bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transforming Activity of AML1-ETO Oncoprotein Is Independent of CBFβ and ETO but Requires Homo-Oligomeric Interaction.
Authors: Zeisig Bernd B , Kwok Colin , Qiu Jihui , Dong Shuo , So Chi Wai Eric 
Published: October 2019

Abstract
AML1/RUNX1 and CBFb encode two critical transcription factors essential for the generation of hematopoietic stem cells (HSC). In acute myeloid leukemia, where leukemic stem cells (LSCs) have been functionally identified, AML1 and CBFb also represent the most commonly mutated targets. While animal models indicate that AML1 fusions per se are not sufficient to induce full-blown leukemia, they enhance self-renewal and expand targeted HSC and early progenitors, a property also reported for other oncogenic transcription factors involved in acute leukemia. Although attempts had been made to identify the critical domains required for AML-ETO (AE) mediated transformation, conflicting results were presented from most of these studies using exploited wellestablished cell lines, which suffer from the pitfall of carrying irrelevant genetic aberrants that may not reflect the normal biology of the disease. The only available structure/ function data on primary cells was limited to NHR2 of the ETO portion of the fusion, but it does not distinguish the functional contribution between homo-oligomerization and hetero-oligomerization. The lack of comprehensive structure/function data has not only significantly impeded the progress of understanding the biology of the disease, but also hinders the development of specific therapeutics. To this end, we performed extensive functional analysis to identify the key components essential for AE-mediated transformation of primary hematopoietic cells. In spite of the critical role of CBFb for wild type AML1 functions and its direct involvement in chromosomal translocation, we demonstrate that multiple AE single point mutants defective in CBFb interaction were still capable of transforming primary hematopoietic cells. Consistently, shRNA mediated knockdown of the endogenous expression of CBFb in primary cells did not compromise the transforming activity of AE, strongly suggesting a dispensable function of CBFb in AE mediated transformation. On the other hand, we demonstrate that NHR2 as the only domain in the ETO portion of the fusion is essential for transformation, but its heterooligomeric function including interaction with transcriptional repressor ETO family proteins is dispensable for the transforming activity. In contrast, synthetic FKBP homooligomerization modules could functionally replace NHR2, indicating that AE mediated transformation is critically dependent on homo-oligomeric property of the resultant fusion. Moreover, the transformation can also be abolished by a small molecule inhibitor that specifically dissociates homo-oligomerization. Together, these results not only identify the essential components and refine potential avenues for therapeutic targeting of AE oncogenic complexes, but also strongly endorse a common homo-oligomerization dependent mechanism shared by the most prevalent leukemia associated transcription factors.
Identification of MicroRNA Markers of Sensitivity to the Novel Bruton’s Tyrosine Kinase (BTK) Inhibitor PCI-32765 in Non-Hodgkin’s Lymphoma.
Authors: Balasubramanian Sriram , Chen Jun , Honigberg Lee , Tee Lillian , Mamuszka Hannah 
Published: October 2019

Abstract
PCI-32765 is a novel, first-in-class small molecule inhibitor of Bruton’s tyrosine kinase (BTK), currently in preclinical development and shortly entering Phase I clinical trials for the treatment of hematological malignancies. BTK is essential in B-cells for signalling via the B-cell receptor (BCR), and mutations in BTK have been shown to cause x-linked agammaglobulinemia in humans and x-linked immunodeficiency (xid) in mice. We have found that PCI-32765 has demostrated significant growth inhibitory activity in vitro in a number of non-Hodgkin’s lymphoma (NHL) cell lines as well as primary human NHL tumors cultivated ex-vivo. However, certain subsets of NHL cell lines were found to respond better to the inhibitor, and in keeping with this, a wide variance was also observed in the response among primary human tumors. PCI-32765 had GI50 <3uM in 7/15 cell lines (46%), including 3 follicular lymphoma (FL) and 3 diffuse large B-cell lymphoma (DLBCL) lines. Based on these results, we tested primary tumors including 15 FL and 7 DLBCL, utilizing a modified version of Oncotech’s EDR Assay. We found that while only 1 of the DLBCL tumors showed a similar response, PCI-32765 had a response rate of 40% in the primary FL samples. The reasons for this variation are still under investigation but one reason may be that that not all tumors are equally dependent on BTK activation for their survival. In order to obtain a predictive marker for response to PCI-32765 in human tumors, we utilized a microRNA (miRNA) profiling approach in the primary FL tumors using a commercially available miRNA array platform. We hypothesized that miRNA might be a better diagnostic tool than mRNA because miRNA function is more conserved and it is more stable than mRNA (thus easier to harvest successfully from paraffin embedded blocks). Further, it has been shown that miRNA signatures can predict responsive and resistant phenotypes based on basal miRNA levels, and have been found to have essential functions in lymphomas. When the miRNA profiles from the FL tumors were analyzed, we found that a signature consisting of 4 miRNAs could predict the sensitive tumors with a p-value <0.01. Of these, 2 were upregulated and 2 were downregulated in the sensitive samples compared to the resistant ones. It is likely therefore that these are implicated in the mechansisms of sensitivity and resistance to PCI-32765 in the FL tumors, and the connections between genes regulated by these miRNAs to pathways controlled by BTK are being explored.
Complications of Hysterectomy in Women with Von Willebrand Disease.
Authors: James Andra H , Myers Evan R , Cook Chad , Pietrobon Ricardo 
Published: October 2019

Abstract
Background: Case reports and small case series suggest that women with von Willebrand disease (VWD) are at a very high risk of bleeding complications with hysterectomy. Because the procedure may be beneficial to women who suffer from heavy menstrual bleeding, an understanding of the true risks involved is essential for appropriate decision making.
Objectives: To estimate the incidence of bleeding and other complications in women with VWD who undergo hysterectomy.
Methods: The United States Nationwide Inpatient Sample (NIS) from the Healthcare Cost and Utilization Project of the Agency for Healthcare Research and Quality for the years 1988–2004 was queried for all hysterectomies for nonmalignant conditions. Data were analyzed based on the NIS sampling design. Bivariate analyses were used to examine the differences between women with and without VWD. Multivariate analysis was used to adjust for potential confounders among women who underwent hysterectomy for heavy menstrual bleeding.
Results: 545 of the 1,358,133 hysterectomies were to women with VWD. Women with VWD were significantly more likely to experience intraoperative and postoperative bleeding (2.75% versus 0.89%, p < 0.001) and require transfusion (7.34% versus 2.13%, p < 0.001) than women without VWD. One woman with VWD died (odds ratio = 28.49).
Conclusions: While the risk of bleeding complications from hysterectomy in women with VWD is smaller than previously reported, women with VWD did experience significantly more bleeding complications than women without VWD. Nonetheless, for women who have completed childbearing, the risks of hysterectomy may be acceptable.
Excluding HIT Diagnosis by a Particle Gel Immunoassay.
Authors: Schallmoser Katharina , Drexler Camilla , Rohde Eva , Strunk Dirk , Groselj-Strele Andrea , Lanzer Gerhard , Kroll Hartmut , Panzer Simon 
Published: October 2019

Abstract
Heparin-induced thrombocytopenia (HIT) is a prothrombotic condition, characterized by a platelet drop of >50% and/or thrombosis with a temporal relationship of 1–2 weeks after initiation of heparin treatment. These surrogate markers are useful for the clinical assessment but are hardly applicable in multi-morbid patients with clinical conditions mimicking HIT. Platelet activation assays (heparin-induced platelet aggregation assay, HIPA; serotonin release assay, SRA) and platelet factor-4/polyanion enzyme immunoassays (PF4-ELISA) confirm HIT. HIPA and SRA are highly specific but laborious requiring selected donor platelets and extended experience. High titer IgG antibodies correlate with clinical HIT, but ELISA is also time-consuming. The alternative H/PF4-antigen particle gel immunoassay (ID-PaGIA H/PF4®) is easy, provides results within one hour and detects mainly IgG, but also IgA/M antibodies. We evaluated specificity and sensitivity of the PaGIA in comparison to HIPA and ELISA (PF4-ENHANCED®) in 285 patients (median 71yrs, range 1–97, 45% cardiovascular surgery; f/m 56/73, and 55% medical, f/m 78/78) with undetermined likelihood for HIT and 89 healthy controls (median 42yrs, range 26–64, f/m 49/40) to validate it as a rapid assay to exclude HIT. HIPA was positive in 12% of patients. Based on ROC curves (ELISAs vs HIPA), OD-cutoff values for the IgG/A/M- and IgG-ELISA were 0.761 and 0.564, respectively. In controls the OD of IgG-ELISA was 0.066 (0.028–0.500) and lower than from patients’ samples negative by PaGIA and IgG/A/MELISA (n=158; 0.074; 0.009–0.339; p=0.017). PaGIA was positive in 70 patients (25%). In both patient groups both ELISA ODs were higher if PaGIA and IgG/A/MELISA were positive compared to negative PaGIA but positive IgG/A/MELISA (p<0.001; data are shown as median; range). Analyzed by ROC curves (ELISAs vs HIPA), IgG/A/MELISA (OD-cutoff 0.761) and IgG-ELISA (OD-cutoff 0.564) had sensitivities of 81% and 86%, and specificities of 75% and 81%. Positive and negative predictive values were 31% and 97% (IgG/A/MELISA) and 39% and 98% (IgG-ELISA), respectively. PaGIA, IgG/A/M- and IgG-ELISA were significant predictors for the results of HIPA (p<0.001) and the IgG-ELISA had the highest explained variance (41%). Based on our results the PaGIA may serve as a rapid test to exclude HIT since a negative PaGIA is rarely associated with high titer IgG PF4-antibodies or a positive HIPA. In cases of positive PaGIA results and in patients highly suspicious for HIT but with negative PaGIA, alternative anticoagulation should be considered until results from functional testing are available.
Platelet-Targeted Expression of Coagulation Factor VIII (FVIII) Shows Efficacy for Using the Dog as a Large Animal Model for Gene Therapy of Hemophilia a
Authors: Wilcox David A. , Du Lily M. , Haberichter Sandra L. , Jacobi Paula M. , Fang Juan , Jensen Eric S. , Shi Qizhen , Montgomery Robert R. 
Published: October 2019

Abstract
In order to develop a large animal model for platelet-targeted gene therapy of Hemophilia A, we transduced normal canine bone marrow with a lentivirus vector under the transcriptional control of integrin αIIb gene promoter driving expression of human BDD-FVIII. Bone marrow was collected from normal dogs and transduced in the presence of recombinant canine c-SCF, c-IL-6, human h-TPO, and h-flt-3/flk-2 ligand differentiation factor. Immunofluorescence analysis using antibodies against human FVIII, and the megakaryocyte-specific marker integrin αIIb revealed synthesis of FVIII within tissue cultured megakaryocytes derived from lentivirus transduced bone marrow cells. This result appeared similar to synthesis, trafficking and storage of FVIII trafficked to α-granules of the murine “small animal” model for hemophilia A and human megakaryoyctes in vitro. To examine the effect of FVIII expression in platelets, in vivo, FVIII-transduced canine bone marrow was xenotransplanted into immune-compromised “NOD-SCID” mice treated with a sublethal dose (350 cGy) of irradiation. Flow cytometric analysis using antibodies specific for canine glycoproteins Ibα and integrin αIibβ3 revealed that circulating canine platelets comprised approximately 20–30% of the total platelet population in whole blood isolated from the mice within four weeks after xenotransplant. Immunofluorescence confocal microscopy detected a punctuate staining for FVIII that co-localized with a platelet α–granule protein, P-selectin, within a subpopulation of canine platelets isolated from murine whole blood. In contrast, FVIII was not detected in platelets from control samples. In addition, chromogenic analysis of platelets isolated from mice transplanted with FVIII-transduced bone marrow demonstrated the presence of a biologically active form of FVIII, FVIII:C at 2 mU/ml/lysate of 1×108 platelets. These results indicate that canine megakaryocytes are able to synthesize and store a biologically active form of FVIII that can be retained within progeny platelets. We speculate that dogs affected with hemophilia A should serve as an ideal “large animal” model to test if FVIII can undergo regulated release from platelets following physiologic hemostatic response to vessel injury. This raises the possibility of developing a locally inducible secretory pool of FVIII in platelets of patients with Hemophilia A following autologous transplantation of FVIII-transduced hematopoietic stem cells.
Directed Evolution of Immune-Escaping Adeno-Associated Virus Vectors
Authors: Maersch Stephan , Huber Anke , Hallek Michael , Buening Hildegard , Perabo Luca 
Published: October 2019

Abstract
Efficiency of therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity against the natural virus. Genetic engineering by rational design or directed evolution has been employed in the last 3 years to generate capsids that escape antibody neutralization and has led to identify several amino acid residues of the capsid proteins that can be mutated in order to decrease antibody recognition (Perabo et al., 2006; Maheshri et al, 2006; Lochrie et al., 2006). In this novel study, we aimed to exploit the comprehensive knowledge gathered so far by generating novel capsid variants that carried multiple point mutations at these previously identified sites. Capsid libraries were generated by codon randomization of several immunogenic residues and screened to isolate mutants that most efficiently infected human cells despite the presence of anti-AAV2 neutralizing antibodies. Besides testing novel combinations of concomitant mutations at these sites, this approach allowed for the first time an exhaustive scanning of combinations of all 20 natural amino acids at each position. We identified several novel capsid mutants that remain highly infectious even when incubated with serum concentrations that completely neutralize wild type AAV2. Our results demonstrate that combining mutations at several sites it is possible to improve the immune-escaping ability of the capsid. In addition, we show that escaping ability and other biological characteristics of these mutants are strongly dependent on the type of amino acid substituted, demonstrating that an exact choice of substituted amino acids is essential to maximize stealth properties and minimize loss of packaging ability, particle stability and transduction efficacy. These vectors can be used for therapeutic gene transfer to patients with pre-existing immunity, or for repeated treatment after antibodies are generated upon first application.
Production of High Titer cGMP-Grade SIN Gamma-Retroviral Vectors by Transfection in a Closed System Bioreactor
Authors: Van der Loo Johannes C.M. , Swaney William , Nordling Diana , Schambach Axel , Baum Christopher , Williams David A. , Reeves Lilith , Malik Punam 
Published: October 2019

Abstract
The need for gamma-retroviral vectors with self-inactivating (SIN) long terminal repeats for clinical trials has prompted a shift in the method with which large scale GMP-grade vectors are produced, from the use of stable producer lines to transient transfection-based techniques. The main challenge of instituting this methodology was to develop SIN retrovirus vectors that produced high amounts of genomic vector RNA in packaging cells, and to design scalable processes for closed system culture, transfection and virus harvest. Using improved expression plasmids, the Vector Production Facility, an academic GMP manufacturing laboratory that is part of the Translational Cores at Cincinnati Children’s Hospital, has developed such a method based on the Wave Bioreactor® production platform. In brief, cells from a certified 293T master cell bank are expanded, mixed with transfection reagents, and pumped into a 2, 10 or 20 Liter Wave Cell Bag containing FibraCel® discs. Cells are cultured in DMEM with GlutaMax® and 10% FBS at 37°C, 5% CO2 at a rocking speed of 22 rpm and 6° angle. At 16–20 hrs post-transfection, the media is changed; virus is harvested at 12-hour intervals, filtered through a leukocyte reduction filter, aliquoted into Cryocyte freezing containers, and frozen at or below −70°C. Several processing parameters, including the confluency of cells harvested prior to transfection, the timing of transfection, the amount of plasmid DNA, exposure of cells to PBS/TrypLESelect, and the timing of the media change post-transfection affected vector titer. Mixing cells with plasmid and transfection mixture prior to seeding onto FibraCel, as compared to transfecting cells 1-day post-seeding (as is standard when using tissue culture plastic) increased the titer from 104 to 4 × 105 IU/mL. Similarly, increasing the amount of plasmid DNA per mL from 4.6 to 9.2 μg doubled the titer in the Wave, while it reduced titer by 20–40% in tissue culture flasks (Fig. 1). Using an optimized protocol, six cGMP-grade SIN gamma-retroviral vectors have now been produced in support of the FDA’s National Toxicology Program (NTP), with unconcentrated vector titers ranging from 1 × 106 to as high as 4 × 107 IU/mL. Using similar processing, we have produced a large scale SIN gamma-retroviral vector (GALV pseudotyped) for an international X-linked SCID trial with average unconcentrated titers of 106 IU/mL in all viral harvests. In summary, the process developed at the Cincinnati Children’s Hospital Vector Production Facility allows for large scale closed-system production of high-titer retroviral vectors for clinical trials using transient transfection.
Figure Figure
Coating of AAV Vectors with Human Albumin Blocks Antibody Binding and Enables Transduction of Human Hepatocytes in the Presence of Neutralizing Antibodies
Authors: Wright J. Fraser 
Published: October 2019

Abstract
Neutralization of recombinant adeno-associated virus (AAV) gene transfer vectors by pre-existing antibodies is a significant barrier to clinical gene transfer using systemic administration. In a recent clinical trial for hemophilia B, while efficient gene transfer and therapeutic levels of hFIX were achieved in a human subject with low pre-existing antibodies to AAV2, no gene transfer was observed in a subject with a modest pre-existing AAV2 antibody titer of 1:17 who received the same dose (Manno et al. Nature Med.2006;12:342–347). With the objective to achieve consistent and efficient transduction in the presence of AAV antibodies, ‘humanized’ AAV vectors were generated by covalent attachment of human albumin (HSA) to the surface of purified AAV2 by cross linking the single free thiol (Cys34) of HSA to lysine residues on the vector surface using suflosuccinimidyl 4 [N-maleimidomethyl] cyclohexane-1-caboxylate (Sulfo-SMCC). Variable levels of HSA substitution up to 65 HSA molecules per virion were achieved, corresponding to a maximum of approximately one HSA molecule per virion capsid protein. Analysis by SDS-PAGE of the modified vector revealed the presence of a 130kDA polypeptide species in the conjugated vector, not observed in the unmodified vector, that was consistent with predicted molecular weight obtained by covalent attachment of HSA with AAV capsid protein VP3. Dynamic light scattering demonstrated an average radius of 16.6 nm for the HSA-conjugated vector compared with 11.6 nm for unconjugated vector, consistent with coating of the vector surface with HSA. Analysis by flow cytometry demonstrated that the binding of AAV2-specific monoclonal antibody A20 to unmodified vectors was progressively reduced to background levels in HSA-conjugated vectors with increasing HSA substitution. Quantitative analysis by Biacore indicated that the association rate and available sites per virion for antibody binding were reduced > 10- fold in HSA-conjugated vectors. The conjugated vectors were significantly more resistant than unmodified vectors to neutralization by AAV antibody, demonstrating comparable transduction in the presence of 16-fold higher concentration of MAb A20 following transduction of cultured human hepatocytes (HepG2). In conclusion, covalent coupling of HSA to the surface of recombinant AAV2 is demonstrated as a feasible method to modify AAV vectors to increase their resistance to neutralizing antibodies. ‘Humanized’ AAV vectors such as HSA-coated AAV that effectively mask viral epitopes recognized by antibodies and display human epitopes may enable more consistent transduction following systemic administration in vivo, and enable vector readministration.