFPhi) and are mature thrombocytes. There is also a small population of immature cells, which express GFP weakly (GFPlo), that are of uncertain lineage. Peripheral blood and splenic cell suspensions contain only GFPhi cells. Studies in humans and mice have shown that CD41 is transiently expressed on the earliest hematopoietic progenitors and is then silenced, suggesting that the GFPlo cells might be hematopoietic progenitors or HSCs. To test this hypothesis, we transplanted flow-sorted GFPhi and GFPlo cells into gamma-irradiated adult AB strain zebrafish. The GFPhi cells were no longer detected 7 days after transplantation. In contrast, we have observed long-term engraftment and multi-lineage reconstitution for over 6 months after transplantation of GFPlo cells into zebrafish conditioned with gamma irradiation at 25 Gray or 40 Gray. Multi-lineage reconstitution persisted following serial transplantation of the GFPlo cells. Initial transplants were carried out with 105 GFPlo cells. To test the self renewal properties of GFPlo cells, transplants were carried out with injections of 1000 to 1 GFPlo cell(s)/fish. Multi-lineage reconstitution was seen with all doses and was achieved in 5 of 54 fish after transplantation of a single GFPlo cell. Flow cytometric analysis of whole kidney marrow from fish examined six months after transplantation demonstrated the persistence of GFPlo cells. Taken together, these studies suggest that GFPlo cells are capable of long-term hematopoietic reconstitution and are true HSCs. We believe that the zebrafish HSC transplant model holds great promise for the in vivo study of HSCs and may provide a valuable tool to track HSC homing, proliferation and differentiation.
α4-Integrin+ Endothelium Derived from Primate Embryonic Stem Cells Generates Both Primitive and Definitive Hematopoietic Cells.
Authors: Shinoda Gen , Umeda Katsutsugu , Heike Toshio , Arai Masato , Niwa Akira , Ma Feng , Suemori Hirofumi , Luo Hong Yuan , Chui David H.K. , Torii Ryuzo , Shibuya Masabumi , Nakatsuji Norio , Nakahata Tatsutoshi 
Published: April 2020

Abstract
The mechanism of commencement of hematopoiesis in blood islands of the yolk sac and the aorta-gonad-mesonephros (AGM) region during primate embryogenesis remains elusive. We previously showed the development of both primitive and definitive hematopoiesis when cynomolgus monkey embryonic stem cells were co-cultured with OP9 stromal cells. In this study, we examined the hematopoietic potential of endothelial cells developing in our coculture system and demonstrated that VE-cadherin+CD45− endothelial cells derived from embryonic stem cells were able to generate primitive and definitive hematopoietic cells sequentially, as revealed by immunostaining of floating erythrocytes and colony-forming assay in cultures. All floating erythrocytes which emerged initially expressed ε- and ζ-globins, while β-globin expression was hardly detected. The percentage of floating erythrocytes positive for β-globin gradually increased thereafter, and almost all erythrocytes were positive by day 40. Meanwhile, expression of ε- and ζ-globins declined gradually. Clonal analysis revealed that single bipotential cells for hematopoietic and endothelial lineages were included in this endothelial cell population. Hemogenic activity of endothelial cells was observed exclusively in the α4-integrin+ subpopulation. RT-PCR data showed that Runx1, a transcriptional factor associated with definitive hematopoiesis, was expressed in the hemogenic α4-integrin+ subpopulation, but not the non-hemogenic α4-integrin− subpopulation among embryonic stem cell-derived endothelial cells. The kinetics of this hemogenic subpopulation was similar to that of hemogenic endothelial cells previously reported in the yolk sac and the AGM region in vivo, in that they emerged only for a limited time. On the other hand, VE-cadherin−CD45−α4-integrin+ cells gave rise to more primitive erythrocytes than VE-cadherin+CD45−α4-integrin+ cells, but hardly contributed to definitive hematopoiesis. These results indicate that VE-cadherin+CD45−α4-integrin+ endothelial cells generate primitive and definitive hematopoietic cells sequentially, while VE-cadherin−CD45−α4-integrin+ cells are primary sources for primitive hematopoiesis. It seems that precursors of primitive and definitive erythropoiesis arise simultaneously but that the definitive precursors require a period of maturation before they differentiated into blood cells. We suggest that a subset of endothelial cells is involved in primitive as well as definitive hematopoiesis during primate embryogenesis, and that α4-integrin marks the hemogenic subpopulation in primates.
Active Immunotherapy with FavId® (Id/KLH) Following Rituximab Induction: Long-Term Follow-Up of Response Rate Improvement (RRI) and Disease Progression in Follicular Lymphoma Patients (pts).
Authors: Koc Omer N. , Redfern Charles , Wiernik Peter H. , Rosenfelt Fred , Winter Jane N. , Guthrie Troy H. , Kaplan Lawrence , Holman Peter , Densmore John , Hainsworth John , Lin Thomas S. , Castillo Rene A. , Janakiraman Nalini , Melink Teresa , Liu Song , Bender John 
Published: April 2020

Abstract
Background: Studies of Id/KLH immunotherapy in treatment naïve (TN) fNHL pts following first remission from chemotherapy have demonstrated a prolonged duration of remission. We expanded this approach in a PhII trial to include TN and relapsed/refractory (R/R) pts with stable disease (SD) or PR or CR/CRu following 4 wkly doses of rituximab. We report here long term follow up of RRI, defined as the % of pts who converted from SD to PR or PR to CR/CRu following initiation of Id/KLH treatment, and analysis of disease progression.
Treatment: Pts received rituximab (375mg/m2 i.v. wkly x 4 during wk1–4) and those with stable or responsive disease received Id/KLH (1 mg s.q. mo x 6) starting on wk 12 along with GM-CSF (250 mcg, s.q.) on days 1–4. Pts continued to receive booster injections on a reduced schedule until disease progression. Radiological scans, performed every 3 mos, were reviewed centrally. Disease progression and response were assessed using modified Cheson criteria. This data analysis was performed 32 mo from end of enrollment.
Results: 103 pts were enrolled and received rituximab; 89 pts had ≥SD and received Id/KLH + GM-CSF of whom, 54 were R/R, and 35 were TN. The overall response rate (ORR) was 47% (42/89) at mo 3. After the initiation of Id/KLH dosing, ORR improved to 63% (56/89). In RRI pts, median time to best response was 9.1 mos from start of rituximab treatment (5.2 – 29.6 mos). 12 of 39 PR pts (31%) converted to CR/Cru. 14 of 43 SD pts (33%) converted to PR.
Pt Groups # of Pts % Prog Free Median TTP Est by K-M (mo) Pts w/RRI 26 69% 37.9 TN rituximab responders 23 70% Not Reached (NR) Rituximab responders (All) 42 60% NR Non Responders (All) 47 36% 14.9 All Pts 89 47% 18.2 TN Pts (All) 35 66% NR Relapsed Pts (All) 54 37% 16.8
Conclusion: RRI following FavId is seen in nearly one third of pts. The magnitude of this improvement, the number of conversions to CR/CRu and median time to best response (9 mo and as late as 29.6 mo) suggests this is likely to be a FavId effect rather than late response to rituximab. Responders and TN pts show more durable responses compared to previously published data for rituximab therapy. FavId may also confer a benefit for nonresponders and R/R pts relative to rituximab alone. To confirm benefit of FavId following rituximab induction, a placebo controlled PhIII study was initiated; it completed enrollment in Jan 06. RRI data from this study is expected to be available in Nov 06.