bluuebunny/crossref_metadata_2025_split huggingface dataset:
AAV8-Factor IX Hepatic Gene Transfer with Transient Immunosuppression Is Safe but Is Abrogated by Low Titers of Neutralizing Antibody in Rhesus Macaques.
Authors: Jiang Haiyan , Patarroyo-White Susannah , Liu Tongyao , Yang Bin , Nagy Dea , He Lin , Scallan Ciaran D. , Sommer Jurg , Zhou Shangzhen , Couto Linda B. , High Katherine A. , Pierce Glenn F. 
Published: April 2020

Abstract
AAV8 vectors have potential in human gene transfer due to improved transduction efficiencies as demonstrated in small animal models, and are also reported to show less cross-reactivity to AAV2 neutralizing antibodies prevalent in humans. To assess this hypothesis, the efficacy and safety of AAV8-human Factor IX (hFIX) was evaluated in rhesus macaques. At doses of 5–20x1012 vg/kg, AAV8-hFIX achieved therapeutic to supraphysiological levels of circulating human FIX via intra-hepatic artery delivery in AAV8-naïve macaques. However, transduction was abrogated in macaques with pre-existing AAV8 neutralizing antibodies at titers as low as 1:5. Intrahepatic delivery of AAV8-hFIX resulted in gene transfer primarily in liver, and was well distributed among individual hepatic lobes. Two macaques that experienced traumatic catheterization developed immediate and delayed (week 4–6) transient transaminitis post surgery. Nevertheless, a significant percentage of AAV8-hFIX transduced hepatocytes persisted despite the liver damage and partial loss of hFIX expression. Transduction was well tolerated in all other macaques. Transient immunosuppression with tacrolimus and mycophenolate mofetil did not impair AAV8-hFIX transduction. The results [1] support AAV8-mediated gene transfer in humans but indicate that both the efficacy achieved and the resistance to pre-existing neutralizing antibodies of AAV8-hFIX are unlikely to be significantly enhanced over AAV2 vector, [2] suggest that a peri-operative insult and likely triggering of innate immunity may result in a later inflammatory response that results in a decrease in transgene protein production, and [3] provide evidence that a short course of immunosuppression does not alter liver transduction by AAV vectors.
Severe Impairment of Dendritic Cell Allostimulatory Activity by Sendai Virus Vectors Is Overcome by Matrix Protein Gene Deletion.
Authors: Armeanu Sorin , Bitzer Michael , Smirnov Irina , Bossow Sascha , Appel Silke , Ungerechts Guy , Bernloehr Christian , Kanz Lothar , Neubert Wolfgang J. , Lauert Ulrich M. , Brossart Peter 
Published: April 2020

Abstract
Delivery of antigens to dendritic cells (DCs) plays a pivotal role in the induction of efficient immune responses ranging from immunity to tolerance. The observation that certain viral pathogens are able to infect DCs has led to a concept where applications of recombinant viruses are employed for antigen delivery with the potential benefit of inducing potent antigen specific T-cell responses directed against multiple epitopes. As a prerequisite for such an application, the infection of DCs by recombinant viruses should not interfere with their stimulatory capacity. In this context we could show, that an emerging neagtive-strand RNA viral vector system based on Sendai virus (SeV) is able to efficiently infect monocyte derived human DCs (moDCs). However, after infection with SeV wildtype the responses of DCs to bacterial lipopolysaccharide (LPS) as a powerful mediator of DC maturation characterized by upregulation of CD83, MHC and costimulatory molecules, enhanced secretion of cytokines and the T cell stimulatory capacity were severly impaired. This was not due to a higher production of IL-10 or inhibition of the nuclear localized expression of Rel-B and c-rel, members of the NF-kB transcription factor family, but was rather the result of an increased apoptosis induction of DC. Interestingly, using various recombinant SeV vectors being devoid of single viral genes we were able to identify the SeV matrix (M) protein as a key component in moDC functional impairment after viral infection. Consequently, usage of M-deficient SeV vectors preserved the allostimulatory activity in infected moDCs despite an efficient expression of all other virally encoded genes, thereby identifying M-deficient vectors as a highly potent tool for the genetic manipulation of DCs.
Stable Gene Transfer and Expression in Human Primary T-Cells by the Sleeping Beauty Transposon System.
Authors: Zhou Xianzheng , Huang Xin , Wilber Andrew C. , Bao Lei , Tuong Dong , Tolar Jakub , Orchard Paul J. , Levine Bruce L. , June Carl H. , McIvor Scott , Blazar Bruce R. 
Published: April 2020

Abstract
The Sleeping Beauty (SB) transposon system is a non-viral DNA delivery system in which a transposase directs integration of an SB transposon into TA-dinucleotide sites in the genome. To determine whether the SB transposon system can mediate integration and long-term transgene expression in human primary T-cells, freshly isolated peripheral blood lymphocytes (PBLs) without prior activation were nucleofected with SB vectors carrying a DsRed reporter gene. Plasmids containing the SB transposase on the same (cis) (n=10) or separate molecule (trans) (n=8) as the SB transposon mediated long-term and stable reporter gene expression in human primary T-cells. We observed that delivery of SB transposase-encoding plasmid in trans effectively mediated stable gene expression in primary T-cells, exhibiting about a 3-fold increase (11% vs. 3% with 10 microgram plasmid on day 21) in potency in comparison with the cis vector (p&lt;0.0001). In addition, a transposase mutant construct was incapable of mediating stable gene expression in human PBLs (n=6, p&lt;0.0001), confirming that catalytic DDE domain is necessary for transposition in human primary T-cells. Immunophenotyping analysis in transposed T-cells showed that both CD4 and CD8 T-cells were transgene positive. SB-mediated high level of transgene expression in human T-cells was maintained in culture for at least 4 months without losing observable expression. Southern hybridization analysis showed a variety of transposon integrants among the 6 DsRed positive T-cell clones and no transposon sequences identifiable in the 2 DsRed negative clones. Sequencing of transposon:chromosome junctions in 5 out of 6 transposed T-cell clones confirmed that stable gene expression was due to SB-mediated transposition. In other studies, PBLs were successfully transfected using the SB transposon system and shown to stably and functionally express a fusion protein consisting of
a surface receptor useful for positive T-cell selection and a “suicide” gene useful for elimination of transfected T-cells after chemotherapy.
This study is the first report demonstrating that the SB transposon system can mediate stable gene transfer in human primary PBLs, which may be more advantageous for T-cell based gene therapies over widely used virus-based or conventional mammalian DNA vectors in terms of simplicity, stability, efficiency and safety.
Clonal Marking of Mouse Hematopoietic Stem and Lymphoid Progenitor Cell Populations.
Authors: Mojica-Henshaw Mariluz P. , Pierce L. Jeanne , Phillips John D. , Planelles Vicente , Spangrude Gerald J. 
Published: April 2020

Abstract
We have developed a method to clonally mark hematopoietic stem and lymphoid progenitor cell populations using a novel sequence tag approach. A library containing an 11-base random sequence tag is cloned into a lentivirus vector, packaged using the VSV-G glycoprotein and HIV-1 capsid, and transduced into freshly isolated mouse hematopoietic stem cell (Thy-1.1lowc-kithigh) or progenitor cell (Thy-1.1negc-kithigh) populations. To minimize artifacts introduced by prolonged culture, we have utilized a 3-hour spinoculation protocol performed in the absence of cytokines. Transduction efficiency was evaluated in vitro by methylcellulose colony assay and liquid cultures, and in vivo by transplanting the transduced cells into lethally irradiated mice. A bicistronic lentivirus vector with a CMV promoter driving expression of a transcript encoding Thy-1.2-IRES-GFP was used to optimize the transduction protocol. Liquid culture assays demonstrated 57% transduction efficiency after 5 days of growth, based on expression of the Thy-1.2 and GFP reporter proteins. Mice transplanted with transduced Thy-1.1negc-kithigh progenitor cells were sacrificed after 16 days, a time at which we have previously observed robust progenitor cell engraftment in the thymus while progeny of Thy-1.1lowc-kithigh HSC have not yet appeared. In 4 of 4 transplanted mice, we observed donor-derived cells in the bone marrow, lymph nodes and thymus. The percentage of total cells expressing the lentivirus-derived transgene ranged from 1.6% of bone marrow cells to 20% of thymocytes. Peripheral blood from mice transplanted with transduced HSC were analyzed and monitored every 4 weeks for transgene expression. We observed that although the Thy-1.2 marker was expressed and maintained up to 14 weeks after HSC transplant, GFP transgene expression was minimal. Based on these preliminary results, we have engineered a new lentivirus vector containing random sequence tags and the Thy-1.2 marker. This strategy provides a simple and efficient way of tracking the progeny of individual cells within a transplanted population, using PCR amplification of the random tags found within mature cell populations derived from the transduced cells. Sequence analysis of individual clones derived from different lineages of cells will enable us to better define the lineage potentials of specific progenitor cell subpopulations.
Provision of Peripheral Blood Stem Cell Service in Developing World: Experience from a Private Medical Center in Malaysia.
Authors: Chin Ng Soo , Teh Alan , Tan K.M. 
Published: April 2020

Abstract
Stem cell transplants (SCT) are potentially life-saving procedures but the availability of such services is limited in the government sector in Malaysia. The cost and logistics of having SCT abroad are daunting. This paper outlined our experience in setting up a SCT unit in a private hospital and reporting on the first hundred cases done over a 4 and a half year period from 1999 to 2004. All cases of SCT were performed with peripheral blood stem cells and in 5 cases (5%) back- up marrow was necessary as &lt;2X10^6 per kg of CD34 positive PBSC was harvested. Autologous SCT was performed on 66 cases for the following indications i.e. Non Hodgkin’s lymphoma (37), Acute myeloid Leukemia (13), multiple myeloma (10) and Hodgkin’s lymphoma (6). Thirty four allogeneic SCT were performed for AML (16), chronic myeloid leukemia (8), acute lymphoblastic leukemia (4), Non Hodgkin’s lymphoma (4), Myelodysplastic syndrome (1) and aplastic anaemia (1). Twenty of the allografts were conventional SCT while 14 had reduced intensity allografts. Transplant related mortality (TRM) was recorded in 4 cases (13.6%) of allogeneic SCT. Three of the deaths were sepsis related. No TRM was seen in autologous SCT. The direct medical cost (included stem cell harvest and the first 3 months post transplant care) of autologous SCT was a mean of US$ 16000 (ranged from US$ 9000 to US$ 20000) while allogeneic SCT cost a mean of US$ 25000 (ranged from US$ 20000 to US$ 60000). Our experiences indicated that SCT is feasible in a private medical center in a developing nation—the SCT results were comparable with established centers but with substantial cost saving as compared with SCT done abroad.
Hypertension and Obesity in Long Term Survivors of Childhood and Adolescent Cancer.
Authors: Haddy Theresa B. , Mosher Revonda B. , Reaman Gregory H. 
Published: April 2020

Abstract
As a retrospective, single institution study of blood pressure and obesity in survivors of childhood and adolescent cancer, the records of 103 consecutive patients seen in a Long Term Follow Up Clinic from January 1 through June 30, 2004, were examined. Fifty-one patients (49.5%) were female, 52 (50.5%) male. The mean age was 14.6 yr, range 4–33 yr. Ethnic backgrounds were Caucasian 59 (57.3%), AfroAmerican 24 (23.3%), Hispanic 12 (11.6%), Asian 5 (4.9%), and MidEastern 3 (2.9%). Blood pressures for 1–17 yr of age were considered normal if the systolic or diastolic was &lt;90 percentile, prehypertensive if 90–95 percentile, and hypertensive if ≥95 percentile for age, sex, and height. Blood pressures for 18 yr and older were considered prehypertensive if the systolic was 120–139 and/or the diastolic pressure 70–89, and hypertensive if the systolic was ≥140 and/or the diastolic pressure ≥90. Child and adolescent subjects were considered underweight if the BMI was &lt;5 percentile, nonobese if &lt;85 percentile, overweight if 85–97 percenntile, and obese if &gt;97 percentile for age and sex. Adult subjects were considered underweight if the BMI was &lt;18.5, nonobese if 18.5–24.9, overweight if 25–29.9, and obese if ≥30. A total of 29 (28.2%) patients were hypertensive or prehypertensive, and 74 (71.8%) were normal. A total of 38 (36.9%) patients were obese or overweight, 60 (58.3%) normal, and 5 (4.8%) underweight. Most Wilms’ tumor patients, 8 of 10 (80.0%), had hypertension or prehypertension, but only 4 of 10 (40.0%) were obese or overweight. Four of 5 (80.0%) patients with acute myeloid leukemia were obese or overweight, but only 2 of 5 (40.0%) had hypertension or prehypertension. Hispanic (75.0%) and AfroAmerican subjects (66.6%) were likely to be obese or overweight, but only 33.3% of both groups had hypertension or prehypertension. Although the number of study patients is small, our results emphasize the importance of identifying risk factors for hypertension and obesity in childhood and adolescent cancer survivors, particularly in survivors of Wilms’ tumor.
Adverse Events (AEs): Are They Factored into Costs of Hemophilia Care?.
Authors: Aledort Louis M. , Bohn Rhonda 
Published: April 2020

Abstract
Much of the high cost of hemophilia treatment ignores the cost of care for potential AEs related to treatment. Data have been published regarding the thrombotic AEs associated with the use of therapeutic biologics in the management of hemophilia patients with inhibitors. One objective was to quantify the incremental costs of these thrombotic AEs associated with either the available plasma-derived or recombinant products used in managing these patients. Although these occur at low frequency, myocardial infarction, stroke, and pulmonary emboli are reported.
Methods: A review of the literature revealed that no data exist for the cost of these events in hemophilia patients. Using the PharMetrics longitudinal database, it provided us with the frequency and charges associated with thrombotic AEs in hemophilia patients with and without inhibitors. These changes are not inclusive of total replacement product.
Demographic and Clinical Characteristics of the Hemophilia Cohort 1997–2004 Characteristic(Statistic) Hemophilia Patients without Inhibitors (n=5,042) Hemophilia Patients with Inhibitors (n=190) *Myocardial infarction: codes 410.X, 411.X, 412, 429.7 **Pulmonary embolism: codes 415.1X ***Cerebrovascular accident: codes 434.X and 436 Myocardial infarction* 182 patients 26 patients 230 episodes 44 episodes Mean $34,427 $40,110 Median $6,983 $7,070 S.D. $69,680 $64,254 Min-Max $0 – $497,096 $206 – $266,305 Pulmonary Embolism** 78 patients 21 patients Mean 111 episodes 31 episodes Median $18,095 $13,647 S.D. $6,069 $3,186 Min-Max $30,537 $24,738 $4 – $235,638 $63 – $106,082 Cerebrovascular accident*** 128 patients 26 patients Mean 201 episodes 44 episodes Median $27,528 $53,890 S.D. $6,069 $12,554 Min-Max $52,164 $103,017 $0 – 304,714 $32 – $567,055
Thrombotic complications of inhibitor therapy have been recognized for many years. Essentially, little data exist for the costs of these events and none for potential requirements for rehabilitation, loss of employment, and impact on the family. Data from clinical trials may well be a future key factor in determining reimbursement for therapeutics. There is a growing need for longitudinal data to assess the long-term safety as well as the associated costs of potential AEs. Knowledge of these costs will aid treaters in making therapeutic decisions. Coupled with the incidence of these events, incremental costs will aid health care planners to better understand the costs of care associated with hemophilia patients with inhibitors.
Mesenchymal Stem Cells Regulate the Cell Division Symmetry of Hematopoietic Stem Cells by an Integrin Dependent Mechanism.
Authors: Gottschling Sandra , Saffrich Rainer , Seckinger Anja , Krause Ulf , Miesala Katrin , Horsch Kerstin , Wuchter Patrick , Ho Anthony D. 
Published: April 2020

Abstract
Asymmetric cell division is the basal assumption for the generation of a hematpoietic system. On the basis of the murine stromal cell line AFT024 we could demonstrate that cell division symmetry is governed by intercellular contacts between hematopoietic stem cells (HSC) and niche cells. In the adult bone marrow the niche is primarily represented by osteoblasts, whereas during fetal hematopoiesis and pathological extramedullary hematopoiesis in the adulthood mesenchymal stem cells (MSC) are thought to hold this function. Nevertheless data demonstrating an alteration of cell division symmetry by MSC as well as an determination of the type of cellular interaction regulating this process are lacking so far. Therefore the cell division symmetry of individual human PKH-26 labelled CD34+/133+ HSC in contact and non-contact cultures with MSC was monitored for 7d by means of time lapse fluorescence microscopy. For comparison, cultures without feeder layer were monitored. The experiments were performed both, in the absence and presence of early acting cytokines (LIF, IL-6, MIP-1α, G-CSF, TPO, SCF, Flt-3L) promoting the maintenance of primitive progenitors. We found that the vitality and number of dividing cells was exclusively dependent on soluble factors with the highest vitality and number of dividing cells in cytokine augmented MSC cultures (89.9 ± 6.4% vital cells and 98.8 ± 2.7% dividing cells) and the lowest in cultures with neither stromal nor cytokine support (67.9 ± 0.7% vital cells and 41.8 ± 2.5% dividing cells). Nevertheless the percentage of asymmetrically dividing cells was twice as much in MSC contact cultures (22.7 ± 0.7%) than in all other culture conditions (9.1 ± 3.4 to 12.2 ± 2.3%). This demonstrates for the first time that MSC can alter cell division symmetry by a contact dependent mechanism. To identify the type of cellular interactions between HSC and MSC regulating the cell division symmetry, we monitored the latter in prescence of a function blocking β1-integrin antibody that reduced the percentage of asymmetrically dividing cells in MSC contact cultures from 22.7 ± 0.7% to 8.9 ± 4.2%. This was accompanied by a reduction of adherent HSC from 31.6 ± 6.1% to 9.7 ± 4.1% and a loss of functionally primitive cells as determined by a LTC-IC assay. Taken together these data demonstrate for the first time that MSC can alter the cell division symmetry of HSC by a mechanism that is partially dependent on β1-integrin mediated cell contacts.
Reconstitution of Regulatory T Cells after Selective Depletion of CD25+ Host-Reactive Donor Lymphocytes from Allografts and Association with Acute Graft-Versus-Host-Disease.
Authors: Mielke Stephan , Rezvani Katayoun , Solomon Scott R. , Kilical Yasemin , Savani Bipin N. , Wish Laura , Carter Charles S. , Schindler John , Hensel Nancy , Kurlander Roger , Read Elizabeth J. , Vitetta Ellen S. , Barrett A. John 
Published: April 2020

Abstract
Ex-vivo selective depletion (SD) of alloreactive donor lymphocytes by anti-CD25 immunotoxin is a novel method to reduce the severity of graft-versus-host-disease (GVHD) in elderly patients undergoing matched-sibling transplantation (Blood 2005, 106 (3), 1123–9). Nevertheless, there is concern that the concurrent removal of CD4+CD25+ regulatory T cells (Tregs) with the alloactivated CD25-expressing T cells could precipitate more GVHD if depletion of alloreacting cells were incomplete. We, therefore, studied the recovery of Tregs in 11 patients within the first 150 days after SD transplantation by surface phenotyping for CD4 and CD25 and real time PCR for forkhead protein3 (foxp3) gene, shown to be specific for characterisation of Tregs. At transplant patients received a median of 1.0 (0.5–1.5) x108/kg selectively depleted (CD25−) CD3+ T cells and a stem cell product containing 0.25 x104/kg (0.1–5.0) residual unselected CD3+ T cells. Three patients received SD-DLIs on day +60 or day +100. Low average levels of foxp3 gene expression appeared around day +30 post-transplant and remained almost stable until day +150 (Figure A). GVHD occurred in 7 patients (2 grade I and 5 grade II). Acute grade I-II GVHD was restricted to 7 patients with significantly lower foxp3 gene expression at day +30 (for one patient the earliest available sample was from day +60) (Figure B). In contrast to the relatively steady state of foxp3 gene expression average CD4+CD25+ cell numbers were highest during the first 60 days declining thereafter and suggesting the presence of relatively more effector cells in the early post-transplantation period. Chimerism analysis on selected CD4+CD25+ cells showed them to be predominantly of donor origin. These results suggest that foxp3+ Tregs delivered with the stem cell product may undergo modest expansion post-transplant. The absence of high grade aGVHD in these 11 patients probably relates to the efficient removal of host-alloreactive T cells by the SD process. Nevertheless, the occurrence of low-grade aGVHD in patients with the lowest foxp3 gene expression indicates that Tregs are also involved in GVHD protection after SD transplants. As the SD approach is at a very early clinical stage of development this possibility remains to be explored in further studies.
Figure Figure