Artículos de revistas: "Ficoll-Hypaque method"

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Quach, Alex y Antonio Ferrante. "The Application of Dextran Sedimentation as an Initial Step in Neutrophil Purification Promotes Their Stimulation, due to the Presence of Monocytes". Journal of Immunology Research 2017 (2017): 1–10. http://dx.doi.org/10.1155/2017/1254792.

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The purification of human neutrophils forin vitrostudies is challenging as they are easily activated through ex vivo manipulations. The technique of erythrocyte sedimentation combined with density gradient centrifugation remains widely practiced and was the subject of this study. Since in the sedimentation step the leukocytes are incubated with dextran, we have raised the likelihood that cellular activation would occur with mediator release leading to neutrophil activation. By comparing the activity of neutrophils purified from whole blood by the classical 2-step method of dextran sedimentation followed by low-density Ficoll-Hypaque (1.077 g/mL) medium, and the 1-step high-density Ficoll-Hypaque (1.114 g/mL) gradient centrifugation, we found that neutrophils from the 2-step method had a significant increase in cell surface CD11b expression and CD62L shedding and a marked increase in adhesion. Decreased random migration and chemotaxis and raised baseline oxidative burst activity were also observed. The effect was not specific to dextran, as using Ficoll for erythrocyte sedimentation replicated the elevated neutrophil adherence. Through the depletion of monocytes, lymphocytes, and platelets prior to sedimentation, we deduced that monocytes were responsible for the neutrophil activation. Our findings suggest that care needs to be exercised in choosing the method of neutrophil purification for functional studies.

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Wang, Jian-Ping, Justin P. Tyler, Surayya I. Siddiqui, Shobha J. Patel y Uma Kundu. "High Speed Ficoll-Hypaque Gradient Centrifugation Method for Processing Blood Contaminated Cytology Fluid Specimens". Journal of the American Society of Cytopathology 2, n.º 1 (octubre de 2013): S80. http://dx.doi.org/10.1016/j.jasc.2013.08.215.

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Bignold, L. P. y A. Ferrante. "Mechanism of separation of polymorphonuclear leukocytes from whole blood by the one-step Hypaque-Ficoll method". Journal of Immunological Methods 96, n.º 1 (enero de 1987): 29–33. http://dx.doi.org/10.1016/0022-1759(87)90363-2.

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Pattan, Shivanand S., Kishore G. Bhat, Geeta D. Pattar y Manjula Kuntagi. "COMPARISON OF THREE DIFFERENT TECHNIQUES FOR ISOLATION OF NEUTROPHILS FROM BLOOD AND THEIR UTILITY IN PERFORMING NITROBLUE TETRAZOLIUM TEST". International Journal of Research -GRANTHAALAYAH 7, n.º 12 (8 de junio de 2020): 115–22. http://dx.doi.org/10.29121/granthaalayah.v7.i12.2019.305.

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Background & Objectives: Assays for neutrophils constitute an important component of screening tests in clinical immunology. There are no standard protocols for performing many of these tests and procedures vary from one laboratory to another. In addition, normal ranges for these assays in healthy Indian population have not been defined. Hence, an attempt is made to evaluate and present a simple technique for WBC isolation and NBT test. Methods: The study involved participation of 30 healthy adult volunteers. Ten of blood sample collected from each subject was subjected to three different procedures for isolation of WBCs - Ficoll-Hypaque gradient, dextran sedimentation and gelatin sedimentation methods. Cells isolated from these procedures were then used to perform NBT test. Smears were prepared, stained with Giemsa and results were expressed as % of stimulated and unstimulated cells. Results: The mean cell yield from both dextran and gelatin methods was comparable (2921.67cells vs 2806.67cells/cu mm). The cell yield from Ficoll-Hypaque method was much lower (1408.33 cells/cu mm). In NBT test, the mean readings of stimulated (61%) and unstimulated cells (18%) were almost similar in all three procedures of cell isolation. Conclusions: Comparison of procedures show that gelatin and dextran sedimentation methods yield high amount of relatively purified WBCs. The efficacy of cells isolated from all three procedures in NBT test was almost similar. The range of stimulated and unstimualted cells in the subjects were within expected levels. Gelatin sedimentation is economical, easy to perform and can be adopted to any clinical laboratory for WBC isolation.

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RAYAMAN, PERVİN, ERKAN RAYAMAN, ADİLE ÇEVİKBAŞ, REFİK DEMİRTUNÇ, AHMET ÖZER ŞEHİRLİ, ŞEYDA GÜL ALAGÖZ y ÜMRAN SOYOĞUL GÜRER. "Effect of antibiotics on polymorphonuclear leukocyte functions and myeloperoxidase activity, glutathione and malondialdehyde levels in allergic asthma". Polish Journal of Microbiology 64, n.º 1 (2015): 69–72. http://dx.doi.org/10.33073/pjm-2015-010.

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We investigated the effect of ciprofloxacin, rifampicine and doxycycline on myeloperoxidase (MPO) activity, glutathione (GSH) and malondialdehyde (MDA) levels in allergic asthma patients and healthy volunteers. Polymorphonuclear leukocytes (PMNs) were isolated with ficoll-hypaque gradient centrifugation method. MPO activity was assayed with modified o-dianisidine, GSH by Ellman's and MDA levels by Beuge's method. PMN functions and MDA levels of patients significantly decreased when compared with healthy volunteers. Ciprofloxacin significantly increased PMN functions, MPO activity and MDA levels of both groups. We have demonstrated that ciprofloxacin has beneficial effects on MPO activity and PMN functions in allergic asthma patients and healthy volunteers.

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Milne, D. B., N. V. Ralston y J. C. Wallwork. "Zinc content of cellular components of blood: methods for cell separation and analysis evaluated." Clinical Chemistry 31, n.º 1 (1 de enero de 1985): 65–69. http://dx.doi.org/10.1093/clinchem/31.1.65.

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Abstract Platelets, mononucleated cells, polymorphonucleated cells, and erythrocytes were separated from whole blood by use of discontinuous gradients of colloidal polyvinylpyrrolidone-coated silica ("Percoll"). We measured the zinc content of these cells by flame atomic absorption spectrophotometry, using a modified technique for micro-samples that obviated matrix interferences. Thus, results obtained by conventional flame atomic absorption and by the micro-method were identical. Inter-comparisons of separation methods indicated that separation of platelets and mononucleated cells by a two-gradient system of "Ficoll-Hypaque" (a synthetic polymer of sucrose) or Percoll was relatively poor, whereas there was a good separation when a tertiary gradient system of Percoll was used. The apparent zinc content of mononucleated cells depended on the degree of separation from the platelets, with contamination by platelets resulting in artificially high values for mononucleated cells.

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Elin, R. J. y J. M. Hosseini. "Magnesium content of mononuclear blood cells." Clinical Chemistry 31, n.º 3 (1 de marzo de 1985): 377–80. http://dx.doi.org/10.1093/clinchem/31.3.377.

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Abstract The magnesium content of mononuclear blood cells may provide a better assessment of intracellular magnesium or total body magnesium status than does measurement of magnesium in plasma or erythrocytes. We describe a method for determining this, and report results for 20 normal volunteers. The mononuclear cells are separated with a discontinuous Ficoll-Hypaque gradient, washed, centrifuged at 400 X g, and lysed by sonication in 10 mmol/L NaCl. The magnesium in the cell lysate, with 2.93 g of added lanthanum oxide per liter, is determined by atomic absorption spectrometry. The mean mononuclear cell magnesium content in our volunteers was 70.7 (SD 14.1) fg per cell and 9.29 (SD 1.85) mg/g of DNA. The CVs for the determinations of magnesium, DNA, and cell count were 3.0%, 5.0%, and 8.7%, respectively. There was a significant correlation (r = 0.67, p less than 0.001) between results by the two methods of expressing magnesium content of mononuclear cells. However, by either method there were no significant correlations among results for magnesium concentration of mononuclear cells, plasma, or erythrocytes.

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Gallacher, R. E., M. C. Browning, C. G. Fraser, S. P. Wilkinson y W. J. MacLennan. "A method for simultaneously estimating plasma, erythrocyte, and leukocyte sodium, potassium, and magnesium: reference values and considerations from biological variation data." Clinical Chemistry 33, n.º 8 (1 de agosto de 1987): 1326–30. http://dx.doi.org/10.1093/clinchem/33.8.1326.

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Abstract We describe a method for measuring plasma, erythrocyte, and leukocyte sodium (Na+), potassium (K+), and magnesium (Mg2+) concentrations in 10-mL blood specimens. After separating cells with Ficoll-Hypaque and washing with isotonic choline chloride, erythrocytes and leukocytes are counted, so that results can be expressed as amount of substance per cell and also to monitor cell integrity and possible contamination. Plasma and cell lysates were analyzed (CV less than or equal to 7.0%) with flame photometry and atomic absorption spectrometry. Reference intervals for an elderly population with values for plasma electrolytes within reference intervals are similar to those for younger healthy subjects. From data on biological variation, reference values for erythrocyte cations are not of much use, and analytical goals for precision are not met, but the results might be useful for monitoring disease progression in individual patients. In contrast, reference values for leukocyte cations are theoretically of use and goals are achieved, but large changes are required before consecutive results can confidently be said to be significantly different.

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Moriguchi, Keiichi y Kei-Ichi Hirai. "Phagocytosis and hydrogen peroxide production of Eosinophils in the rats spontaneously infected with Mycoplasma pulmonis". Proceedings, annual meeting, Electron Microscopy Society of America 48, n.º 3 (12 de agosto de 1990): 314–15. http://dx.doi.org/10.1017/s0424820100159126.

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The ability of hydrogen peroxide (H2O2) production was cytochemically compared in eosinophils (EP) between specific pathogen free (SPF) and spontaneously Mycoplasma pulmonis-infected male rats (Wistar strain).Peritoneal cells including EP (PEP) were harvested with a cold Hanks' balanced salt solution containing 0.1% glucose (HBSSG). Simultaneously, blood granulocytes with eosinophils (BEP) were isolated from the same animals by a Ficoll-Hypaque method. Cells were incubated with latex particles in HBSSG for 60-90 min. Thereafter, cells were washed and transferred into a cerium (Ce) medium consisting of 0.1 M tris - maleate buffer, pH 7.5, 1mM CeCl3, 10 mM sodium azide, 0.1. glucose and 0.25 M sucrose. The incubation was carried out for 20 min at 37°C with occasional stirring. Some cells were incubated for 60 min at 37 °C in a DAB medium containing 0.1% 3.3’-diaminobenzidine 4HCl with particles in 0.1 M phosphate buffer, pH 7.4. All cells were then fixed with glutaraldehyde and osmium tetroxide before processing for electron microscopy, x-ray microanalysis of the subcellular electron-dense reaction deposits was performed under a Tracor-Northern EDX attached to a JEM-1200EX STEM system.

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Nogueira-Machado, Jose Augusto, Gabriela Rossi Ferreira, Caroline Maria Oliveira Volpe, Pedro Henrique Villar-Delfino y Fabiana Rocha Silva. "Diabetes potentiates ROS production in granulocytes from patients with chronic kidney disease". Endocrinology&Metabolism International Journal 9, n.º 1 (3 de marzo de 2021): 9–14. http://dx.doi.org/10.15406/emij.2021.09.00301.

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Background: Type 2 diabetes (DM2) and chronic kidney disease (CKD) are inflammatory pathologies. Diabetes is characterized by hyperglycemia and CKD by the gradual and irreversible loss of kidney function. Both diseases develop oxidative stress, and reactive oxygen species (ROS) play a pivotal role in the pathogenesis. This study aimed to determine ROS production by granulocytes from renal patients (CKD) with or without diabetes. Methods: Granulocytes from patients with DM2, CKD, CKD-DM2, and healthy controls were purified using the Ficoll-Hypaque gradient method. Granulocyte ROS generation in the absence or the presence of PDB (an activator of NADPH-oxidase) or Concanavalin A (Toll- receptor 3,9 activator) was evaluated in a luminol-dependent chemiluminescence method. The cell-free DNA in the serum of DM2, CKD, and CKD-DM2 patients was measured by the fluorescence method before and after hemodialysis. Results: Our results show a significant increase in ROS production by granulocytes from patients with CKD, DM2, and CKD-DM2 compared to healthy control (p<0.05). CKD-DM2 group produced the most significant ROS levels with or without NADPH-oxidase activation. ROS production showed a significant increase in the presence of ConA. In contrast, mitochondrial (internal) ROS showed a different ROS response. DNA extrusion was higher in the CKD-DM2 group after hemodialysis suggesting cell death. Conclusion: The results demonstrated that CKD-DM2 patients produced high ROS generation levels and increased DNA extrusion after hemodialysis. It may suggest that CKD-DM2 disease is more severe and has a worse clinical prognosis.

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Dervieux, Thierry, Yaqin Chu, Yi Su, Ching-Hon Pui, William E. Evans y Mary V. Relling. "HPLC Determination of Thiopurine Nucleosides and Nucleotides in Vivo in Lymphoblasts following Mercaptopurine Therapy". Clinical Chemistry 48, n.º 1 (1 de enero de 2002): 61–68. http://dx.doi.org/10.1093/clinchem/48.1.61.

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Abstract Background: Mercaptopurine is a prodrug requiring intracellular activation to thiopurine nucleotides to exert antileukemic effect. We developed a reversed-phase liquid chromatographic assay for the quantification of mercaptopurine, thioguanine, and methylmercaptopurine nucleoside and nucleotide concentrations in the target tissue, the leukemic lymphoblast. Methods: Leukemic blasts were isolated from peripheral blood and bone marrow by a standard Ficoll-hypaque procedure. Proteins were removed by ultrafiltration in the presence of dithiothreitol. Thiopurine ribonucleotides were converted into their respective ribonucleosides by treatment of ultrafiltrate with acid phosphatase. Thiopurine nucleosides and bases were measured by direct injection of ultrafiltrate into the chromatographic system. Thiopurine nucleotide concentrations were calculated by subtracting the thiopurine nucleoside concentrations measured after treatment with acid phosphatase from those measured after direct injection of ultrafiltrate in the chromatographic system. Analytes were separated on a C18 Supelco column with ammonium phosphate-methanol eluent coupled with ultraviolet detection. Results: CVs for intra- and interday precision were 1.1–14% (median, 4.9%), and recovery of added analyte was 89–126% (median, 105%) at low and high concentrations of analytes, except for mercaptopurine riboside. The median signal for each of the five metabolites in lymphoblast samples was 98% (range, 80–106%) of that in water. Detection limits for thiopurine bases and nucleosides ranged from 0.5 to 4.5 pmol/5 × 106 cells. Conclusions: This method is suitable for measurement of thiopurine metabolite concentrations in lymphoblasts in children with acute lymphoblastic leukemia following a single dose of intravenous mercaptopurine.

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Ulgen S Yaramis, CP, E. Rayaman, U. Soyogul Gurer, ME Or y AO Sehirli. "Effects of inactivated Parapoxvirus ovis on polymorphonuclear leukocyte function and myeloperoxidase activity in horses". Veterinární Medicína 59, No. 12 (22 de diciembre de 2014): 631–36. http://dx.doi.org/10.17221/7823-vetmed.

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Immunomodulatory products have been used for years in veterinary medicine. Inactivated Parapoxvirus ovis (iPPVO) is currently used in equine medicine as an immunomodulator to improve the immune system and as a prophylactic treatment to prevent or treat infectious diseases. This study was designed to determine the effects of iPPVO on polymorphonuclear leukocyte (PMNL) function (phagocytosis and intracellular killing activity) and the myeloperoxidase (MPO) activity of PMNLs in horses. Twenty-four healthy English thoroughbred horses with an average age of 11 years were included in the study. Venous blood samples (10 ml) were taken before (agent-free controls) and after the administration of iPPVO (2 ml i.m. injection on Days 1, 3, and 5). PMNLs (1 × 107 cells/ml) were isolated from venous blood containing EDTA (0.1 g/ml) with Ficoll-Hypaque gradient centrifugation. Cellular phagocytosis and intracellular killing activities were assayed using a modification of Alexander’s method before and after treatment with iPPVO. MPO activity was also measured. The administration of iPPVO significantly increased the phagocytic, intracellular killing, and MPO activities of equine PMNLs (P = 0.0058, P = 0.0050, and P = 0.0070, respectively). This study demonstrates a strong correlation between MPO activity and PMNL function. The administration of iPPVO to horses has a supportive effect on their cellular immunity and an immunomodulatory effect against equine viral infections.

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Cordero, Yndra, Grecia M. Corao, José A. Cova y Alfredo Usubillaga. "Effect of some ent -Kaurenes on the Viability of Human Peripheral Blood Mononuclear Cells". Natural Product Communications 7, n.º 5 (mayo de 2012): 1934578X1200700. http://dx.doi.org/10.1177/1934578x1200700503.

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The possible cytotoxic activity of some ent-kaurenes on human mononuclear cells, obtained from peripheral blood, was studied having in mind future studies on their antitumor activity. The cells were obtained using the Ficoll-Hypaque method, adjusted to 2×106cells/mL, and incubated with kaurenes for 48 hours at 3×10-5, 30×10−5, 300×10−5 and 3000×10−5μmol/well. Ent-kaurenic acid showed no toxicity at all concentrations studied. The least toxic of all the kaurene derivatives studied was ent -15,16-epoxy-17-acetoxy-(-)-kauran-19-oic acid, with a cellular viability of 99% at 3×10−5μmol/well, and 94% at 30×10−5μmol/well. Another compound that showed low toxicity was the 2,3,4,6-tetra-acetyl-α-D-pyranosyl ester of ent-15-oxo-(-)-kaur-16-en-19-oic acid with 44% viability at 3000×10−5Vmol/well. The most toxic compounds at all concentrations tested were ent-kaur-16-en-19-ol acetate and ent-16α-hydroxy-(-)-kauran-19-oic acid. On the other hand, ent-kaur-9(11)16-dien-19-oic acid, ent-kauran-19-oic acid, and ent-kaur-16-en-19-ol were toxic only at the highest concentration studied. According to these results, and considering the concentrations employed, ent-kaur-16-en-19-oic acid and ent-15,16-epoxy-17-acetoxy-(-)-kauran-19-oic acid could be used for in vivo experiments and possibly for therapeutic purposes on humans, without much risk.

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Dewanti, I. Dewa Ayu Ratna, Pujiana Endah Lestari, Roedy Budirahardjo, Dyah Setyorini, Ristya Widi Endah Yani, Sunlip Wibisono y Maizirwan Mel. "THE EFFECT OF STEEPING ROBUSTA COFFEE BEANS ON MONOCYTES: EXPRESSION OF IL-1β AND TNF-α AGAINST Streptococcus mutans". Coffee Science 14, n.º 4 (9 de diciembre de 2019): 477. http://dx.doi.org/10.25186/cs.v14i4.1619.

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Adhesion, IL–1β, TNF–α are components that affect in inflammation. So, the effect of steeping green and black Robusta coffee beans to adhesion of Streptococcus mutans on this components. This study used monocytes isolated from healthy human peripheral blood using Ficoll-Hypaque centrifugation method. Monocytes were divided into eight groups, i. e. (i) Control group (untreated monocytes), (ii) Streptococcus mutans group (monocytes + S. mutans), (iii) Black Coffee 2.5 % group (monocytes + black coffee beans 2.5 % + S. mutans), (iv) Black Coffee 5 % group (monocytes + black coffee beans 5 % + S. mutans), (v) black Coffee 10 % group (monocytes + black coffee beans 10 % + S. mutans), (vi) Green Coffee 2.5 % group (monocytes + green coffee beans 2.5 % + S. mutans), (vii) Green Coffee 5 % group (monocytes + green coffee beans 5 % + S. mutans), (viii) Green coffee 10 % group (monocytes + green coffee beans 10 % + S. mutans). S. mutans adhesion on monocytes was analyzed using histochemistry method, while immunocytochemical staining was used for analyzing IL–1β and TNF–α. Cells counting was done per 100 monocytes under a light microscope with 400 × magnification. Data were analyzed using ANOVA followed by LSD test. Results showed that steeping green and black Robusta coffee beans increased the adhesion of S. mutans on monocytes, but it decreased of IL–1β, TNF–α expression (P < 0.05). In conclusion, steeping of green and black robusta coffee beans reduced inflammation against S. mutans.

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Wang, Jing, Huiru Wu, Xinli Liu, Huiyu Jia y Hong Lu. "Effect of LPS on Cytokine Secretion from Peripheral Blood Monocytes in Juvenile Idiopathic Arthritis-Associated Uveitis Patients with Positive Antinuclear Antibody". Journal of Immunology Research 2021 (3 de mayo de 2021): 1–8. http://dx.doi.org/10.1155/2021/6691681.

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Objectives. Antinuclear antibody (ANA) positivity is a key finding in JIA-associated uveitis (JIAU), but there are quite a few patients with negative ANA. There is no relevant report on the difference of their clinical manifestations. Previous animal model studies have found that the occurrence of uveitis is related to macrophage activation. In this article, our goal is to investigate changes in the morphology and cytokines of peripheral blood mononuclear cells (PBMCs) in uveitis patients testing positive or negative for ANAs after lipopolysaccharide (LPS) stimulation. Methods. A total of 30 patients were included in this study (10 in each group). They were divided into three groups (the ANA-positive [ANA+] group, ANA-negative [ANA-] group, and control group). There were ten patients (6 females and 4 males) in each group. Peripheral venous blood was collected into a heparinized tube, and PBMCs were isolated as soon as possible by the Ficoll-Hypaque density gradient separation method. Isolated cells were mixed with RPMI-1640 medium, and the cell concentration was adjusted to ensure that each patient had the same number of cells entering the study. After putting the extracted PBMC into the culture plate, LPS was added carefully to the plate. The cell culture supernatants were collected at 0 h, 3 h, 6 h, 12 h, and 24 h after LPS stimulation to detect the concentrations of IL-6, IL-1, TNF-α, and IL-10. Immunofluorescence was used to discover the deformation of macrophages after LPS stimulation. Results. The newly isolated cells were approximately round. 6 h after LPS stimulation, the ratio of noncircular cells/circular cells was the highest in the ANA+ group. Unlike IL-10 that has been rising during the observation period, IL-6, IL-1, and TNF-α peaked at 6 h after LPS stimulation. Conclusion. With LPS motivation, cytokines in the ANA+ group increased the most violently.

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Finbloom, D. S., D. L. Hoover y M. S. Meltzer. "Binding of recombinant HIV coat protein gp120 to human monocytes." Journal of Immunology 146, n.º 4 (15 de febrero de 1991): 1316–21. http://dx.doi.org/10.4049/jimmunol.146.4.1316.

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Abstract Inasmuch as the exact level of CD4 Ag expression on macrophages is controversial and because HIV may interact with macrophages in a manner different from that on T cells, we analyzed the binding of gp120 to freshly isolated and cultured monocytes. rgp120 was iodinated using the lactoperoxidase method to a sp. act. of 600 Ci/mmol. Highly purified monocytes (greater than 90%) were isolated from the leukapheresed blood of normal volunteers by Ficoll-Hypaque sedimentation followed by countercurrent centrifugal elutriation and cultured 7 days in DMEM supplemented with 1000 U/ml macrophage CSF in 10% human serum. Whereas MOLT/4 cells consistently bound freshly prepared 125I-rgp120 at 80% specificity with 5100 +/- 700 mol/cell, MCSF cultured monocytes bound rgp120 at only 0 to 20% specificity and 420 +/- 200 mol/cell. Most of the radioactivity bound by these cells could not be blocked by the addition of unlabeled rgp120. In contrast, the U937 myeloid cell line bound rgp120 with 50% specificity and about 2500 mol/cell. Whereas the antibody OKT4a (anti-CD4) blocked 80% of the binding on MOLT/4 cells and 50% on U937 cells, binding was only inhibited on the average of 6% on cultured monocytes. When soluble rCD4 was used as an inhibitor, binding to MOLT/4 cells was blocked by 80%. In contrast, binding to cultured monocytes was inhibited by 28%. HIV infectivity was blocked by similar concentrations of OKT4a. These observations suggest that although most binding of gp120 to cultured monocytes is not to the CD4 determinant, several hundred molecules do bind to a CD4-like molecule which promotes virus entry and replication.

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Aucella, F., C. Tetta, V. Tessore, C. De Nitti, M. Vigilante, G. Gatta, E. Grandone et al. "Is Steam Sterilization Really Making Any Difference in Dialysis-Induced Cytokine Release?" International Journal of Artificial Organs 25, n.º 9 (septiembre de 2002): 832–37. http://dx.doi.org/10.1177/039139880202500904.

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Ethylene oxide (ETO) is presently the most commonly used sterilization method for medical devices. Although alternative sterilization modes such as steam sterilization have been suggested, the effect of steam on dialysis-induced cytokine release is unknown. We enrolled 9 patients on chronic hemodialysis and evaluated at different intervals IL- 1βproduction while treated with ETO (NC1785–Bellco) and steam sterilized NC 1785S-Bellco) Synthetically Modified Cellulose (SMC). A basal test during treatment with NC 1785 was performed (A); the same test was set up 4 weeks after treatment with NC 1785S (B) and, lastly, 4 weeks after returning to NC 1785 (C). Peripheral blood mononuclear cells (PBMC) were purified before and after the dialysis session, were isolated on a Ficoll/Hypaque gradient and incubated for 24 h. Spontaneous IL-1β release was evaluated in the supernatant and in the lysate. In A, IL-1βlevels were (in pg/ml/106 cells, in supernatant and lysate, respectively): 5.8 ± 4.8 and 7.6 ± 5.2 in pre-HD and 4.68 ± 3.6 and 9.7 ± 6.65 in post-HD. These levels showed a clear reduction in B: 2.5 ± 2.2 and 4.4 ± 3.1 in pre-HD, and 4.35 ± 6.6 and 7.52 ± 7.22 in post-HD. In the C test, 4 weeks after the return to the ETO membrane, IL-1βlevels remained unchanged: 2.9 ± 1.8 and 4.5 ± 3.1 in pre-HD; and 2.6 ± 3 and 5.7 ± 6.6 in post-HD. Statistical analysis showed significant changes in the pre-HD levels both in supernatant (p < 0.04) and in lysate (p < 0.04). Steam sterilization of SMC induced a lower spontaneous IL-1β release, but this effect was not statistically significant due to the large inter-individual variation. Hence, contrary to claims of better biocompatibility, steam sterilization does not result in a reduced production of pro-inflammatory IL-1β.

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Lee, Kyoung-Eun, Hyun-Ae Woo, Seoug-Ha Yang, Jung-Won Huh, Moon Young Choi, Hye Jung Chang, Jee-Young Ahn et al. "Restoration of Peroxiredoxins and Catalase Is Closely Correlated with the Level of Philadelphia Chromosome during Imatinib Therapy in CML." Blood 110, n.º 11 (16 de noviembre de 2007): 4539. http://dx.doi.org/10.1182/blood.v110.11.4539.4539.

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Abstract Background: Several investigators have recently shown that activated growth factor receptors increase the relative levels of intracellular ROS and that bcr/abl kinase induces the production of ROS in hematopoietic cells. In addition, bcr-abl kinase induces self-mutagenesis via ROS to encode IM resistance. Meanwhile, two members of the peroxiredoxin family, Prx 1 or Prx 2, efficiently lowered the intracellular level of H2O2 and blocked the induction of apoptosis by ceramide, suggesting that the Prx enzymes contribute to intracellular signaling by removing H2O2. In this study, we investigated the changse in the levels of H2O2-removing enzymes like Prxs, glutathione peroxidase 1 (Gpx1), and catalase during Imatinib (IM) therapy in CML. Methods: Mononuclear cells(MNC) were isolated by standard Ficoll-Hypaque from the bone marrow aspiration at the time of diagnosis and during treatment with IM (Gleevec, Novartis, East Hanover, NJ). Standard cytogenetics analysis and RT-PCR for Philadelphia chromosomes were performed. For immunoblot analysis of antioxidant enzymes, cell lysates were fractionated by SDS-PAGE, and the separated proteins were transferred electrophoretically to a nitrocellulose membrane (Protran, Germany) and were probed with antibodies specific for Prx I, II, VI, GPx1, or catalase (AbFrontier, South Korea). Results: Samples from the diagnosis CML patients showed significantly decreased levels of Prx I, Prx II, Prx IV, and Gpx I, but also revealed an increased level of catalase. Especially prominent was the diminished ratio of Prx II to catalase in CML patients when compared with those of normal individuals. As the level of Philadelphia chromosomes decreased to that of normal individuals as the result of IM treatment, the expression levels of Prx(s) (P=0.018) and catalase (P=0.009) were restored to the levels of normal individuals. Conclusions: Decreased Prx 2 and elevated catalase levels at the time of diagnosis are closely correlated with the elevated bcr/abl kinase level in CML. The aberrant expression of those antioxidant enzymes returned to normal with IM treatment. Now, we will attempt to develop these method(s) to assess the changes of Prx(s) and catalase on the single cell level using immunohistochemistry with monoclonal Ab(s). Understanding the molecular mechanisms of changes on antioxidant might be a potent tool in developing more effective new drugs in CML.

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Lannert, H., T. Able, S. Leicht, R. Saffrich, V. Eckstein, M. Lenze, R. Hofmann, A. Lenze, T. Franz y A. D. Ho. "Different kinases acting on stathmin (oncoprotein, Op18) in human healthy and malignant hematopoietic stem cells". Journal of Clinical Oncology 25, n.º 18_suppl (20 de junio de 2007): 17527. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.17527.

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17527 Background: Stathmin/Op18 is a cytosolic phosphoprotein which regulates the dynamics of microtubules. This regulation is important in mitosis during cell division and in the migration of cells in modification of the cytoskeleton. The process of tumor proliferation and metastasis is characterized by high rates of mitosis and migration into distant tissues. Stathmin itself is regulated by kinases through phosphorylation of mainly 4 different serin sides. In this study, we investigated stathmin- and its kinases expression in native hematopoietic CD34+ stem cells (HSCs) from bone marrow (BM) in comparison to mobilized peripheral blood stem cells (mPBSCs) from G-CSF stimulated donors and leukemic CD34+ cells from patients with AML. Methods: Mononuclear cells were isolated by a standard Ficoll-Hypaque gradient separation method from the different blood sources. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. In comparative proteome analysis, we detected the protein expression of stathmin in mPBSCs, AML CD34+ cells, and in native HSCs from BM. We performed microarray-based gene expression profiles of these cells and focused on kinases regulating stathmin’s activity. Furthermore, we monitored stathmin and its relevant kinases by FACS analyses of the enriched cell fractions and by fluorescence microscopy of bone marrow smears and cytospins. Results: In this study, we have shown in comparative proteome analysis (Q-TOF-MS/MS) that stathmin is expressed in G-CSF mobilized hematopoietic stem cells for the first time and in AML cells. In microarray analysis we indentified up- and down-regulated kinases: MAPK, PAK1, PKC beta/zeta, MEKK3 and CDKs. Accordingly, we demonstrated in FACS analyses and in immunofluorescence microscopy the high intracellular expression of PKCzeta in AML cells and MEKK3 as well PAK1 in mPBSCs. Conclusions: Our findings show that G-CSF stimulates Stathmin expression in mPBSCs and plays a key role in migration into peripheral blood. Furthermore, we show the different expression of kinases acting on stathmin in mPBSCs and AML cells. Consequently, stathmin and its relevant kinases promise to become a future target in therapies of malignant processes. No significant financial relationships to disclose.

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Jueckstock, J. K., B. Rack, E. Thurner-Hermanns, H. Forstbauer, K. Pantel, H. Ulmer, M. Beckmann, W. Lichtenegger, W. J. Janni y K. Friese. "Detection of minimal residual disease (MRD) in peripheral blood of primary breast cancer patients: Translational research in the SUCCESS study". Journal of Clinical Oncology 25, n.º 18_suppl (20 de junio de 2007): 565. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.565.

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565 Background: Patients with detection of MRD in bone marrow are known to have an increased risk for recurrence and a poorer clinical outcome. However, peripheral blood would be the preferable compartment to monitor treatment efficacy due to increased feasibility. The translational research program of the German SUCCESS-trial was established to evaluate MRD in peripheral blood at 4 different time points during adjuvant systemic treatment of breast cancer patients. Here first results of the detection of MRD at primary diagnosis and after adjuvant chemotherapy will be presented. Patients and Methods: Cells were separated by Ficoll-Hypaque density- gradient centrifugation followed by labelling of epithelial cells with the anti-cytokeratine-antibody A45-B/B3 (directed against cytokeratines 8, 18 and 19) and immunohistochemically staining with neu-fuchsin. All preparations were screened by two independent persons. Results: 328 breast cancer patients were analyzed at primary diagnosis. Among those, 133 patients returned for a 2nd blood sampling after completion of adjuvant chemotherapy. Most of the tumors were small (43% pT1, 51% pT2, 4% pT3, 1% pT4) but of intermdediate or unfavourable grade, (G1 7%, G2 48%, G3 45%). 66% of the patients were node-positive (34% pN0, 38% pN1, 20% pN2, 8% pN3) and a positive hormone receptor status was seen in 71%. In 22% the Her2-status was positive. MRD in peripheral blood was found in 31% of all patients before and in 9% after chemotherapy. The mean number of detected cells was 2 (range 1- 9). In 87,2 % of the patients who showed MRD at the first measurement no MRD was detected after chemotherapy. 16% of patients without detection of MRD at primary diagnosis showed MRD after chemotherapy. Neither tumor size (p= 0.624), lymph node metastases (p= 0.450), histopathological grading (p= 0.168), hormone receptor status (p= 0.270) or Her2/neu status of the primary tumor (p= 0.893) correlated with the presence of MRD. Conclusions: The detection of MRD in peripheral blood can be widely used and is suitable for repeated measurements. Further follow-up will show, if this method can be used for risk stratification and monitoring of treatment efficacy in adjuvant breast cancer. No significant financial relationships to disclose.

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Lannert, Heinrich, Thomas Franz, Volker Eckstein, Reiner Hofmann, Angela Lenze, Kerstin Horsch, Katrin Miesala y Anthony D. Ho. "Quantitative and Qualitative Protein Expression Mapping of Highly Enriched G-CSF Mobilized CD34+ Stem Cells from Peripheral Blood." Blood 104, n.º 11 (16 de noviembre de 2004): 4130. http://dx.doi.org/10.1182/blood.v104.11.4130.4130.

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Abstract Background: Proteome analysis is a direct measurement of proteins in terms of their presence and relative abundance in a defined system. The overall aim of a proteomic study is characterization of the complex network of cell regulation. Different states of a cell can be compared and specific qualitative and quantitative protein changes can be identified. We focused our first proteom-investigations on G-CSF mobilized CD34+ stem cells from peripheral blood (PB). Methods: Mononuclear cells from healthy donors were isolated by a standard Ficoll-Hypaque gradient separation method after leucapheresis from PB. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. Sample preparation, determination of protein concentrations, 2D-gel-electrophoresis with a 1D-separation isoelectric focusing (IEF) with immobilised pH gradient (IPG) strips (17cm), 2D-separation with SDS-PAGE were performed and described in Proteome Works System (BioRad), pH range from 3 until 10 and molecular weight from 5 until 200kDa. Sypro ruby stained gels were used for protein identification by peptide mass fingerprint analysis with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Results: A.) The pattern of the G-CSF mobilized CD34+ fraction from PB cells is complex and shows >1000 protein spots (dependent on protein concentration) in silver stain using PDQuest. B.) The most dominant 150 protein spots were characterized by MALDI-TOF analyses. A sample of identified spots and their functional groups are specified: 1. Cytosketeletal proteins: tubulin, actin, profilin, tropomyosin. 2. Signaling proteins: enolase, rho GDP dissociation inhibitor 2, glutathione S transferase, nucleophosmin. 3. Metabolism: phosphoglycerate kinase I, glyceraldehyde-3P-dehydrogenase. 4. Protein folding: heat shock proteins (HSP 60, HSP70c), molecular chaperones (GRP78). Conclusions: Proteomics is a highly effective method to describe the gen-expression and the cell biology of stem cells on real protein transcription. Highly purified CD34+ cells from PB demonstrate a complex proteom. Our preliminary results show that cell cycle determining chaperones (folding of other protein and responsible for their functional activation or deactivation) are dominantly expressed. Thus cytoskeletal proteins dominate the protein pattern of CB stem cells. Further Proteom-comparison with other subsets of stem cells from different origins (e.g. fetal liver, cord blood, bone marrow) are currently achieved. Figure Figure

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Luque, Diogo G., Ana Carolina S. Ferreira, Flavia C. Vasconcelos, Raquel C. Maia y Jolie K. Kwee. "High Catalase Activity: Another Possible Pathway for Imatinib-Resistant CML Patients." Blood 106, n.º 11 (16 de noviembre de 2005): 4890. http://dx.doi.org/10.1182/blood.v106.11.4890.4890.

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Abstract Imatinib (imatinib mesylate: Novartis Pharmaceuticals, Basel, Switzerland) is a well-known selective inhibitor of BCR-ABL tyrosine kinase activity, a hallmark enzyme in the pathogenesis of chronic myeloid leukemia (CML). Resistance to imatinib in CML patients has been associated with different and complex types of mechanisms ranging from nonspecific multidrug resistance protein, preventing the drug to reach its target, to BCR-ABL kinase domain point mutations, reducing the affinity of the enzyme to Imatinib. More recently, it was described that modulation of reduced glutathione (GSH) intracellular levels restores sensitivity to Imatinib in Imatinib-resistant cell line. This suggests that induction of hydrogen peroxide (H2O2), a well-known apoptotic reactive oxygen species (ROS), might be another Imatinib anti-leukemic mechanism action. Additionally, it is known that catalase activity is enhanced through phosphorilation by the c-abl tyrosine kinase (TK). In a preliminary work, our group found normal levels of GSH but high levels of antioxidant enzyme catalase in CML patients. The aim of this work is to evaluate the possible role of catalase as another pathway to Imatinib resistance. White blood cells from blood donors, CML patients and patients with reactional leukocytosis caused by infectious bacterian diseases were obtained using Ficoll-Hypaque. Catalase activity was measured by monitoring decomposition of 10mM H2O2 at 240nm according to the method described by Aebi. The viability of eritroleukemia cell lineage K562 previously treated or not with 100μM 3-aminotriazole (catalase inhibitor) was evaluated by MTT method with Imatinib 1, 5 and 25μM for 24 and 48 hours. Imatinib induced intracellular ROS generation was monitored by flow cytometry with 40μM 2′, 7′-dichlorofluorescein diacetate (DCFH-DA), a well-known ROS capturing reagent. Catalase activity was increased up to 30x in CML patients but not in reactional leukocytosis. When BCR-ABL positive cell line K562 was treated with Imatinib (50μM), catalase activity levels decreased in 38% compared with untreated cells. Also, we found that 3-aminotriazole can overcome the resistance to Imatinib in K562 cells resulting in 25% enhancement of dead cells. The protection of catalase against Imatinib-ROS generation was confirmed by DCFH-DA/FACs in cells incubated with PEG-catalase (monomethoxypolyethylene glycol covalent attached to catalase). Taken together, these results suggest that catalase might be another attractive target for overcoming resistance to Imatinib in Imatinib-resistant CML cells with high catalase activity.

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Pulido-Méndez, María M., Elvia Azuaje y Alexis Rodríguez-Acosta. "A novel activity on thymocytes cells exerted by the rattlesnake (Crotalus durissus cumanensis) venom". Biomédica 41, n.º 3 (22 de septiembre de 2021): 449–57. http://dx.doi.org/10.7705/biomedica.5599.

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Introduction: The thymus is active mainly during the neonatal and pre-adolescent periods.Objective: To test naïve thymocytes proliferation and monocytes stimulation.Materials and methods: We collected fresh thymus tissue from neonate mice after surgery. Suspension cells were coated onto Ficoll-Hypaque support. The obtained cells (thymocytes) were cultured measuring the proliferation of naïve T cells stimulated by Crotalus durissus cumanensis (Cdc) venom at sub-lethal doses (20 ng). Then, we supplemented the wells with AlamarBlue™ and incubated them for 5 h to test their proliferation. Mononuclear cells from mice peripheral blood were collected and layered onto the support of the Ficoll-Hypaque solution. We added the thymocytes actively dividing (25 x 105 cells) from cultures stimulated with Cdc venom at 20 ng/well to cultured monocytes freshly obtained from the Ficoll-Hypaque separation. Both cell populations were incubated for 36 h until monocytes matured to macrophages.Results: The naïve thymocytes rapidly proliferated after stimulation with the Cdc venom (NTCdc) and these successively induced the maturation and function of monocytes progenitor cells to mature macrophages, which ingested Chinese ink.Conclusions: The naïve thymocytes proliferated by stimulation with the Cdc venom and subsequently the NT/Cdc induced the rapid maturation and function of monocytes progenitor cells becoming mature macrophages with their phenotypic characteristics.

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Huang, Xin, Suxia Geng, Jianyu Weng, Zesheng Lu, Linji Zeng, Minming Li, Chengxin Deng, Xiuli Wu, Yangqiu Li y Xin Du. "Analysis of the Expression of PHTF1 and Related Genes in Acute Lymphoblastic Leukemia". Blood 126, n.º 23 (3 de diciembre de 2015): 4988. http://dx.doi.org/10.1182/blood.v126.23.4988.4988.

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Abstract Background: In the human T-cell acute lymphoblastic leukemia (T-ALL) cell line Molt-4, siRNA-mediated suppression of BCL11B expression was shown to inhibit proliferation and induce apoptosis, which may be related to PHTF1 gene expression, and the FEM1B and Apaf-1 genes may be downstream of PHTF1. In this study, we analyzed the expression level of PHTF1 and related genes in patients with ALL to clarify the role of the PHTF1-FEM1b-Apaf-1 pathway in hematologic malignancies. Methods: Fifteen newly diagnosed and untreated patients with AML, fourteen newly diagnosed and untreated patients with CML in chronic phase, twenty-two newly diagnosed and untreated patients with ALL, and six newly diagnosed and untreated patients with CLL were recruited. Peripheral blood mononuclear cells (PBMCs) from ten healthy individuals (HIs) served as controls. PBMCs were separated using the Ficoll-Hypaque gradient centrifugation method. All procedures were conducted in accordance with the guidelines of the Medical Ethics committees of the Health Bureau of Guangdong Province, China.Real-time PCR was used to determine the gene expression level of PHTF1 in hematologic malignancies. The PHTF1, BCL11B, FEM1B and Apaf-1 gene expression levels and correlations were analyzed in patients with primary ALL and healthy individuals (HIs). Results: PHTF1 overexpression was found in recently diagnosed AML (p <0.001), CML-CP (p<0.001), and ALL (p= 0.016) patients in comparison with HIs. The PHTF1 expression level in CLL patients was not significantly different compared with HIs (p= 0.165). FEM1b and Apaf-1 overexpression was found in recently diagnosed ALL (p<0.005) patients compared with HIs. Positively correlated expression was found for the PHTF1, FEM1b and Apaf-1 genes in patients with ALL (p<0.005) and HIs (p<0.005), and positively correlated expression was found for the PHTF1 and BCL11B genes in HIs (p<0.005). Conclusions: PHTF1 acts as a tumor suppressor gene, and its overexpression might be related to cell proliferation, inhibition and apoptosis. PHTF1 and BCL11B gene disorders may contribute to T-ALL pathogenesis. PHTF1 might be a therapeutic target for triggering the PHTF1-FEM1b-Apaf-1 apoptosis pathway in primary acute lymphoblastic leukemia. Acknowledgment The project was sponsored by grants from National Natural Science Foundation of China (No. 81100384, No. 81270648, No.91129720, and No. 30771980) Disclosures No relevant conflicts of interest to declare.

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Chan, Howard H., Brenda Su, Wei Li, John G. Kelton y Greg Denomme. "Intravenous gamma Globulin Does Not Increase RNA Expression of FcgRIIb in Purified Human Monocytes." Blood 106, n.º 11 (16 de noviembre de 2005): 3888. http://dx.doi.org/10.1182/blood.v106.11.3888.3888.

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Abstract Introduction Intravenous gamma globulin (IVIG) has been shown to modulate immune cytopenia through FcγRIIb in a murine model (1). There are conflicting results from the studies on the protein expression of the FcγRIIb. In that murine model, there is increase in FcγRIIb positive splenocytes following IVIG treatment (1). Yet, in patients with Kawasaki disease, flow cytometric analysis of peripheral CD14+ mononuclear cells shows no change in FcγRIIb expression (2). To study the effect of IVIG on the RNA expression of FcγIIB, we performed RT-PCR using monocytes collected from three healthy volunteers. The monocytes were harvested using a magnetic separation method. The purified monocytes were then cultured with IVIG in vitro. Methods Peripheral blood mononuclear cells (PBMC) from three healthy donors were isolated from heparin anticoagulated blood by Ficoll-Hypaque density separation. The PBMC were washed and the monocytes were isolated using anti-CD14 magnetic bead positive selection. In the first set of experiments, purified monocytes were cultured at 37°C, 0.5% CO2 in media containing 10% fetal calf serum (FCS) (control) and in the same media containing IVIG (0.5 mg/mL and 5.0 mg/mL). In the second set of experiemnts, purified monocytes were cultured at 37°C, 0.5% CO2 in serum free medium (SFM), in SFM containing IVIG (5.0 mg/mL), and in SFM containing 10% FCS. Monocytes were harvested from the culture flasks after an 18-hour incubation. Isolated monocytes were lyzed and the total RNA was extracted with TRIzol reagent according to the manufacturer’s protocol. One μg of RNA was used to generate first strand complimentary DNA using oligo-dT and Superscript II reverse transcriptase. Amplitaq Gold DNA polymerase was used to PCR-amplify FCGR2B cDNA. PCR amplification was performed using 5 μL of cDNA and 1 μM each of an FCGR2B-specific primer pair (3) in a total volume of 50 μL. The PCR products were evaluated using 10% polyacylamide gel electrophoresis. Results There was no change in FCGR2B expression when purified monocytes were incubated with either 0.5 mg/mL or 5mg/mL IVIG for 18 hours (n=3) (Fig1). However, there was a demonstrable increase in FCGR2B expression when monocytes were cultured in the presence of FCS (Fig2). Conclusion IVIG does not directly modulate monocyte FCGR2B gene transcription. Figure Figure

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Chan, Howard H. W., Wei Li, Brenda B. Su y Gregory A. Denomme. "The Effect of IVIG on Fcγ Receptor-Dependent Monocyte Phagocytosis and FcγR Gene Expression." Blood 104, n.º 11 (16 de noviembre de 2004): 3829. http://dx.doi.org/10.1182/blood.v104.11.3829.3829.

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Abstract BACKGROUND The mechanism(s) of intravenous immunoglobulin (IVIG) towards inhibition of monocyte phagocytic activity involves the function and/or the expression of inhibitory FcγRIIb in a murine model. To confirm these findings in human monocytes, we used a human monocyte phagocytic model in vitro to study the effects of IVIG on the phagocytic activity and the expression of FcγR genes. METHODS Part A: Monolayer Monocyte Phagocytosis Assay Normal volunteer’s peripheral blood mononuclear cells (PBMC) were isolated from heparin anticoagulated blood by Ficoll-Hypaque (Pharmacia Biotech) density separation. The PBMCs were washed and the monocytes were purified using a magnetic bead-positive selection method with anti-CD14 antibody (Miltenyl Biotec). 105 monocytes were incubated in a microtiter plate at 37°C for 1 hour before exposed to IVIG 0.5 g/L. Anti-D (WinRho) sensitized Rh positive (R2R2) red cells were added to the monocytes at 0.5 hour and 18 hour post-IVIG treatment. After 1 hour incubation with sensitized RBC, monocytes phagocytic activity is measured by chemiluminescence detection with a LumiCount (Packard). The readings were normalized with maximal chemiluminescence signal achieved by the monocytes without prior exposure to IVIG (positive control). Part B: RT-PCR of FcγRIIa and FcγRIIb After 18 hours of exposure to two different concentrations of IVIG (0.5 and 5 gm/L), monocytes were collected and total RNA was isolated with TRIzol reagent (Invitrogen). 1 μg of RNA was used to generate first strand cDNA using Superscript II RT kit (Invitrogen). FcγRIIa and IIb were amplified with AmpliTaq Gold DNA polymerase system (Applied Biosystems). The PCR products were evaluated by polyacrylamide gel electrophoriesis. RESULTS Part A: Dose-response curves were generated by plotting normalized chemiluminescence against the concentration of anti-D used to sensitize the red cells. Anti-D sensitized red cells were phagocytosed by monocytes in a dose-dependent manner. There is a time-dependent inhibition of monocyte phagocytosis when monocytes were incubated with IVIG at 0.5 gm/L. (Fig. 1) Part B: There is no significant difference in the gene expression of FcRγIIb and FcγRIIa in the adherent monocytes after incubating with either low dose (0.5 gm/L) or high dose (5 gm/L) of IVIG for 18 hours. (Fig 2) CONCLUSION Delayed inhibition of phagocytic activity with18-hour exposure to IVIG is not directly mediated via the modulation of FcγRIIb gene expression in human monocytes. Other mechanisms, such as intracellular signalling or receptor coupling, might be involved in the delayed inhibitory effects of IVIG. Figure Figure Figure Figure

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Chiou, Tzeon-Jye, Tien-Hua Chu, Wei-Hao Wu, Sin-Tak Chu y Woan-Fang Tzeng. "Expansion and Reactivation of Human Naïve T-Cells Transition into CD4+CD25+FoxP3+CD127-Regulatory T Cells, In Vitro, for the Potential Use of Graft-Versus-Host Disease Prevention". Blood 128, n.º 22 (2 de diciembre de 2016): 5729. http://dx.doi.org/10.1182/blood.v128.22.5729.5729.

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Abstract Background Allogeneic hematopoietic stem cell transplantation (HSCT) has been used to treat some of hematological malignancies and inherited or acquired non-malignant diseases. Unfortunately, graft-versus-host disease (GVDH) occurred approximately 15% in transplant recipients and decreases the success of allogeneic HSCT. Currently, isolation of natural regulatory T cells (nTregs) and in vitro-expanded nTregs was shown to be an effective therapy to GVHD patients. However, shortage of nTregs in peripheral blood and time consumption of expansion in vitro may eventually limit the clinical application. Conversely, induction regulatory T cells (iTregs) can be generated in vitro from naïve T cells and to a large number of iTregs in short time. It may improve the GVHD therapeutic efficiency. The stability of FoxP3 in iTregs is a crucial factor for suppression of GVHD. Besides, a functional iTreg cell should be CD127 negative. Because of that, we should provide a large number of effective iTreg cells, the CD4+CD25+FoxP3+CD127-iTregs, to ensure the successful therapy. Aim In order to harvest the large number of effective iTregs for preventing the GVHD, we try to develop a method for induction of more iTreg cells via expansion and repeated activation of naïve T cells. Methods Human PBSC were prepared from peripheral blood of health donor by Ficoll-Hypaque density gradient centrifugation. Naïve T cells were isolated by negative selection. The harvested naïve T cells were cultured and stimulated under cytokines-containing RPMI1640 medium. The flow chart was shown in figure. For cell proliferation, the cells were cultured under IL-2 supplemented medium. The harvested cells were analyzed by flow cytometry with fluorescence-conjugated CD antibodies, including CD4, CD25, CD127 and FoxP3. Results With our purpose, harvest more iTreg cells should be efficient for clinical application. Amplification of the T cell number can obtain more iTreg cells; therefore, cultivation of the T cells under IL-2-containing medium would stimulate the cell proliferation to about 4-fold on the 10th day. After the first cytokines stimulation, we harvested the iTregs, and then, keep the cells in IL-2 only medium for another 5 days to amplify the T cells. On the 10th day, naïve T cell reactivation would be performed with anti-CD3/CD28 antibodies and with cytokines supplement for the second induction. The number of iTregs would increase to about 20-40%. It indicated that we would harvest more iTreg cells for application. If we cultured the naïve T cells under IL-2 medium in the beginning, we would not obtain much more iTregs, comparing with the reactivation method. Conclusion Our study showed that after the first activation and induction of naïve T cells, we could expand more cells under IL-2 containing medium. Then, after the second activation and induction, we could harvest more iTreg cells markedly. The result should develop a novel-cell based approach for potentially reducing the risk of GVHD within short time. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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Mi, Yingchang, Wenbin Wu, Qing Zhang, Yan Li, Xiaoyan Li, Zheng Tian y Min Wang. "Expression of Kindlins in Acute Myeloid Leukemia". Blood 118, n.º 21 (18 de noviembre de 2011): 4910. http://dx.doi.org/10.1182/blood.v118.21.4910.4910.

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Abstract Abstract 4910 The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. They comprise of three evolutionarily conserved members, kindlin-1, kindlin-2 and kindlin-3, they share considerable sequence and structural similarities. A few of study revealed that Kindlin-2 influences solid tumor cell invasion and resistance. With regard to AML, the influence of Kindlins is still unknown. To evaluate the clinical significance of Kindlin-2 in acute myeloid leukemia (AML), we investigated the expression of Kindlin-2, kindling-3 in AML cells. 1. Materials and methods K562, KG-1a, HL60, U937, Jurkat cell lines were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, GIBCO) at 37°C in a humidified atmosphere of 5% CO2. Bone marrow (BM) samples were obtained from 88 patients with de novo AML from Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC). Samples of 9 normal donors and ITP were used as the control group. Bone marrow mononuclear cells (BMMCs) were prepared by Ficoll-Hypaque density gradient centrifugation. Expressions of Kindlin-2, Kindlin-3 were detected by RQ-PCR. The following primers for real-time PCR were used: (a) Kindlin-2 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (b) Kindlin-2 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (c) Kindlin-3 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (d) Kindlin-3 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (e) GAPDH sense primer, 5'-GAAGGTGAAGGTCGGAGTC-3'; (f) GAPDH antisense primer, 5'-GAAGATGGTGATGGGATTTC-3'. Analysis was performed using ABI 7500 Sequence Detection software (Applied Biosystems). The expression of Kindlin-2 and Kindlin-3 were showed as RQ value calculated through ΔΔCt method [ΔΔCt = (CtKindlin □ CtGAPDH)sample □ (CtKindlin □ CtGAPDH)calibrator]. The ΔCt (CtKindlin □ CtGAPDH) of K562 was defined as calibrator, and the RQ of calibrator was 1.000. Relationships between Kindlin-2, Kindlin-3 and the patients' clinical data were analyzed. 2. Results Expression of Kindlins in newly diagnosis AML The level of Kindlin-2 in AML (0.163±1.665) was significantly lower than that in non-AML (1.683±1.395) controls (p=0.010). No significant difference was found between the AML and controls in levels of Kindlin-3 (p=0.216). Out of the 79 patients who accepted treatment, 61 patients achieved complete remission (CR) and 18 patients were NR. Patients with higher expression of Kindlin-2 had a higher CR rate (86.8% vs 68.3%) (p=0.050). Expression of kindling 3 was unrelated to CR rate. Both of kindling-2 and kindling-3 increased after CR. This finding implicates Kindlin-2 as a potential prognostic factor of AML. Disclosures: No relevant conflicts of interest to declare.

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Narducci, Maria Grazia, Maria Cristina Picchio, Cristina Lazzeri, Irene Angelucci, Enrico Scala, Antonella Bresin, Elisabetta Caprini et al. "The B-Cell Chemoattractant Factor CXCL13 Is Expressed in the Malignant Lymphocyte of the Sezary Syndrome." Blood 108, n.º 11 (1 de noviembre de 2006): 2292. http://dx.doi.org/10.1182/blood.v108.11.2292.2292.

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Abstract Sézary Syndrome (SS) is a rare and aggressive form of Cutaneous T-Cell Lymphoma (CTCL) characterised by a distinct metastatic pattern mainly involving blood and skin. Our expression analyses performed by microarrays demonstrated that many chemokines resulted up-regulated in this type of lymphoma. Since these chemoattractant molecules play a critical role in cellular recruitment and homing to tissues and in the metastatic process of several tumors, we focused our attention on one of them named CXCL13, a lymphoid chemokine involved in B-cell compartmental homing within secondary lymphoid organs. Peripheral Blood Mononuclear cells (PBMCs) were isolated from blood obtained from SS patients and controls by Ficoll-Hypaque density gradient centrifugation (Sigma Aldrich). SS cells and healthy resting CD4+ lymphocytes were purified by positive selection using an anti-human-CD4 conjugated dynabeads (Oxoid). Total RNA was extracted using the Trizol reagent (Life Technologies). Quantitative-Real Time RT-PCR analysis was performed on CD4+ sorted from 14 SS patients and 3 controls. CXCL13 primers were designed by means of the Primer Express software package (Applied Biosystems). The qRT-PCR were performed with a SYBR Green I dye chemistry and AmpliTaq Gold DNA Polymerase on an ABI PRISM 7000 machine (Applied Biosystems). Immunohistochemistry analyses for CXCL13 were performed on formalin-fixed, paraffin-embedded skin biopsies from 15 SS, 15 MF, 6 MF-B cell rich patients using streptoavidin-biotin peroxidase labeling method (DAKO). Sections were counterstained with hematoxylin. Plasma CXCL13 levels were determined using a CXCL13 ELISA kit (BD Pharmingen). Results can be summarized as follow: qRT-PCR analysis revealed that 6 out 13 of SS patients showed an high mRNA levels of CXCL13; Immunohistochemistry analysis showed that CXCL13 is abundantly expressed by neoplastic skin-infiltrating lymphocytes of 9 out 15 SS skin biopsies. Conversely, CXCL13 is weakly expressed on scattered neoplastic skin-infiltrating lymphocytes of 1 out 15 MF and 1 out 6 MF-B cell rich biopsies. Plasma CXCL13 concentrations in SS patients (n = 10) were 1362 ± 134 pg/mL. Conversely, those in MF patients (n = 10) and healthy donors (n = 5) were 70 ± 43 and 13 ± 10 pg/mL, respectively. Compared with healthy controls, plasma CXCL13 levels were significantly higher in patients with SS (p<0.001) and with MF (p=0.04). In this study we report that both circulating and skin-infiltrating neoplastic lymphocytes of SS patients abundantly express CXCL13. Furthermore, this chemokine is also detectable at high level on plasma of SS patients. Conversely, CXCL13 is not observable in healthy controls as well as in Mycosis Fungoides, a variant of low grade of CTCL. These findings indicate that CXCL13 could play a role in pathobiology of Sézary Syndrome and that the expression of this chemokine could be used as diagnostic marker for this kind of tumor

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Naim, Mehrnoush R., Rolando Gumatay, Geraldine D. Ong, Qi Wang, Alfredo Santiago, Lesley Cabigon, Sharon Daroy-Atriatico, Ernesto J. R. Bustamante y Xiaohai Zhang. "P259 Comparison of two cell preparation methods: Standard Ficoll hypaque vs. Easysep for flow and CDC crossmatching". Human Immunology 78 (septiembre de 2017): 244. http://dx.doi.org/10.1016/j.humimm.2017.06.319.

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Takamatsu, Hiroyuki, Yoshitaka Zaimoku, Tatsuhiko Ozawa, Shintaro Kawai, Hidenori Tanaka, Hiroyuki Kishi, Atsushi Muraguchi y Shinji Nakao. "Development of Novel Human Anti-HLA-Monoclonal Antibodies for Clinical Applications Using Peripheral Blood B Cells Derived from Anti-HLA Antibody-Positive Donors". Blood 128, n.º 22 (2 de diciembre de 2016): 4723. http://dx.doi.org/10.1182/blood.v128.22.4723.4723.

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Abstract Introduction: The development of monoclonal antibodies (mAbs) such as anti-CD20 mAb (rituximab) and anti-CD38 mAb (daratumumab) has revolutionized the treatment of lymphoid malignancies. Potential efficacy of HLA allele-specific mAbs in treating malignant lymphoma has been shown by several studies. However, treatment of B-cell malignancies with humanized mAbs against HLA-DR alleles is associated with infusion-related toxicities (Lin et al., Leuk Lymphoma, 2009). In addition, previous attempts to develop mAbs specific to some HLA alleles (e.g., HLA-B61) by immunizing mice with recombinant HLA proteins have failed, likely because of a wide variety of polymorphisms of HLA molecules. To overcome these problems, we recently created a novel method to develop mAbs using microarray technology and human peripheral blood (PB) B lymphocytes derived from donors who are positive for anti-HLA antibodies. The process took approximately 1 month to produce human anti-HLA mAbs. Methods: Approximately 20 ml of PB was collected from anti-HLA antibody-positive donors, and mononuclear cells (MNCs) isolated using the Ficoll-Hypaque method were cultured in RPMI-1640 + 10% FCS containing 5 μg/ml R-848, 1000 IU/ml hIL2, 2.5 μg/ml CpG2006, 2.5 μg/ml anti-CD40 antibody, 100 ng/ml hIL21, 2 ng/ml hIL17, 10 ng/ml hIL4, and 100 ng/ml hBAFF for 6 days. CD138+ cells were then isolated using anti-CD138-antibody-conjugated microbeads. A microwell array chip was manufactured using micromachining techniques, as previously described (Jin et al., Nature Medicine, 2009). Microwells (diameter, 10 μm; depth, 15 μm) were formed on a silicon surface. The chip was coated with a PBS-containing purified HLA antigen (10 μg/ml) and incubated overnight at 4°C. After removing the antigen solution, 50 μl of the CD138+ cell suspension was added to the chip, and the mixture was incubated for 4 h at 37°C. After gentle washing with PBS, 2 μg/ml of anti-human IgG Fc-Cy3 solution was added to the microwells, and the plate was left at room temperature for 15 min. Finally, the cells were stained with 1 μM Oregon Green for 2 min. The microwells were screened for the antigen-specific antibodies released from single cells under a fluorescence microscope. Antigen-specific antibody-secreting cells (ASCs) from individual wells were isolated using a micromanipulator fitted with capillaries under the fluorescence microscope and were then expelled to microtubes for reverse transcription. The antibody cDNA fragments for VH and VL fragments were amplified using the single-cell 5′-RACE method and inserted into expression vectors containing the complete constant region of cDNA for heavy or light chains. Thereafter, CHO cells were transfected with both the heavy and light chain expression vectors to obtain a supernatant containing complete antibody molecules. The antigen specificity of the recombinant antibodies was examined using ELISA and flow cytometry (FCM). The frequency of ASCs in the PB-MNCs of donors was quantified using allele-specific oligonucleotide PCR. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of the mAbs were assessed using conventional methods. Results and Discussion : Two novel human mAbs specific to HLA-A24 and HLA-B61 were successfully generated. The antigen specificity of the mAbs was confirmed using ELISA and FCM. The mAbs bound to normal and malignant blood cells that expressed corresponding HLA alleles and showed CDC/ADCC activities against five B lymphoblastoid cell lines but not against lymphocytes/monocytes/granulocytes derived from five healthy donors, probably because of low expression levels of the target HLA alleles. The frequency of ASCs in PB-MNCs of donors was less than 0.001%. Conclusion s : The present method enabled the generation of mAbs specific to HLA alleles, which can be used for detecting minimal residual diseases of hematological malignancies as well as for treating B-cell lymphoma, within 1 month. Disclosures Takamatsu: Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria. Kawai:Wakunaga Pharmaceutical Co., Ltd.: Employment. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

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32

Rambaldi, Alessandro, Gianmaria Borleri, Gianpietro Dotti, Piermario Bellavita, Ricardo Amaru, Andrea Biondi y Tiziano Barbui. "Innovative Two-Step Negative Selection of Granulocyte Colony-Stimulating Factor–Mobilized Circulating Progenitor Cells: Adequacy for Autologous and Allogeneic Transplantation". Blood 91, n.º 6 (15 de marzo de 1998): 2189–96. http://dx.doi.org/10.1182/blood.v91.6.2189.

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Abstract A major obstacle in purifying either autologous or allogeneic hematopoietic stem cells from granulocyte colony-stimulating factor (G-CSF) mobilized circulating progenitor cells (CPC) is represented by the huge cellularity present in each apheretic product. To obtain a significant debulking of unwanted cells from the leukapheresis, we developed a modified protocol of immune rosetting whereby human ABO-Rh– compatible red blood cells (RBCs) are treated with chromium chloride and then coated with murine monoclonal antibodies (MoAbs) against leukocyte antigens. When experiments were performed with leukaphereses obtained from normal donors or from T-cell acute lymphoblastic leukemia (T-ALL) patients, RBCs were coated with murine MoAbs against human mature myeloid cells (CD11b) and T cells (CD6); whereas, in the case of patients with B-precursor ALL, B-cell non-Hodgkin's lymphoma (B-NHL), or multiple myeloma (MM), RBCs were coated with anti-CD11b only. After incubation with CPC, rosetting cells (myeloid precursor cells, granulocytes, monocytes, and T cells) were removed by Ficoll-Hypaque density gradient centrifugation with a blood cell processor apparatus, COBE (Lakewood, CO) 2991. After this step, a significant reduction of the initial cellularity was consistently obtained (range, 72% to 97%), whereas the median absolute recovery of the CD34+ cells was above 85% (range, 64 to 100), with a 10-fold relative enrichment ranging from 3% to 41%. In a second step, CPC can be further purged of contaminating T or B cells by incubation with lymphoid-specific magnetic microbeads (anti-CD2 and -CD7 to remove T cells; anti-CD19 to remove B cells) and elution through a type-D depletion column (composed of ferromagnetic fiber) inserted within a SuperMACS separator device (Miltenyi Biotech, Bergisch-Gladbach, Germany). By this approach, a highly effective (three to four logs) T-cell depletion was achieved in all experiments performed with normal donors or T-ALL patients (median loss of CD3+cells: 99.8% [range 99.2 to 100]) and an equally efficient B-cell depletion was obtained from B-precursor ALL, B-NHL, or MM patients. At the end of the procedure the T- or B-cell depleted fraction retained a high proportion of the initial hematopoietic CD34+ stem cells, with a median recovery above 70% (range 48% to 100%) and an unmodified clonogenic potential. In five patients (two follicular NHL and three ALL) the purified fraction of stem cells was found disease free at the molecular level as assessed by polymerase chain reaction (PCR) analysis of the t(14;18) chromosome translocation or clono-specific DNA sequences of IgH or T-cell receptor γ and δ chain genes. Purified autologous and allogeneic CPCs were transplanted in three and six patients, respectively, who showed a prompt and sustained hematologic engraftment. In conclusion, this method represents a simple and reproducible two-step procedure to obtain a highly efficient purging of T or B cells from G-CSF expanded and mobilized CPCs. This approach might lead to the eradication of the neoplastic clone in the autologous stem cell inoculum as well as for T-cell depletion during allogeneic transplantation.

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33

Rambaldi, Alessandro, Gianmaria Borleri, Gianpietro Dotti, Piermario Bellavita, Ricardo Amaru, Andrea Biondi y Tiziano Barbui. "Innovative Two-Step Negative Selection of Granulocyte Colony-Stimulating Factor–Mobilized Circulating Progenitor Cells: Adequacy for Autologous and Allogeneic Transplantation". Blood 91, n.º 6 (15 de marzo de 1998): 2189–96. http://dx.doi.org/10.1182/blood.v91.6.2189.2189_2189_2196.

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A major obstacle in purifying either autologous or allogeneic hematopoietic stem cells from granulocyte colony-stimulating factor (G-CSF) mobilized circulating progenitor cells (CPC) is represented by the huge cellularity present in each apheretic product. To obtain a significant debulking of unwanted cells from the leukapheresis, we developed a modified protocol of immune rosetting whereby human ABO-Rh– compatible red blood cells (RBCs) are treated with chromium chloride and then coated with murine monoclonal antibodies (MoAbs) against leukocyte antigens. When experiments were performed with leukaphereses obtained from normal donors or from T-cell acute lymphoblastic leukemia (T-ALL) patients, RBCs were coated with murine MoAbs against human mature myeloid cells (CD11b) and T cells (CD6); whereas, in the case of patients with B-precursor ALL, B-cell non-Hodgkin's lymphoma (B-NHL), or multiple myeloma (MM), RBCs were coated with anti-CD11b only. After incubation with CPC, rosetting cells (myeloid precursor cells, granulocytes, monocytes, and T cells) were removed by Ficoll-Hypaque density gradient centrifugation with a blood cell processor apparatus, COBE (Lakewood, CO) 2991. After this step, a significant reduction of the initial cellularity was consistently obtained (range, 72% to 97%), whereas the median absolute recovery of the CD34+ cells was above 85% (range, 64 to 100), with a 10-fold relative enrichment ranging from 3% to 41%. In a second step, CPC can be further purged of contaminating T or B cells by incubation with lymphoid-specific magnetic microbeads (anti-CD2 and -CD7 to remove T cells; anti-CD19 to remove B cells) and elution through a type-D depletion column (composed of ferromagnetic fiber) inserted within a SuperMACS separator device (Miltenyi Biotech, Bergisch-Gladbach, Germany). By this approach, a highly effective (three to four logs) T-cell depletion was achieved in all experiments performed with normal donors or T-ALL patients (median loss of CD3+cells: 99.8% [range 99.2 to 100]) and an equally efficient B-cell depletion was obtained from B-precursor ALL, B-NHL, or MM patients. At the end of the procedure the T- or B-cell depleted fraction retained a high proportion of the initial hematopoietic CD34+ stem cells, with a median recovery above 70% (range 48% to 100%) and an unmodified clonogenic potential. In five patients (two follicular NHL and three ALL) the purified fraction of stem cells was found disease free at the molecular level as assessed by polymerase chain reaction (PCR) analysis of the t(14;18) chromosome translocation or clono-specific DNA sequences of IgH or T-cell receptor γ and δ chain genes. Purified autologous and allogeneic CPCs were transplanted in three and six patients, respectively, who showed a prompt and sustained hematologic engraftment. In conclusion, this method represents a simple and reproducible two-step procedure to obtain a highly efficient purging of T or B cells from G-CSF expanded and mobilized CPCs. This approach might lead to the eradication of the neoplastic clone in the autologous stem cell inoculum as well as for T-cell depletion during allogeneic transplantation.

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34

Friedman, Daphne R., Yvonne Mowery, Margaret Kennedy, Karen M. Bond, J. Brice Weinberg y George J. Cianciolo. "The Anti-Inflammatory Investigational Agent LMP-420 Demonstrates in Vitro Cytotoxic Activity against Chronic Lymphocytic Leukemia Cells." Blood 114, n.º 22 (20 de noviembre de 2009): 2358. http://dx.doi.org/10.1182/blood.v114.22.2358.2358.

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Abstract Abstract 2358 Poster Board II-335 Background: Chronic Lymphocytic Leukemia (CLL) is a common incurable hematologic malignancy. When therapy is required, maximizing durable responses is often at the risk of increasing toxicity. Thus, developing novel therapeutic agents that have minimal overlapping toxicity with currently used chemotherapy would be advantageous. To this end, we investigated LMP-420, a boronic acid containing purine nucleoside analogue, that potently inhibits tumor necrosis factor alpha (TNF) transcription in stimulated peripheral blood mononuclear cells (PBMCs) without affecting cell viability. Since TNF has been implicated in promoting CLL cell viability and can be produced by CLL cells themselves, we hypothesized that LMP-420 would be cytotoxic for CLL lymphocytes, either alone or in combination with fludarabine. Methods: To test the activity of LMP-420, we negatively selected circulating CLL cells from blood collected from patients using the RosetteSep B-cell enrichment cocktail (StemCell Technologies) and a Ficoll-Hypaque gradient. This method yields greater than 95% purity of malignant lymphocytes, determined by immunophenotyping CD5+CD19+ cells. Prognostic markers such as IgVH mutation status, CD38 and ZAP70 expression, and interphase cytogenetics were determined as previously described. We assessed the fractional toxicity and 50% effective dose (ED50) of LMP-420, fludarabine, or the combination, with the MTS colorimetric cytotoxicity assay, in which CLL cells were incubated for three days in Hybridoma media + 10% fetal bovine serum with serial dilutions of drug alone or in combination. Apoptosis was measured via annexin V flow cytometry methods and caspase 3/7 activity assays. We determined the effect of LMP-420 compared to fludarabine on the normal hematopoietic system by testing serial dilutions of both agents in killing normal PBMCs and in suppressing erythroid and myeloid colony formation. Results: The median ED50 of LMP-420 for CLL cells was 423 nM (range 0.01 to 2224 nM, n = 21). Two patients had high-risk cytogenetics (17p or 11q deletions), and their ED50 values for LMP-420 were 691 and 90 nM, respectively. The cytotoxic effect of fludarabine was potentiated on average 80 or 261 fold with the addition of LMP-420 at concentrations of 62 or 250 nM, respectively (ranges 1.14 – 947 and 1.19 – 2754). This agent killed malignant lymphocytes by apoptotic mechanisms in a dose-responsive fashion, as demonstrated by both Annexin V staining and caspase 3/7 activity assays. While LMP-420 has potent anti-CLL activity, it has minimal effects on normal hematologic cells. For example, fludarabine suppresses erythroid and myeloid colony formation by greater than 50% at a concentration of 1 uM, while this level of inhibition is seen for LMP-420 at a concentration of 90 uM. The average cytotoxic ED50 of LMP-420 on normal PBMCs using the MTS assay was greater than 90 uM, whereas for fludarabine, it was 5.3 uM. This finding was confirmed with apoptosis assays. Conclusions: The results of these experiments demonstrate that LMP-420, a novel inhibitor of TNF expression, has cytotoxic activity against CLL cells, including those with high-risk features. LMP-420 appears to increase the cytotoxic effect of the chemotherapy agent fludarabine, while imparting minimal increase in hematologic toxicity. Thus, LMP-420 is promising new therapeutic agent in CLL. Disclosures: No relevant conflicts of interest to declare.

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35

Christensen, Dale J., Karen M. Bond, Alicia D. Volkheimer, Jessica Oddo, Youwei Chen, Jon P. Gockerman, Joseph O. Moore et al. "The SET Oncogene, a Potent PP2A Inhibitor, Is Elevated in CLL and Antagonism of SET Induces Apoptosis." Blood 114, n.º 22 (20 de noviembre de 2009): 802. http://dx.doi.org/10.1182/blood.v114.22.802.802.

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Abstract Abstract 802 Background and Significance: Even though we have treatments for CLL, it remains an incurable leukemia. We need new and better treatments for this disease. The Akt kinase is usually constitutively activated (phosphorylated) in CLL, and it acts to maintain CLL cell viability. The tumor suppressor protein phosphatase 2A is important in deactivation of Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and NFkB (through IkK). We developed apoE-mimetic peptides that potently decrease phosphorylation of Akt and MAPKs, decrease TNF and nitric oxide synthase expression, and display anti-inflammatory activity in vitro and in vivo by antagonism of SET, a potent physiological inhibitor of PP2A. Others have reported that PP2A activity is reduced and that SET is overexpressed in cells of chronic myelocytic leukemia patients. Increased SET expression with consequent decreased PP2A activity leads to dysregulated kinase signaling. We show here that SET is also overexpressed in CLL cells, and that SET antagonist apoE-mimetic peptides kill CLL cells. Methods: Patients were from the Duke University and V.A. Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CD19+ CLL or normal B cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. ApoE-mimetic peptides were prepared by chemical synthesis. Western blotting was used with anti-SET and anti-beta-actin antibodies. Apoptosis assays were performed with the annexin-V:propidium iodide staining method. Results: Samples from 17 CLL patients and 5 normal volunteers were examined by Western blotting. SET protein levels were 6.2-fold higher in CLL cells than in normal B cells. The apoE-mimetic COG compounds are peptides of 17 to 34 amino acids derived from the ligand-binding region of the apolipoprotein E holoprotein, and they act by binding to SET and preventing the inhibition of PP2A. This results in a net increase in PP2A activity in CLL cells. We have examined 11 COG peptides. Each of the 11 peptides displayed some cytotoxicity for CLL cells in vitro, irrespective of the patients' stages and other good or bad prognostic findings. 12 of 17 CLL patients were Rai stage 0 at presentation, and 5 were stage 1 or 2. They had been followed 4.2 yr (median; range 1.0 – 24.1 yr). 2 of 16 were CD38 positive, and 6 of 15 were Zap-70 positive. Of 15 analyzed, 6 had unmutated IgVH gene. 11 of 17 patients had not been treated. Peptide COG449 was the most potent, while a control peptide with an inverted apoE sequence had no activity. COG449 induced cell death in a dose-dependent fashion in all patients' samples, with a mean ED50 of 80 nM. The ED50 of COG449 for normal B cells was very high (> 10,000 nM). Annexin-V staining indicated that apoptosis was induced at concentrations in good agreement with the ED50 for cytotoxicity of the compounds tested. In vivo studies in normal mice using COG449 show no toxicity even at doses of 100 mg/kg when delivered by subcutaneous injection. Conclusions: We demonstrated SET overexpression in CLL cells and that apoE-mimetic peptides bind SET to de-inhibit PP2A. This results in apoptosis and death of CLL cells in vitro with high efficacy and potency (low nanomolar ED50s). CLL cells are killed preferentially compared to normal PBMC and B cells. Preliminary studies show that the peptide is nontoxic in normal mice. Trials in CLL patients will help determine the efficacy in vivo. Disclosures: Christensen: Cognosci Inc.: Employment. Oddo:Cognosci Inc.: Employment. Vitek:Cognosci Inc.: Employment, Equity Ownership.

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36

Ito, Yoshinori. "Abstract A30: Intravenous infusion of aGalCer loaded CD14-positive monocytes efficiently promotes amplification of circulating NKT-like cells, resulting immune responses and anti-tumor effects in malignant tumor patients". Cancer Immunology Research 10, n.º 12_Supplement (1 de diciembre de 2022): A30. http://dx.doi.org/10.1158/2326-6074.tumimm22-a30.

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Abstract Natural Killer T (NKT) cells are unique lymphocyte lineage that present both NK and T cell phenotype. NKT cells hold strong direct cytotoxic ability toward tumor cells through glycolipid/CD1d complex on tumor cells and NKG2D pathway. NKT cells secrete large amounts of IFNg and stimulate CD8 and NK cells as indirect cytotoxic effects. To elicit NKT cells responses in cancer patients, adoptive transfer of a-galactosyl ceramide (aGalCer) loaded APCs was exploited in a decade. iNKT cancer therapy developed by Taniguchi M, et al. is an immunotherapy using aGalCer loaded CD14-positive monocytes (CD14-Mo) infusion. This culture method is quite unique and convenient. It requires only 2 days culture. We completed the treatment of 14 malignant tumor patients with aGalCer loaded CD14-Mo (aGalCer CD14-Mo) and evaluated immune responses and anti-tumor effects. Methods: Autologous mononuclear cells were isolated by leukapheresis and then separated by Ficoll-Hypaque centrifugation. Monocytes were purified by CD14 positive selection. CD14-Mo was cultured for 2 days with GM-CSF and aGalCer. After cultivation, all cells were frozen and stored in liquid nitrogen until use. Usually above 95% of aGalCer CD14-Mo is expressing CD40. For tumor treatment, patients were injected at least twice with aGalCer CD14-Mo intravenously (IV) (0.5-2.0 x 108 cells per injection) with 3-4 weeks interval. One of the patients was injected with aGalCer CD14-Mo subcutaneously (SC). We evaluated safety and efficacy of iNKT cancer therapy by CTCAE, tumor maker, RECIST and immunological analysis. Fourteen patients, including NSCLC (3 cases), renal cancer (2 cases), ovarian cancer (2 cases), DLBCL (2 cases), urothelial (2 cases), cervical, colorectal and H&N cancer, were enrolled in this immunotherapy between 2020-2021. Results: In all cases, we could not observe any therapy-related adverse effects above CTCAE Grade 2. Several anti-tumor effects such as tumor marker reduction and/or SD were observed in 54.5% of evaluated cases and also increased numbers of CD8 cells and/or NK cells were observed in 83.3% of evaluated cases. Number of CD3+CD56+ positive cells in peripheral blood usually called as circulating NKT-like cells (cNKT) was amplified in 81.8% of patients whom injected aGalCer CD14-Mo IV. Among patients with amplified cNKT, CD8/NK upregulation and anti-tumor effects were observed in 100% and 75% of patients respectively after aGalCer CD14-Mo IV infusion. In the patients treated by aGalCer CD14-Mo IV with 5-FU/CDDP infusion or regular radiotherapy and the patient treated by aGalCer CD14-Mo SC infusion, any of cNKT amplification was not observed. Conclusion: aGalCer CD14-Mo IV infusion (iNKT cancer therapy) efficiently promote cNKT amplification. cNKT amplification clearly related with immune responses and anti-tumor effects. cNKT amplification seems to be abolished by combination therapy such as regular radiotherapy or chemotherapy. However, it is required more accumulation of cases to confirm. cNKT status is quite useful for the evaluation of iNKT cancer therapy. Citation Format: Yoshinori Ito. Intravenous infusion of aGalCer loaded CD14-positive monocytes efficiently promotes amplification of circulating NKT-like cells, resulting immune responses and anti-tumor effects in malignant tumor patients [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A30.

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37

Toyama, Kohtaro, Norifumi Tsukamoto, Akio Saito, Hirotaka Nakahashi, Yoko Hashimoto, Hiromi Koiso, Akihiko Yokohama et al. "JAK2 Mutation Status in Granulocytes, Platelets and Erythrocytes Differs Between Polycythemia Vera and Essential Thrombocythemia". Blood 112, n.º 11 (16 de noviembre de 2008): 5228. http://dx.doi.org/10.1182/blood.v112.11.5228.5228.

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Abstract Background The gain-of-function point mutation in Janus kinase 2 exon 14 gene (JAK2-V617F) influences the diagnosis of bcr/abl-negative chronic myeloproliferative disorders (CMPDs). We previously reported that analyzing platelets is advantageous in detecting the JAK2-V617F mutation, particularly in essential thrombocythemia (ET), when compared to granulocytes. However, there have been few reports analyzing the JAK2-V617F mutation in erythroid lineage cells, and comparing the mutation status in all three lineages. Method Study protocols were approved by the Institutional Review Board of Gunma University Hospital, and written informed consent was obtained from all the patients. Heparinized peripheral blood was obtained from 113 patients with CMPDs (82 with ET, 25 with polycythemia vera (PV), and 6 with primary myelofibrosis (PMF). After centrifugation, platelets were collected from the upper plasma layer. Remaining blood was mixed with Hank’s Balanced Salt Solution and was subjected to Ficoll-Hypaque density gradient centrifugation. Granulocytes were obtained from the pellet. Mononuclear cells were resuspended in RPMI 1640 medium; 5 × 105 cells were plated in duplicate in 1 ml of methylcellulose medium and cultured in a humidified atmosphere of 5 % of carbon dioxide at 37°C for 14 days in the presence of erythropoietin to obtain erythroid colonies (BFU-E). T-cells were obtained from the remaining mononuclear cells using anti-CD3 immunoconjugated magnetic beads. After extraction of DNA from granulocytes, T-cells and BFU-E, and RNA extraction from granulocytes and platelets, PCR amplification and sequencing of exon 14 of the Jak2 gene was performed to confirm the presence of JAK2-V617F mutations. To confirm the mutation status of granulocytes, T-cells and BFU-E, allele-specific PCR (AS-PCR) was performed. Results For ET, 57 out of 82 patients (69.5%) had the JAK2-V617F mutation. In the 57 patients with the JAK2-V617F mutation, 38 (67%) had the mutation in all three lineages, 5 had the mutation in granulocytes and platelets, 2 had the mutation in platelets and BFU-E, 10 patients had the mutation only in platelets and 2 patients had the mutation only in BFU-E. In contrast, for PV, 22/25 patients (88%) had the JAK2-V617F mutation. Of note, in 22 patients having JAK2-V617F mutation, 20 (91%) were JAK2-V617F mutation-positive in all three lineages; the remaining two patients had the mutation in either platelets or BFU-E. The frequency of JAK2-V617F in all three lineages was significantly higher in PV than in ET (p < 0.05). For PMF, 5 of 6 patients had the mutation in granulocytes, and 3 of these had it in all three lineages. Conclusion Among JAK2-V617F mutation-positive CMPDs, most PV patients had the JAK2-V617F mutation in all three lineages, thus suggesting that the JAK2-V617F mutation occurs in progenitor cell(s) common to granulocytes, platelets and erythrocytes. In contrast, only 67% of ET patients had the JAK2-V617F mutation in three lineages; in the remaining cases, not all of the three lineages have the mutation. This difference in lineages showing the JAK2-V617F mutation between the ET and PV may be related to the pathophysiological differences in ET and PV. Furthermore, the heterogeneous mutation status in ET may be related to its heterogeneous clinical manifestation.

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Buda, Gabriele, Enrico Orciuolo, Giancarlo Carulli, Sara Galimberti y Mario Petrini. "Molecular Remission After VTD or TAD As Induction for Multiple Myeloma: Results with Two Different Methods of Analysis." Blood 120, n.º 21 (16 de noviembre de 2012): 2929. http://dx.doi.org/10.1182/blood.v120.21.2929.2929.

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Abstract Abstract 2929 The high rate of complete response (CR) effected by novel agents as induction has also renewed interest in the evaluation of minimal residual disease (MRD) in Multiple Mieloma (MM) [1]. For this purpose, In our hospital we conducted a retrospective study to compare the activity of VTD induction versus TAD. Using two different methods, as flow cytometric (FC) assay and polymerase chain reaction (PCR) technology we evaluated in the bone marrow the molecular residual disease (MRD), in the subgroup of patients that obtained CR. We evaluated 87 multiple myeloma patients treated between February 2009 and April 2012 and eligible for autologous transplant. Patients (table I) had untreated, newly diagnosed and symptomatic MM. All patients provided written informed consent. Patients were treated with 4 cycles of TAD or VTD. TAD consisted of four cycles of intravenous doxorubicin on day 1 every 28 days in day-hospital, dexamethasone 40 mg orally on days 1 through 4 and 9 through 12, and thalidomide 100 mg/day continuously and orally administered. VTD consisted of four 3-week cycles of bortezomib 1.3 mg/m2administered intravenously on days 1, 4, 8, and 11 plus dexamethasone 40 mg days 1–2 and days 3–4, 8–9, 11–12 (all cycles) and thalidomide 100 mg/day continuously and orally administered. Our target was the evaluation of minimal residual disease (MRD) in patients that obtained CR. Immunophenotyping was carried out by a FacsCanto II cytometer equipped with 3 lasers (405, 488, 633 nm). A seven-color method was used, with monoclonal antibodies (MoAb) conjugated with the following fluorochromes: FITC, PE, PercCP-Cy5.5, Pe-Cy.7, APC, APC-Cy.7, AmCyan [2]. PCR analyses were performed on mononuclear cells separated by Ficoll/Hypaque gradient. High molecular weight DNA was extracted and suitable aliquots were utilized for PCR assays to identify BM infiltration represented by clonal IgH rearrangement. Capillary electrophoresis and fluorescence detection with a virtual filter C was performed using a ABI Prism 310 Genetic Analyzer (Applied Biosystems). Runs were executed with the module GS STR POP 4 (1 ml) C with 10-second and 15 kV injection and run voltage, 60°C constant temperature, 24 min run time, using polymer POP 4 and the running buffer Genetic analyzer 1X (Applied Biosystem). Genescan 2.1 software was then used to analyze the PCR products, with accurate sizing and quantification of the peak areas, according to our previously published method [3]. After 4 cycles, The overall response rate was 91% in the VTD group versus 84% in the TAD group (Table 2). However, the CR plus VGPR rate was significantly higher in the VTD arm (51% vs 18%, P= .001). The difference in CR rate was 28% (30% in VTD group vs 2% in TAD group). In the only patient that obtained CR in the TAD group FC and PCR were able to still detect MRD but in ten of thirteen (77%) patients that achieved CR in VTD group both additional assays confirmed absence of MRD. VTD regimen was able to induce a very high rate of CR including undetected MRD even if evaluated with two different methods. In conclusion, in comparison with TAD induction, VTD significantly increased the rate of molecular remissions. Table I: Patients characteristics TAD VTD Total 44 43 87 Sex Male 18 (41%) 24 (56%) 42 Female 26 (59%) 19 (44%) 45 Age of diagnosis Median age 61 60 61 Range 35–73 29–72 29–73 MM Subtype IgA 8 11 19 IgG 32 28 60 LCD 4 3 7 NS 0 1 1 ISS I 27 25 52 II 7 8 15 III 10 10 20 Stage( Durie and Salmon) I 6 6 12 II 8 2 10 III 30 35 65 Karyotype Normal 35 36 71 Abnormal 9 7 16 Table II: Response rates TAD VTD CR 1 (2%) 13 (30%) VGPR 7 (16%) 9 (21%) PR 29 (66%) 17 (40%) SD 5 (11%) 3 (7%) PD 2 (5%) 1 (2%) Disclosures: No relevant conflicts of interest to declare.

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Serratì, Simona, Carla Minoia, Anastasia Chilla, Anna Laurenzana, Francesca Margheri, Maria Antonia Frassanito, Nicola Sgherza et al. "Cancer Associated Fibroblasts in Multiple Myeloma: The Urokinase Receptor System in Tumor Growth Regulation". Blood 124, n.º 21 (6 de diciembre de 2014): 5687. http://dx.doi.org/10.1182/blood.v124.21.5687.5687.

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Abstract BACKGROUND: The multiple myeloma (MM) represents a process in which an asymptomatic stage of monoclonal gammopathy of undetermined significance (MGUS) precedes virtually all cases of MM. It is known that tumor progression are determined by a favorable tumor microenvironment (TME) and in this scenario fibroblasts represent the principal cellular component in the TME. A particular subpopulation of fibroblasts, cancer associated fibroblasts (CAFs), has recently raised the interest of many researchers due to their active participation in tumor growth and invasion and their association with higher malignancy grade, and poor prognosis. Recent findings indicate that the urokinase plasminogen activator (u-PA), and the urokinase receptor (u-PAR) are critical in cell invasion and degradation processes. Degradation and remodeling of the surrounding tissues are crucial in the early steps of tumor progression by facilitating expansion of the tumor mass, tumor cell proliferation, migration, and invasion. uPAR is expressed by multiple tumor associated cell types found in tumors. Targeting uPAR expressed on tumor-associated cells may be as important as targeting uPAR expressed on tumor cells and may lead to enhanced antitumor activity especially in those tumor types expressing uPAR on both types of cells. METHODS: Cell purification and cultures: BM mononuclear cells (BMMCs) were isolated by Ficoll-Hypaque gradient from heparinized bone marrow (BM) aspirates from 10 patients with relapse/refractory MM, 7 patients with asymptomatic MM, 7 with remission MM, 10 with MGUS. Fibroblasts were purified from BM stromal cells BMSCs through anti-fibroblasts-microbeads, and culture in DMEM medium with 10% FBS. Cell phenotype analysis: CAFs were analyzed on heparinized bone marrow aspirates and they were identified by FSP1 and α-smooth muscle actin (α-SMA) expression on gated CD45-population. Expression of alpha-SMA in BM purified fibroblasts was also demonstrated by immunofluorescence staining. Immunofluorescence: CAFs were cultured in DMEM medium, fixed in paraformaldehyde, and permeabilized according to routine methods. The primary antibodies were anti–uPAR, and anti–α-SMA. Fibroblast nuclei were stained with DAPI. Quantitative PCR analysis: Complementary DNA was prepared from 1 ug total RNA using a GoScript reverse transcription system. The relative quantity of uPA, uPAR, MMP-2, α-SMA and vimentin messenger RNA were measured using the Applied Biosystems 7500 Fast Real-Time PCR System and determined by the comparative Ct method using 18S ribosomal RNA as the normalization gene. The study was approved by the local Ethics Committee and all patients provided their informed consent in accordance with the Declaration of Helsinki. RESULTS: Cell phenotype analysis:Flow cytometry analysis showed that CAFs were increased in patients with relapse-MM compared to patients with asymptomatic, remission MM and MGUS suggesting that CAFs expansion is involved in MM progression. CAFs activation: The increased frequency of alpha-SMA in CAFs of relapsed MM patients was demonstrated by the immunofluorescence analysis. Overall, these results suggest that MM activation is associated with the overexpression of uPAR. (Fig.1) CAFs activation was also demonstrated by Real Time PCR and the figure 2 shows the overexpression of activation molecules as well as proinvasive systems in CAF of relapsed MM in comparison of MGUS and asymptomatic MM. CONCLUSIONS: In MM developement and progression the BM niche appears to play an important role in differentiation, migration, proliferation, and drug resistance of the malignant PCs. The main goal of this proposal was to globally approach the expression of CAFs’ activation and proinvasive systems in the initiation and progression of MM. Our results highlight an important mile stone on the phisiopatology of MM progression demonstrating that the CAF-activated phenotype is associated with an over-expression of the most important pro-invasive systems, and in particular the relapsed-MM CAFs seem to overexpress the fibrinolytic pattern of invasive tumor-like cells. On the basis of these results we aim to develop a biological model which can become a potential therapeutic target. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Russo, Roberta, Maria Rosaria Esposito, Daniela Spano, Luigia De Falco, Roberta Asci, Carmelo Piscopo, Immacolata Andolfo y Achille Iolascon. "COPII Complex Characterization During Erythroid Differentiation and Its Involvement in CDAII Disease." Blood 114, n.º 22 (20 de noviembre de 2009): 3009. http://dx.doi.org/10.1182/blood.v114.22.3009.3009.

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Abstract Abstract 3009 Poster Board II-985 Congenital dyserythropoietic anemia type II (CDA II) is an autosomal recessive disorder affecting the normal differentiation-proliferation pathway of the erythroid lineage. It comprises an anemia of variable severity, jaundice, and variable splenomegaly. Erythroid hyperplasia with binuclearity or multinuclearity involving late erythroblasts in the bone marrow (BM) is a key feature of the diagnosis (Iolascon, 2001). In addition, on electron microscopy, vesicles of endoplasmic reticulum (ER) appear to be running beneath the plasma membrane (Alloisio, 1996). The principal biochemical feature is the hypoglycosilation of several proteins, such as transferrin and band 3 (Anselstetter, 1977). Recently, we identified SEC23B as the CDA II causative gene (Schwarz, 2009). The SEC23B gene encodes the SEC23B component which is part of the cytoplasmic coat protein (COP)II complex. COPII coated vesicles bud from the endoplasmic reticulum to export newly synthesized proteins to the trans Golgi. In yeast, COPII coated vesicles form by the sequential binding of Sar1-GTP, the inner complex proteins Sec23-Sec24 and the outer complex components Sec13-Sec31 on the endoplasmic reticulum (ER) (Fromme, 2008). Our aim was to characterize the COPII complex in CD34+ progenitor cells during erythroid differentiation by gene expression analysis. Mononuclear cells from the peripheral blood of normal subjects were isolated on a Ficoll-Hypaque gradient and CD34+ progenitors were separated on immuno-affinity columns. For erythroid differentiation, CD34+ cells of three different pools were separately plated on plastic culture dishes in methylcellulose medium containing 3U/mL erythropoietin (EPO) (Pinho, 2008). The cells have been cultured for 7 and 14 days after EPO treatment. Quantitative real time (qRT)-PCR on CD34+ during erythroid differentiation was performed to assess the gene expression level. The relative gene expression was calculated by 2*(-ΔCt) method (Livak, 2001), using GAPDH gene as internal control (figure 1). Mammalian orthologues have been identified for each of the five proteins involved in COPII coat formation. In humans, two isoforms of Sec23, Sar1 and Sec31 and four mammalian isoforms of Sec24 have been reported (Kuge, 1994; Paccaud, 1996; Wendeler, 2007; Mancias, 2008; Shugrue, 1999; Tang, 2000; Stankewich, 2005). We already demonstrated that during normal erythropoiesis a SEC23A down regulation is associated to SEC23B upregulation. These data suggest that SEC23B mutants could disrupt the COPII complex in erythroid lineage, and consequently induce CDA II (Schwarz, 2009). On the contrary, the two isoforms of SAR1 showed the same trend during erythroid differentiation: however, SAR1A isoforms has an higher expression when compared to SAR1B isoform. Among the four SEC24 isoforms, SEC24A, B and C showed the same increased expression after EPO treatment; SEC24D, indeed, showed a decreasing trend during differentiation time. Only one form of mammalian SEC13 has been described (Shaywitz, 1995), and it showed an increased expression after EPO treatment. Between SEC31 mammalian isoforms, SEC31A showed overall an higher gene expression compared to SEC31B gene. The gene expression analysis of SEC12, the transmembrane guanine nucleotide exchange factor that catalyzes the COPII vesicle formation by GDP-GTP exchange on Sar1 (Sato, 2004), revealed an higher mRNA level at 14 days when compared to 7 days after EPO treatment. Here, we have identified the isoforms of COPII complex most expressed during erythroid differentiation. This study could allow us to clarify the role of each COPII gene in erythroid lineage, and to identify other genes potentially involved in CDA II pathogenesis.Figure 1.Gene expression profile during CD34+ erythroid differentiation. Data are presented as mean ± standard deviation. *p value < 0.05 calculated on day 0 by Student t test corrected by Bonferroni method.Figure 1. Gene expression profile during CD34+ erythroid differentiation. Data are presented as mean ± standard deviation. *p value < 0.05 calculated on day 0 by Student t test corrected by Bonferroni method. Disclosures: No relevant conflicts of interest to declare.

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Bora, Komal, Kavita Natarajan, Ferdane Kutlar, Hanfang Zhang, Hongyan Xu, Betsy Clair, Kathleen M. McKie y Abdullah Kutlar. "In Vitro Exploratory Studies of Haptoglobin Polymorphisms and Their Effect on Cytokine Release from Cultured Mononuclear Cells in Sickle Cell Disease." Blood 112, n.º 11 (16 de noviembre de 2008): 1441. http://dx.doi.org/10.1182/blood.v112.11.1441.1441.

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Abstract The deleterious effects of hemolysis through its end product cell free hemoglobin(Hb) as a nitric oxide (NO) scavenger is well established and has been incriminated in the pathogenesis of some complications of SCD such as pulmonary hypertension, leg ulcers, renal dysfunction, and possibly stroke. These observations have led some investigators to hypothesize that these complications form a subphenotype of SCD related predominantly to hemolysis and endothelial dysfunction. In hemolytic states Hb released from RBC complexes with Haptoglobin (Hp) and is removed from the circulation by macrophages and monocytes through binding CD163, the Hb scavenger receptor expressed on these cells. When the binding capacity of Hp is exceeded, the concentration of free Hb rises in the plasma. Hp is a polymorphic protein encoded by a gene on chromosome 16q2.2; there are two allelic variants, Hp 1 and Hp 2. Hp 2 is believed to have resulted from an intragenic duplication event, leading to an elongated Hp a-chain. Individuals homozygous for the long a2 chain express large multimeric molecules (Hp 2-2). During the past decade, a considerable body of evidence has accumulated suggesting that Hp-2 allele is a major susceptibility gene for the development of vascular complications (coronary artery restenosis and development of cardiovascular disease) especially in diabetic patients. It has been hypothesized that the Hb-Hp2 complexes have a 10-fold greater affinity for the CD 163 receptor, and the binding of Hb-Hp2 complexes generates a more powerful inflammatory response with a more prominent cytokine release. Recently, we performed a preliminary analysis of the distribution of Hp1 and Hp2 alleles among pediatric and adult SCD patients and reported a significantly higher allele frequency for Hp2 among pediatric patients, suggesting a survival advantage for carriers of Hp1 allele (Yaun et al, Blood, 2005). We now report the results of an exploratory in vitro study of cytokine release from purified mononuclear cells obtained from a normal control and an SCD patient following exposure to Hb-Hp1-1 and Hb-Hp2-2 complexes. Mononuclear cells (106/well) isolated by Ficoll-Hypaque density gradient were incubated with Hb A-Hp1-1, Hb A-Hp2-2, Hb S Hp1-1, and Hb S-Hp2-2 complexes with a 1:1 ratio (wt/wt) at a final concentration of 1 mg/ml. After 24 hr incubation at 37°C, the supernatants collected after centrifugation were used for cytokine assays by a multiplex bead method. A blank (medium only), Hb A and Hb S without Hp were also incubated with mononuclear cells. Multiplex bead assays showed that cytokine release (GM-CSF, IL-1b, IL-6, IL-10, and TNFa) was much higher (3–12 fold) from both the control and SCD mononuclear cells upon exposure to Hb-Hp2-2 complexes, but much less or no effect by Hb-Hp1-1. The fold induction of TNFa and IL-1b was much higher in SCD cells than in control cells. There was no significant difference between Hb A and Hb S in terms of cytokine release when they were complexed with either Hp1-1 or Hp2-2, suggesting that the cytokine release was predominantly related to Hp type but not to Hb. Pure Hb A and Hb S increased cytokines over the control (blank) but to a significantly smaller extent than Hb (A or S) Hp-2-2 complexes. These preliminary results are confirmatory of a deleterious effect of the Hp2-2 genotype through a more pronounced inflammatory response and are suggestive of a potential novel mechanism whereby hemolysis could result in adverse outcomes related to Hp polymorphisms. If confirmed in larger studies and through phenotypic associations, attenuation of this response via anti-inflammatory modalities may provide a therapeutic strategy. Figure Figure

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Friedman, Daphne R., Patricia H. Davis, Mark C. Lanasa, Joseph O. Moore, Jon P. Gockerman, Taylor Nelson, Karen M. Bond et al. "Pre-Clinical and Interim Results of a Phase II Trial of Perifosine In Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia (CLL)". Blood 116, n.º 21 (19 de noviembre de 2010): 1842. http://dx.doi.org/10.1182/blood.v116.21.1842.1842.

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Abstract Abstract 1842 Perifosine (Keryx Biopharmaceuticals) is an oral alkylphospholipid that has been shown to have clinical activity in multiple myeloma and Waldenstrom's macroglobulinemia. Pre-clinical data suggest that its activity is due to inhibition of the Akt signal transduction pathway. The Akt pathway is known to be important for viability in CLL, another B-cell malignancy. Therefore, we investigated the in vitro activity of perifosine in freshly isolated primary CLL cells. CLL patients at the Duke University and Durham VA Medical Centers were enrolled in an IRB-approved research protocol for blood sample collection. CLL cells were negatively selected using the RosetteSep B-cell enrichment cocktail (StemCell Technologies) and a ficoll-Hypaque gradient. This method yields greater than 95% purity of malignant lymphocytes, determined by flow cytometry. Prognostic markers such as IgVH mutation status, CD38 and ZAP70 expression, and interphase cytogenetics were determined. We found the 50% effective dose (ED50) of perifosine for inducing cytotoxicity in CLL cells after a three-day incubation using the MTS colorimetric assay to be 510 nM (n = 29, range 120 – 1540 nM). CLL cells were obtained from patients with generally poor prognostic markers: 52% CD38+, 93% ZAP70+, 78% IgVH unmutated, 42% 17p deletion, 8% 11q deletion, 27% trisomy 12, 12% normal, and 12% 13q deletion as a sole abnormality. There were no statistical differences in ED50 between cells obtained from patients in high or low risk prognostic groups. Perifosine induced apoptosis in a dose- and time-dependent manner, measured by Annexin V and PI staining (n = 4). Based upon these pre-clinical results, we initiated a phase II study of perifosine (50 mg orally, twice daily) in relapsed or refractory CLL/small lymphocytic lymphoma (SLL) (NCT00873457). The primary objective of this study is to assess the response rate at 3 and 6 months of perifosine treatment in patients with relapsed or refractory CLL/SLL. Secondary objectives are to monitor toxicity, evaluate overall survival, progression-free survival and response duration, and perform laboratory correlates. Early interim results of this study are presented. Since trial initiation in September 2009, 13 patients have been enrolled. Nine patients had Rai stage III/IV disease at the time of therapy, and 4 patients were fludarabine-refractory. Patients had extensive prior treatment (median 4, range 1 – 11). Many patients had high-risk prognostic features: 9/11 IgVH unmutated, 10/13 CD38+, and 11/13 ZAP70+. Evaluation of interphase and metaphase cytogenetics demonstrated 4 patients with 17p deletion, two with 11q deletion, 2 with trisomy 12, 4 normal, and 4 with other complex cytogenetic anomalies. Of 12 patients who began therapy, 5 patients withdrew from the study prior to 3 months, 6 patients received at least 3 months of therapy, and 1 patient completed 6 months of therapy. Of the patients who received at least 3 months of therapy, there were 5 patients with stable disease, and one patient with partial response, using iwCLL response criteria. Grade 3/4 toxicities included anemia (n=2), fatigue (n=2), dehydration (n=1), febrile neutropenia (n=1), hyperbilirubinemia (n=1), hyponatremia (n=1), cough (n=1), and dyspnea (n=1). One patient required a dose reduction to 50 mg daily and two patients required dose delays due to toxicities. In conclusion, perifosine has potent in vitro activity against primary CLL cells. Preliminary results of this ongoing phase II study of oral perifosine in relapsed or refractory CLL/SLL demonstrate mostly disease stabilization in a group of very high-risk patients and an acceptable toxicity profile. Completion of this clinical study is necessary to determine if perifosine monotherapy has a potential role in the treatment of CLL/SLL. Disclosures: Sportelli: Keryx Biopharmaceutical: Employment, Equity Ownership. Weinberg:Keryx Biopharmaceuticals: Research Funding.

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Nagata, Yasuyuki, Itsuko Ishizaki, Michihiko Waki, Yoshimi Ide, Md Amir Hossen, Kazunori Ohnishi y Mitsutoshi Setou. "Palmitic Acid, As Verified Using Lipid Profiling By Secondary Ion Mass Spectrometry, Demonstrates Anti-Tumor Activity Against Multiple Myeloma". Blood 124, n.º 21 (6 de diciembre de 2014): 5683. http://dx.doi.org/10.1182/blood.v124.21.5683.5683.

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Abstract Introduction Many recent studies have examined lipid metabolic changes in multiple myeloma (MM). Changes in lipid metabolism affect the survival of MM cells. Developments in imaging mass spectrometry (IMS) have facilitated research on the lipid profiles of tumors. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an IMS technique that uses a focused ion beam as the primary source for ionization. TOF-SIMS imaging is used to analyze the surface of specimens at sub-micrometer resolution, enabling analyses of the subcellular distribution of molecules in individual cells. TOF-SIMS analysis has enabled the detection of multiple fatty acid groups from single cells. Therefore, we applied this method to human clinical specimens to analyze the membrane fatty acid composition and determine candidate molecules for MM therapies. Using the different lipid profiles for MM cells and normal plasma cells (PCs), we conducted a cytocidal assay with MM cell lines supplemented with the fatty acids screened out by the profiles to assess lipotoxicity against MM. The molecules demonstrating distinct differences among cell types (i.e., MM and PC) were considered candidates for which supplementation leads to imbalanced lipid metabolism and cell death in a tumor-specific manner. We further evaluated the induction of apoptosis. Methods Primary patient MM cells and normal PCs were isolated from the bone marrow aspirates of two patients and two healthy volunteers using fluorescence-activated cell sorting. These separated cells were analyzed with PHI TRIFT V (ULVAC-PHI, Inc.). Analyses were performed in negative ion mode, and signals in the mass range of m/z 0 to 1850 were monitored. We performed pairwise comparisons of mean signal intensities for five types of fatty acids between MM cells and PCs. MM cell lines (U266 and RPMI-8226) were treated with 0–1000 µM of palmitic acid, palmitoleic acid, linoleic acid, oleic acid, and stearic acid. The number of viable cells in suspension at 72 hours after treatment was determined by the trypan blue exclusion test. HS-5, a human bone marrow stromal cell line, was used in the co-culture experiment. Healthy volunteers’ normal peripheral blood mononuclear cells (PBMCs) were purified by Ficoll-Hypaque density-gradient centrifugation. The distribution of apoptotic and necrotic cells were analyzed by measuring AnnexinV binding and propidium iodide uptake. Results The amounts of MM cells and PCs relative to the total nucleated cells were 3.38%, 35.9% for MM cells, 0.0368% and 0.246% for PCs. Multiple ions, including phosphoric acid, and five species of fatty acids (palmitoleic acid, palmitic acid, linoleic acid, oleic acid, and stearic acid) were detected. The mean signal intensities of palmitoleic acid and palmitic acid of MM cells were significantly lower than those of normal PCs (P = .00081 and .0018, respectively). These results were replicated in a second pairwise comparison. We did not observe statistically significant differences in intensities for linoleic acid, oleic acid, or stearic acid. In the cytocidal assay, palmitic acid reduced U266 cell viability dose-dependently for doses of 50–1000 μM. High concentrations of the other fatty acids also reduced cell viability; however, the effect on cell death was not observed at the low dose of 50–100 µM, as it was for palmitic acid. Even in co-culture experiments, palmitic acid decreased the viability of MM cells. Moreover, the proportions of both apoptotic and necrotic cells increased and the proportion of viable cells decreased 24 hours after palmitic acid treatment in MM cells. Palmitic acid also reduced the viability of RPMI-8226 cell lines. Meanwhile, cell viabilities of normal PBMCs were not affected by palmitic acid, even at 100–500 µM. Conclusion We applied the single-cell TOF-SIMS lipid analysis effectively to a very small population of cells. Significantly smaller intensities of palmitoleic acid and palmitic acid were observed in MM cells compared to normal cells. We also demonstrated an inhibitory effect of palmitic acid on the survival of MM cells. Palmitic acid is a potential candidate for novel therapeutic agents that specifically attack MM and should be considered in future studies of MM in a lipid biology framework. Disclosures No relevant conflicts of interest to declare.

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Barbarito, Giulia, Beruch Dejene, David C. Shyr, Gopin Saini, Linda Oppizzi, Sruthi Mantri, Hye-Sook Kwon, Matthew H. Porteus, Kenneth I. Weinberg y Alice Bertaina. "Heterogeneity of Hematopoietic Stem and Progenitor Cell (HSPC) Composition in Αβ T-Cell/CD19 B-Cell Depleted Peripheral Blood Cell Stem Cell (PBSC) Transplant Grafts and Correlation with Immune and Hematopoietic Recovery". Blood 136, Supplement 1 (5 de noviembre de 2020): 12–13. http://dx.doi.org/10.1182/blood-2020-143214.

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INTRODUCTION. While total CD34 counts in PBSC graft products have correlated with overall likelihood of hematopoietic recovery after allogeneic hematopoietic stem cell transplantation (HSCT), analyses of the HSPC composition and its relationship to relevant post-transplant clinical outcomes are lacking. In fact, the biological basis for different dynamics of hematopoietic/immune recovery, the risk of infection, and graft-versus-host disease (GvHD) is not fully understood. We have performed the first analysis of HSPC graft composition in 6 αβ T-cell/CD19 B-cell depleted haploidentical (αβhaplo) HSCT. Additionally, we correlated the HSPC graft composition with the distribution of the same HSPC subsets in serial post-HSCT bone marrow aspirates performed at Days 30, 60, and 90, with the peripheral blood counts [neutrophils, monocytes, platelet (PLT)] and with the immune recovery (CD3+, CD3+CD4+, CD3+CD8+, αβT, γδT, NK cells) at the same time points. The patients were divided in two groups: 3 patients had a robust and sustained hematopoietic recovery (Group 1) while 3 patients experienced mild cytopenia after Day 60 (Group 2). All patients were transplanted for acute leukemia and received a myeloablative TBI-based conditioning regimen. See Table 1 for details about patients and graft composition. METHODS. All patients were enrolled in the Stanford IRB approved BMT Protocols 179/351/361 and had peripheral blood (PB) and bone marrow (BM) evaluated at Day 30, 60 and 90 post HSCT for the primitive CD34+ Lin- HSPC subsets: HSC (CD38-CD45RA-CD90+), MPP (CD38+CD45RA+), CMP (CD38+CD45RA-CD123+), GMP (CD38+CD45RA+CD123+), MEP (CD38+CD45RA-CD123+) and CLP (CD38+CD127+). Aliquots of αβhaplo-HSCT grafts were cryopreserved for later analyses. Mononuclear cells were isolated from PBSC, PB and BM by Ficoll-Hypaque (Sigma-Aldrich) density gradient centrifugation. FACS analyses were performed on either fresh or frozen cells on Becton Dickinson (BD) Aria II flow cytometer. At least 5x105 events were acquired and analyzed using FlowJo software (BD). RESULTS. Despite consistent levels of αβT-cell depletion and CD34 enrichment, the frequency of the HSPC subsets varied between the grafts. Notably, while CMP and GMP were very consistent across the 6 grafts, the frequency of HSC, CLP, MEP and MPP showed a 2-fold range of variation (Fig1A). No significant correlation was observed between the HSC frequency in the graft and the hematopoietic/immune recovery. However, the frequency of HSC in the BM at Day 30 is statistically correlated (P=0.027) with the PLT at Day 90 (Fig1B). In these preliminary results, the different distribution of CMP, GMP, MEP and MPP did not impact on the hematopoietic/immune recovery. However, there was a significant correlation (P=0.02) between CLP and γδ T cells reconstitution at Day 90 in Group 1 patients (Fig1C). Additionally, the neutrophils, monocytes and phagocytic cells recovery at Day 90 is statistically correlated with the GMP frequency in the BM at Day 30 (P=0.017; P=0.018; P=0.0132, respectively) (Fig1D). Interestingly, the same strong correlation is observed between the CMP in the BM at Day 60 and the recovery of neutrophils and phagocytic cells (P=0.016, P=0.019) at Day 90 (Fig1E), but the CMP at day 30 are already predictive of a robust engraftment in the Group 1 patients (data not shown). CONCLUSIONS. In αβhaplo-HSCT, previously identified factors influencing hematopoietic recovery have been mainly limited to the enumeration of bulk CD34 counts and of mature effector cells, such as αβ/γδ T and NK cells. On the other hand, the presence of GvHD and thymic injury have been correlated to the kinetics of immune reconstitution. We hypothesized that the HSPC composition of the graft would impact lymphohematopoietic recovery in αβhaplo-HSCT recipients. Although preliminary, our data indicate that even with a consistent method of graft manipulation, the HSPC graft composition is heterogeneous. Variations in HSPC subsets frequency and number can contribute to significant differences in lymphohematopoietic recovery and, therefore, clinical outcome. The evaluation of a larger number of patients with longer follow up after HSCT are required. Comparative studies with unmanipulated T-cell replete and cord blood grafts are ongoing. Such analyses will be instrumental not only for prediction of clinical outcome, but also for optimization of novel graft engineering strategies. Disclosures No relevant conflicts of interest to declare.

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Meszaros, K., J. Bojta, A. P. Bautista, C. H. Lang y J. J. Spitzer. "Glucose utilization by Kupffer cells, endothelial cells, and granulocytes in endotoxemic rat liver". American Journal of Physiology-Gastrointestinal and Liver Physiology 260, n.º 1 (1 de enero de 1991): G7—G12. http://dx.doi.org/10.1152/ajpgi.1991.260.1.g7.

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There are several types of glucose-consuming, immunologically active nonparenchymal cells interspersed among the glucose-producing parenchymal liver cells. Combining the in vivo 2-deoxyglucose tracer technique with cell separation methods enabled us to investigate the effect of Escherichia coli endotoxin on the rate of glucose utilization by the nonparenchymal cells. Rats were injected with [14C]deoxyglucose, and intracellular 2-deoxyglucose 6-phosphate was determined in different liver cell fractions. Parenchymal, Kupffer, and endothelial cells as well as polymorphonuclear leukocytes (PMN) were separated from the liver by centrifugal elutriation followed by Ficoll-Hypaque density gradient. The number of PMN obtained from the liver was increased severalfold 3 h after endotoxin and was comparable to the number of Kupffer cells. Glucose utilization by the liver of fasted rats was due predominantly to nonparenchymal cells. Endotoxin enhanced the rate of glucose utilization by Kupffer (6.7-fold) and endothelial (2.7-fold) cells and by the infiltrated hepatic PMN (5.4-fold). Enhanced glucose metabolism of immunologically active cells is part of the hepatic immune response and subserves the antibacterial defense of the body. The activated cells, however, may also have the potential of causing tissue damage by releasing harmful toxic metabolites.

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Payton, Jacqueline E., Guido Marcucci, Michael D. Radmacher, Kati Maharry, Christian Langer, Claudia D. Baldus, Clara D. Bloomfield y Timothy J. Ley. "Multiplexed Digital Quantification of mRNA Abundance: a Highly Reproducible, PCR–independent Assessment of BAALC, ERG, and MN1 mRNA Levels in Acute Myeloid Leukemia (AML) Bone Marrow (BM) and Peripheral Blood (PB) Samples." Blood 114, n.º 22 (20 de noviembre de 2009): 1599. http://dx.doi.org/10.1182/blood.v114.22.1599.1599.

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Abstract Abstract 1599 Poster Board I-625 The greatest obstacle to routine clinical testing of gene expression levels has been the lack of reproducibility of currently used methodologies, such as quantitative reverse transcriptase PCR (qRT-PCR) and microarray expression profiling. While these assays are useful for retrospective analyses of batched samples, they cannot be used for upfront evaluation of individual patients (pts) for molecular risk and treatment assignment. To overcome this barrier, we tested a recently developed, high throughput, PCR-independent, digital quantification technology, the nCounter system (Nanostring® Technologies). This system counts individual mRNA molecules, rather than measuring non-linear fluorescence generated by PCR-amplified targets (eg qRT-PCR). Using 72 AML samples and spike-in controls, we and collaborators demonstrated that the nCounter system is highly reproducible, sensitive, and accurate to femtomolar concentrations (Payton, J, et al. JCI 119:1714-26; Geiss, G, et al. Nat Biotech 26:317-25). Here we validated this technology using an independent set of 101 pts with a diagnosis of de novo cytogenetically normal AML. At diagnosis, pts presented with FAB subtypes M0, M1, M2, M4, M5(A, B), had a median age of 43 years (range 19-59), median white blood count of 28.5× 103/μL (range 1.4-273.0), median of 69% BM blasts (range 22-95) and median of 65% PB blasts (range 0-97). Paired BM and PB specimens were available for 27 pts; blast percentages were ≥ 20% for all paired specimens. We used the nCounter system to measure mRNA abundance (‘counts‘) of 27 genes whose expression correlates with clinical and/or pathological criteria, including 3 genes associated with prognosis (BAALC, ERG, MN1), and control/housekeeping genes (GAPDH, ABL, Actin). Briefly, mononuclear cells from pretreatment BM or PB were enriched on Ficoll-Hypaque gradients and RNA was isolated using Trizol reagent; 100ng of total RNA was assayed in triplicate by nCounter according to the manufacturer's protocols. The nCounter results demonstrated substantial reproducibility, with a median CV [coefficient of variation, (standard deviation/mean *100)] <6% across replicates. In addition, the nCounter counts for BAALC, ERG, and MN1 normalized to ABL were highly correlated with the ABL-normalized qRT-PCR results. Significant correlation was observed for all 3 genes, with the following Spearman correlation coefficients: BAALC r = 0.9, ERG r = 0.7, and MN1 r = 0.8 (all p<0.001). Correlation of BAALC, ERG, and MN1 nCounter counts with the expression levels measured by Affymetrix® HG-U133 plus 2.0 microarrays were also tested. Summary measures of microarray gene expression levels were computed using the Robust Multichip Average method, which incorporates quantile normalization of arrays. Significant correlation of nCounter and microarray results was observed, with Spearman correlation coefficients as follows: BAALC r = 0.96, ERG r = 0.8, and MN1 r = 0.8 (all p<0.001). For the 27 sets of paired samples, nCounter results for BM and PB were also significantly correlated, with Spearman correlation coefficients of BAALC r = 0.9, ERG r = 0.7, and MN1 r = 0.6 (all p<0.001). Because RNA quickly degrades if not promptly isolated from PB or BM, and degraded RNA often fails qRT-PCR assays, we determined whether RNA quality affected nCounter performance by assessment of standard quality parameters, including ratio of absorbance at 260 and 280 nm (260:280, a measure of RNA purity, acceptable 1.8-2.0) and RNA Quality Index (RQI, which assesses 18S:28S rRNA ratio and RNA degradation, 7-10 acceptable). Quality ranged from very high, with 260:280 ratios >1.9 and RQI scores >9, to relatively low, with 260:280 ratios <1.8, RQI scores <4, and degraded RNA visible on the Experion® RNA chip. Such a range of RNA quality is consistent with our experience with clinical specimens, which may be delayed in transit to the laboratory. Nevertheless, fewer than 3% of nCounter assays failed to generate acceptable results (11/393 assays), likely because no PCR step is required. Our results show that the nCounter system is a rapid, relatively inexpensive ($0.72/assay), and highly reproducible methodology that will be very useful for routine diagnostic testing of prognostic gene expression and upfront molecular-risk assessment for treatment guidance in AML pts. Disclosures No relevant conflicts of interest to declare.

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Navarini, Luca, Marta Vomero, Stefano Di Donato, Damiano Currado, Onorina Berardicurti, Annalisa Marino, Pietro Bearzi et al. "2-Arachidonoylglycerol Reduces the Production of Interferon-Gamma in T Lymphocytes from Patients with Systemic Lupus Erythematosus". Biomedicines 10, n.º 7 (12 de julio de 2022): 1675. http://dx.doi.org/10.3390/biomedicines10071675.

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Background: the endocannabinoid 2-arachidonoylglycerol (2-AG) plays a pivotal role in immune cells regulation. The plasma levels of 2-AG are increased in patients with systemic lupus erythematosus (SLE) and correlate with disease activity. Moreover, in plasmacytoid dendritic cells from SLE patients, 2-AG is able to control the production of type 1 interferon (IFN) through CB2 activation. The aim of this study was to evaluate the potential role of 2-AG on T lymphocytes from SLE patients. Methods: peripheral blood mononuclear cells (PBMCs) from SLE participants and age- and sex-matched healthy donors (HD) were isolated by Ficoll–Hypaque density-gradient centrifugation. The PBMCs were treated with increasing concentrations of 2-AG, and AM251 and AM630 were used to antagonize CB1 and CB2, respectively. Flow cytometry was used to assess the expression of CD3, CD4, CD8, CD25, IFN-ɣ, IL-4, and IL-17A. Results: 2-AG (1 μM) decreased IFN-ɣ expression (p = 0.0005) in the Th1 lymphocytes of SLE patients. 2-AG did not modulate the cytokine expression of any other T lymphocyte population from either SLE or HD. Treatment with both 2-AG and AM630 increased the IFN-ɣ expression in Th1 lymphocytes of SLE patients (p = 0.03). Discussion: 2-AG is able to modulate type 2 IFN production from CD4+ T lymphocytes from SLE patients through CB2 activation.

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Cerejo, Sofia de Amorim, Sheila Canevese Rahal, João Ferreira de Lima Neto, Fabiana Azevedo Voorwald y Fernanda da Cruz Landim e. Alvarenga. "Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culture". Acta Cirurgica Brasileira 26, n.º 5 (octubre de 2011): 333–38. http://dx.doi.org/10.1590/s0102-86502011000500002.

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PURPOSE: To evaluate three methods to isolate rats MSCs and to analyze the potential of a castor oil polyurethane base membrane as a scaffold for MSCs. METHODS: Four male Wistar rats, aged 20-30 days were used. Bone marrow aspirates from femur and tibia were harvested using DMEM high glucose and heparin. The cell culture was performed in three different ways: direct culture and two types of density gradients. After 15 days, was made the 1st passage and analyzed cell viability with markers Hoerscht 33342 and propidium iodide. The MSCs were characterized by surface markers with the aid of flow cytometry. After this, three types of castor oil polyurethane membranes associated with the MSCs were kept on the 6-well plate for 5 days and were analyzed by optical microscopy to confirm cell aggregation and growth. RESULTS: Separation procedures 1 and 2 allowed adequate isolation of MSCs and favored cell growth with the passage being carried out at 70% confluence after 15 days in culture. The cells could not be isolated using procedure 3. When the 3 castor oil polyurethane membrane types were compared it was possible to observe that the growth of MSCs was around 80% in membrane type 3, 20% in type 2, and 10% in type 1. CONCLUSION: Both Ficoll-Hypaque densities allow isolation of rat MSCs, and especially castor oil-based membrane type 3 may be used as a scaffold for MSCs.

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Paoliello-Paschoalato, A. B., A. E. C. S. Azzolini, M. F. C. Cruz, L. F. Marchi, L. M. Kabeya, E. A. Donadi y Y. M. Lucisano-Valim. "Isolation of healthy individuals' and rheumatoid arthritis patients' peripheral blood neutrophils by the gelatin and Ficoll-Hypaque methods: Comparative efficiency and impact on the neutrophil oxidative metabolism and Fcγ receptor expression". Journal of Immunological Methods 412 (octubre de 2014): 70–77. http://dx.doi.org/10.1016/j.jim.2014.07.001.

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Babiker, Nihad Elsadig, Alsadig Gassoum, Mohamed Abdelrahman Arbab, Sawsan Ahmed Hamed ALDeaf, Imad Fadl-Elmula, Nahla E. Abdelraheem, Mohamad Ahmed Ali El-Sheikh y Hassan Hussein Musa. "Production of insulin producing cells from cord blood mesenchymal stem cells and their potential in cell therapy". Journal of Drug Delivery and Therapeutics 9, n.º 6-s (15 de diciembre de 2019): 65–71. http://dx.doi.org/10.22270/jddt.v9i6-s.3739.

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Introduction: Mesenchymal stem cells (MSCs) were described as adherent cells with a fibroblast-like appearance, have a great capacity for self-renewal while maintaining their multipotency and differentiation into multiple tissues in vivo and in vitro. Methods: MSCs were isolated from cord blood of Sudanese donors using Ficoll-Hypaque gradient density protocol, and differentiate into β- like cells using 3-step protocol. STZ induced diabetic rats were injected intraperitoneally with the differentiated islet β- like cells and blood glucose levels were monitored for seven days. Results: The adherent cell appeared round and sphere after one-week of incubation, and the fibroblast-like colony was strongly attached after three weeks of seeding. The phenotyping of cells showed positivity for CD13, and negativity for CD34, CD45 and HLADR. MSCs were induced into islet-like cells using a 3-step (15-days) protocol. The differentiated cells showed positive diathizone stain and positive imuno-reactivity to anti-human insulin antibody. Secretion of insulin by insulin-producing cells showed positive result with >3.4 u/ml scale reading in high glucose concentration medium. After one-week of transplantation the level of blood glucose was reduced from 410 to 225 mg/dl in the experimental rat. Conclusion: Human UCB-MSCs can be differentiated into insulin-secreting cells invitro, and are able to produce and secrete insulin in response to high glucose concentration in vivo and in vitro. Keywords: Cord blood, Mesenchymal stem cell, islets β-like cells

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