bluuebunny/crossref_metadata_2025_split huggingface dataset:
Noninvasive Approach to Detection of T-Cell Mediated Graft Rejection through Antibody-Tagged Nanoparticles Using SQUID Detection.
Authors: Jaetao Jason E. , Lovato Debbie M. , Sergatskov Dmitri A. , Bergemann Christian , Bryant Howard C. , Flynn Edward R. , Larson Richard S. 
Published: October 2019

Abstract
Organ transplantation is a life-saving procedure for patients. However, acute T-cell mediated graft rejection occurs in a significant quantity of recipients, which is a potentially life-threatening condition. Current diagnostic criteria for transplant rejection rely on invasive tissue biopsies and relatively non-specific clinical features. We hypothesized that nanoparticles could be developed that specifically bind to T-cells. Furthermore, we postulated that these nanoparticles could be detected non-invasively by a Superconducting Quantum Interference Device (SQUID). Nanoparticles were conjugated through either amino or carboxy termini to either anti-CD2 or anti-CD3 antibodies. All three cell types were found to bind exclusively to specifically targeted antibody-tagged nanoparticles as identified through light microscopy and SQUID detection. Using receptor site densities determined by flow cytometry and the magnetic moment per nanoparticle results, SQUID is capable of determining the number of labeled cells in a sample. Our results indicate that T-cells bind approximately 1×104 nanoparticles per cell. A SQUID sensor array also allows for in vitro localization of discrete sources of labeled T-cells. Using a phantom as a surrogate for organ or soft tissue, we also demonstrate that discrete signals of 105 T- cells can be detected at 10 cm from the SQUID detector. These results demonstrate the potential utility of antibody-tagged nanoparticles as a contrast agent and SQUID as a noninvasive and sensitive detection device in patients who may be undergoing T-cell mediated graft rejection. In addition, the extreme sensitivity of this technique may provide not only a means of early, non-invasive detection of impending T-cell mediated rejection, but also an approach to titrating chemotherapeutics for treating rejection.
Rapid Expansion of Acute Myeloid Leukemia-Reactive Cytotoxic T Cells from CD8+CD62L+ Blood Lymphocytes of HLA-Matched Healthy Donors In Vitro
Authors: Distler Eva , Woelfel Catherine , Pesth Sylvia , Kraus Nina , Wehler Thomas C. , Meyer Ralf G. , Nonn Marion , Huber Christoph , Woelfel Thomas , Herr Wolfgang 
Published: October 2019

Abstract
Allogeneic cytotoxic T-lymphocyte (CTL) therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of growing leukemia-reactive CTLs from healthy donors in vitro. We established an allogeneic mini-mixed lymphocyte-leukemia culture (MLLC) approach by stimulating comparably small numbers (104/well) of CD8+ T cells isolated from healthy donors against irradiated primary AML blasts in 96-well plates. Prior to use, CD8+ T cells were immunomagnetically separated into a CD62L(high)+ subset enriched for naive precursors and central memory cells as well as a CD62L(low)+/negative subset containing effector memory cells. The culture medium contained IL-7, IL-12, and IL-15. After 2 weeks, IL-12 was replaced by IL-2. Mini-MLLCs were performed in seven different healthy donor-AML pairs that were matched for HLA class I according to high-resolution molecular typing. Following 2 weekly re-stimulations with primary AML blasts, mini-MLLC responder populations were analyzed for reactivity on day 19 of culture using split-well IFN-gamma ELISPOT assays. AML-reactive CD8+ T-cell responders were obtained from all 7 donor-AML pairs. The majority of reactive cultures originated from the CD62L(high)+ subfractions. In 4 out of 7 pairs MLLC responder populations mainly recognized AML blasts, but not Epstein-Barr virus transformed B-lymphoblastoid cell lines of donor and patient origin. The AML-reactive CD8+ T cells were restricted by single HLA class I alleles as determined by blocking experiments using a panel of HLA allele-specific monoclonal antibodies. Representative mini-MLLC responders demonstrated strong cytotoxicity against primary AML blasts in 51Chromium-release assay. Cross-reactivity testing identified an HLA-A*0201-restricted CTL population that recognized AML blasts much stronger than non-malignant monocytes of the same patient. This CTL neither recognized recipient-derived primary fibroblasts nor other hematopoietic cells suggesting a leukemia-associated rather than a minor histocompatibility antigen as the target structure. Several MLLC-derived CTL populations expressed unique T cell receptor Vbeta chains consistent with clonal origin from AML-reactive precursors. Multiple CTL responders reached a cell yield exceeding 108 after 6 to 10 weekly re-stimulations with AML blasts. Our results suggest that in healthy individuals most AML-reactive CD8+ CTLs originate from the CD62L(high)+ peripheral blood subpopulation containing naive precursor and central memory T cells. This mini-MLLC approach allows the rapid expansion of AML-reactive CD8+ CTLs from HLA-matched healthy donors to cell numbers sufficient for antigen identification strategies or adoptive immunotherapy trials.
Vaccination with Host Dendritic Cells Induces Graft-Versus-Leukemia Responses without Severe Graft-Versus-Host Disease in a Preclinical Mouse Model for Allogeneic Stem Cell Transplantation.
Authors: van der Voort Robbert , Ruben Jurjen , Vos Annemieke , Philippens Marielle , de Witte T.M. , Dolstra Harry 
Published: October 2019

Abstract
Allogeneic stem cell transplantation is a potent treatment for patients with leukemia. This therapeutic effectiveness is largely attributed to the graft-versus-leukemia (GVL) response during which alloreactive donor CD8+ T cells eradicate minor histocompatibility antigens (MiHA)-expressing leukemic cells. Unfortunately, recognition of MiHA expressed in healthy tissues, including the liver, skin and gut, often results in morbidity and even mortality due to the development of graft-versus-host disease (GVHD). Antigen presenting cells (APC) play an essential role in the activation of alloreactive T cells and thus in the induction of GVL responses and GVHD. Using several in vivo models it has been suggested that host APC, which present endogenous MiHA to donor CD8+ T cells, are more potent inducers of alloreactive responses than donor APC. Here, we compared the ability of host APC versus donor APC to activate alloreactive T cells in vitro and to induce alloreactions in vivo. For this purpose, we isolated T cells and cultured bone marrow-derived dendritic cells (DC), the most potent APC, from B6.SJL ‘host’ mice and MHC (H-2b)-matched but MiHA-mismatched C3.SW ‘donor’ mice. Following their activation by the TLR4 ligand lipopolysaccharide for no longer than 5 hours, both host and donor DC showed a fully mature phenotype (MHCIhigh MHCIIhigh CD40+ CD80high CD86high), and comparable migratory behavior towards lymph node-specific chemokines. In primary mixed leukocyte reactions host DC were 2–3 times more potent than donor DC in inducing donor CD8+ T cell proliferation. To determine the role of host and donor DC in the development of GVL and GVHD, we established a preclinical mouse model (C3>B6) for allogeneic stem cell transplantation. Repopulation analyses demonstrated that T cells, B cells and myeloid cells from blood and spleen of transplanted mice were for more than 95% of donor origin. Transplanted mice were subsequently vaccinated intravenously with 3 weekly doses of 3 × 105 host or donor DC. DC vaccination, which did not lead to significant GVHD, was followed by an intravenous challenge with 106 GFP-expressing mouse AML (C1498) cells. While sham-vaccinated mice died quickly due to leukemia progression, vaccination with both host and donor DC resulted in diminished leukemia growth. However, only vaccination with host DC, and not donor DC, resulted in a (partial) rescue from challenge. These results suggest that host DC are the most potent inducers of GVL reactions without causing significant GVHD. We believe that these data will be instrumental in designing appropriate DC-based immunotherapies in transplanted leukemia patients.
BCR-ABL Specific Sequences Are Required To Prevent Ph+ Acute Lymphoblastic Leukemia in Syngeneic Mice by DNA Vaccination.
Authors: Köchling Joachim , Prada Javier , Bahrami Masoud , König Merediz Sven A. , Stripecke Renata , Seeger Karl , Henze Günter , Wittig Burghardt , Schmidt Manuel 
Published: October 2019

Abstract
Although 70 – 80 % of children with childhood acute lymphoblastic leukemia (ALL) can be cured by poly-chemo-therapy, the prognosis of patients with Philadelphia chromosome positive (Ph+) ALL remains poor. Therefore, new relapse prevention strategies are needed for patients with Ph+ ALL during remission. We have shown previously, that vaccination of mice with leukemia cell lines modified to express costimulatory molecules and cytokines induce a systemic immunity against the syngeneic BCR-ABLp185 expressing cell line BM185. However, the difficulties to culture and transfect human leukemia cells limit the clinical application of leukemia cell based vaccines. Thus, we evaluated the pre-immunization of mice with DNA based vaccines subsequently challenged by the cell line BM185. Ballistic transfer of minimalistic immunogenically defined gene expression (MIDGE) vectors encoding a BCR-ABLp185 fusion specific peptide or GM-CSF were used for in vivo transfection of murine skin. In addition, we used double stem-loop immunomodulators (dSLIM), containing three CpG-motifs as non-specific immune adjuvant. We provide survival data that shows specific immunization and protection of mice which received the complete vaccine BCR-ABL/GM-CSF/dSLIM. Mean tumor-free survival (p=0.019) and overall survival (p=0.008) were significantly longer compared to non-vaccinated mice and 26.7% survived and never developed leukemia. In contrast, tumor-free and overall survival of mice immunized with either dSLIM or GM-CSF alone or both dSLIM and GM-CSF was not significantly longer compared to non-vaccinated mice. Similarly, substitution of BCR-ABL by irrelevant TEL-AML1 sequences abolished the vaccine efficacy of the BCR-ABL/GM-CSF/dSLIM vaccine. The biometrical data were confirmed by CTL assays which showed that specific lysis was significantly higher after vaccination with BCR-ABL/GM-CSF/dSLIM compared to GM-CSF/dSLIM (p<0.05) and to naïve mice (p<0.005). Previously, we have shown in T-cell depletion studies that CD8+ T cells were the effector cells in the BM185 cell vaccine model and we suggest that CD8+ T cells also play an essential role in the BM185 DNA vaccine model. Together, we provide biometrical and functional data that DNA-based vaccination with BCR-ABLp185 fusion specific sequences, GM-CSF and dSLIM can protect mice against a lethal Ph+ ALL challenge.
Transduction of Macaque Hematopoietic Repopulating Cells with Lenti and Foamy Retroviral Vectors with MGMT Selection Cassettes To Evaluate AIDS Gene Therapy Strategies.
Authors: Trobridge Grant D. , Beard Brian C. , Dickerson David , Gooch Christina , Olsen Philip , Fletcher James , Li Ming-Jie , Hu Shiu-Lok , Rossi John J. , Kiem Hans-Peter 
Published: October 2019

Abstract
AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lenti and foamy retroviral vectors that inhibit HIV replication by RNAi in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. However inefficient gene delivery to hematopoietic stem cells has limited progress for AIDS gene therapy. We have developed both lenti and foamy retroviral vectors that contain methylguanine-DNA-methyltransferase (MGMT) expression cassettes to allow for in vivo selection, and have transduced macaque (M. nemestrina) long term repopulating cells with both vector systems. Following transplantation we observed rapid engraftment and levels of gene marking in the peripheral blood that should allow us to in vivo select both lenti and foamy-marked hematopoietic repopulating cells. In one animal transplanted with a lentiviral vector we obtained marking at 265 days post-transplant of over 30% in peripheral blood granulocytes and 20% in peripheral blood lymphocytes prior to in vivo selection. Anti-SHIV/HIV transgene cassettes targeting tat and rev that allow for potent inhibition of SHIV and HIV replication in vitro have been incorporated into both lenti and foamy vectors and we have transduced macaque long term repopulating cells with lenti vectors containing an anti-HIV cassette. We are currently developing protocols for efficient in vivo selection and future studies will investigate the ability of macaque hematopoietic repopulating cells transduced with lenti and foamy MGMT anti-HIV vectors to inhibit SHIV infection ex vivo and in vivo.
Current Practice of Testing for Hereditary Thrombophilia in the Netherlands.
Authors: Coppens Michiel , van Mourik Jan A. , Eckmann Carel M. , Middeldorp Saskia 
Published: October 2019

Abstract
Inherited thrombophilia is known to increase the risk for venous thromboembolism (VTE). Furthermore, an association between recurrent pregnancy loss and hypertensive pregnancy complications (including preeclampsia and HELLP syndrome) appears present. A relationship between thrombophilia and arterial cardiovascular disease could not be demonstrated in most studies. Since thrombophilia is prevalent in patients with VTE, testing for these abnormalities often reveals positive test results which may lead to widespread testing. This is also the case in patients with various other conditions. However, there is little convincing evidence about effectiveness of testing due to the uncertainty with respect to therapeutic consequences of a positive thrombophilia test results.
To assess the current practice of thrombophilia testing in The Netherlands and to get an impression of the therapeutic implications of testing for patients, simple questionnaires were sent out to physicians that ordered tests for protein S, C, or antithrombin activity, factor V Leiden (or APC resistance) or the prothrombin 20210A mutation in 1998 consecutive individuals who were investigated between November 2003 and March 2004. The tests were performed in a laboratory that serves as a diagnostic facility for mainly non-academic hospitals and general practitioners throughout the Netherlands. Of a total of 1265 returned questionnaires, 1130 were suitable for analysis (response rate 63%). Of the tested individuals, 64% were female (median age 38 years, inter-quartile-range [IQR] 30–50) and 35% were male (median age 51, IQR 41–59). Tests were ordered by internists (37%), gynaecologists (20%), neurologists (15%) and general practitioners (13%). Only 42% of the tested individuals had experienced VTE, 23% arterial cardiovascular disease, and 17% had had pregnancy complications. A known carrier or VTE in the family was the reason for testing in 180 asymptomatic individuals (16%). Testing had had no therapeutic consequences in 869 tests (77%). In 32% of these patients, physicians stated that had the test revealed a thrombophilic defect, this would not have altered management of the patient.
This study shows that testing for thrombophilia occurs very often in patients with various conditions. In the majority of cases, the results do not change therapeutic management of tested patients. Widespread thrombophilia testing is costly and may cause unnecessary concern in carriers. This study underscores the need to acquire high level evidence about its effectiveness that should include clinical outcomes as well as quality of life and costs.
Discrimination of Health States in Follicular Lymphoma with Utilities Derived from the EuroQOL EQ5D Instrument.
Authors: Friedlich Jessica D. , Cheung Matthew C. , Imrie Kevin R. , Hales Brigette , Mittmann Nicole , Buckstein Rena 
Published: October 2019

Abstract
Introduction: Health utilities (HU) elicited directly from patients have immediate application in facilitating medical decision-making and cost-effectiveness determinations. Collection of HU using generic health status scales enable comparisons across diseases, but may not be sensitive to variations in health states within a particular disease. The EuroQOL EQ5D is a generic scale that has never been used to generate utilities in a broad spectrum of follicular/indolent lymphoma patients and has not been validated in this context.
Methods: A consecutive, cross-sectional cohort of patients attending an outpatient malignant hematology clinic at a major cancer centre (Toronto, Canada) represented the eligible study population. Patients with a diagnosis of FL or other indolent NHL who consented to the study were asked to complete demographic and disease specific questionnaires in addition to the EuroQOL EQ-5D and Functional Assessment of Cancer Therapy (FACT)-Lymphoma quality of life assessment tools.
Results: Eighty-four patients completed the survey study (>95% response rate). Mean age was 58.7 (+/− 13.8 SD) and 55% were male. Diagnoses included FL (55%), CLL (25%), and other indolent NHL (20%). The majority of patients presented in advanced stage (stage III–IV; 65%) and had received some therapy to date, although 29% were still being observed at the time of survey administration. The mean utility score for the population was 0.84 (+/− 0.24; range 0–1). We evaluated the construct that patients receiving active treatment and those who were not in remission would have lower utility scores. Indeed, utilities were higher in patients being observed (0.91 +/− 0.16) compared to those in first remission (0.84 +/− 0.25), subsequent remissions (0.81 +/− 0.20), or those who were receiving active chemotherapy (0.75 +/− 0.27; p=0.049). Patients who were being followed in ongoing remission also trended to higher health status values (mean 0.88 +/− 0.21) compared to those who were not in remission (0.80 +/− 0.22; p=0.15). Utilities elicited from the EQ5D showed a moderate correlation with a criterion measure of quality of life, the FACT-Lymphoma scale (Spearman correlation coefficient 0.54, p<0.0001).
Conclusions: Utilities from the EQ5D are able to discriminate various health states in patients with follicular and other indolent lymphomas and the scale demonstrates construct and criterion validity in this population. HU scores are particularly sensitive to changes in patient remission and treatment status.
Regulation of Cathpsin L by the Serpin Protein C Inhibitor.
Authors: Fortenberry Yolanda , Bialas Ryan , Mitchell Candace , Church Frank C. 
Published: October 2019

Abstract
Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation and fibrinolysis including thrombin and activated protein C (APC). The physiological role of PCI, however, remains under investigation since PCI both inhibits and promotes thrombin generation. The cysteine protease, cathepsin L, has been shown to play a role in many physiological processes including cardiovascular disease, atherosclerosis, blood vessel remodeling, and tumor cell invasion. Recently, several serpins have been described to inhibit both serine and cysteine proteases and they are termed “cross-class” inhibitors. The goal of this project was to determine if PCI inhibits cathepsin L activity and if so, does this inhibition process mimic the mechanism of serine proteases. Previous studies have shown that the prototypical serpin, antithrombin (AT), inhibits the cysteine proteases papain and cathepsin L. We found that PCI is a more efficient inhibitor of cathepsin L than AT with an inhibition rate (k2) of 1.5 × 106 M−1min−1 compared to 5.2 × 104 M−1min−1 for AT. Also, PCI is a more efficient inhibitor of cathepsin L than either thrombin or APC whose inhibition rates are 5.7 × 105 M−1min−1 and 3.4 × 104 M−1min−1, respectively. In contrast to AT, PCI does not inhibit papain. Thrombin inhibition by AT and PCI is accelerated in the presence of glycosaminoglycans such as heparin and heparan sulfate. The inhibition of cathepsin L by PCI is not accelerated in the presence of heparin suggesting either that cathepsin L does not bind heparin or that heparin is not required for accelerated inhibition of cysteine proteases. Interestingly, a reactive site P1 mutant (R354A) of PCI does not inhibit thrombin but does inhibit cathepsin L at rates comparable to wild-type PCI. This implies that the P1 residue of PCI does not determine specificity for inhibition of cathepsin L unlike for thrombin and APC. We believe that the specificity is primarily determined by the hydrophobic Phe residue located at the P2 position since other serpins that inhibit cathepsin L contain either a Phe or Val at the P2 position. Mutating the P14 residue (T341R; mutation in the hinge region) of PCI results in the conversion of PCI from an inhibitor to a substrate. As expected, the PCI-P14 mutant does not inhibit either thrombin or cathepsin L. Another characteristic of the serpin inhibition mechanism is formation of a bi-molecular SDS stable complex. We found that wild-type PCI and PCI-P1 mutant both form a stable complex with cathepsin L under non-reducing conditions. Lastly, the wild-type PCI-cathepsin L interaction has a stoichiometry of inhibition (SI) value of 1.6. This indicates that PCI is an effective and possibly a physiologically relevant inhibitor of cathepsin L. Regulating cathepsin L by serpins like PCI may be a novel and new pathway of regulation of hemostasis-thrombosis, cardiovascular and metastatic diseases.