bluuebunny/crossref_metadata_2025_split huggingface dataset:
Erythrocyte Adhesion and Phosphatidylserine Exposure in HbSC Disease: Baseline Data from the CHAMPS Study
Authors: Telen Marilyn J. , Delahunty Martha , Snyder Cathie , Wang Winfred C. 
Published: April 2020

Abstract
Red blood cells (RBCs) in sickle cell anemia are characterized by increased adhesion to laminin and endothelial cells, due both to increased adhesion receptor expression as well as receptor activation. However, these RBC properties have not been comprehensively evaluated in HbSC disease. In the on-going multi-center CHAMPS trial, all subjects have a diagnosis of HbSC disease and have had at least one significant vaso-occlusive event in the previous 12 months (but none in the previous 4 weeks). All subjects are at least 5 years of age (median 13.4 yr, range = 5.2–53.3 yr) at enrollment. Subjects receive hydroxyurea (HU) and/or magnesium pidolate (Mg) (or their placebos); endpoints include the effect of study drugs on erythrocyte density and RBC-endothelial interactions. To characterize the adhesive properties of RBCs at baseline in these patients, we performed the following analyses on blood samples shipped to a central lab at Duke University from 11 participating sites: adhesion to laminin at 1 dyne/cm2 shear stress; adhesion to endothelial cells after epinephrine (epi) stimulation of RBCs at shear stress of 1 dyne/cm2; and phosphatidylserine exposure, as measured by mean fluorescence intensity after binding of fluoresceinated annexin V. Most subjects had two pre-treatment specimens analyzed. Analysis of the first 83 samples (47 patients) showed that a mean of 1.64% (± 0.15% SEM) of circulating HbSC RBCs expressed phosphatidylserine, or a little less than half as many as in HbSS RBC samples (4.12% positive ± .91% SEM, n = 29) tested as controls (HbSC vs HbSS p=.0009). However HbSC RBCs were also different from HbAA RBCs (1.076% positive, n = 5), and all three sets of values were significantly different by ANOVA (p = .0017) (Figure). The two baseline visits for HbSC subjects resulted in nearly identical means of 1.64% and 1.65%, respectively. Adhesion of HbSC red cells to laminin at baseline was quite variable among patients, with a mean of 36.1 adherent cells/mm2 (range 2–256). This was slightly but not significantly lower than the values obtained using HbSS control RBCs (mean 58.8 cells/mm2, range 0.2–258). After epi stimulation of RBCs, 9.6% of cells adhered to endothelial monolayers at 1 dyne/cm2, whereas 14.8% of HbSS RBCs were adherent (p = .186). HbAA RBCs showed minimal adhesion in both assays (&lt; 5 cells/mm2 and &lt;3%, respectively). Finally, no correlation was found by linear regression analysis between phosphatidylserine exposure and either adhesion to laminin or adhesion to endothelial cells. In summary, HbSC RBCs show appreciable adhesion to both laminin and endothelial cells, as well as phosphatidylserine exposure. While in general these abnormalities are less pronounced than for HbSS RBCs, they are still well outside the normal range and provide potential targets for therapeutic intervention.
Figure Figure
Statins Inhibit the Function of Human Antigen-Presenting Cells
Authors: Shimabukuro-Vornhagen Alexander , Liebig Tanja , Zoghi Shahram , Wendtner Clemens M , von Bergwelt-Baildon Michael S 
Published: April 2020

Abstract
Statins are lipid-lowering drugs that reduce cholesterol production by inhibition of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway. It recently has been shown that statins, apart from their lipid-lowering action, have pleiotropic effects on the immune system. They possess immunomodulatory and anti-inflammatory activity. Our analysis of gene expression data comparing resting B cells with CD40-activated B cells, which are potent antigen-presenting cells (APC), showed that following CD40-activation expression of the enzymes in the mevalonate pathway was up-regulated. These results together with the known ability of some statins to directly interfere with the interaction of lymphocyte function-associated antigen 1 (LFA-1), an adhesion molecule involved in activation of APCs, with its ligands provided the rational for our investigation of the effects of HMG-CoA reductase inhibition on the function of human APCs. Addition of simvastatin led to inhibition of the proliferation and adhesion of B cells activated by via CD40 or TLR9. Likewise, the expression of MHC class II (HLA-DR) and of the costimulatory molecules CD80 and CD86 was reduced. The antigen presenting capacity of simvastatin-treated B cells was reduced as was shown by a reduction of T cell proliferation induced by allogeneic simvastatin-treated B cells. Simvastatin inhibited activation of resting B cells as well as already activated B cells. The inhibition was dose-dependent and doses as low as 10nM Simvastatin led to signification reduction of B cell activation. We observed similar effects on human dendritic cells. Interestingly, the concentration of simvastatin required for inhibition of T cell proliferation in response to stimulation by CD3/CD28-coated beads was 1–2 logs higher than for B cells. The inhibitory action of statins was only partially dependent on the mevalonate pathway since addition of excess mevalonate did not fully restore B cell activation. Our findings of the effects of statins on human APCs in conjunction with the observation made by others that preemptive treatment with a HMG-CoA reductase inhibitor protected mice from graft-versus-host-disease (GVHD) without affecting the graft-versus-leukemia effect provide the rational for further testing of statins in patients undergoing allogeneic stem cell transplantion. Since APCs are required for the development of acute GVHD the modulation of antigen presentation by DCs and B cells might be an important mechanism of action of statins. The exquisite sensitivity of APCs to statin-mediated inhibition could synergize with the T cell-directed immunosuppression of conventional immunosuppressants. In summary, statins represent a promising new immunomodulatory drug for the treatment immune-mediated diseases such as GVHD and autoimmune disease.
Soluble CD14 Protects Human Lymphocytes from Apoptosis
Authors: Naparstek Elizabeth , Sredni Benjamin , Zigman Eti , Senyor Gali , Tartakovsky Boris 
Published: April 2020

Abstract
CD14, a 56 Kd glycoprotein, typically present on myeloid cells, has been traditionally associated with innate immunity and pattern recognition. Recently its membrane bound form has been shown to be involved in apoptosis, as a tethering receptor for apoptotic cells on the surface of phagocytes-in this case with the purpose of removing apoptotic cells, and also as a surface molecule involved in protection from apoptosis of monocytes, neutrophils and recently on enterocytes, challenged with LPS.
Our aim was to evaluate the possible involvement of the soluble CD14 in the apoptotic pathway of human lymphocytes.
Methods: Freshly obtained human peripheral blood lymphocytes were cultured in vitro with gliotoxin, an apoptotic inducer. Human recombinant CD14 was added to the culture at physiological concentrations (10μg/ml-0.5 μg/ml) and apoptosis was assessed by cell membrane integrity using 7AAD, mitochondrial membrane potential by DiOC6(3) and cytoplasm shrinkage by cell size scatter analysis.
Results: Using DiOC6(3) we were able to show that human lymphocytes cultured in the presence of gliotoxin contained 63.8%±21 apoptotic cells, as opposed to 12.2%±11.5 in control cultures. Addition of recombinant human CD14 at a concentration of 10 mg/ml neutralized the apoptotic effect of gliotoxin back to 20.2%±10 (p&lt;0.003). This inhibitory effect was blocked by CD14-specific monoclonal antibodies, but not by control antibodies. We then identified and synthesized the fragment within the CD14 molecule that was responsible for this apoptosis protective effect, and demonstrated its comparable protective efficacy in vitro as shown in figure 1. The figure clearly reveals that this specific peptide, as opposed to the scrambled peptide, protected the lymphocytes form apoptosis, similarly to the full CD14 protein. Same results were obtained using 7AAD and cytoplasm shrinkage.
Conclusion: Our data thus suggest that circulating CD14 may play an important role in the prevention of apoptosis of lymphocytes and perhaps of other cells.
Figure Figure
Suppression of Interferon Regulatory Factor 8 Modulates Toll Like Receptor 2 and Toll Like Receptor 7/8 Induced Dendritic Cell Activation
Authors: Werth Daniela , Bringmann Anita , Brauer Katharina , von Schwarzenberg Karin , Held Stefanie , Brossart Peter 
Published: April 2020

Abstract
Interferon regulatory factor 8 (IRF-8) is a member of the IRF family of transcription factors, which are stimulated through interferon mediated pathways. In mice, IRF-8 seems to play an essential role in the development and maturation of dendritic cells (DCs). However, very limited knowledge is available about the potential role of IRF-8 in the human system. To bridge this gap we analyzed function and activation of human monocyte-derived dendritic cells (mDCs) lacking IRF-8 expression. To knockdown IRF-8 protein levels, we electroporated mDCs with different siRNAs against IRF-8. Additionally, we stimulated the electroporated mDCs with the Toll like receptor (TLR) 2 ligand Pam3Cys or the TLR 7/8 ligand R848. IRF-8 knockdown in mDCs was verified constantly by Western Blot analysis using an anti-IRF-8 antibody. We found that IRF-8 knockdown clearly diminished the expression of the human lymphocyte antigen molecules HLA-ABC and HLA-DR in Pam3Cys and R848 stimulated mDCs. To gain functional data, we performed ELISAs to determine cytokine and chemokine secretion. The electroporation of mDCs with IRF-8 specific siRNA resulted in profound inhibition of secretion of the cytokines IL-6, IL-12 and TNF-a as well as the chemokines MIP-1a (CCL3), MCP-1 (CCL2) and RANTES (CCL5). To get additional information on IRF-8 function in human mDCs, the regulation of signal transduction pathways was determined by Western Blot analysis. The suppression of IRF-8 diminished the nuclear translocation of the NF-kB family member’s c-Rel and RelB as well as PU.1 and IRF-3 in activated mDCs. In addition, we showed that the suppression of IRF-8 caused a reduced phosphorylation of ERK and JNK, but had no effect on the expression of STAT3. In summary, the knockdown of IRF-8 reduced the capability of mDCs to develop appropriate phenotype and functions in response to activating stimuli. Our results indicate that these effects are mediated via the ERK, NF-kB and PU.1 signalling pathways. IRF-8 plays an important role in the activation and function of human mDCs.
Enhanced B Cell Activation in the Absence of CD81
Authors: Sanyal Mrinmoy , Fernandez Rosemary , Levy Shoshana 
Published: April 2020

Abstract
CD81 is a component of the CD19/CD21 coreceptor complex in B cells. This tetraspanin molecule was previously shown to enable membrane reorganization in B cells responding to complement-bound antigens. Here we stimulated B cells via their B cell receptor (BCR) and demonstrate that Cd81−/− B cells fluxed higher intracellular free calcium ion along with increased phosphorylation of PLCγ2 and Syk. The stimulated Cd81−/− B cells also proliferated faster and secreted higher amounts of antibodies. Moreover, activation of the TLR4 pathway in Cd81−/− B cells induced increased proliferation and antibody secretion. Furthermore, Cd81−/− mice mounted a significantly higher immune response to T-cell independent antigens than their wildtype counterparts. Finally, analysis of Cd81−/− B cells that were generated by bone marrow transplantation into Rag1−/− mice confirmed a cell intrinsic hyperactive phenotype. Taken together, these results indicate that CD81 plays a negative role in B cell activation in vitro and in vivo.
Cyclin D3 Is Required for the Germinal Center Reaction
Authors: Peled Jonathan U. , Yu J. Jessica , Ding Beibei Belinda , Shaknovich Rita , Sicinski Piotr , Scharff Matthew D. , Ye B. Hilda 
Published: April 2020

Abstract
Germinal Centers (GC) of secondary lymphoid tissues are critical to mounting a high-affinity humoral immune response. B cells within the GC undergo rapid clonal expansion and selection while diversifying their antibody genes through class switch recombination and somatic hypermutation. Although it is generally believed that GC B cells employ a unique proliferative program to accommodate these processes, very little is known about how the GC-associated cell cycle is orchestrated. The D-type cyclins are important regulators of the G1 phase of the cell cycle and are the ultimate targets of many mitogenic and oncogenic stimuli. The Cyclin D3 gene is rearranged and over-expressed in certain mature B cell malignancies, and its overexpression has been reported to predict poor clinical outcome in patients with diffuse large B cell lymphoma. It has been observed that during their development, B cells switch from expressing cyclin D2 to cyclin D3 when they are recruited into the GC response. It is unclear, however, whether this switch simply reflects a change in the transcription factors that govern cyclin expression or serves a biological mandate. Here we report that mice deficient in cyclin D3 are profoundly impaired in their ability to form GCs as measured by immunohistochemistry and flow cytometry. Production of antigen-specific antibodies and affinity maturation, as ascertained by ELISA, are concomitantly reduced in these animals. These phenotypes can be at least partially explained by a significant block in the G1-phase of the cell cycle of GC B cells in vivo. Interestingly, this block in the G1-S transition is observed despite an apparent compensatory increase in cyclin D2 expression. In addition, naive B cells activated in vitro by either LPS or LPS and IL-4 display only minor changes in cell-cycle profile, suggesting that a specific requirement for cyclin D3 is unique to GC B cells. We also find moderately reduced Bcl6 mRNA expression in both naïve and GC B cells from the cyclin D3 knockout mice. Since Bcl6 is a master regulator of the GC response, decreased activity of this transcriptional repressor may further contribute to the severity of the GC phenotype. This is the first demonstration that cyclin D3 plays a unique role during the GC response in that it is required for its optimal structure and function. In addition to expanding appreciation for the cell type- and tissue-specific functions of the three D-type cyclin molecules, our findings have implications for understanding the role of Cyclin D3 in human B cell lymphomas.
Sulforaphane and PEITC Augment Activity of Conventional and Novel Anti-Myeloma Drugs
Authors: Jakubikova Jana , Sedlak Jan , Klippel Steffen , Leiba Merav , Tai Yu-Tzu , Hideshima Teru , Podar Klaus , Chauhan Dharminder , Raje Noopur , Richardson Paul G. , Anderson Kenneth C. , Mitsiades Constantine S. 
Published: April 2020

Abstract
Isothiocyanates (ITCs) represent a family of phytochemicals found in cruciferous vegetables. Several epidemiological studies indicated that high intake of diet-derived ITCs may provide chemopreventive effect associated with a reduced risk of renal, prostate, pancreatic and colorectal carcinoma. ITCs have also been reported to enhance the chemosensitivity of diverse types of tumor cells. We therefore evaluated the cytotoxic effect of ITC, sulforaphane (SFN) and phenylethyl isothiocyanate (PEITC) on a panel of human MM cell lines, including cells resistant to doxorubicin (RPMI-Dox40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5), and dexamethasone (MM.1R, OPM1 and OPM2); as well as cells with low sensitivity to thalidomide derivatives (RPMI 8226-S) and sensitive cell lines (MM.1S). We evaluated the anti-MM activity of these compounds using both MTT and flow cytometric assays. Our results suggest that all tested MM cell lines are susceptible to the cytotoxic effect of both ITCs at concentrations in the same order of magnitude as those achieved in vivo by dietary consumption of cruciferous vegetables. PEITC (IC50 of 3.5–8.2 μM) was more potent than SFN (IC50 of 5–15 mM) at 48 h. ITCs induce apoptotic death of MM cells, evidenced by Annexin V-FITC staining, increased sub-G1 population measured by flow cytometry, and cleavage of PARP and caspase-3 by western blot analysis, ITCs also induced G2/M cell cycle arrest and depletion of mitochondrial potential in JC-1 probed MM cell lines. Multiplex analysis of phoshorylation signaling pathways, using the Luminex system and confirmed by conventional western blot analysis, revealed that PEITC at 2hrs triggers MAPK activation (MEK1 (4-fold), ERK1/2 (4.4-fold), JNK (25.4-fold) and p38MAPK (6-fold)) at 2h, which likely are stress responses of cells to PEITC treatment. SFN induced less pronounced but a more sustained MAPK activation (up to 24 h) than PEITC. Concentration-dependent increases in phosphorylation of GSK3α/β, c-jun, p70S6 kinase, and p90RSK were also observed at early time-points after ITCs-treatment. In contrast, decreased phoshorylation of Akt was observed in MM cells treated with SFN at 2 h and PEITC at 24 h. Chou-Talalay analysis of the effects of combinations of ITCs with anti-MM drugs (dexamethasone, melphalan and bortezomib) revealed all combinations to have synergistic MM cytotoxicity. Importantly, ITCs treatment, both alone and in combination with the aforementioned agents, also significantly suppressed proliferation of CFSE-labeled MM cell lines co-cultured with the human bone stromal cell line HS-5. These results indicate that SFN and PEITC suppress survival and proliferation of MM cells, both alone and in combination, and suggest their therapeutic potential in MM.
Arsenic Trioxide Plus ABT-737 Is Synergistic to Induce Apoptosis in AML Cells by Repression of Mcl-1 Protein and Inactivation of Bcl-2 Protein
Authors: Xia Lijuan , Wang Rui , Qian Hao , Gabrilove Janice , Waxman Samuel , Jing Yongkui 
Published: April 2020

Abstract
Arsenic trioxide as a single agent induces remission in acute promyelocytic leukemia (APL) patients with minimal toxicity but not in other types of acute myeloid leukemia (AML). In vitro, APL as compared to AML cells are more sensitive to arsenic trioxide-induced apoptosis. Arsenic trioxide-induced apoptosis in APL cells results from activation of a mitochondria-mediated pathway. The Bcl-2 antiapoptotic protein family members Bcl-2, Bcl-XL, Bfl-1 and Mcl-1 block mitochondria-mediated apoptosis. In studies of several AML cell lines, we detected high levels of Bcl-2 and Mcl-1 protein, but lower or no expression of Bcl-XL and Bfl-1. Arsenic trioxide treatment decreased the levels of Mcl-1 without inducing apoptosis in AML cells suggesting that Bcl-2 would be a key factor causing arsenic trioxide resistance. We therefore hypothesize that inhibitors of Bcl-2 might restore arsenic trioxide-induced programmed cell death. In this study, the apoptotic effects of arsenic trioxide, ABT-737 (a potent Bcl-2 inhibitor) and their combination were investigated in NB4, HL-60, U937 and K562 cells. Arsenic trioxide at 1–2 μM induced apoptosis only in NB4 cells but decreased the levels of Mcl-1 in all of the four cell lines. ABT-737 at concentrations lower than 5 μM induced apoptosis in NB4, HL-60 and U937 cells but not in K562 cells which had undetectable Bcl-2 levels. Arsenic trioxide (2 μM) plus ABT-737 (0.05–0.5 μM) synergistically induced apoptosis in HL-60 and U937 but not in K562 cells as determined by PARP cleavage and Annexin V staining. Our data suggest that inhibition of Mcl-1 expression by arsenic trioxide and inactivation of Bcl-2 activity by ABT-737 leads to the synergistic apoptosis observed with this combination and is the basis for a novel treatment for AML.
Hyperglycemia Promotes in Vitro All Blast Resistance to Steroid Induced Blast Lysis
Authors: Papadakis Vassilios , Tsitsikas Konstantinos , Vasdekis Vassilios , Parcharidou Agapi , Paisiou Anna , Papargyri Sofia , Polychronopoulou Sophia 
Published: April 2020

Abstract
Clinical patient observations led to the hypothesis of increased ALL blast resistance in hyperglycemia (HG) states in pediatric ALL patients, either with preexisting diabetes mellitus (DM) or with steroid induced diabetes (SID). To evaluate the above, the chromometric MTT (methyl thiazole tetrazolium) assay was used Kaspers GJ et al, Leuk Res1995;19:175–81.Ficoll separated nucleated bone marrow cells from newly diagnosed pediatric ALL patients were cultured in usual conditions (NL) and in higher glucose levels (233–295 mg/dl), with the addition of titrated prednisolone (PDN) concentrations. The PDN concentration that lyses 50 % of the cultured blast cells (LD50) is the measure of PDN drug sensitivity. PDN concentrations of 0.02–2.0, 2.1–150, above 150 μg/uL indicate high, intermediate and low blast sensitivity to PDN, respectively. Hence, patient blasts with LD50 at &gt;150 μg/uL are resistant to PDN. The MTT assay results have been related to patient outcome (remission induction and event free survival, Pieters R et al Pediatr Oncol1994;22:299–308). Twelve newly diagnosed pediatric patients, with median age 6.4 years (range, 1.7 to 16.5 years) with common/CD10+ (N=8), pre-B (N=2), early pro-B (N=1) and maturing B (N=1) ALL were studied. All patients exhibited &gt;80% ALL blast BM infiltration. Sensitivity (LD50 &lt; 150 μg/uL) in both NL and HG conditions was evident for 2 patients, while resistance to both conditions was seen in 3 patients. Patients resistant at NL conditions were resistant in HG, too. In contrast, 7 of 12 patients sensitive to PDN in NL conditions proved resistant in HG conditions (glucose 233–295 mg/dL). All 9 sensitive in NL conditions patients (in HG conditions 2 sensitive and 7 resistant) exhibited a median ×800 fold increase of the MTT LD50 measured (range 6–29.000 fold). For all patients (N=12) median MTT LD50 under NL conditions was 0.15 μg/uL and in HG conditions 167.5 μg/uL (× 116.6 fold increase). This change was statistically significant (Mc Nemar’s test, p=0,016) and this change was not simply related to the change in glucose concentration. These findings of greatly increased ALL blast resistance in hyperglycemia environment, can support the notions given earlier (Weiser MA et al, Cancer2004;100:1179–85, Pieter’s R EHA 2008) that hyperglycemia by itself can promote PDN resistance. Given that steroids is a mainstay in ALL treatment and that steroid induced hyperglycemia is a common occurrence, blood glucose monitoring and hyperglycemia correction appears to be of paramount significance in delivering an effective ALL treatment.