Human Blood Dendritic Cells
Brian J. Nickoloff in Dermal Immune System, 2019
The human peripheral blood dendritic cell is a rather rare and phenotypically distinct cell that comprises from 0.2 to 0.5% of peripheral blood mononuclear cells. The dendritic cell is quite distinct morphologically, phenotypically, and functionally from the other recognized types of antigen-presenting cells, such as monocytes/macrophages and B lymphocytes. Although a specific dendritic cell marker in the murine system has been reported,17 there is no unique and specific marker as yet described that is expressed exclusively on the human dendritic cell. A number of attempts in several laboratories to raise specific monoclonal antibodies to the peripheral blood dendritic cell have succeeded only in confirming that dendritic cells express very high levels of known integrins, adhesins, and histocompatibility antigens. Therefore, it is possible that the phenotypic component of the unique functional capabilities of the dendritic cell results from the combination of high levels of adhesins, integrins, surface histocompatibility molecules, and other antigens rather than any one distinctive surface marker (see Figure 3).
Gene Therapy Clinical Trials for Adenosine Deaminase Deficiency/Severe Combined Immunodeficiency
Eric Wickstrom in Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
The peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugádon, washed, counted and cultured in AIM-V nutrient medium containing IL-2, a T-cell growth factor, and OKT3 monoclonal antibody to activate the Τ cells. Proliferating Τ lymphocytes were incubated with the LASN vector supernatant, in the presence of protamine and IL-2, two times daily for 2-3 days. The cells were replenished with fresh AIM-V medium, harvested, washed and resuspended in normal saline, filtered through a platelet filter and infused through a peripheral vein after a test dose was given. An initial infusion of ~6 × 107 cells/kg body weight was given in a volume of 10 ml/kg body weight administered over a period of 2 hrs. Later (see Figure 5 for details), the number of ADA gene-corrected Τ lymphocytes was progressively increased and given at intervals.
Combination Cytokine Therapy in Cancer
Ronald H. Goldfarb, Theresa L. Whiteside in Tumor Immunology and Cancer Therapy, 2020
Natural killer cell activity, T-cell phenotype (CD4, CD8, CD56, CD16, CD4/HLA-DR, CD8/HLA-DR, CD56/HLA-DR) and 2’5’ oligoadenylate synthetase (2’5’As) were measured prior to therapy, during therapy and following completion of treatment; complete immune data were available in 28/36 patients (24). For immunologic monitoring, blood samples were drawn into preservative-free heparinized syringes between 8:00 and 11:00 am. Peripheral blood mononuclear cells (PBMNC) were separated by centrifugation on Ficoll-Hypaque gradients, washed and counted in a trypan blue dye. Natural Killer Activity (NK) was measured in a standard short-term (4 hr) cytotoxicity assay using cultured K562 cells as targets (25). T-cell populations were evaluated by two-color flow cytometry performed on a FACScan. 2’5’ oligoadenylate synthetase (2’5’As) were determined by the method previously described by Merritt et al (26). The data were combined to create five time points for statistical analyses. Time effect, treatment effect, effect of initial dose level and relationship between clinical response and potential explanatory factors were evaluated.
Challenges in the management of HIV infection: update on the role of probiotic supplementation as a possible complementary therapeutic strategy for cART treated people living with HIV/AIDS
Published in Expert Opinion on Biological Therapy, 2019
Giancarlo Ceccarelli, Maura Statzu, Letizia Santinelli, Claudia Pinacchio, Camilla Bitossi, Eugenio Nelson Cavallari, Vincenzo Vullo, Carolina Scagnolari, GabrieIla d’Ettorre
Interestingly, in another study that explored how the pathway of tryptophan metabolism influences the gut–brain axis in HIV-infected patients, a correlation between high levels of CSF immune activation markers and the over-expression of IDO at the gut-mucosal surface was described. In particular, a significant reduction of neopterin and IDO mRNA levels was found after six months of a dietary management with multi-strain probiotic supplementation (Vivomixx® in EU, Visbiome® in USA, and the DeSimone Formulation® in Korea) [139]. To confirm this possible mechanism of gut–brain interaction and to investigate the link between probiotic supplementation, tryptophan pathway, and serotonin (a neuro-active metabolite of tryptophan) levels, a pilot study was recently conducted in HIV-positive subjects. The serum concentrations of serotonin and tryptophan and the expression of immune activation markers (CD38 and HLA-DR on peripheral CD4 + T lymphocytes) were measured in HIV-infected patients on effective cART at baseline and after 6 months of probiotic supplementation. After probiotic supplementation with Vivomixx®, a significant increase in concentration of serum serotonin, a decreased level of tryptophan in plasma, a reduction in CD38 and HLA-DR expression on CD4 + T cells and a reduced expression of IDO mRNA on peripheral blood mononuclear cells were observed, confirming the possible existence of a tryptophan pathway-mediated probiotic modulation on CNS activity [140].
Ankylosing Spondylitis Patients Display Aberrant ERAP1 Gene DNA Methylation and Expression
Published in Immunological Investigations, 2022
Yubo Ma, Dazhi Fan, Shanshan Xu, Jixiang Deng, Xing Gao, Shiyang Guan, Xu Zhang, Faming Pan
Peripheral blood mononuclear cells were isolated from peripheral blood using density gradient centrifugation. Then, total RNA were extracted and purified from peripheral blood mononuclear cells by TRIzolTM LS reagent. Total RNA was reversely translated as complementary DNA after the degradation of mixed genomic DNA using PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara Bio Inc., Japan). SYBR Green kit (Takara Bio Inc., Japan) was used in quantitative real-time PCR process based on QuantStudioTM 7 Flex (Life Technologies, USA). The expression data of ERAP1 was normalized to internal reference β-actin, and the relative expression level of ERAP1 was calculated by comparative 2−ΔΔCt method. Primers sequence of ERAP1 and β-actin were listed as following: β-actin, forward: 5ʹ-CTCCATCCTGGCCTCGCTGT-3ʹ, reverse: 5ʹ-GCTGTCACCTTCACCGTTCC-3ʹ; ERAP1, forward: 5ʹ-TTTGAACTTGGCTCATCTTCC-3ʹ, reverse: 5ʹ-AATTGTCTGTTGGACACAACG −3ʹ.
Nanovesicles released by OKT3 hybridoma express fully active antibodies
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Mariantonia Logozzi, Rossella Di Raimo, Francesca Properzi, Stefano Barca, Daniela F. Angelini, Davide Mizzoni, Mario Falchi, Luca Battistini, Stefano Fais
Peripheral blood mononuclear cells were isolated from whole blood by density gradient centrifugation using standard procedures (Ficoll-Paque Plus, GE Healthcare, Chicago, IL, USA). PBMC were plated at 0.5 × 106 cells for well and cultured in the presence of decreasing dilutions (20, 10, 5, and 2,5 × 109) of OKT3 exosome preparation, OKT3 supernatant and OKT3 supernatant without exosomes. As positive controls, anti-CD3 antibody (OKT3 clone, Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the cells at 1 µg/ml in a soluble state or plate bound. As negative control decreasing dilutions of P3X63 exosome preparation were used. In all conditions, 10 υg/ml of Brefeldin A (Sigma Aldrich, Saint Louis, MO, USA) was added. After 7 h, PBMC were washed twice and labelled superficially with the following anti-human antibodies with the addition of LIVE/DEAD Fixable Aqua Dead Cell Stain (ThermoFisher, Waltham, MA, USA): CD3 (Beckman Coulter, Indianapolis, IN, USA), CD4 (Becton Dickinson, Franklin Lakes, NJ, USA), CD8 (Milteniy, Bergisch Gladbach, Germany) conjugated, respectively, with Pe-Cy7, Brilliant Violet 605 and APC-Vio770. The cells were then washed, fixed (0.5% formaldehyde) and permeabilized with 0.5% of Saponin solution for the intracellular staining. PBMC were labelled intracellularly with the following anti-human cytokines antibodies: IL2 (PE-dazzle-594 conjugated, Biolegend, San Diego, CA, USA), IFN-γ (Violet 450 conjugated, Becton Dickinson, Franklin Lakes, NJ, USA) and TNF-α (Fitc conjugated Milteniy, Bergisch Gladbach, Germany).
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