bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Comparison of Freshly Isolated and Ex-Vivo Expanded CD4+CD25+Foxp3+ Regulatory T Cells in Suppressing Murine Acute GvHD
Authors: Sega Emanuela I , Leveson-Gower Dennis , Nguyen Vu H. , Negrin Robert 
Published: October 2019

Abstract
Graft versus host disease (GVHD) is a major complication of hematopoietic stem cell transplantation resulting from donor T cell reactivity against host tissue antigens. CD4+CD25+Foxp3+ regulatory T cells (Treg) are known to be important in maintaining self tolerance and preventing autoimmunity. Using murine models of acute GVHD in which allogeneic bone marrow cells are transplanted into lethally irradiated hosts, we and others have shown that donor Treg are able to suppress GVHD induced by donor allogeneic T cells and dramatically improve survival. Treg are rare and suppression of GVHD requires adequate numbers of Treg in relation to the number of conventional T cells (Tcon). To overcome this problem, expansion of Treg has been performed, however there has not been a head to head comparison of the function of expanded vs fresh Treg. Highly purified CD4+CD25+Foxp3+ T cells (>98% purity) were expanded using anti-CD3/anti-CD28 dynabeads and 1000 U/ml IL-2. Under these conditions, after five days Treg expanded up to 13 fold while maintaining high Foxp3 expression levels (85–90%). Longer expansion periods result in more T cell expansion but an overgrowth of Foxp3 negative T cells. In a mixed lymphocyte reaction assay, the ex-vivo expanded Treg efficiently suppressed the proliferation of alloreactive T cells. The expanded Treg were evaluated in an in vivo acute GVHD mouse model in direct comparison with freshly isolated Treg using a novel bioluminescent imaging assay that allowed for assessment of Tcon proliferation in addition to traditional metrics of GVHD severity including weight gain, survival and GVHD score. Initial experiments show that, similar to freshly isolated Treg, the ex-vivo expanded Treg suppress GVHD symptoms and improve survival, although a greater number of expanded Treg were required comparable to freshly isolated Treg. The mean GVHD score for the Tcon alone group was 5.8±1.02. Fresh Treg added at 1:1 ratio decreased the GVHD score to 0.75±0.25 (p=0.0036). Ex-vivo expanded Treg demonstrated a dose-dependent decrease in GVHD score, although four times more expanded Treg were needed to obtain a similar reduction in GVHD score (0.50±0.5, p=0.0036). This observed difference in potency was not due to the ex-vivo expanded Treg being short-lived when infused in mice. Bioluminescence imaging of luciferase positive (luc+) cultured Treg showed the same in vivo persistence as freshly isolated Treg. The ability to expand ex-vivo generated Treg is greater than the difference in potency, making ex-vivo expanded Treg potentially a viable option for treatment of GVHD, however, increased ratios of Treg:Tcon are likely to be required.
Engineering CD19-Redirected T Lymphocytes to Enhance Their Safety and Efficacy
Authors: Hoyos Valentina , Savoldo Barbara , Vera Juan F , Quintarelli Concetta , Rooney Cliona M , Heslop Helen E. , Brenner Malcolm K , Dotti Gianpietro 
Published: October 2019

Abstract
Modification of primary T cells to express chimeric antigen receptors (CARs) is an attractive strategy for the generation of tumor-specific T cells for adoptive therapies. However, their efficacy is limited by poor expansion within the tumor microenvironment. The addition of co-stimulatory endodomains, such as CD28, to the CAR may enhance cell expansion in response to the antigen, but cell growth and survival remain suboptimal since the co-stimulation provided through these endodomains cannot recapitulate the spatiotemporal sequence of signals T cells receive during physiologic activation. To further potentiate the expansion and survival of CAR-modified T lymphocytes, we generated a new vector encoding 3 molecules: CAR.19 incorporating the CD28 endodomain, codon optimized hIL15 to enhance cell survival and growth, and an inducible suicide gene based on the expression of Caspase9 (iCasp9) to increase the margin of safety associated with transgenic expression of an autocrine growth factor. These three sequences were linked using 2A-like peptide sequences. We compared the proliferative capacity, cytotoxic activity and in vivo anti-tumor effects of T lymphocytes expressing either CAR.19-28z alone or CAR.19-28z, IL15 and the suicide gene. T lymphocytes were activated with OKT3/CD28 antibodies and then transduced with retroviral supernatants. Phenotypic analysis showed 70±10% and 75±5% transduction efficiency for iCasp9/CAR19-28z/IL15 and CAR19-28z T cells, respectively. Only the iCasp9/CAR19-28z/IL15 T cells produced IL15 (>100pg/mL) after stimulation with CD19+ tumor cells. In co-culture experiments, T cells expressing CAR.19-28z alone or iCasp9/CAR19-28z/IL15 both completely eliminated CD19+ tumor cells within 72 hours. However, labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that the proliferation of the iCasp9/CAR19-28z/IL15+ T cells in response to CD19+ tumor cells was greater than that of CAR.19-28z control+ T cells. Finally, the activation of the suicide gene iCasp9 with a small-molecule dimerizer (CID 50ng/mL) rapidly induced >90% apoptosis of T cells expressing iCasp9/CAR19-28z/IL15. Hence all three transgenes were functional. To assess the antitumor effects of the modified cells in vivo, we used a xenograft SCID mouse model and an in vivo bioluminescence system. CD19+ Daudi cells (1×106) expressing firefly Luciferase (FL) were injected i.p., and on day 4, mice received i.p iCasp9/CAR19-28z/IL15+ or CAR19-28z+ or control T cells (10×106). By day 30 the tumor signal was significantly reduced in mice receiving iCasp9/CAR19-28z/IL15+ T cells (ROI<7×107) compared to mice receiving CAR19-28z or control T cells (ROI>2.0×109). In conclusion, our data indicate that a tricistronic vector can effectively be expressed in tumor-redirected human T cells, improving their survival and allowing their destruction should unwanted effects occur.
Pentostatin, Cyclophosphamide, and Rituximab (PCR) Achieve High Response Rates in Indolent B-Cell Lyphoma without Prolonged Myelosuppression
Authors: Samaniego Felipe , Fanale Michelle , Pro Barbara , Hagemeister F.B. , McLaughlin Peter , Romaguera Jorge , Neelapu Sattva , Rodriguez Maria Alma , Fayad Luis , Younes Anas , Kwak Larry W. 
Published: October 2019

Abstract
Background: The addition of rituximab to combination chemotherapy has improved treatment outcomes of indolent lymphomas. Combination therapy with purine analogs, akylating agents, and monoclonal antibodies is a promising approach for treating indolent B-cell lymphoma. Nucleoside analog-based regimens selectively target lymphoid cells, making them attractive drugs for lymphoid cancers. Pentostatin is a nucleoside analog that compared with other nucleoside analogs is reported to have less bone marrow cell toxicity. The combination of pentostatin, cyclophosphamide, and rituximab (PCR) is an effective regimen for relapsed chronic lymphocytic leukemia.
Methods:In this study, we examined the efficacy of pentostatin, cyclophosphamide, and rituximab for the treatment of B-cell lymphoma. Pentostatin (4 mg/m2), cyclophosphamide (600 mg/m2), and rituximab (375 mg/m2) were given on day 1 of a 21-day cycle with planned 6 or 9 cycles and restaging after every 3 cycles. Patients received prophylaxis with acyclovir 400 mg/po bid and trimethoprim-sulfamethoxazole 3 times per week. Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) tissues were stained for ZAP70 protein.
Results: At the time of abstract submission, 80 patients with a median age was 60 years were treated, 43 patients with follicular lymphoma, 27 with SLL/CLL, and 9 with mucosa-associated lymphoid tissue. We observed an overall response of 77 out of 80 (96%), CR/CRu in 65 out of 80 (81%), PR in 12 out of 80 (15%), and mixed response or progression in 3 out of 80 (4%). Neutropenia (49% < 1000/uL), thrombocytopenia (2% < 100,000/uL), nausea (15% grade 3), fatigue (37% grade 3 and 4), and muscle pain (21% grade 3) was observed. ZAP70 staining and response will be reported. Two patients experienced prolonged pancytopenia that resolved and no cases of myelodysplasia were observed.
Conclusions: PCR therapy is an effective regimen in indolent B cell lymphoma with tolerable toxicity.
Small Molecule XIAP Inhibitors Cooperate with TRAIL to Trigger Apoptosis in Childhood Acute Leukemia Cells and Overcome Bcl-2-Mediated Resistance
Authors: Fakler Melanie , Löder Sandra , Vogler Meike , Schneider Katja , Jeremias Irmela , Debatin Klaus-Michael , Fulda Simone 
Published: October 2019

Abstract
Children with high risk acute lymphoblastic leukemia (ALL) do not respond well to current treatments. This failure is, at least in part, due to defects in apoptosis programs. Therefore, new strategies are required that counter apoptosis resistance in order to improve the poor prognosis of high risk pediatric acute leukemia. Since XIAP, a member of “Inhibitor of Apoptosis Proteins” (IAPs), is expressed at high levels in acute leukemia and blocks apoptosis at a central point of the apoptotic machinery, XIAP may present a suitable molecular target for therapeutic intervention. Here, we report that neutralizing XIAP by small molecule inhibitors is a novel and effective approach to sensitize childhood acute leukemia cells for TRAIL- or chemotherapy-induced apoptosis. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, synergize with TRAIL to induce apoptosis in acute lymphoblastic leukemia cells. Also, XIAP inhibitors act in concert with TRAIL to reduce clonogenic growth of ALL cells demonstrating that they suppress long-term survival. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases, loss of mitochondrial membrane potential and cytochrome c release in a caspase-dependent manner, indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Intriguingly, XIAP inhibitors overcome Bcl-2-mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Thus, XIAP inhibitors combined with TRAIL even break Bcl-2-imposed resistance, a defect in the apoptotic pathway that is common in acute leukemia and associated with poor prognosis. Further, XIAP inhibitors prime ALL cells for apoptosis induced by various anti-leukemic drugs, e.g. cytarabine, doxorubicin, etoposide and 6-mercaptopurine, or by agonistic anti-CD95 antibody. Notably, XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In contrast to malignant cells, XIAP inhibitors at equimolar concentrations alone or in combination with TRAIL are non-toxic to normal peripheral blood mononuclear cells despite expression of the apoptosis-inducing TRAIL receptors on the cell surface, pointing to a therapeutic window. Most importantly, XIAP inhibitors significantly reduce leukemic burden in vivo in a mouse model of pediatric ALL engrafted in NOD/SCID mice. Thus, small molecule XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood acute leukemia.
Combination of Lenalidomide, Melphalan, Prednisone and Thalidomide (RMPT) in Relapsed/Refractory Multiple Myeloma: Results of a Multicenter Phase II Clinical Trial
Authors: Palumbo Antonio , Falco Patrizia , Sanpaolo Grazia , Falcone Antonietta , Ferderico Vincenzo , Canepa Letizia , Crugnola Monica , Baldini Luca , Larocca Alessandra , Magarotto Valeria , Petrucci Maria Teresa , Boccadoro Mario 
Published: October 2019

Abstract
Background: In newly diagnosed multiple myeloma (MM) patients the addition of lenalidomide or thalidomide or bortezomib to the standard oral melphalan and prednisone (MP) combination significantly increased response rate and event-free survival. In advanced MM, the 4 drug combination VMPT further improves response rate. In this multicenter open label phase I/II trial the safety/efficacy profile of the 4 drug combination, lenalidomide, melphalan, prednisone and thalidomide (RMPT) was evaluated in patients with relapsed/refractory myeloma.
Methods: Oral lenalidomide was administered at 10 mg/day on days 1–21, oral melphalan at 0.18 mg/kg on days 1–4, oral prednisone at 2 mg/kg on days 1–4. Thalidomide was administered at 50 mg/day (Arm A) or 100 mg/day (Arm B) on days 1–28. Each course was repeated every 28 days for a total of 6 courses. Aspirin 100 mg/day was given as a prophylaxis for thrombosis. Maintenance therapy included lenalidomide alone at 10 mg/day on days 1–21.
Results: Forthy-four patients, median age 69 years (range 47–80), with relapsed or refractory MM were enrolled. Twenty-six patients received RMPT as second line of therapy, 18 as third line. Twenty patients received prior autologous transplant, 10 thalidomide-based regimen, 9 bortezomib-based regimen and 3 allogeneic stem cell transplant. After a median of 2 courses, 75.8% of patients achieved at least a partial response (PR), including 30% very good partial response (VGPR). Among patients who received RMPT as second line therapy the PR rate was 81.8%, including VGPR 36.4%.Among patients who received thalidomide 100 mg, the PR rate was 93.3% (including VGPR 46.7%) compared to 64.7% of thalidomide 50 mg. The 1-year-progressionfree survival was 48.6% and the 1-year survival from study entry was 90%. Grade 3–4 hematologic adverse events included: neutropenia (66.6%), thrombocytopenia (36.3%) and anemia (30.2%). Grade 3–4 non hematologic adverse events included: infections (21.2%), neurological toxicity (6%) and fatigue (9%). No thromboembolic events were reported.
Conclusion: Initial results showed that RMPT is an effective salvage therapy with a high proportion of responses. Toxicities were manageable. No thromboembolic complications were reported.
The SH2-B Adaptor Protein Negatively Regulates EPO-Dependent Signalling Via Interaction with Erythropoietin Receptor Ptyr-343
Authors: Javed Mojib Javadi , Tschirch Edda , Beattie Bryan K , Stickle Natalie , Huang Kai , Jaster Robert , Carter-Su Christin , Barber Dwayne 
Published: October 2019

Abstract
Erythropoiesis is a developmentally important process, whereby multipotent hematopoietic stem cells differentiate into mature erythrocytes. Erythropoietin (EPO) is a critical regulator in this process and mediates its signal via the erythropoietin receptor (EPO-R) and the primary associated tyrosine kinase, JAK2. EPO, EPO-R and JAK2 play a crucial role in erythropoiesis, as deficiency in any of these proteins results in an embryonic lethal anemia. Structural-functional studies and murine knock-in models have shown EPO-R pTyr-343 to play a critical role in EPO mediated signalling. STAT-5 is activated by EPO-R pTyr-343, but STAT5ΔN mice do not have any profound erythroid abnormalities. Such evidence has led our group to hypothesize that other SH2 containing effectors interact with EPO-R pTyr-343. Cloning of Ligand Target screening was utilized to demonstrate that EPO-R pTyr-343 binds to adaptor protein SH2-Bβ. SH2-B contains multiple protein-protein interaction domains including multiple proline-rich regions, a PH domain and an SH2 domain. Although SH2-B does play a role in a number of signaling pathways, it is not required for embryonic development. Since SH2-B is a potent regulator of JAK2 in context of Growth Hormone and Leptin signaling, and it can directly interact with EPO-R pTyr-343, we hypothesize that SH2-B functions as an important adaptor protein downstream of the EPO-R.H2-B constitutively associates to the inactive EPO-R, an interaction that is independent of JAK2 binding to the EPO-R. Upon EPO stimulation, enhanced SH2-dependent binding of SH2-B to pTyr-343 and pTyr-401 of the EPO-R was confirmed utilizing a panel of EPO-R truncation mutants. The EPO mediated interaction between SH2-B and activated EPO-R is both dose and time dependent. EPO stimulation also results in SH2-B serine and threonine phosphorylation. Importantly, the interaction of SH2-B and EPO-R was observed in erythroid cell lines and primary murine splenocytes. The function of SH2-B in EPO signaling was investigated via knocking down SH2-B in Ba/F3-EPO-R cells. Knock down of SH2-B results in hypersensitive EPO-dependent phosphorylation of multiple targets including the EPO-R, JAK2, STAT5 and Erk1/2. It is evident that SH2-B is a global negative regulator of EPO-dependent signaling via its ability to affect EPO-dependent JAK2 activation.
Lnk Controls Hematopoietic Stem Cell Self-Renewal through Direct Interactions with JAK2 and Contributes to Oncogenic JAK2-Induced Myeloproliferative Diseases in Mice
Authors: Bersenev Alexey , Wu Chao , Balcerek Joanna , Tong Wei 
Published: October 2019

Abstract
Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2) that triggers a cascade of downstream signal transduction pathways that regulates many aspects of cell development. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. Surprisingly, we found that Lnk−/− HSCs have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeated 5-Florouracil (5-FU) treatments in vivo compared to wild type HSCs. We further provided genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal predominantly through Mpl. Consistent with this observation, Lnk deficiency in HSCs potentiates JAK2 activation in response to TPO. Biochemical experiments reveal that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Dysregulation of cytokine receptor signaling pathways leads to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML). Recently, the JAK2 V617F mutation has been observed at high frequencies in several myeloproliferative diseases (MPDs). The JAK2V617F mutant retains Lnk binding ability, suggesting Lnk status could modify MPD development. Indeed, we found that loss of Lnk accelerates oncogenic JAK2- induced CML/MPD in the mouse transplant models. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may contribute to leukemic transformation conferred by oncogenic JAK2.
No Influence on the Incidence of Osteonecroses When Dexamethasone Replaces Prednisone during Induction Treatment for Childhood ALL: Results of Trial ALL-BFM 2000.
Authors: Möricke Anja , Zimmermann Martin , Schrauder Andre , Stanulla Martin , Schmid Hansjörg , Fengler Rüdiger , Frühwald Michael , Mann Georg , Schrappe Martin 
Published: October 2019

Abstract
Osteonecroses (ON) are common complications associated with the treatment of acute lymphoblastic leukemia (ALL). For symptomatic ON, incidences of 1.6% to 9.3% have been published in the literature. The range of incidence rates may be related to treatment, data acquisition methods and patients’ age distribution. Between 09/99 and 06/06, 3,048 protocol patients with ALL (age 1–17 years) were enrolled into the multicenter trial ALL-BFM 2000 (Germany, Austria, Switzerland). Data on symptomatic ON were prospectively collected and were reported in 111 patients (3.6%, 95% confidence interval (CI) 3.0–4.4%). The median age of the patients with ON was 12.8 years (range 1.3–17.9 years), the median time from ALL diagnosis until diagnosis of ON was 15 months (range 2–73 months; data available from 92 patients). The incidence in adolescents, in particular adolescent girls, was significantly higher than in younger children (age <10 years: girls 0.8%, boys 0.7%, p=0.68; ≥ 10 years: girls 18.4%, boys 7.6%, p<0.001). The corticosteroid preparation used in the induction therapy (randomization 60 mg/m2/day prednisone vs. 10 mg/m2/day dexamethasone for three weeks plus tapering) did not influence the ON rate (prednisone 3.2%, dexamethasone 3.0%, p=0.77, 95% CI for difference: −1.2 – 1.6%). Various treatment attempts of ON were reported; 10 patients have been documented to be free of symptoms. At least 38 patients underwent surgical interventions, mainly core decompressions. Total endoprostheses of one or both hips were implanted in at least 10 patients. Final evaluation of all patients in particular with regard to long-term sequelae is not yet possible. The existing data demonstrate the far-reaching consequences of ON for the quality of life in a considerable proportion of the patients. Comparing these results with the ON data of study ALL-BFM 95 (Bürger et al., Pediatr Blood Cancer, 2005) which were collected in a predominantly retrospective way, the data suggest an increase in ON incidence in the current trial ALL-BFM 2000 (ALL-BFM 95: 1.6%). As the control treatment arms of the two trials were quite similar and the randomized treatment modifications in ALL-BFM 2000 had no influence on the ON rate, the higher incidence may be attributed to the prospective data collection in the current study and possibly to a higher attention to these complications by the attending doctors.
Therapy Outcome in Childhood ALL: Experience from a Tertiary Care Centre in North India.
Authors: Marwaha Ram Kumar , Kulkarni Ketan , Trehan Amita , Bansal Deepak 
Published: October 2019

Abstract
Survival in childhood ALL, in resource challenged nations, has not kept pace with cure rates of over 80% in developed countries. This descriptive study was designed to assess survival data and identify risk factors for adverse outcome. Data obtained from case records of 762 patients with ALL was analyzed. Information regarding the clinical-demographic profile, therapy and course of illness were recorded. Status and duration at last follow-up were utilized to generate Kaplan-Meier survival curves. The mean age at presentation was 5.7±0.23 years (M:F::3.2:1). Caretakers of 230 (30.2%) patients opted for no therapy. There were 68 and 60 deaths in induction and remission phases respectively. Besides these, 111 children either defaulted therapy (23) or were lost to follow up (88). Relapsed disease was documented in 125 cases. The 5 year overall and event-free survival was 46 and 34% respectively. The p values for survival analysis using the Log-rank test for groups formed according to the gender, age, symptom-diagnosis interval, TLC and platelet count were 0.19, 0.02, 0.08, <0.001, 0.001 respectively. Bulk disease (p=0.047), mediastinal adenopathy (p=0.045), TLC (p=0.016), platelet count (p=0.031) and administration of 2 intensification blocks (p=0.012) were found to be significant predictors of outcome by univariate and multivariate analysis. The holistic management of ALL in children requires financial resources, a strong psychosocial structure and access to quality supportive care. Almost one third of our patients opted for no therapy. The other problem areas identified were a high proportion of therapy defaulters, lost to follow up and infection related deaths during induction and remission phases. TLC >50000/microliter and platelet count <30000/microliter had an adverse impact on survival. The administration of both early and delayed intensification improved survival. The introduction of remedial measures for tapping the difficulties identified, would undoubtedly improve cure rates in children with ALL in developing nations.