bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulatory T Cells Do Not Affect Human Hematopoietic Stem Cell Engraftment and Prevent T Cell Alloreactivity Against CD34+ Cells: A Preclinical Study.
Authors: Mahmud Dolores , Chunduri Sandeep , Mahmud Nadim , Van Den Dries Lennert , Maciejewski John J. , Rondelli Damiano 
Published: October 2019

Abstract
We have previously demonstrated that allogeneic blood T cells stimulate cord blood (CB) CD34+ cell differentiation into professional antigen presenting cells (APC) in-vitro and in-vivo (Abbasian J, Blood2006:108:203–208). In this study we immunomagnetically selected human CD4+CD25+ regulatory T cells (Tregs) and showed that >80% of these cells were positive for FoxP3 intracellular expression. Then we tested whether Tregs may affect CB CD34+ cell clonogenic activity in-vitro an in-vivo, and if co-incubation of Tregs and CD34+ cells may modify the phenotype and function of Tregs. A colony-forming cells (CFU-C) assay performed with CD34+ cells mixed with allogeneic Tregs at 1:2 ratio resulted in comparable numbers of Granulocyte- Macrophage CFU (CFU-GM), burst-forming unit-erythroid (BFU-E) and CFU-Mix as compared to cultures with CD34+ cells alone (p=0.2, p=0.5 and p=0.5, respectively)(n=3 exps). Human CD34+ cells were co-transplanted with human CD4+CD25+ allogeneic Tregs into NOD/SCID mice at 1:1 and 1:2 ratio. After 6 weeks mice marrow was harvested and showed a 1.3±1.1% (n=3 mice) and 1.6±0.8% (n=4 mice) engraftment of huCD45+ cells, respectively, which was comparable to the engraftment observed in control animals transplanted with CD34+ cells alone (1.4±0.4). In addition, among the engrafted huCD45+ cells similar proportion of CD33+ myeloid cells, CD14+ monocytes and CD1c+ dendritic cells were observed in the three groups of animals. Mixed lymphocyte culture (MLC) experiments showed that irradiated CD34+ cells stimulated brisk proliferative responses of CD4+CD25- cells (S:R=1:2), but did not induce any proliferation of Tregs (n=3 exps). After incubation with CD34+ cells in the presence of IL2, on average >80% CD4+CD25+ cells maintained the intracellular expression of FoxP3 and surface expression of CD62L and CD152 (n=3 exps). Then, Tregs autologous to CD34+ cells were isolated from the CB CD34- cell fraction while allogeneic Tregs were isolated from healthy individuals’ peripheral blood. When 2.5 x 104 autologous or allogeneic Tregs were added to an MLC with 2.5 x 104 irradiated CD34+ stimulator cells and allogeneic responders at 1:2 ratio, they suppressed T cell alloreactivity to CD34+ cells on average by 68±14% and 41±16%, respectively (n=3 exps). Our findings suggest that co-transplantation of CD34+ cells and autologous or allogeneic Tregs may allow normal stem cell engraftment while limiting T cell alloreactivity. These results will prompt the design of new strategies in allogeneic hematopoietic stem cell transplantation, particularly in an HLA incompatible setting.
A New Mice Model of Graft Versus Host Disease Based on Chemotherapy Conditioning.
Authors: Sadeghi Behnam , Hassan Zuzana , Aghdami Nasser , Rozzel Bjorn , Abedi-Valugerdi Manuchehr , Hassan Moustapha 
Published: October 2019

Abstract
Background: Transplantation related complications such as graft versus host disease (GVHD) are limiting factors for allogeneic haematopoietic stem cell transplantation (HSCT). The majority of mice models of GVHD are based on the use of radiotherapy as a conditioning regimen. However, these models may not explain mechanisms of HSCT related complications following chemotherapy-based conditioning regimen.
Aims: The aim of this study was establishment of a mice model of GVHD using the chemotherapy as conditioning regimen.
Methods: Recipient female BALB/c (H-2Kd) were conditioned using busulfan (Bu) in total dose of 80 or 100 mg/kg followed by cyclophosphamide (Cy) in total dose of 200 or 300 mg/kg. Twenty millions of bone marrow (BM) cells and 30×106 spleen cells of male C57BL/6 (H-2Kb) were injected into the recipient mice in allogeneic setting. As a control, syngeneic HSCT using female BALB/c as recipients and donors and the same conditioning regimen and cell doses as in allogeneic setting was used. Acute GVHD manifestations and histo-pathological changes in skin, liver and intestine were evaluated at different time points after BMT. The chimerism and tissue mapping were studied by FACS, immunohistochemistry and FISH analysis. Affected skin was examined by FACS analysis to find donor T cells. Cytokines level were measured by using luminex assay.
Results: Infusion of 20×106 BM cells and 30×106 spleen cells from C57BL/6 mice to BALB/c mice conditioned with 80 or 100mg/kg Bu plus 200mg/kg Cy induced lethal acute GVHD between days 5–7 post transplantation. Histological and clinical signs of GVHD were found in 85% and 100% of the allogeneic transplanted mice based on the intensity of conditioning. About 35% of mice in allogeneic group died due to GVHD progression. The number of donor T cell in the recipient skin increased with GVHD progression and was correlated to the level of chimerism in the spleen but not in the BM. CD3+ CD8+ T cells infiltration and apoptosis were found in the skin, intestine and liver of mice suffering from GVHD. None of the syngeneic transplanted mice died or developed any sign of GVHD even in long term follow up, and CD3+CD8+ T cells were absent in their tissue samples. Increasing the dose of Cy in conditioning regimen in allogeneic transplanted mice increased mortality of animals.
Conclusion: We have established a new mouse model of acute GVHD using chemotherapy based conditioning. This model can be utilized to study the basic mechanisms underlying GVHD triggered by cytostatics used in the conditioning regimen and affecting different cell sub-populations and cytokine storm.
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Delivery of mRNA into Dendritic Cells Using Novel Transfer Peptides Leads to Prolonged Antigen Expression and Potent Immune Responses In Vivo.
Authors: Woan Karrune , Heiser Axel , Dahm Philipp , Vieweg Johannes , Su Zhen 
Published: October 2019

Abstract
We have previously shown that vaccination with RNA-transfected DC is a potent strategy to stimulate CTL and antitumor immunity in cancer patients. In this study, we investigated whether novel transfer peptides derived from the RNA-binding region of the HIV-1 nucleocapsid protein could be utilized for effective delivery of mRNA into human monocyte-derived dendritic cells (DC). Here we show that both peptide-mediated mRNA delivery and electroporation of DC with mRNA resulted in efficient gene transfer. However, the use of transfer peptides led to prolonged antigen expression and did not negatively affect the viability of DC, the migratory capacity of matured DC, and the production of cytokines by these cells in vitro. In murine studies, DC loaded with transfer peptide-mRNA complexes were clearly superior, compared to mRNA-electroporated DC, in stimulating antigen-specific CTL, CD4+ T cell, and antibody responses. Importantly, no transfer peptide-specific cellular or humoral immune responses were detected in vaccinated mice. Our data suggest that vaccination with transfer peptide-mRNA-loaded DC may represent a promising strategy to stimulate potent anti-tumor immune responses in a vaccination setting.
Lenalidomide Enhancement of NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) Is Mediated by Granzyme B and FasL and Is Associated with Modification of SHIP-1, PLC-g2 and pERK and Enhanced Chemokine Production.
Authors: Wu Lei , Adams Mary , Schafer Peter , Muller George , Stirling David , Bartlett Blake J. 
Published: October 2019

Abstract
Lenalidomide is an oral anti-angiogenic, anti-proliferative and immunomodulatory drug that is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of previously treated multiple myeloma patients. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo and we have previously shown that in an in vitro ADCC system lenalidomide is able to directly enhance human NK cell and monocyte function in response to rituximab (chimeric anti-CD20 mAb) and other IgG1 antibodies. We have now addressed the effect of lenalidomide on NK cell signaling pathways and have shown that compared to the levels seen with FC gR stimulation (with IgG) plus IL-12, there is an inhibitory effect of lenalidomide on SHIP-1 with concomitant enhancement of PLC g2 and pERK. This is associated with increased secretion of a variety of chemokines as well as GM-CSF and decreased IL-6 production. In functional ADCC assays, NHL cell lines were pre-coated with rituximab and exposed to either NK cells or monocytes pre-treated with lenalidomide. After 4 (NK cells) or 15 (monocytes) hours in culture the viability of the tumor cells was assessed. The consistent enhancement of ADCC in the presence of lenalidomide is associated with greatly increased expression of granzyme B and FasL, although perforin is maximally elevated without lenalidomide. We found that ADCC is totally blocked by an inhibitor of granzyme B and is partially blocked by anti-FasL. In conclusion, we have shown that the ability of lenalidomide to enhance ADCC-mediated killing of rituximab treated CD20+ve NHL cell lines is mediated by granzyme B and FasL and is associated with clear effects on NK cell signaling and chemokine production.
Monosodium Urate Crystal as a Carrier for Idiotype Tumor Vaccine.
Authors: Sakamaki Ippei , Inai Kunihiro , Ueda Takanori , Tsutani Hiroshi 
Published: October 2019

Abstract
Monosodium urate (MSU) crystal works as a danger signal to evoke specific cell immunity. It stimulates dendritic cells (DCs) maturation and enhances the generation of cytotoxic T cells (CTL), so that it could help specific immune therapy for tumor. On the other hand, MSU crystals are found to attach IgG in gouty arthritis and has been reported that the site of attachment is the Fab moiety of IgG. We focused on the application of MSU as a carrier of IgG idiotype protein to induce cytotoxic T cells to cell line producing the idiotype protein. Multiple myeloma cell line, IM-9, which is characterized by having HLA-A2 and producing monoclonal IgG-k protein, was used as a target cell. Monoclonal Fab fragments were used as idiotype protein; those were made by digesting with papain of IgG collected from the medium harvesting IM-9 cells. MSU crystals were made by sitting at refrigerator of uric acid dissolved by sodium hydroxide solution with boiling. Allogenic dendritic cells were derived from monocytes in peripheral blood of HL-A matched volunteer by treatment with GM-CSF and IL-4. They were pulsed by MSU attached with Fab fragments (MSU-Fab), by Fab fragments (Fab), or by none. Following education by pulsed DCs to CD3+ T cells collected from peripheral blood of same volunteer, CD8+ T cells were collected and were used as effector cells. Cell cytotoxic ability of the effector cells was determined by lactate dehydrogenase release assay under mixed culture of the target cells and the effector cells (CD8+ cells: IM-9 cells = 5: 1). Compared to Fab, MSU-Fab were easily phagocytosed by DCs. DCs pulsed with MSU-Fab or MSU were induced to express CD83 molecule, a marker for mature human blood dendritic cells, whereas neither Fab-pulsed nor untreated-control DCs did not express CD83 molecule. CD8+ T cells educated by DCs pulsed with MSU-Fab significantly killed more IM-9 myeloma cells than CD8+ T cells educated by DCs pulsed with MSU, DCs pulsed with Fab or DCs alone. These cytotoxic effect of CD8+ T cells educated by DCs pulsed with MSU-Fab were reduced when MSU crystals were bathed 10% fetal bovine serum solution before mixing with Fab in order to block MSU to be attached with Fab. The present study revealed that MSU crystals work as carrier of idiotype tumor vaccine not only by facilitating the utilization of the vaccine protein in DCs but also by enhancing the ability to induce tumor specific CTL. The safety and efficacy of MSU-Fab idiotype vaccine will be determined by clinical setting in patients with multiple myeloma.
Cyclosporine A (CSA) Significantly Reduces the Cytotoxic Effects of In Vitro Expanded NK Cells: Implications for Adoptive NK Cell Therapy in the Setting of Allogeneic Hematopoietic Stem Cell Transplantation (HCT).
Authors: Yokoyama Hisayuki , Berg Maria , Lundqvist Andreas , McCoy J. Philip , Srivastava Shivani , Smith Aleah , Childs Richard 
Published: October 2019

Abstract
The ability to expand NK cells in vitro has led to the recent initiation of protocols incorporating adoptive NK cell infusions after HCT. Calcineurin inhibitors such as CSA are commonly used to prevent graft versus host disease (GVHD) in HCT recipients. Recently, Hong et al found the phenotype and function of fresh NK cells cultured in vitro with CSA was altered, with CSA treated NK cell cultures having enhanced cytotoxicity against tumor targets. However, the impact of CSA on in vitro expanded NK cell function and phenotype has not been explored. We analyzed cell proliferation, IFN-gamma production, cell surface immunofluorescent staining and cytotoxicity against K562 and renal cell carcinoma cell lines by in vitro expanded vs freshly isolated NK cells cultured in physiological doses of CSA (40ng/ml, 200ng/ml, 1000ng/ml for 18hrs). Fresh NK cells were obtained from the PBMC of healthy donors using immunomagnetic beads to isolate CD56+/ CD3− cells. NK cells were expanded in vitro using irradiated EBV transformed B cells as feeder cells in media containing IL-2 [500U/ml] for 12–14 days. Comparing CSA containing cultures to controls, there was a significant reduction in IL-2 stimulated fresh NK cell proliferation (stimulation index 0.51± 0.1) and TRAIL expression (MFI 10.4 vs 3.01). Furthermore, an ELISA assay showed fresh NK cells treated with CSA had a significant reduction in IL-2 induced IFN-g production compared to controls (median 231 vs 57 pg/ml, p=0.025). In contrast, in vitro expanded NK cells cultured in CSA showed no significant reduction of proliferation or TRAIL expression. At the highest doses of CSA (1000ng/ml), minimal inhibition of K562 killing of freshly isolated NK cells was observed. In contrast, expanded NK cells cultured in CSA for 18 hours compared to controls had a significant reduction in the killing of K562 cells (E:T=10:1, median 66 vs 43% lysis, p=0.011) and RCC tumor cells (E:T=20:1, 14.8 vs 8.8%, p=0.043).
Figure Figure
These data confirm CSA alters the phenotype and function of CD3−/CD56 + NK cells. Importantly, CSA appears to have a deleterious effect on expanded NK cell tumor cytotoxicity that was not observed with fresh NK cells. These finding suggest the anti-tumor effects of in vitro expanded NK cells could be hindered when adoptively infused in HCT patients receiving CSA.
Cure of Murine Medulloblastoma by Adoptive Transfer of Tumor-Specific T Cells.
Authors: Nicholson Cameron L. , Plautz Gregory E. 
Published: October 2019

Abstract
Medulloblastoma is the most common malignant brain tumor of childhood. Although the current overall survival has increased due to more aggressive therapy, the involved surgery, radiation and in some diseases high-dose chemotherapy have significant acute and long-term toxicities. T cell immunotherapy is a promising adjuvant for medulloblastoma because the immune response is specific for targeted antigens and is highly anatomically focused, occurring at the level of cell-to-cell interactions, thereby sparing adjacent normal brain tissue. Adoptive transfer of tumor-sensitized T cells has demonstrated therapeutic efficacy in animal tumor models, and clinical trials involving transfer of T lymphocytes are ongoing. Previous strategies have employed cultured human medulloblastoma cell lines as therapeutic targets, however priming of T cells using a spontaneously-arising medulloblastoma cell line in an immunocompetent mice model may be more representative of human cancer and more applicable to clinical practice. Spontaneous Murine medulloblastoma cell lines were created from knock-out Ptc+/−p53−/− mice and passaged in vivo. Brain tumors were established by transcranial inoculation of a single-cell suspension of murine medulloblastoma tumor cells in immunocompetent, C57BL/6N mice. Murine Medulloblastoma cell lines were rapidly tumorigenic and consistently fatal following intracranial inoculation in a dose-dependent manner. T-cells were obtained surgically from tumor draining lymph nodes (TDLN) in syngeneic mice and after initial purification, antigen-stimulated CD62Llow T cells were isolated by depletion of CD62Lhigh cells using MACS beads. T-cells were further activated ex vivo by incubation with anti-CD3 monoclonal antibody and exposure to media containing Interleukin-2 and Interleukin-7. Mice carrying 3-day established tumors receive systemic transfer of T cells by injection through the tail vein after 10 days of ex vivo culture. Mice typically receive sub-lethal total body irradiation (5 Gy) from a Cesium irradiator for lympho-depletion several hours before adoptive transfer. Mice were followed for survival and sacrificed when neurological symptoms were apparent as per Institutional Animal Care and Use Committee established guidelines. The activated T cells adoptively transferred to irradiated hosts with 3-day established intracranial MM1 tumors resulting in complete regression of established tumors (see figure). In addition, the above therapy demonstrated a dose-dependent effect, and the mice were cured without any evidence of neurological dysfunction caused by the anti-tumor response. This research suggests anti-medulloblastoma T cell vaccines may be promising for subsequent clinical testing.
Figure 4. Active Immunology of 3-day MMI medulloblastoma Figure 4. Active Immunology of 3-day MMI medulloblastoma
Hematopoietic Stem Cells Mobilization by G-CSF and LTB4 Was Significantly Suppressed by an Oxygen Radical Scavenger or an Inhibitor of NADPH Oxidase.
Authors: Mun Yeung-Chul , Oh Soo Ah , Lee Kyoung-Eun , Yoo Eun-Sun , Chang Hye-Jung , Choi Moon-Young , Ahn Jee-Young , Kim Jae-Hong , Seong Chu-Myong 
Published: October 2019

Abstract
Introduction: We previously had reported that LTB4 was able to mobilize HSC within 4 hours without significant side effects and that LTB4 receptor was involved in both of the pathways of mobilization induced by G-CSF and LTB4 in the murine model. Though the roles of protease (ie. NE, MMPs) seem to be important, the precise mechanisms are still unknown. ROS (reactive oxygen species) have cell signaling roles that are involved in signal transduction cascades of numerous growth factor-, cytokine-, and hormone-mediated pathways, and regulate many biological systems. Because a variety of inflammatory cytokines and chemokines, including G-CSF and LTB4, induce a rapid increase of intracellular ROS, we hypothesized that the role of ROS on HSC mobilization induced by G-CSF and LTB4 might be important.
Methods: MS5, murine stromal cell line cells, or bEnd3, murine microvascular cell line cells, were grown to confluence on the microporous transwell membrane. Murine marrow cells were placed on top of the prepared transwell membrane. The transwells were then seated in wells containing media and G-CSF or LTB4 with or without pretreatment of NAC, an oxygen free radical scavenger, or DPI, an inhibitor of NADPH oxidase-like flavoproteins. Cells that migrated through the stromal or endothelial layer into the wells were assayed for mobilization. NAC and DPI were given to C57BL/6 mice followed by rhG-CSF (5μg, IV) or LTB4 (1μg, IV) after a period of 2 hours. 24 hours after the rhG-CSF injection or 4 hours after the LTB4 injection, peripheral blood samples were obtained via cardiac puncture. The samples were analyzed for TNC using a trypan blue stain and FACS analysis were performed using Sca-1 and lineage markers, including CD45R (B220), CD116, Gr-1 and TER119.
Results: The numbers of migrated cell through the MS5 or bEnd3 were increased by treatment of G-CSF or LTB4. However, increasing effects of G-CSF or LTB4 to the transmigration through the MS5 or bEnd3 were inhibited by pretreatment of NAC or DPI. Comparing the control arm, the numbers of WBC and HSC (Sca-1+Lin−) in blood were decreased in the G-CSF or LTB4 mobilized mice (N=4), in which NAC or DPI were pretreated (Sca-1+Lin− fraction in peripheral blood: 1.91±0.80% in G-CSF alone, 0.55±0.37% in G-CSF with NAC, 0.36±0.09% in G-CSF with DPI, P<0.05; 1.92±0.49% in LTB4 alone, 0.55±0.29% in LTB4 with NAC, 0.67±0.52% in LTB4 with DPI, P<0.05).
Conclusions: Through our data, it is suggested that ROS are involved in the HSC mobilization induced by G-CSF and LTB4 in the murine model. It would be very interesting to test the effect of G-CSF or LTB4 on ROS K/O mice in the future. As for now, we will try to find the critical signal transduction pathway, through which we may find more effective and efficient molecules for HSC mobilization.
AV513 Enhances Tissue Factor Activity by Inhibiting TFPI in Hemophilia A Plasma: Potential TFPI Target Sites and Potentiation of Factor VIII Activity.
Authors: Knappe Sabine , Kripalani Nicholas , Patarroyo-White Susannah , Johnson Kirk W. , Prasad Srinivasa 
Published: October 2019

Abstract
AV513, a sulfated polysaccharide prepared from brown seaweed and enriched for fucoidan accelerates clotting in hemophilia A and B plasma. Prior studies have demonstrated that AV513 functions mainly by inhibiting TFPI activity. In this report, we have further studied AV513 mechanism of action using human plasma thromboelastography (TEG) model and a thrombin generation assay in FVIII deficient plasma. In the TEG assay, AV513 enhanced tissue factor (TF) induced clotting in human hemophilia A pooled plasma in a dose-dependent manner. A dose-dependent potentiation effect by AV513 was observed with reduction of TEG R time to normal levels at 0.5 μg/mL of AV513. An AV513 dose dependent increase in thrombin generation was observed in hemophilia A pooled plasma with a 3-fold increase in peak thrombin generation at 2.5 μg/mL AV513 (basal: 40 nM vs. treated: 140 nM). When TFPI was immunodepleted from hemophilia A plasma (FVIII/TFPI deficient), sub-optimal concentrations of TF enhanced thrombin generation which was not further enhanced by addition of AV513. The increase in thrombin generation in FVIII /TFPI deficient plasma was inhibited by 93% after the addition of exogenous full-length TFPI or by 47% after the addition of truncated rTFPI (amino acids 1–161) that lacks the Kunitz 3 domain and the C-terminal tail. AV513 at low concentrations reversed exogenous full length TFPI activity almost completely in FVIII/TFPI-deficient plasma but had only a small effect on truncated TFPI. In a human plasma TEG combination study of AV513 and low concentrations of rFVIII (ReFacto), AV513 at a concentration of 0.25 μg/mL reduced the TEG R time by 35% in severe hemophilia A plasma compared to 10 mU/mL of rFVIII alone. These results indicate that AV513 functions as a pro-coagulant by inhibiting TFPI at least in part via targeting the C-terminal region. Moreover, AV513, in the presence of low rFVIII (ReFacto) levels, improves severe hemophilia A plasma clotting - as measured by TEG - beyond that observed with either agent alone.