bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fas Down-Modulation in Epstein Barr Virus (EBV)-Specific Cytotoxic T-Lymphocytes (CTLs) Reduces Their Sensitivity to Fas/Fasl-Induced Apoptosis.
Authors: Dotti Gianpietro , Savoldo Barbara , Pule Martin , Straathof Karin C. , Biagi Ettore , Eric Yvon , Vigoroux Sthephane , Rooney Cliona M. , Brenner Malcolm K. 
Published: April 2020

Abstract
Adoptive immunotherapy with Epstein Barr Virus (EBV)-specific CTL has been successfully used to treat patients with EBV related malignancies including Post Transplant Lymphoproliferative disorder, Hodgkin’s lymphoma and Nasopharyngeal carcinoma. However, these and other potentially immunogenic tumors have evolved evasion strategies that subvert the effectiveness of the immune response. One such strategy involves expression of FasL, which likely impedes the host immune response by accelerating the apoptotic death of Fas-expressing tumor infiltrating T-cells. EBV-specific CTLs, like most effector memory T cells express high levels of Fas and are highly sensitive to cross-linking of the ligand. We therefore determined whether EBV-specific CTLs could be genetically modified to resist FasL-mediated immune evasion. We used retroviral siRNA (pSUPER.retro) directed against the Fas gene product to knockdown receptor expression in tumor-antigen specific CTLs. Transduction of CTLs with siRNA targeting Fas mRNA significantly knocked down Fas expression compared to control cells (Fas MFI 107 ± 47 vs. 402 ± 47, p&lt;0.001). This effect was paralleled by a significant reduction of apoptosis induced by the Fas agonistic antibody (clone CH-11) in modified CTLs compared to control cells (23% ± 3% vs. 44% ± 7% p=0.006). Fas down-modulation was stable over time in CTLs modified, and addition of CH-11 antibody to the culture selected a uniformly Faslow CTL population (Fas MFI 79 ± 29) that was entirely resistant to FasL mediated lysis. However, germ line deletions as well as function-impairing mutations of Fas and FasL can induce T-lymphoproliferation and autoimmune diseases. We therefore compared the growth characteristics and the antigen specificity of unmodified and Fas knockdown CTLs. Survival and proliferation of genetically modified CTLs remain completely dependent on antigen specific stimulation and the presence of other physiological growth signals. Moreover, as assessed by the analysis of the Vb TcR repertoire, IFN-g-release (Elispot) and tetramer staining the modified CTLs remained polyclonal and conserved their antigen specificity. These data suggest that responsiveness to this single death signal may be removed from an effector-memory population of CTLs without adversely affecting their safety. Tumor-antigen-specific CTLs with lower expression of Fas receptor should have a selective functional and survival advantage over unmodified CTLs in the presence of tumors expressing FasL, and may be of value for adoptive cellular therapy of such malignancies.
PLCβ Is Critical for T-Cell Chemotaxis In Vivo.
Authors: Bach Tami L. , Chen Qing-Min , Jordan Martha S. , Choi John K. , Wu Dianqing , Koretzky Gary A. , Zigmond Sally H. , Abrams Charles S. 
Published: April 2020

Abstract
Chemokines acting through G-protein coupled receptors play an essential role in both the immune and inflammatory responses. Phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) are two distinct signaling molecules that have been proposed as potential candidates in the regulation of this process. Studies with knockout mice have demonstrated a critical role for PI3Kγ, but not PLCβ, in Gαi-coupled receptor-mediated neutrophil chemotaxis. We compared the chemotactic response of peripheral T-cells derived from wild type mice with mice containing loss-of-function mutations of either PI3Kγ, or both of the two predominant lymphocyte PLCβ isoforms (PLCβ2 and PLCβ3). In contrast to neutrophils, loss of PI3Kγ did not significantly impair T-cell migration in vitro, although PI3K pharmacologic inhibitor experiments suggest that another isoform of this enzyme might contribute to T-cell migration. However, loss of PLCβ2β3 decreased chemokine-stimulated T-cell migration in vitro. Chelation of intracellular calcium by BAPTA-AM and Quin-2 AM decreased the chemotactic response of wild type lymphocytes, but pharmacologic inhibition of PKC isoforms by GF109203x did not impair T-cell migration. This suggests that the T-cell migration defect seen in the PLCβ2β3-null T-cells may be due to an impaired ability to increase the cytoplasmic calcium concentration, while there appears to be little requirement for PKC activity. Indeed, SDF-1α-induced calcium efflux was not detected in the PLCβ2β3-null lymphocytes. Compared to fluorescently labeled wild type T-cells, labeled PLCβ2β3 knockout T-cells migrated less efficiently into secondary lymphoid organs of recipient mice. This demonstrates that PLCβ is also required for migration in vivo. PLCβ2β3-null mice develop spontaneous skin ulcers starting around 3 months of age. Histological examination of the lesions revealed a dense inflammatory infiltrate composed of neutrophils, macrophages, and plasma cells, consistent with acute and chronic inflammation. Remarkably, lymphocytes, typical of chronic inflammation, were rare to absent by histology and by paraffin immunohistochemistry for CD3, also consistent with an in vivo migratory defect of T-cells.
These results show that phospholipid second messengers generated by PLCβ and isoforms of PI3K, other than PI3Kγ, play a critical role in lymphocyte chemotaxis. Collectively, our data demonstrate that although PLCβ-mediated signaling plays no role in neutrophil chemotaxis, it makes a substantial contribution to this process within T-lymphocytes.
CCL19 and CCL21 Chemokines Induce Endocytosis and Augment Antigen Presentation in Human Mature Dendritic Cells.
Authors: Ogasawara Masahiro , Tanaka Junji , Imamura Masahiro , Kasai Masaharu 
Published: April 2020

Abstract
Dendritic cells (DCs) are potent antigen presenting cells capable of regulating immune responses. DCs lose the ability to capture and process antigens during maturation. In the present study, we examined the effects of CCR7 ligands, CCL19 and CCL21, on endocytosis and antigen presentation in human mature dendritic cells. Immature DCs were generated from peripheral blood monocytes by culturing with GM-CSF and IL4 for 2–3 days. For maturation, immature DCs were cultured with the addition of TNFα, IL1β, IL6 and prostaglandin E2 for another 24 hours. Immature or mature DCs were incubated with FITC-dextran with or without CCL19. Immature DCs internalized FITC-dextran efficiently independent of the presence of CCL19 after 1 hour incubation. On the other hand, mature DCs scarcely internalized FITC-dextran without CCL19. In the presence of CCL19, however, mature DCs internalized FITC-dextran significantly (approximately 60% positive). The effect of CCL19 on the uptake of FITC-dextran in mature DCs was dose and time dependent. CCL21 exerted a similar effect on mature DCs. Next, we examined whether CCL19 facilitates antigen presentation in mature dendritic cells. CD4+ T cells were cultured with irradiated autologous mature DCs which had been incubated with leukemia cell lysate with or without CCL19. Marked proliferation of CD4+ T cells occurred only when these cells were cultured with mature DCs loaded with leukemia cell lysate in the presence of CCL19. This is the first demonstration that chemokines have a pivotal role in endocytosis and antigen presentation by human monocyte-derived dendritic cells to the best of our knowledge. These results demonstrated that generation of potent antigen-loaded mature DCs in relatively short term culture using various cytokines and chemokines may have an important clinical implication to facilitate DC-based immunotherapy.
Reduced Inhibitory KIR Receptors in Patients with NK-Lymphoproliferative Disease of Large Granular Lymphocytes (NK-LDGL) Leads to Dysregulated Killing.
Authors: Epling-Burnette P.K. , Bai Fanqi , Painter Jeffrey S. , Wei Sheng , Djeu Julie Y. , Loughran Thomas P. 
Published: April 2020

Abstract
NK-LDGL is associated with the expansion of CD3−, CD16+ and/or CD56+ lymphocytes, which express a skewed repertoire of NK receptors. (Epling-Burnette et al, Blood103:3431, 2004) Single anti-KIR antibody reactivity was noted in seven patients (54%) and “defective” KIR reactivity in four patients (31%) (n=13). Defective KIR expression was characterized by the loss of reactivity to all available antibodies. These patients expressed a large number of activating KIRs by genotype analysis.
Semi-quantitative RT-PCR demonstrated that lower than normal levels of RNA of the inhibitory KIR was present in some patients in contrast to normal NK cells. Consistent with a high level of activating receptors, we found the NK-LDGL cells have potent cytolytic function in both direct and redirected cytotoxicity assays. Overexpression of activating receptors in the absence of the appropriate inhibitory receptors may contribute to the bone marrow suppression commonly observed in these patients. We first performed Class I typing of the HLA-C locus of five patients with NK-LDGL using PCR-SSP (One Lambda, Inc, Canoga Park, CA). Phenotypes included Cw05/Cw16, Cw15/15, Cw07/12, Cw04/08, and Cw02/03. Each of the five patients had lower then normal levels of inhibitory KIR (KIR2DL1 and KIR2DL2/3) by flow-cytometry and by RT-PCR. In one patient, we examined the functional ability of these inhibitory receptors to block killing of tumor cells expressing cross-reactive Class-I ligands.
Homozygous Cw15/15 was expressed by NK-LDGL5, which is a cross-reactive epitope to Cw3 and recognized by KIR2DL2/3. NK cells from NK-LDGL5 were capable of killing both the class I-negative tumor target 721.221 cells and HLA-Cw3-transfected 721.221 cells suggesting that deficient levels of inhibitory KIR were expressed on the patients NK cells to block activating NK receptor-mediated killing. These results suggest that NK cells from patients with NK-LDGL could mount dysregulated autoimmune-mediated killing, which may impact disease pathogenesis.
Analysis of Clonal Contributions to T Lymphoid and Myeloid Lineages during Early Hematopoiesis Following Autologous Transplantation in the Rhesus Macaque.
Authors: Adler Rima L. , Hematti Peiman , Calmels Boris , Ferguson Cole J. , Keyvanfar Keyvan , Wu Colin , Follmann Dean , Dunbar Cynthia E. 
Published: April 2020

Abstract
Hematopoietic stem cells are defined by their ability both to self-renew and to differentiate into cells that repopulate all hematopoietic lineages. A common lymphoid progenitor (CLP) from mouse marrow has been phenotypically purified and shown to only transiently repopulate all lymphoid lineages (T, B, and NK) but not myeloid lineages when transplanted into lethally irradiated recipients (Kondo et al, 1997). Using the rhesus macaque as a model for human hematopoiesis, we asked whether lineage-restricted repopulating cells contribute to hematopoietic output and for how long following engraftment. In order to follow the progeny of progenitor or stem cell clones in vivo, retroviral vectors were used to mark primitive hematopoietic cells before reinfusion into lethally irradiated autologous recipients. The conditions utilized result in high level of stable retroviral marking in all lineages. We have previously reported use of the sensitive Linear Amplification Mediated - Polymerase Chain Reaction (LAM-PCR) showing that a highly polyclonal set of stable clones accounts for myelopoiesis (Schmidt et al, 2002). In the current study, detailed characterization of the full array of clones contributing to granulocytes, T-lymphocytes, and B-lymphocytes post-engraftment was performed, via sequence analysis of individual clones delineated by individual insertion site sequences at various time points following transplantation of CD34+ cells. Thus far, we have identified 59 granulocyte and 148 T-lymphocyte clones at 1 month following transplantation. Most clones detected at this time point uniquely contribute to only the lineage from which they were identified, only three clones were common to both lineages (1.5% overlap, 95%CI=(0–3.1%)), suggesting independent cells are responsible for hematopoietic reconstitution at this early time point. Six-months following transplantation fewer clones were detected to contribute to hematopoiesis. Interestingly, of the 22 granulocyte and 46 T-lymphocyte clones identified, only two were shared between both lineages (3.0% overlap, 95%CI= (0–7.2%)), further supporting the hypothesis that lineage committed progenitors are responsible for both early and potentially more stable hematopoiesis in this model. To our knowledge, no other study has provided such an extensive characterization of clones contributing to early or more stable in vivo hematopoiesis in the primate model. Preliminary sequence specific tracking experiments reveal that the clones contributing at six-months remain lineage specific over time. Further tracking of clones is being conducted to fully assess the role of such lineage specific progenitor cells in B-lymphopoiesis and long-term hematopoiesis.
Entry Bias: Order of Patient Entry Influences Outcome in Trials of Allogeneic Transplant for Renal Cell Carcinoma.
Authors: Artz Andrew S. , Kocherginsky Masha , Van Besien Koen 
Published: April 2020

Abstract
Early trials evaluating nonmyeloablative allogeneic hematopoietic transplant (NST) for metastatic renal cell carcinoma (RCC) have reported widely discordant results. We performed a meta-analysis of the published literature to identify prognostic factors for NST. However, known RCC prognostic factors are not often detailed in these studies, hindering the identification of patient differences among studies. We explored the order of patient entry as a surrogate marker for patient selection, hypothesizing that patient selection, rather than treatment differences, accounted for early promising results. Patient specific data and adequate follow-up were available for 76 patients from six studies. The rank of patient entry on each individual trial was recorded as well as ≥ grade 2 acute graft-versus-host disease (aGVHD), recipient age, recipient sex, response (partial plus complete), and survival. Multivariable analyses for response and survival were modeled using logistic and cox proportional hazards regression, respectively. The mean overall response rate was 22/76 (29%), with responses across individual studies significantly varying from 0% to 57% P =.009 by Chi-square). Median Kaplan-Meier survival was 263 days. Neither age nor sex was significantly ( associated with response or survival. Acute GVHD occurred in 42% of individuals and correlated with response (OR=9.9, P =0.012) but not survival (HR=1.53, P=0.28). When adjusting by study, later patient entry rank reduced the probability of response (OR=0.30, P =0.007) and survival (HR=1.89, P =0.006). Alternatively, being among the first five patients enrolled in a given study relative to subsequent patients, increased the probability of response (OR=6.69, 95% CI 1.95–39.1, P=0.005), even when adjusting for aGVHD, and afforded a survival benefit (HR= 0.45, 95% CI 0.022 −0.92, P=0.028). The prognostic strength of patient entry rank strongly suggests “entry bias” in patient selection. This bias potentially accounts for the large variation in outcome among studies and for the promising results in early studies. Entry bias analysis offers a novel method to assess early phase trials for selection bias, when detailed individual prognostic information is lacking. Further study is warranted to determine to what extent, if any, entry bias occurs in other clinical trial settings.
Figure Figure
Importance of Transplantation Variables in Marrow to Muscle Conversion.
Authors: Abedi Mehrdad , Greer Deborah A. , Foster Bethany A. , Colvin Gerald A. , Harpel Jashua A. , Dooner Mark S. , Quesenberry Peter J. 
Published: April 2020

Abstract
We have previously shown that conversion of marrow to skeletal muscle is increased by cardiotoxin injury and we have demonstrated levels of up to 12% conversion. We have now used the same model of cardiotoxin injury to determine variables that affects this conversion. Muscle fibers were defined by their morphology and presence of desmin and dystrophin staining and absence of CD45 staining. In a GFP to B6 transplant model, there was a significant increase in the incidence of muscle chimersim with increasing the dose of marrow donor cells unrelated to the level of hematopoietic chimersim. Radiation was a critical factor and a combination of 500cGy of whole body and 500cGy of lower extremity radiation was found as the optimal model. However radiation impact was mostly due to its effect on hematopoietic chimerism and there was very little local effect on muscle. Higher doses of radiation were detrimental and resulted in significant muscle atrophy. Transplantation of marrow cells to NOD/Scid mice without radiation resulted in muscle engraftment although this was a rare event. Furthermore, timing of the cardiotoxin injury relative to the transplantation was an important factor since injection of cardiotoxin before or right after transplant resulted in minimal conversion while injury seven days or more after transplant resulted in significant conversion events. When sublethal doses of cardiotoxin were injected systemically after marrow transplant GFP positive muscle fibers were seen but the incidence was much lower than direct intramuscular injections of cardiotoxin. Multiple cycles of mobilization and injury also improved the incidence of GFP positive fibers in the regenerated muscle. We conclude that in addition to injury, cell mobilization and cell selection, improvements in the efficiency of this process can be achieved by applying specific doses and distribution of radiation (i.e. local and systemic), by giving the appropriate doses of marrow cells and by selecting the appropriate time for injury. Combining these models gives quantitatively significant fiber conversions approaching levels that would be therapeutically effective in the treatment of muscular dystrophies.
Variation in Transfusion Rates amongst Canadian Academic and Community Medical Centers.
Authors: Hutton Brian , Fergusson Dean , Hebert Paul C. , Tinmouth Alan , McIntyre Lauralyn , Kmetic Andrew 
Published: April 2020

Abstract
Purpose: Few recent studies have examined approaches to transfusion practice in high-risk patients. We therefore documented practice variation following repair of hip fracture or cardiac surgery, as well as those requiring intensive care following a surgical intervention or multiple trauma.
Methods: We documented rates of red cell transfusion in 41,568 patients admitted to 11 hospitals across Canada between August 1998 and August 2000 as part of a retrospective observational cohort study. Adjusted odds of transfusion, median number of transfusions per patient and median number of transfusions per patient day were compared across sites. We also compared mean nadir hemoglobin concentrations from center to center in the subgroup of 7,552 patients that received red cells.
Results: The overall rate of red cell transfusion was 38.7 %, and ranged from 23.8% to 51.9% across centers among the 41,568 peri-operative and critically ill patients. Women were more likely to be transfused (43.7% versus 35.3%, p&lt;0.0001), with higher rates of transfusion in 8 of 11 centers. Following adjustment for the effects of age, gender, severity of illness and the presence of twelve different comorbidities, the odds of transfusion ranged from 0.44 to 1.53 overall, from 0.42 to 1.22 in patients undergoing a hip fracture repair, from 0.72 to 3.17 in cardiac surgical patients undergoing cardiac surgery, and from 0.27 to 1.11 in critically ill and trauma patients (figure 1). Examination of both median number of units transfused and median number of units transfused per patient day also revealed between-site variation (figure 2). In the 7,552 transfused patients, the mean adjusted nadir hemoglobin was 74.0±4.83g/L overall, and ranged from 66.9±1.7g/L to 84.5±1.6g/L across centers. Similar differences among centers were observed amongst hip fracture patients (71.2±2.9g/L to 82.8±1.7g/L), cardiac surgical patients (65.7±1.1g/L to 77.3±1.0g/L) and critically ill and trauma patients (66.1±3.04g/L to 87.5±2.5g/L).
Conclusions: We noted significant differences in the rates of red cell transfusion and nadir hemoglobin concentrations in various surgical and critical care settings. Such variation provides the impetus for investigators to explore its potential causes, and furthermore to attempt to define optimal transfusion practice in a variety of clinical settings by conducting randomized controlled trials.
Mutation Status of Imatinib Mesylate-Resistants CML Patients and Clinical Outcomes: A French Multicenter Retrospective Study for the fiLMC Group.
Authors: Corm Selim , Nicollini Franck , Borie Dominique , Sorel Nathalie , Leguay Thibault , Hayette Sandrine , Facon Thierry , Roy Laurence , Giraudier Stephane , Marit Gerald , Preudhomme Claude , Michalet Mauricette , Mahon Francois-Xavier , Thuilliez Michel , Guilhot Francois , Roche-Lestienne Catherine 
Published: April 2020

Abstract
Imatinib mesylate (IM) is highly effective in newly diagnosed CML. Unfortunately, acquired abl-kinase domain mutations are commonly identified to be associated to resistance or relapse in patients with advanced disease. Distinct mutation exhibit varying degrees of resistance, and mutation occurring in the ATP-binding loop (P loop) or in positions in direct contact with IM (i.e. T315) may be correlated with subsequent disease progression.
In this study, we investigated the bcr-abl mutation status of imatinib-resistant CML patients and correlated to the disease outcome.
Patients and methods: From CML patients diagnosed in 5 French centers, 67 IM-resistants patients presenting mutations by direct sequencing of the abl kinase domain at time of resistance or relapse were included in this study. When starting IM monotherapy (J1) at 400 to 600 mg per day, 45 patients were in chronic phase of CML, 12 patients were in accelerated phase of the disease and 7 patients were in blast crisis. The median duration of IM is 29 months (range 3–54) and the median delay between diagnosis and J1 is 34 months (range 0–168). At the end of the study, 23 patients were dead. According to the mutation status, a Kaplan-Meier curve was construct for time to death to J1, and survival were compared using log rank test.
Results: By direct sequencing, 17 patients presented mutations in the P-loop (residues 252 to 255), 10 patients presented the T315M mutation alone, 1 patient had the T315 and the M351 mutations, and 39 patients presented mutations in other kinase domains. According to the mutation status, survival difference between patients presenting P-loop mutations or mutations in an other abl-kinase (including T315), no significant difference was observed (p=0.07). However, survival difference between patients presenting P-loop mutations, T315 mutation and mutations in an other abl-kinase domains was highly significant (p=0.0031, figure). The median survival for the P-loop and T315 mutated patients was of 39 months and 29 months respectively, and median survival not reached for others.
Conclusion: By this study we confirm that either the T315 mutation or mutations in the P-loop had a poor prognosis. Detailed correlation analysis of mutation status according to clinical features and response to increased IM-dose will be reported.
Figure Figure