bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.
Authors: Qian Hong , Jacobsen Sten Eirik W. , Ekblom Marja
Published: October 2019

Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.
Cytogenetics in Adult De Novo Acute Myeloid Leukemia: An Institutional Experience of a Predominantly Minority Population.
Authors: Berko Ebenezer , Osafo David , Tamkus Deimante , Griza Decebal S. , Sloan Steven , Jajeh Ahmad , Kovarik Paula , Catchatourian Rose
Published: October 2019

Cytogenetic findings at diagnosis correlate with prognosis in adult AML. Data have been largely derived from Caucasian populations. The purpose of this study was to define the frequency of cytogenetic abnormalities in de novo AML in a predominantly minority population and compare the data with that reported in the literature. We performed a retrospective review of cytogenetic findings at diagnosis in 40 adult AML patients diagnosed between 1999 and 2005. 17 African Americans, 11 Hispanics, 7 Caucasians, 1 Asian and 4 unknown ethnicity made up this population (72.5% minority). The age range was between 15 and 82 years. 28 patients (70%) were less than 55 years and 15 (37.5%) were less than 35 years of age. 75% of patients had abnormal karyotypes and 25% had normal karyotypes. Prognostic group categorization showed that 45% of patients were in the adverse group, 35% in the intermediate group and 20% in the favorable group. The most common cytogenetic finding was complex abnormalities occurring in 37.5% of patients. The frequency of complex abnormalities with adverse prognosis found in this predominantly minority population is higher than that reported in the literature (12% in SWOG/ECOG study; 6% in MRC AML 10 trial). Our study is limited by its small sample size. Further studies with a larger sample size would be needed to clarify the role of race/ethnicity in cytogenetic aberrations in AML.
Homoharringtonine Induced Apoptosis of Leukemic Cell Line MUTZ-1 Cells Is Mediated by the Endoplasmic Reticulum Stress Pathway.
Authors: Cai Zhen , Hu Jie , He Donghua , Wu Wenjun , Gao Liang , Bao Hanying
Published: October 2019

The endoplasmic reticulum (ER) is the major organelle for protein synthesis and maturation as well as the regulation of intracellular calcium (Ca2+) homeostasis. The accumulation of unfolded proteins in the ER lumen or depletion of Ca2+ from the ER lumen leads to the ER stress response known as unfold protein response. This response enhances the ability of protein folding by inducing ER resident chaperones such as glucose-regulated stress protein 78 or immunoglobulin heavy-chain binding protein (GRP78/BIP) mRNA and protects the cell from ER stress by splicing of X-box binding protein 1 (XBP-1) mRNA, and synthesis of phosphatidylcholine, the primary phospholipid of the ER membrane. However, when the ER function is severely impaired, the organelle elicits apoptotic signals. One of the components of the ER stress-mediated apoptosis pathway is C/EBP homologous protein (CHOP), also known as growth arrest- and DNA damage-inducible gene 153. Homoharringtonine (HHT), one of the alkaloids from a Chinese natural plant, Cephalotaxus, has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. But the mechanism of how HHT causes apoptosis remains unknown. In this study, we investigated the relationship between ER stress and apoptosis of MUTZ-1 cells (cell line of MDS) treated with HHT. HHT induced growth inhibition of MUTZ-1 cells was measured by MTT method. Apoptosis was detected by FACS and observed by light and electron microscopy. Concentration of cystosolic calcium was measured with the spectrofluorometer. The mRNA expression level of CHOP, BIP and XBP1 was detected by semi-quantitative RT-PCR. We found that after exposure of MUTZ-1 to HHT (rang 0.02~0.4ug/ml) for 24 to 72 hours, the IC50 and the percentage of apoptotic MUTZ-1 cells increased in a time and dose dependent manner. Typical apoptotic cells and release of Ca2+ from the cytosolic Ca2+ storage were observed. RT-PCR analysis revealed that mRNAs for ER stress-associated proapoptotic factor CHOP and an ER chaperone BiP, XBP1 are markedly increased at 4 hours after 0.05ug/ml HHT treated and reach the top level at 12hours, then decreased to the level before treatment at 24 hours. Induction of ER stress-responsive proteins (BIP and CHOP) was also observed at 12 hours. These results indicate that HHT could not only inhibit MUTZ-1 growth but also induce MUTZ-1 apoptosis and HHT-induced MUTZ-1 cell death is likely mediated by the ER stress pathway.
Myelofibrosis in a Patient with Sarcoidosis.
Authors: Abbasi Elham , Liu Enli , Mims Martha P. , Udden Mark M.
Published: October 2019

Secondary bone marrow fibrosis can be seen in several conditions such as hematological malignancy, metastatic malignancy and autoimmune disorders like lupus. We report here a 50 year old Middle Eastern female with a history of pulmonary sarcoidosis diagnosed by lung biopsy in January of 2004, beta thalassemia trait, hypertension and hypothyroidism. At the time of diagnosis she was started on bronchodilating inhalers. Nine months later she was referred to hematology for left lower quadrant pain and possible enlarged spleen on examination. CT scan revealed extensive mediastinal, retroperitoneal and inguinal adenopathy but no splenomegaly. Hemoglobin was 12.3 with a hematocrit of 38 and an MCV of 61, WBC was 7.8with a normal differential and Platelet count was 238. Her peripheral blood smear revealed moderate anisocytosis, polychromasia and some teardrops. Bone marrow biopsy showed significant fibrosis confirmed by reticulin staining. Biopsy of an inguinal node was consistent with her lung biopsy showing granulomatous disease. In the course of follow up she developed fever, fatigue, anemia, and splenomegaly proven by ultrasound (16cm). Her labs also revealed increased ACE and ESR levels. We started her on prednisone at 1mg/kg and her hemoglobin rose from 8.0 to 12.5. X-chromosome transcription-based clonality assays were employed to deteremine whether this patient had primary myelofibrosis with myeloid metaplasia, a myeloproliferative disorder characterized by clonal hematopoiesis or secondary myelofibrosis which is characterized by polyclonal hematopoiesis. Six exonic X-chromosome polymorphisms were tested to determine the clonality of the patient’s platelets and granulocytes (Liu, BLOOD 2003). Unfortunately, this patient was not informative in any of six exonic X-chromosome polymorphism markers, and we could not determine the clonality of hematopoiesis. Peripheral blood CD34 level; however, was low normal, consistent with reactive fibrosis rather than a primary myeloproliferative disease. To our knowledge this is the first report of secondary bone marrow fibrosis in sarcoidosis.
Therapy-Associated Immunosuppression and Opportunistic Infections in Multiple Myeloma Patients.
Authors: Schutt Philipp , Brandhorst Dieter , Stellberg Werner , Poser Miriam , Ebeling Peter , Muller Siemke , Buttkereit Ulrike , Opalka Bertram , Lindemann Monika , Grosse-Wilde Hans , Seeber Siegfried , Moritz Thomas , Nowrousian Mohammad R.
Published: October 2019

This study evaluates cellular and humoral immune parameters in myeloma patients focusing on the effect of treatment and the risk of opportunistic infections. Peripheral blood lymphocyte subsets and serum levels of nonmyeloma immunoglobulins (Ig) were analyzed in 480 blood samples from 77 myeloma patients. Untreated myeloma patients exhibited significantly reduced CD4+/45RO+, CD19+, CD3+/HLA-DR+, and natural killer (NK) cells as well as nonmyeloma IgA, IgG, and IgM. Conventional-dose chemotherapy (conv-CTX) resulted in significantly reduced CD4+ and even further decline of CD4+/CD45RO+ and CD19+ cells, most notably in relapsed patients. Patients treated with conv-CTX in combination with thalidomide showed significantly increased counts of monocytes as well as serum levels of nonmyeloma IgA and IgM as compared to patients on conv-CTX without additional thalidomide. Following high-dose chemotherapy (HD-CTX) prolonged immunosuppression was observed. While CD8+, NK, CD19+, and CD4+/CD45RO+ cells recovered to normal values within 60, 90, 360, and 720 days, respectively, CD4+ counts remaining reduced even thereafter. Nine opportunistic infections were observed including 5 cytomegalovirus (CMV) diseases, 1 pneumocystis carinii pneumonia (PCP), and 3 varicella zoster virus (VZV) infections with CMV diseases and PCP occurring exclusively after HD-CTX. Opportunistic infections were correlated with severely reduced CD4+ as well as CD4+/CD45RO+, and CD19+ counts. Thus, myeloma patients display cellular and humoral immunodeficiencies, which increase following conv- as well as HD-CTX and constitute an important predisposing factor for opportunistic infections.
Predictive Potential of Minimal Residual Disease Level after the First Imatinib Cycle on the Outcome of Allogeneic Stem Cell Transplantation in Adults with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia.
Authors: Lee Seok , Kim Yoo-Jin , Min Chang-Ki , Cho Byung-Sik , Kim Sung-Yong , Eom Ki-Seong , Kim Heeje , Cho Seok-Goo , Kim Dong-Wook , Lee Jong-Wook , Min Woo-Sung , Kim Chun-Choo
Published: October 2019

Purpose: Previously, we demonstrated the positive impact of first-line imatinib interim therapy on the outcome of allogeneic stem cell transplantation (SCT) in adults with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL) (Blood2005;105:3449). Here, we analyzed for risk factors that affect transplantation outcome, and focused particularly on the prognostic relevance of minimal residual disease (MRD) during treatment course.

Patients and Methods: Fifty-two consecutive adults with Ph-positive ALL who completed SCT following imatinib therapy were enrolled in this prospective study. MRD assessment was performed using real-time quantitative polymerase chain reaction.

Results: Forty-three (87.8%) of the 49 evaluable patients showed a decrease in MRD after imatinib therapy. Molecular remission rates were 18.4% and 44.4% after the first and second imatinib cycles, respectively. Forty-eight (92.3%) of the 52 patients received SCT during first complete remission. With a median follow-up of 42 months after SCT, the actuarial 3-year relapse and disease-free survival (DFS) rates were 22.9% ± 6.6% and 67.3% ± 7.2%, respectively. An MRD level of ≥ 10−3 after the first imatinib cycle was found to be the most powerful predictor of relapse (47.5% ± 14.3% versus 11.4% ± 6.4%, P = .009) and DFS (45.0% ± 13.2% versus 80.9% ± 8.0%, P = .016). The presence of chronic graft-versus-host disease was also found to be associated with a lower relapse (5.3% ± 5.1% versus 37.6% ± 10.8%, P = .029) and better DFS (82.0% ± 9.5% versus 62.4% ± 10.8%, P = .039).

Conclusion: In the setting of allogeneic SCT following imatinib therapy, prospective MRD monitoring may allow us to identify subgroups of Ph-positive ALL patients at high risk of relapse at an earlier treatment stage.
High Engraftment Rate after Second Stem Cell Transplantation for Thalassemia: A Prospective Study.
Authors: Gaziev Javid , Lucarelli Guido , Sodani Pietro , Polchi Paola , Paciaroni Katia , Marziali Marco , Isgro Antonella , Alfieri Cecilia , Simone Maria Domenica , Roveda Andrea , Montuoro Aldo , De Angelis Gioia , Gallucci Cristiano
Published: October 2019

Unlike hematological malignancies patients with thalassemia have an increased risk of graft failure or rejection occurring in up to 15% of patients after myeloablative stem cell transplantation from HLA identical related donors. Patients who reject their grafts and have a return of thalassemic hematopoiesis could benefit from second transplantation with the prospect of cure. Our previous experiences of second SCT using BUCY conditioning regimen alone or in combination with antilymphocyte globulin or total lymphoid irradiation showed a higher graft failure rates (43% to 69%). In 2003 we devised a new preparative regimen in an attempt to improve engraftment rate after second transplantation for thalassemia. The treatment protocol (Protocol 26.1) consisted of pre-conditioning immunosuppression-cytoreduction with hydroxiurea 30 mg/kg/day, azathioprine 3 mg/kg/day (day -45 to -12) and fludarabine 30 mg/m2/day (day -17 to -13) and conditioning regimen with BU 14/16 TT10 CY200 ATG (Thymoglobulin)12,5/10. Thirteen patients with median age of 9 years (range, 4–20 years) were given a second SCT according to this protocol. The median time between the first and second transplant was 29 months (range, 8–204 months). As a stem cell source 4 patients received bone marrow and 9 patients unmanipulated peripheral blood stem cells (PBSC). All but two patients received stem cells from the same donor. Twelve out of 13 patients (92%) had sustained full donor engraftment. One patient had early graft failure and died from cerebral bleeding due to refractory thrombocytopenia despite an autologous back-up. Other two patients died from acute or chronic GvHD -related complications. The probability of survival, thalassemia-free survival, transplant related mortality and rejection were 76%, 76%, 18% and 8% respectively with a median follow-up of 26 months (range, 8–47 months). Four patients developed grade II–III and 1 patient grade IV acute GvHD responsive to steroids and 3 patients had extensive chronic GvHD. Both acute and chronic GvHD occurred in patients who received PBSC. The incidence of CMV and EBV reactivation was 62% and 38% respectively. None of these patients developed EBV related lymphoproliferative disorders. Six patients had BK virus- related hemorragic cystitis (2 moderate, 2 severe and 2 mild cystitis). In conclusion, the high engraftment rate observed in this study suggests that this new preparative regimen is effective curative treatment for second transplant in patients with thalassemia. Disclosure: No relevant conflicts of interest to declare.
Circulating Endothelial Cells as Noninvasive Marker of Angiogenesis in Patients with Chronic Lymphocytic Leukemia.
Authors: Gora-Tybor Joanna , Krawczynska Anna , Lech-Maranda Ewa , Szmigielska-Kaplon Anna , Jesionek-Kupnicka Dorota , Blonski Jerzy Z. , Robak Tadeusz
Published: October 2019

There is accumulating evidence that circulating endothelial cells (CECs) play an important role in angiogenesis. An increased neovascularisation has been documented in bone marrow and lymph nodes of patients with chronic lymphocytic leukemia (CLL). The aim of our study was to compare the number of CECs in peripheral blood of CLL patients and healthy donors and to correlate these numbers with bone marrow microvessel density (MVD). In addition we checked if the number of CECs correlates with the response to treatment. We analyzed the total (CECs), activated endothelial cells (aCECs), apoptotic endothelial cells (apoCECs) and endothelial precursors (CPECs) count by the four colour flow cytometry in the peripheral blood of 80 untreated CLL patients and 29 healthy donors. We also evaluated the MVD in the bone marrow biopsy in 21 CLL patients and 11 controls, using the hot spot analysis of vessels density (x200 HPS). In 34 patients treated with purine analogs (Cladribine or Fludarabine) as first line treatment, we analyzed the influence of the initial number of CECs on treatment results. We found significantly higher numbers of CECs, aCECs, apoCECs and CPECs in CLL patients than in healthy controls (p=0.0001, p=0.001, p=0.0001 and p=0.001 respectively). The total number of CECs was significantly higher in the group of patients with advanced disease (Rai stage 3–4) as compared to patients in Rai stage 0–2 (p=0.02). A correlation was found between CECs and apoCECs number and the β2-microglobulin level (r=0.4, p=0.01 and r=0.3, p=0.01 respectively). The MVD was significantly higher in the bone marrow of CLL patients (median 60, range 27–147) as compared to controls (median 20, range 10–60) (p=0.01). In addition, the number of aCECs correlated with the MVD in CLL patients (r= 0.5, p= 0.02). Moreover, the patients with CECs numbers above median had significantly lower probability to achieve response to treatment with purine analogs (p=0.048). In conclusion, the number of CECs is increased in CLL patients and correlates with vessel density and tumor stage. These findings indicate that analysis of CECs may be a suitable, noninvasive marker of angiogenesis in CLL patients. Evaluation of CECs could be useful to identify CLL patients with low probability of response to purine analogs that may benefit from alternative therapy including anti-angiogenic agents.
Importance of PI3 Kinase Family for Crosstalk between Chronic Lymphocytic Leukemia B Cells and the Stromal Microenvironment: Therapeutic Implications.
Authors: Niedermeier Matthias T.W. , Rawluk Justyna , Knight Zachary , Shokat Kevan , Wierda William G. , Keating Michael J. , Burger Jan A.
Published: October 2019

Nontumoral accessory cells such as marrow stromal cells (MSC) or nurselike cells (NLC), which constitute the leukemia microenvironment, constitutively secrete the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). CXCL12 transduces signals via its receptor CXCR4, which is expressed at high levels by Chronic Lymphocytic Leukemia (CLL) B cells. Via the CXCL12-CXCR4 axis, CLL cells migrate and adhere to stromal cells. Adhesion to stromal cells protects CLL cells from spontaneous and drug-induced apoptosis in a contact-dependent fashion. Signaling pathways regulating these processes in CLL B cells are largely unknown. Here, we examined the importance of phosphatidyl-inositide 3-kinases (PI3-K) for migration and viability of CLL B cells using non-specific and isoform-specific PI3-K inhibitors. The importance of PI3-K for migration of CLL cells to CXCL12 was determined by transwell chemotaxis and pseudoemperipolesis (PEP) assays. Inhibition of PI3-K resulted in a significant reduction of CLL cell migration in chemotaxis and PEP assays. In comparison to untreated CLL cells, Ly 294002 inhibited chemotaxis to 65 ± 4.6% of untreated controls. Using a panel of isoform-specific PI 3-K inhibitors (PI-103, PIK-90, IC87114, TGX-115, ZK-75), we observed inhibition of chemotaxis by the multi-targeted compounds PI-103 (51.4 ± 0.2%) and PIK-90 (57.5 ± 8.9%), whereas p110beta and delta inhibition had no effect. We conclude from this part of the study that PI3-kinases play an important role for CXCR4 signaling in CLL B cells, mediating migratory responses and protection from apoptosis. Experiments with inhibitors of PI3-K with higher target selectivity suggest a dominant role for the class I PI3-K p110alpha for migration in response to CXCL12. Because adhesion to stroma mediates protection from chemotherapeutic drugs, we tested PI3-K inhibitors alone and in combination with fludarabine in CLL-stroma co-cultures. Pre-treatment of CLL cells with the PI3-K inhibitors Ly 294002, PI-103, and PIK-90 resulted in a significant decrease in viability of CLL cells co-cultured with and without stroma. Moreover, PI3-K isoform specific inhibitors enhance the cytotoxicity of Fludarabine and partially reverse the protective effect of stromal cells on fludarabine-induced apoptosis. Collectively, this study establishes that PI3-Ks play an important role in CXCR4 signaling for CLL cell migration and adhesion to stromal cells. New, isoform-specific PI3-K inhibitors enhance the cytotoxicity of fludarabine in suspension cultures and in co-cultures with stromal cells. Therefore, the therapeutic potential of PI3-K inhibitors alone or in combination with fludarabine should further be investigated.

Figure. Figure.
Activation of Nucleic Acid-Sensing Toll-Like Receptors Induces Proliferation, Cytokine Production, Immunogenic Phenotype, and Plasma Cell Differentiation of CLL Cells and Immunoglobulin Production.
Authors: Paul Santanu , Chu Charles C. , McCarthy Brian A. , Boyle Erin , Steinberg Bettie M. , Kaufman Matthew , Kolitz Jonathan , Allen Steven L. , Rai Kanti R. , Chiorazzi Nicholas
Published: October 2019

Weak immune function of chronic lymphocytic leukemia (CLL) cells may contribute to disease progression and inhibit effective immunotherapy. Accordingly, agents that enhance the function of CLL cells may be useful in immunotherapeutic approaches to this disease. Since Toll-like receptors (TLRs) are major regulators of B-cell function in innate and adaptive immunity, we asked to what extent stimulating two nucleic acid-sensing TLRs affected the activation and differentiation of CLL B cells. Isolated CLL cells were treated with either the TLR7-activating ligand, gardiquimod, or the TLR9-activating ligand ODN 2006-G5. CLL cells were also treated with the contents of apoptotic leukemic B cells, produced by repeated freezing and thawing. Ability to induce proliferation, as defined by CFSE dilution studies, cytokine production, differentiation, and altered surface molecule expression was measured. Both TLR 7 and TLR 9 ligands individually induced high levels of proliferation and plasma cell differentiation of CLL B cells. A dramatic increase in percentage of dividing cells was seen with simultaneous TLR7 and TLR9 activation, while blocking the TLR 7/9 pathway by pre-treating CLL B cells with chloroquine or bafilomycin reduced the proliferative capacity by 90%. Furthermore, stimulating CLL B cells with TLR7 and 9 ligands significantly up-regulated activation (CD38, CD69 and CD83), adhesion (CD54), and other immunophenotypic (CD40, CD80 and CD86) markers. Enzyme immunoassays of culture supernatants after TLR 7 and TLR 9 stimulation indicated that the levels of soluble IgM increased considerably, although levels of cytoplasmic IgM were unchanged. Production of IL6 and IL10 was also induced. We propose that ligands for TLRs 9 and 7 stimulate CLL B cells to proliferate and differentiate into plasma cells in the absence of T-cell help. In addition these stimuli alter CLL cell surface phenotype to a more immunogenetic profile, providing a rationale for CpG oligonucleotides and gardiquimod as humoral vaccine adjuvants for both normal immune reactions and potentially for anti-leukemic therapy.
Secondary Prophylaxis in a Cohort of Adolescent and Adult Italian Hemophiliacs.
Authors: Tagliaferri Annarita , Rivolta Gianna Franca , Coppola Antonio , Santoro Cristina , Rossetti Gina , Dragani Alfredo , Feola Giulio , Zanon Ezio , Iannaccaro Pier Giorgio , Radossi Paolo , Franchini Massimo
Published: October 2019

Although primary prophylaxis is a well-established and widely recommended practice for the management of patients with severe hemophilia, there are limited data in the literature documenting the benefit of secondary prophylaxis initiated in adolescence or adulthood. In order to address this issue we have conducted a retrospective study among the 10 Italian Hemophilia Centers on adolescent and adult patients with severe hemophilia switched from on-demand treatment to a long term secondary prophylaxis regimen. On the whole, we collected information on 73 consecutive patients with severe hemophilia (66 hemophilia A and 7 hemophilia B). Their median age started prophylaxis was 12 (range 10–17) for 23 adolescents and 30 (17.4–72) for the 50 adults and continued for a median of years 5.8 (2–14.7) in adolescents and 4 years (1.5–12) in adults. The clinical impact of secondary prophylaxis with respect to the last year on-demand regimen before switching is showed in the following table for each group of patients.

Adolescents (n=23) Adults (n=50) On-Demand Prophylaxis On-Demand Prophylaxis Values are reported as mean (standard deviation) Bleedings 32.4(24.8) 2.8 (3.2) 42.7 (24.5) 5.5 (5.2) Joint bleedings 25.6 (22.1) 1.9 (2.3) 36.4 (24.5) 4.2 (4.1) Orthopaedic score 8.4 (7.5) 3.7 (2.4) 21.6 (12.2) 17.7 (12.2) Pettersson score 5.1 (3.4) 5.0 (3.3) 14.7 (13.8) 15.2 (14.3) Work/school days lost 35.1 (34.5) 1.0 (1.5) 33.8 (20.9) 2 (0–17) Concentrate consumption U/Kg/y 2449.8 (1761.9) 3508.3(803.1) 3222.6 (2241.8) 4058.5 (952.3)

The decreased joint bleeding, better orthopaedic score, less work/school days lost, delayed progression of arthropathy and improved well-being observed with secondary prophylaxis show the benefit of this therapeutic approach and appear to counterbalance its higher clotting factor consumption. In conclusion large, prospective, long-term randomized studies are needed to definitevely assess the clinical impact of this strategy in adolescent and adult hemophiliacs and to identify those groups of patients who would benefit from this therapeutic approach.
Flt3-Ligand Plays a Critical Role in Restoring Function of NOD Facilitating Cells.
Authors: Huang Yiming , Fugier-Vivier Isabelle J. , Miller Thomas , Elliott Mary J. , Tanner Michael K. , Xu Hong , Atay Safinur , Hussain Lala-Rukh , Bozulic Larry D. , Chilton Paula M. , Ildstad Suzanne T.
Published: October 2019

CD8+/TCR− facilitating cells (FC) enhance engraftment of purified hematopoietic stem cells (HSC) in syngeneic and allogeneic recipients. FC also induce the production of regulatory T cells (Treg) in vivo and in vitro. The B220+/CD11c+/CD11b− precursor plasmacytoid dendritic cell (p-preDC) subpopulation in FC (p-preDC FC) is critical to FC function. However, p-preDC FC are significantly less efficient in function compared to FC total. In this study, we evaluated the phenotype and function of FC from diabetes-prone nonobese diabetes (NOD) mice. We found that NOD FC contain subpopulations similar to those previously described in B6 FC, including p-preDC, CD19+, NK1.1+DX5+ and myeloid cells. P-preDC represent the major FC subpopulation in NOD mice. The CD19+, DX5+ and B220−/CD11c+/CD11b+ subpopulations were significantly decreased in NOD FC compared to those from B6 or NOR mice (Figure 1a; * = P< 0.05; ** = P< 0.007). To test the function of NOD FC, 500 HSC (c-Kit+/Sca-1+/Lin−) were sorted and transplanted with or without 30,000 FC into conditioned 950 cGy recipients. MHC-matched diabetes-resistant NOR mice were served as a control strain for NOD mice. 5 (31%) of 16 recipients of NOR HSC were engrafted and survived up to 130 days. 7 (70%) of 10 recipients of NOR HSC plus FC have long-term engraftment and survival over 130 days, indicating that the NOR FC significantly enhanced engraftment of NOR HSC compared to HSC alone. In striking contrast, NOD FC were functionally impaired and did not enhance HSC engraftment in NOD recipients as evidenced by similar engraftment of HSC with FC (31%, n = 13) compared to the HSC alone (24%, n = 17; P = 0.579). Notably, when NOD mice were treated with Flt3 ligand (FL; 10 μg/ subcutaneous/daily, 10 days), FC were expanded in peripheral blood (PB). The DX5+ and B220−/CD11c+/CD11b+ subpopulations were significantly increased (Figure 1b). The FL-PB FC significantly facilitate engraftment of allogeneic HSC in vivo in the NOD → B10 model (Figure 1c; P = 0.02). These data demonstrate that NOD FC exhibit significantly impaired function and FL plays an important role in regulation and development of FC function. We propose that the defective function of NOD FC is mechanistically due either to the abnormal activation status of the p-preDC FC population and/or the absence of collaborative subpopulations in FC such as NK FC. This hypothesis offers an attractive explanation for the mechanism of FC to enhance HSC engraftment and to induce tolerance. Studies are underway to evaluate the mechanism by which FC contribute to diabetes-pathogenesis and/or prevention.

Figure Figure
Feasibility of Rapidly Generating Sufficient Autologous Human Marrow Stromal Cells for Cellular Therapy.
Authors: Lane Thomas A. , Garls Davina , Mackintosh Ellen , Kohli Sanjivan , Cramer Steven C.
Published: October 2019

Marrow stromal cells (MSC) are increasingly employed for human cellular therapy protocols. In some cases, it may be advantageous to use autologous MSC, but the feasibility of quickly generating sufficient MSC for autologous therapy has not been established. We investigated the feasibility of rapidly generating large numbers of autologous, early passage, MSC as a prerequisite for the clinical investigation of MSC in human ischemic stroke. Bone marrow (25–35 mL) was collected from 8 healthy volunteers under an IRB-approved protocol; a mononuclear cell (mnc) fraction prepared by ficol density gradient, a portion of the mnc fraction was suspended in low glucose DMEM with 20% pre-screened FBS and plated in T175 culture flasks at 0.2–0.4 × 106 mnc/cm2; maintained at 37 C in a 5% CO2 incubator; non-adherent cells were removed at 48 h and a portion of the cells were passaged when 80% confluent into additional flasks (12–14 days) at 5700 cells/cm2 and MSC were harvested with trypsin at 21 days. The MSC were washed x3 and resuspended in sterile saline for enumeration and suitability testing. MSC were identified according to ISCT criteria (Dominici M, Cytotherapy2006;8(4):315), including plastic adherence, morphology, growth characteristics, and phenotype by flow cytometry (CD73, CD90, CD105 positive; CD14, CD34, CD45, HLA-DR negative) and differentiation into adipocytes. The expected total cell yield was calculated based upon actual cell yields minus the volume of cells required for testing based upon a 70 kg patient.

Results: In 8 experiments using different donors, a median of 40 (range 20–225) × 106 mnc were available for plating from the original marrow. The median mnc recovery after ficol density gradient was 37% (range 23–130), but the median CFU-F recovery after ficol from the original marrow was 96% (94–96). The calculated MSC yield after primary culture was a median of 25 (10–53) × 106 cells. The calculated yield of MSC after 1st passage at 21 days was a median of 2.1 (0.9–4.4) × 106 MSC/kg (70 kg patient weight), and the median cell viability was 98.5% (92–97.9). At the end of 1st passage all samples yielded negative gram stains and cultures for mycoplasma, bacteria, fungal growth and the median endotoxin level was 0.027 EU/mL (0.02 – 0.10). The viability of the harvested MSC was well maintained in saline at 1 × 106 MSC/mL for 24 h at 4 C (85% +/− 9%) but not after 48 h (37% +/− 9%) or if stored at room temperature. The effects of supplementation with 5% human serum albumin on MSC storage for 24 h at 4 C were variable, ranging from 27% to 95% viability, depending on the albumin manufacturer (3 mfrs; n=4 each).

Conclusion: These studies suggest that sufficient MSC that are suitable for autologous human cellular therapy can be rapidly and reliably generated under GTP conditions, that the resultant MSC may be preserved for up to 24 h if stored at 4 C in either saline or pre-screened human serum albumin.
Enrichment of CD34+ Hematopoietic Stem and Progenitor Cells from Human Bone Marrow Using a P-Selectin-Coated Microtube.
Authors: Narasipura Srinivas D. , Liesveld Jane L. , Wojciechowski Joel C. , Charles Nichola , Rosell Karen , King Michael R.
Published: October 2019

Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematological disorders and for basic stem cell research. Primitive CD34+ HSPCs have demonstrated stronger rolling adhesion than mature CD34- mononuclear cells on selectins (Blood2000; 95:478–486). We have exploited this differential rolling behavior to capture and purify HSPCs from bone marrow, by perfusing mononuclear cells through selectin-coated microtubes. Bone marrow mononuclear cells were perfused through the cell capture microtubes coated with adhesion molecules. These utilized a parallel plate flow chamber (Glycotech), and the P-selectin was adsorbed with laboratory tubing of appropriate lengths attached to the inlet, outlet, and vacuum ports of the gasket chamber. After perfusion, the device lumen was washed and captured cells were visualized and estimated by video microscopy. “Rolling” cells were defined as cells translating at less than 50% of the calculated hydrodynamic free stress velocity. Velocities of single cells were determined using a MATLAB program designed to measure the change in position of the cell centroid in a given time period. Adherent cells were eluted by high shear, calcium free buffer and air embolism. Immunofluorescence staining followed by flow cytometry was used to analyze CD34+ HSPCs. CD34+ HSPC purity of cells captured in adhesion molecule-coated devices was significantly higher than the fraction of CD34+ cells found in bone marrow- mononuclear cells (2.5 ± 0.8%). P-selectin coated surfaces yielded 16–20% CD34+ cell purity, while antibody coated surfaces yielded 12–18%. Although the CD34+ cell purities were comparable between selectin and antibody surfaces, the total number of CD34+ HSPCs captured was significantly higher in P-selectin devices (∼5.7–7.1 × 104) when compared to the antibody device (∼1.74–2.61 × 104). Furthermore, analysis for cells positive for CD133, a surface marker for more primitive HSPCs, depicted approximately 10–14 fold enrichment in P-selectin samples over control bone marrow mononuclear cells. The captured cells were viable and exhibited in vitro colony forming capabilities. Thus, P-selectin can be used in a compact flow device to capture and enrich HSPCs. This study supports the hypothesis that flow-based adhesion molecule-mediated capture may be a viable physiologic approach to the capture and purification of HSPCs.
Reciprocal Activation of GATA-1 and PU.1 Marks Initial Specification of Hematopoietic Stem Cells into Myelo-Erythroid and Myelo-Lymphoid Lineages.
Authors: Arinobu Yojiro , Mizuno Shin-ichi , Chong Yong , Shigematsu Hirokazu , Iino Tadafumi , Iwasaki Hiromi , Graf Thomas , Mayfield Robin , Chan Susan , Kastner Philippe , Akashi Koichi
Published: October 2019

Understanding how multipotent cells commit to each of their terminal fate potentials is an important aspect of stem cell biology. In adult murine hematopoiesis, HSCs with long-term self-renewal potential reside within the Lin −Sca-1+c-Kit+ (LSK) fraction having CD34−, Thy1lo, and Flt3/Flk2−phenotypes. The LSK cells having CD34+, Thy1−, and/or Flt3+ phenotypes are capable of multi-lineage reconstitution only for a short-term, and therefore should contain multipotent progenitors (MPPs). In terms of developmental steps downstream of MPPs, there has been a controversy. The existence of prospectively isolatable progenitors capable of generating only myeloerythroid cells (common myeloid progenitor: CMP) or only lymphocytes (common lymphoid progenitors: CLP) outside the LSK fraction suggests that the first commitment step after the MPP stage is the strict bifurcation of lymphoid vs. myeloid pathway. Recently, however, several studies have suggested that the lineage commitment could occur within the LSK fraction, preceding the proposed bifurcation of myeloid and lymphoid pathways. For example, MPPs expressing Flt3 at a high level retained granulocytes/monocytes (GM) but not megakaryocyte/erythrocyte (MegE) potential together with lymphoid potential, suggesting the existence of common progenitor for GM and lymphoid lineages within the LSK fraction. Based on this data, the coupled loss of self-renewal activity and MegE potential in the early HSC commitment has been proposed. How can we reconcile the controversy in the early hematopoietic lineage map? By utilizing mice having GATA-1 or PU.1 transcriptional reporters, we here present a high-resolution map containing new lineage-restricted progenitor populations within the MPP population of CD34+ LSK phenotype. Initial upregulation of GATA-1 and PU.1 occurred independently at the MPP stage. The GATA-1+ MPP displayed potent myeloerythroid potential without giving rise to lymphocytes, whereas the PU.1+ MPP showed granulocyte/monocyte/lymphoid-restricted progenitor activity without megakaryocyte/erythroid differentiation. Furthermore, GATA-1+ and PU.1+ MPPs possessed huge expansion potential, and differentiated into the original CMPs and CLPs, respectively. Thus, the reciprocal activation of GATA-1 and PU.1 primarily organizes hematopoietic lineage fate decision to form the earliest hematopoietic branchpoint that comprises new, isolatable myeloerythroid and myelolymphoid progenitor populations.
Myeloid Gene Activation and T Cell/Natural Killer Cell Gene Repression in Cells Expressing Two Distinct PU.1 Concentrations.
Authors: Kamath Meghana , Houston Isaac , Janovski Alexander , Zhu Xiang , Gowrisankar Sivakumar , Jegga Anil , DeKoter Rodney
Published: October 2019

The Ets transcription factor PU.1 (encoded by the gene Sfpi1) functions in a concentration-dependent manner as a hematopoietic cell fate determinant. PU.1 levels are uniform in early hematopoiesis, increase during myeloid differentiation, and decrease after erythrocyte and T cell/natural killer cell commitment. It is unknown how downstream target genes respond to changes in PU.1 concentration. To address this, we generated mice with two distinct hypomorphic alleles of Sfpi1 and analyzed interleukin-3 dependent cell lines from fetal liver cells homozygous for either allele. PU.1 was produced in these cells at ∼20% (Sfpi1BN/BN) or ∼2% (Sfpi1Blac/Blac) of wild type. These cells fail to terminally differentiate as a consequence of low PU.1 expression and can be maintained as cell lines. To determine what groups of genes are expressed in response to two distinct PU.1 concentrations, we performed whole-genome microarray analysis and compared gene expression in Sfpi1BN/BN and Sfpi1Blac/Blac cell lines to Sfpi1−/− cell lines. Groups of downstream target genes were activated or repressed in four modes in response to the two discrete concentrations of PU.1:

at higher but not lower PU.1 concentration, at lower but not higher PU.1 concentration, at both lower and higher concentration, and in a gradient fashion.

We decided to focus on genes regulated in a gradient manner, because dose-dependency suggests that these may be direct targets of PU.1. Genes activated in a gradient manner were mostly myeloid-specific and enriched for target genes of PU.1. Genes repressed in a gradient manner included erythroid-specific genes and, unexpectedly, T cell and natural killer cell-specific genes. T cell genes were also repressed by PU.1 in cultured progenitor-B cells. With this unique allelic system, we can study three discrete concentrations of PU.1 at 20%, 2%, and 0% to examine concentration-dependent effects of PU.1 on target genes and lineage decisions. Overall, our results suggest that PU.1 functions in a concentration-dependent manner to promote myeloid differentiation and repress T cell or natural killer cell development.
Inhibition of VPAC1 Signaling Raises Platelet Numbers after Myelosuppressive Therapy and in GATA1 Congenital Thrombocytopenia.
Authors: Peeters Karen , Freson Kathleen , Wittevrongel Christine , Thys Chantal , Stassen Jean-Marie , Hoylaerts Marc F. , Van Geet Chris
Published: October 2019

Thrombocytopenia is a serious complication in patients undergoing myelosuppressive chemotherapy. Therefore, the development of new strategies to reduce thrombocytopenia or accelerate platelet recovery without adverse effect on the bone marrow (BM) is still warranted. Previously we found that the pituitary adenylate cyclase activating peptide (PACAP) is a physiological inhibitor of platelet activation (JCI, 2004). The receptor for PACAP on megakaryocytes (MKs) and platelets is the Gs coupled receptor VPAC1. We have presently investigated whether the neutralization of the regulatory role of VPAC1 can affect thrombopoiesis, independently of thrombopoietin (TPO). We found that inhibition of VPAC1 signaling stimulates in vitro megakaryopoiesis, since more MKs could be generated from Sca1+ cells, isolated from normal mouse BM by incubation with a neutralizing anti-VPAC1 antibody (Ab) (23A11). 23A11 was also capable of enhancing the later stages of MK proliferation of human cord blood derived CD34+ cells. These in vitro findings were validated in two in vivo animal models of myelosuppressive therapy. The subcutaneous injection of a neutralizing Ab directed against PACAP (PP1A4) or 23A11 (1 mg/kg on days 0,3 and 7) boosted platelet recovery in mice after chemotherapy-induced thrombocytopenia (20 mg/kg busulfan intraperitoneally on days 7 and 10) in a TPO-independent manner. Platelet recovery in mice, injected with PP1A4, was also significantly increased compared to non-treated mice exposed to 8 Gy total body irradiation. Plasma TPO concentrations did not differ between the two groups. We next questioned whether VPAC1 inhibition could rescue the GATA1-defective phenotype due to a developmental arrest in the megakaryoblast stage. FACS analysis of BM Sca1+ cells from GATA1 deficient mice cultured during 12 days in MK differentation medium in vitro showed an increased MK DNA ploidy when 23A11 was added to the culture. A similar observation was made with CD34+ cells from BM of a GATA1 deficient patient with severe thrombocytopenia. GATA1 deficient mice are thrombocytopenic, with a platelet count of 50 +/− 15 x 10(9) plt/L. This platelet number increases to 150 +/− 40 x 10(9) plt/L after subcutaneous injection with 23A11 whereas injection with PBS had no effect. Again, TPO plasma levels in these injected GATA1 deficient mice showed no association between anti-PACAP or anti-VPAC1 treatment. Thus, our data indicate that VPAC1 signaling tempers normal megakaryopoiesis and that elimination of this pathway facilitates platelet recovery after myelosuppressive therapy and in GATA1 deficiency.
The Indoleamine 2,3-Dioxygenase Pathway Is Essential for Human Plasmacytoid Dendritic Cell-Induced Adaptive T Regulatory Cell Generation.
Authors: Chen Wei , Liang Xueqing , Peterson Amanda J. , Munn David H. , Bruce Blazar R.
Published: October 2019

Plasmacytoid dendritic cells (PDCs) are a unique DC subset that plays a critical role in regulating innate and adaptive immune responses. Recently, we have shown that human PDCs activated by CpG oligodeoxynucleotide (CpG ODN) can drive naive, allogeneic CD4+CD25− T cells to differentiate into CD4+CD25+Foxp3+ regulatory T cells (Tregs). However, the intracellular mechanism(s) underlying PDC-induced Treg generation is unknown. Here we show human PDCs express high levels of indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme that catabolizes tryptophan degradation. Triggering of Toll-like receptor 9 (TLR9) with CpG oligodeoxynucleotides activates PDCs to upregulate surface expression of B7 ligands and HLA-DR antigen, significantly increases the expression of IDO, and results in the generation of inducible Tregs from CD4+25− T cells with potent suppressor cell function. Blocking IDO activity with a pharmacologic inhibitor 1-methyl-D-tryptophan (1MT) significantly abrogates PDC-driven inducible Treg generation and suppressor cell function. Adding kynurenine (KYN), the immediate downstream metabolite of tryptophan, bypasses the 1MT effect, and restores PDC-driven Treg generation. Our results demonstrate that IDO pathway is essential for PDC-driven Treg generation from CD4+25− T cells, and implicates the generation of KYN-pathway metabolites as the critical mediator of this process.
Protein Phosphatase1 gamma (PP1cγ) Is Required for Thrombin-Induced Integrin αIIbβ3 Activation.
Authors: Gushiken Francisca C. , Patel Vimal , Rambuat Rolando , Varmuza Susannah , Vijayan K. Vinod
Published: October 2019

Integrin αIIbβ3 activation is critical for platelet physiology and is controlled by agonists- induced signal transduction through kinases and phosphatases. Compared to kinases, a role of phosphatases in platelet integrin αIIbβ3 signaling and activation is less understood. We have previously demonstrated that the catalytic subunit of protein phosphatase 1 (PP1c) associates constitutively with the integrin αIIbβ3 and regulates myosin light chain phosphorylation. Platelets express three isoforms of PP1c namely PP1cα, PP1cβ/δ and PP1cγ1, however their role in platelet physiology is unclear. In this study, we explored a role for the PP1cγ in integrin αIIbβ3 activation using the PP1cγ null mice that lacks both PP1cγ1 and PP1cγ2 splice variants. Deletion of PP1cγ did not alter the expression of PP1cα and PP1cβ/δ in platelets. Compared to the wild type mice, PP1cγ null mice had significantly (P=0.03) more platelets but not other blood cells and increased megakaryocytes in an in vitro bone marrow culture system, suggesting that PP1cγ may regulate platelet production. Compared to the wild type platelets, PP1cγ null platelets exhibited a ∼50% decreased activation (P=0.007) of integrin αIIbβ3 in response to low doses of thrombin as measured by the binding of soluble Alexa-flour 488-conjugated fibrinogen or Jon/A, an antibody to high affinity integrin αIIbβ3. Consistent with this observation, PP1cγ null platelets showed decreased aggregation in response to thrombin or protease activated receptor activating peptide. Soluble fibrinogen binding and aggregation to ADP, collagen and cross linked collagen -related peptide was not different between the PP1cγ null and wild type platelets. Surface expression of integrin αIIbβ3 was comparable in the PP1cγ null platelets indicating that the decreased activation of αIIbβ3 in PP1cγ platelets was not due to decreased integrin expression. These studies suggest that PP1cγ is required for thrombin-induced αIIbβ3 inside-out signaling process. In contrast, αIIbβ3 outside-in signaling process was not altered in PP1cγ null mice as revealed by normal adhesion to immobilized fibrinogen. Intravital microscopy revealed that the thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly (P=0.03) delayed in PP1cγ null mice compared to wild type mice. These studies illustrates that PP1cγ is an essential component of thrombin signaling that is downstream of protease activated receptors and promotes thrombus formation in part by supporting soluble fibrinogen binding and platelet aggregation.