bluuebunny/crossref_metadata_2025_split huggingface dataset:
CpG Oligonucleotides Induce Anti-Leukemia Activity in a Syngeneic Murine Model of Acute Lymphoblastic Leukemia.
Authors: Seif Alix E. , Bruno Marlo D. , Hall Junior , Brown Valerie I. , Grupp Stephan A. , Reid Gregor S.D. 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) accounts for 80% of all pediatric leukemias and is the most common form of childhood cancer. While most children with ALL are cured by current therapies, refractory and relapsed ALL comprise a significant proportion of all pediatric cancers. Furthermore, nearly half of all ALL diagnoses occur in adults, who carry a much poorer prognosis, with the majority dying of relapsed disease. Relapsed ALL generally requires intensive therapy with significant associated morbidity and mortality. Development of novel therapies is essential to improving outcomes. DNA oligodeoxynucleotides containing CpG motifs (CpG ODN) stimulate anti-tumor immune activity via Toll-like receptor 9 (TLR9) activation and are currently in clinical trials for a variety of solid tumors. We have previously reported that CpG ODN stimulation alters antigen presentation by human ALL cells, enhancing allogeneic Th1 responses. In addition, we have shown that CpG ODN administration in vivo reduces the leukemic burden of primary human ALL xenografts in Nod-SCID mice, and that this activity is mediated in part by NK cells. To further the development of CpG ODN as a novel therapeutic agent for ALL, we have investigated the induction of anti-ALL activity by CpG ODN in a syngeneic ALL setting. CpG ODN did not exhibit direct toxicity against cell lines derived from leukemic Eμ-ret transgenic mice in vitro, nor did it alter CD40 or CD86 expression or cytokine production. However, using a flow cytometry-based in vitro killing assay we observed CpG ODN-induced elimination of leukemia cells when cultured with splenocytes or bone marrow cells from Eμ-ret transgene-negative mice (P=0.0388). The difference between CpG ODN-treated and untreated controls became more pronounced with increasing effector:target ratios (P<0.0001). Preliminary data show that depletion of NK cells markedly decreases the magnitude of the observed effect, supporting the hypothesis that this cell type is involved in targeted control of ALL in this model. The ability of CpG ODN to exert anti-leukemia activity in a syngeneic setting suggests that it may have utility as an adjuvant therapy. To test this hypothesis we administered CpG ODN (or PBS) to syngeneic leukemia-bearing mice 2 days after completion of a chemotherapy regimen used to reduce leukemia burden. When mice were sacrificed 3 weeks after treatment, we found significantly reduced leukemia burden in bone marrow (P=0.0019), spleen (P<0.00001) and blood (P=0.00028) of CpG ODN-treated mice. Cell-depletion and cytokine-neutralization assays are currently ongoing to define the mechanism of action of CpG ODN in these settings. To our knowledge, this is the first demonstration of CpG ODN-induced anti-ALL activity in a post-chemotherapy syngeneic model, suggesting that this agent has the potential to treat minimal residual disease and to reduce the incidence of relapse.
CLL-1 Is a Potential Target for the Treatment of Acute Myeloid Leukemia.
Authors: Korver Wouter , Zhao Xiaoxian , Singh Shweta , Hsi Eric D. , Abo Arie 
Published: October 2019

Abstract
CLL-1 (C-type Lectin-Like Molecule-1) is an inhibitory receptor expressed on myeloid cells. We have generated a series of monoclonal antibodies (mAbs) against CLL-1 and used these mAbs to assess expression of the receptor on normal and AML cells and their therapeutic potential in in vitro and ex vivo cytotoxicity assays. By Immunohistochemistry, normal tissues lacked expression of CLL-1, with the exception of spleen. Using flow cytometry, expression was demonstrated on monocytes, granulocytes and dendritic cells, but not on lymphocytes and platelets. Tissue micro-arrays revealed CLL-1 expression in 97.3% (37/38) of AML cases. Flow cytometry of AML blasts demonstrated expression of CLL-1 on 81% (17/21) of patient samples. No detectable expression was detected in ALL blasts (n=5). Selected anti-CLL-1 mAbs mediated dose-dependent Complement Dependent Cytotoxicity (CDC) in various AML-derived cell lines, while no cytotoxicity was observed in CLL-1 negative K562 (CML) or CA46 (B lymphoma) cells. Human embryonic kidney 293 cells only became susceptible to anti-CLL-1 mAb mediated killing after transfection with CLL-1, demonstrating specificity. Furthermore, anti-CLL-1 mAbs showed CDC activity against all AML blasts tested in ex vivo assays (n=10), while no activity was observed against ALL blasts. Our results demonstrate restricted expression of CLL-1 on cells from myeloid origin and AML blasts and specific cytotoxic activity in in vitro and ex vivo assays. We are currently undertaking xenograft models to evaluate the therapeutic potential of these mAbs in vivo.
Interactions of Midostaurin (PKC412), Rapamycin and Ara-C in AML Cell Lines: Role of FLT3 Signalling.
Authors: Seedhouse Claire H. , Shang Shili , Russell Nigel , Das-Gupta Emma 
Published: October 2019

Abstract
Treatment options for patients with AML who are unfit for intensive chemotherapy are currently very limited. Commonly used treatment regimens such as low dose Ara-C result in complete remission rates of 17% and these remissions are not sustained (Burnett et al 2007). There is a rationale to examine the effect of combining small molecule inhibitors of signalling pathways with low dose Ara-C with the aim to improve its antileukaemic activity. To this end we have studied the cytotoxic interactions of the FLT3 inhibitor PKC412 and the mTOR inhibitor rapamycin with Ara-C in isobologram analysis (CalcuSyn). 4 cell lines were studied: 2 with FLT3 ITDs (MV4-11 and MOLM13) and 2 wild type for FLT3 (HL-60 and BV173). As expected synergy was seen between Ara-C and PKC412 in the FLT3 ITD positive cell lines (CI50: 0.47 MOLM13 and 0.68 MV4-11), but not in the wild type cell lines where antagonism was noted (CI50: 1.21 BV173 and 1.7 HL-60). In contrast, the Ara-C and rapamycin combination was synergistic in all cell lines, irrespective of FLT3 status. When the combination of PKC412 and rapamycin was studied, high levels of synergy were observed in the FLT3 ITD cell lines (CI50: 0.16 MOLM13 and 0.26 MV4-11) and in the BV173 cell line (CI50: 0.37) which has been shown to have autocrine activation of wild type FLT3 (Zheng et al, 2004) but not in HL-60 cells, where the combination was antagonistic (CI50: 3.4). FLT3 signalling pathways converge with the mTOR pathway via AKT and therefore our results suggest that a double hit on FLT3 signalling is likely to be beneficial in the treatment of FLT3 positive leukemias. In contrast rapamycin augments the cytotoxicity of AraC independently of Flt3 status. The combination of all 3 drugs produced no further benefit compared to the use of Ara-C and rapamycin alone. These promising drug combinations warrant further investigation in clinical studies.
A First-In-Human Clinical Trial of Adoptive Therapy Using CD19-Specific Chimeric Antigen Receptor Re-Directed T-Cells for Recurrent/Refractory Follicular Lymphoma.
Authors: Jensen Michael C. , Popplewell Leslie , DiGiusto David , Kalos Michael , Raubitschek Andrew , Forman Stephen J. 
Published: October 2019

Abstract
The immunotherapeutic ablation of lymphoma is a conceptually attractive treatment modality that is the subject of intense translational research activities. One approach being piloted through the City of Hope Lymphoma SPORE is the application of cellular immunotherapy employing cytolytic T-lymphocyte (CTL) grafts genetically engineered for CD19-specific effector functioning. This has been achieved by the construction of a chimeric T-cell antigen receptor (CAR) composed of a CD19-specific single chain immunoglobulin variable fragment (scFv) extracellular targeting domain molecularly coupled to the CD3 complex zeta chain. Cytolytic T-cells that are genetically modified to express this CAR display HLA-independent CD19-specific recognition and killing of lymphoma target cells. Using clinically applicable plasmid gene transfer/selection systems our group has demonstrated that anti-CD19 CAR+/HyTK+ CD8+ effectors can be generated from CTL precursors present in the peripheral blood and expanded to clinically relevant cell doses. We have designed an FDA-authorized (BB-IND#11411) clinical trial in which patients with recurrent/refractory follicular lymphoma undergo leukapheresis and a polyclonal CTL product derived that a.) expresses the anti-CD19 CAR, b.) kills CD19+ lymphoma targets, c) remains dependent on exogenous IL-2 for survival and proliferation, and d.) is sensitive to ganciclovir ablation by virtue of co-expression of the selection-suicide transgene HyTK. The trial is designed to assess the safety of intra-patient dose escalation beginning at a cell dose of 100x106 cells/m2 escalating to a dose of 2,000x106 cells/m2. In addition, this trial employs the use of rituximab to clear circulating B-cells prior to the first T-cell dose and fludarabine following the first T-cell infusion to both induce temporary lymphopenia and limit anti-transgene rejection responses. The protocol employs low-dose subcutaneous rhuIL-2 to support the numerical and temporal persistence of transferred CTLs. Treated patients are monitored with immunologic correlative studies that include Q-PCR for tracking persistence of transferred CTLs, RT-QPCR for following the levels of CD19+ B-cells in the peripheral blood, bone marrow, and lymph nodes, as well as, CT and PET imaging studies to follow disease responses. Here, we report on our initial clinical experience treating patients on this trial.
Analysis of Factors Influencing Skin Toxicity after Autologous Hematopoeitic Stem Cell Transplant.
Authors: Mahindra Anuj , Bolwell Brian , Sobecks Ronald , Rybicki Lisa , Brown Stacey , Pohlman Brad , Kalaycio Matt , Dean Robert , Andresen Steve , Sweetenham John , Copelan Edward 
Published: October 2019

Abstract
Skin toxicity is a known but understudied complication of autologous stem cell transplantation (ASCT). Its assessment is complicated by the development of graft-vs-host disease following allogeneic transplantation. We chose therefore to study skin toxicity following autologous transplantation. We retrospectively reviewed the records of 392 patients undergoing ASCT to analyze risk factors for skin toxicity, its association with mucositis and its effect on survival. Skin toxicity was assessed three times a week during the transplant admission and was classified as none, mild, moderate, severe or life threatening. The most severe skin toxicity is used in this analysis. Mucositis was assessed using the modified oral mucositis assessment scale (OMAS). Median patient age was 53 years; 63% of patients had non-Hodgkin s lymphoma (NHL), 17.6% multiple myeloma, 13.5% Hodgkin disease (HD) and 5.9% acute leukemia. Peripheral blood progenitor cells were mobilized with G-CSF alone (36%) or the combination of etoposide plus G-CSF (64%). The preparative regimen was busulfan (Bu)/cyclophosphamide (Cy) /etoposide in 82% and Bu/Cy alone in 18%. Two hundred and sixty patients (67%) developed skin toxicity of which 143 patients (36.5%) had mild, 105 (26.8%) had moderate and 16 (3.9%) had severe skin toxicity. Factors associated with the development of any skin toxicity in univariable analysis were male gender (p=0.028), diagnosis of NHL (p<0.001) or HD (p=0.001), higher number of prior chemotherapy regimens (p<0.001), mobilization regimen containing etoposide (p< 0.001) and ASCT preparative regimen containing etoposide (p<0.001). Moderate skin toxicity correlated with the above as well. Only the inclusion of etoposide in the mobilization regimen was associated with an increased risk of severe skin toxicity (p=0.035). Etoposide in the preparative regimen remained the most significant risk factor for the development of skin toxicity in multivariable analysis. Higher OMAS score was associated with increased severity of skin toxicity (p<0.001) as shown in the boxplot below:
Patients with severe skin toxicity appeared to have worse overall survival (p=0.054) and relapse-free survival (p=0.08), but these findings did not reach statistical significance.
Conclusion: A substantial proportion of patients develop skin toxicity following autotransplantation. The inclusion of etoposide in the preparative regimen is associated with a significant increased frequency in multivariable analysis. The severity of skin toxicity correlates closely with the severity of mucositis.
Figure Figure
Integrin αvβ3 Is Involved in the Stabilization of Ultra Long von Willebrand Factor Strings under Fluid Shear Stress on Human Endothelial Cells.
Authors: Huang Jing , Heuser John E. , McEver Rodger P. , Sadler J. Evan 
Published: October 2019

Abstract
Von Willebrand factor (VWF) multimers attached to endothelial cells can provide a platform for thrombosis, especially when accompanied by ADAMTS13 deficiency. We characterized the structural features of ultralarge VWF (ULVWF) molecules acutely secreted from Weibel-Palade bodies and identified a receptor responsible for their binding to the surface of cultured human umbilical vein endothelial cells (HUVECs). Using fluorescence microscopy on live cells, VWF multimers formed extended strings within minutes after stimulation. String formation did not require exogenous platelets and occurred over a range of shear stress from 2.5 dyn/cm2 to 40 dyn/cm2. A subset of ULVWF strings spontaneously bound formalin-fixed platelets via platelet GPIb. Quick-freeze, deep-etch electron microscopy showed that ULVWF strings often merged to form bundles and networks. Each string was tethered to the endothelial membrane by a limited number of anchorage sites, many of them located on small membrane projections, suggesting a specific mode of interaction. Several independent approaches implicated integrin αvβ3 in anchoring ULVWF strings to the HUVEC surface. Either “RGDS” peptide or function blocking antibody (LM609) to integrin αvβ3 specifically and dose dependently inhibited ULVWF string formation 62 ± 0.7% and 53 ± 4%, respectively. Furthermore, integrin αv was seen decorating the extending ULVWF strings using a non-functional blocking antibody (LM142) in live-cell immunofluorescence. In addition, a lentiviral vector encoding shRNA against integrin αv resulted in approximately 70% reduction in cell surface αv expression by FACS analysis with antibody LM609. HUVEC infected with this lentivirus were significantly impaired in their ability to form ULVWF strings compared to cells infected with a control virus. In multiple experiments, shRNA knockdown of αv expression reduced ULVWF strings 75.3 ± 4.4% at 2.5dyn/cm2 and 81.6 ± 2% at 7.5dyn/cm2. These results indicated that ULVWF strings bind tightly to endothelial cells via very few anchorage sites and are relatively resistant to fluid shear stress. Although integrin αvβ3 does not appear to be required to stabilize ULVWF strings on mouse endothelium, these data suggest that integrin αvβ3 may participate in the stabilization of ULVWF strings on human endothelial cell surfaces.
Reversible Self-Assembly of Weibel-Palade Body-Like Tubules from Recombinant N-Terminal Domains of von Willebrand Factor.
Authors: Huang Ren-Huai , Wang Ying , Roth Robyn , Yu Xiong , Purvis Angie R. , Heuser John E. , Egelman Edward H. , Sadler J. Evan 
Published: October 2019

Abstract
Weibel-Palade bodies (WPBs) are elongated secretory granules of endothelial cells that are packed with tubules composed of von Willebrand factor (VWF), a multimeric protein required for hemostasis. Disruption of tubular packing prevents the orderly secretion of VWF multimers and blocks the subsequent binding of platelets. The cigar-like shape and tubular cross section of WPBs are conserved in all vertebrates, but little is known about how VWF specifies this packing arrangement. Starting from recombinant 82 kDa VWF propeptide (domains D1D2) and 114 kDa disulfide-bonded D’D3 dimer, we now have assembled tubules reversibly in vitro with the same dimensions as VWF tubules in WPBs. Assembly was induced at pH 6.2, reversed at pH 7.4, and required Ca2+. Recombinant D’D3 dimers did not self-associate at pH 7.4 or pH 6.2, with or without Ca2+. Without Ca2+, VWF propeptide did not bind to D’D3 dimers. At pH 7.4, with Ca2+, VWF propeptide formed noncovalent 160 kDa dimers and, when mixed with D’D3 dimers, assembled a 280 kDa complex of two propeptides and one D’D3 dimer as shown by gel filtration chromatography and multi-angle light scattering. Lowering the pH to 6.2 caused the formation of >3 MDa aggregates with the same stoichiometry, which dissociated upon adding EDTA or raising the pH to 7.4. Quick-freeze deep-etch EM showed that the large aggregates are hollow right-handed tubular helices. The iterative helical real space reconstruction method was used to make 3D reconstructions of the tubules at 22 Å resolution from negative stain EM images (Figure, left). Tubules consist of a right-handed helix with axial rise of 26.2 Å and twist of 85.6 degrees per subunit, or 4.2 subunits per 11 nm turn. The dimensions (outside diameter 25 nm, inside diameter 12 nm) are similar to those of tubules in WPBs in thin sections of endothelial cells by transmission EM (Figure, right and its insert). Each subunit contains one D’D3 dimer flanked by two D1D2 propeptides (Figure, center). Each D’D3 dimer makes a total of six contacts with D1D2 domains. Each D1D2 propeptide makes three contacts with D’D3 and just one end-to-end homotypic contact. The spatial arrangement of these building blocks and inter-domain contacts in tubules suggest a model by which decreasing pH along the secretory pathway coordinates the formation of intersubunit disulfide bonds with the tubular packaging of VWF multimers. Within the WPB, Ca2+-dependent and pH-dependent binding of D1D2 to D’D3 domains stabilizes the packing of VWF multimers into tubules, which behave as constrained springs. Upon secretion, the increased pH weakens these constraints and permits the helical tubules to unfurl into flowing blood without tangling.
Figure Figure
Trend in Prevalence of Human T-Lymphotropic Virus Type-1 (HTLV-1) Infection in Japanese Blood Donors, Nagasaki, 1999 to 2006.
Authors: Iwanaga Masako , Chiyoda Shin , Kusaba Eisuke , Kamihira Shimeru ,   (Intr. by Yasuaki Yamada )
Published: October 2019

Abstract
To evaluate time-trend of HTLV-1 prevalence and the effect of preventative measure against the viral transmission are important in the virus endemic regions. In Nagasaki, Japan, an endemic area of HTLV-1, a routine serological virus screening for blood donors and a prefecture-wide intervention project (the ATL Prevention Program; APP) to prevent milk-borne transmission for the virus carrier mothers have been conducted since 1986 and 1987, respectively. However, the effects of both projects on the virus seroprevalence have not been well evaluated. In this study, we conducted trend analyses of age-specific, birth-year-specific, and period-specific seroprevalence of HTLV-1 for first-time blood donors who donated between January 1999 and December 2006. Among 55668 first-time donors (age at donation; 16–65 years, birth year; 1934–1990), 718 were test positive for HTLV-1, indicating that the overall seroprevalence was1.29% (95%CI, 1.20–1.39). Prevalence was significantly higher in women than men (1.53% vs. 1.13%; OR; 1.36, 95%CI; 1.17–1.57). Seroprevalence increased significantly with increasing age at donation from 0.70% at 16–25 years to 7.34% at over 56 years (Chi-square test, P < 0.0001). The annual prevalence was 1.32 in 1999, 1.31 in 2002, and 1.37 in 2006, indicating that there was no significant secular trend during 1999–2006 (P for trend=0.99). In analyses by age at donation, trends of HTLV-1 prevalence significantly declined among age over 56 years (P for trend=0.02) and age 16–25 years (P for trend=0.0007), whereas in birth-year-specific analyses, there was no apparent change of the prevalence over time, except in birth year 1981–90 group in which the prevalence declined from 1.22% in 1999 to 0.44% in 2006 (P for trend < 0.0001). In analyses for limited birth year from 1985 to 1990, the seroprevalence declined from 0.75% in birth year 1985–86 group, 0.31% in 1987–88 group, to zero% in 1989–90 group (P for trend =0.0002). HTLV-1 seroprevalence was significantly lower among donors born in 1987–90 (after APP) than 1985–86 (before APP). These results indicate that a birth-year-specific analysis for HTLV-1 prevalence may be appropriate to evaluate secular trend since the virus mostly transmit during infancy, and that a prefecture-wide intervention, the refraining from breast-feeding by the virus carrier mothers, contributes a declining HTLV-1 seroprevalence in our region.
Targeting 14-3-3 Sensitizes Native and Mutant BCR-ABL to Inhibition with U0126, Rapamycin and Bcl-2 Inhibitor GX15-070.
Authors: Dong Shaozhong , Kang Sumin , Lonial Sagar , Khoury Hanna Jean , Viallet Jean , Chen Jing 
Published: October 2019

Abstract
Recurring chromosomal abnormalities in human leukemias result in expression of a wide spectrum of constitutively activated fusion tyrosine kinases, including BCR-ABL associated with t(9;22)(q34;q22) chronic myelogenous leukemia (CML). Small molecule tyrosine kinase inhibitors, such as imatinib, are effective therapies for BCR-ABL-mediated human leukemias. However, clinical drug resistance occurs, which warrants development of alternative and/or complementary therapeutic strategies to target critical downstream signaling molecules. We recently demonstrated that disrupting 14-3-3/ligand association by a peptide-based 14-3-3 competitive antagonist, R18 induces significant apoptosis, partially through reactivation of AKT-inhibited pro-apoptotic FOXO3a in FGFR1 fusion transformed hematopoietic cells [Dong et al, Blood, 2007, 110(1):360–9]. Here we report that targeting 14-3-3 by R18 effectively induced significant apoptosis in Ba/F3 and K562 cells expressing BCR-ABL, through liberation and reactivation of FOXO3a, but not by affecting 14-3-3/BCR-ABL association or BCR-ABL kinase activity. In addition, co-immunoprecipitation experiments revealed that R18 was not able to disrupt 14-3-3/BAD association, suggesting a model that targeting 14-3-3 by R18 induces apoptosis in BCR-ABL transformed cells, at least in part, through liberation and reactivation of FOXO3a phosphorylated and inhibited by activated AKT, but not BAD inhibited by ERK. Thus, we hypothesize that targeting 14-3-3 by R18 may potentiate the inhibition of BCR-ABL transformed cells induced by blocking the parallel MEK1/ERK pathway, pro-survival Bcl-2 proteins, or other signaling effectors downstream of AKT. Indeed, R18 sensitized BCR-ABL transformed cells to inhibition with MEK1 inhibitor U0126, Bcl-2 inhibitor GX15-070, or mTOR inhibitor rapamycin, and the combined treatment of R18 with these anti-cancer reagents synergistically induced apoptotic cell death. Moreover, treatment of these reagents potentiates R18-induced reactivation of pro-apoptotic FOXO3a with enhanced expression of downstream transcription targets including p27kip1 and Bim1, suggesting a potential molecular mechanism of the combined therapy. Furthermore, R18 induced apoptotic cell death in cells expressing diverse imatinib-resistant BCR-ABL mutants, including T315I. This inhibition was enhanced by treatment of R18 in combination with U0126 and rapamycin. Together, our findings suggest a novel therapeutic strategy that targeting 14-3-3 may potentiate the effects of conventional therapy for BCR-ABL associated hematopoietic malignancies, and overcome drug-resistance.