Early Erythroid Development Is Enhanced with Hypoxia and Terminal Maturation with Normoxia in a 3D Ex Vivo Physiologic Eythropoiesis Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2453-2453
Author(s):  
Susana Brito dos Santos ◽  
Mark C. Allenby ◽  
Athanasios Mantalaris ◽  
Nicki Panoskaltsis
Keyword(s):  
Mononuclear Cells ◽  
Cytokine Production ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Erythroid Progenitors ◽  
Variable Intensity ◽  
Erythroid Progenitor ◽  
Tnf Α ◽  
The Impact

Abstract Reproduction of dynamic physiologic erythropoiesis in vitro requires a three-dimensional (3D) architecture, erythroblast-macrophage interactions and cytokines such as erythropoietin (EPO). The role of oxygen concentration gradients in this process is unclear. We have created a 3D bone marrow (BM) biomimicry using collagen-coated polyurethane scaffolds (5mm3) to expand cord blood mononuclear cells (CBMNCs) in a cytokine-free environment for 28 days (D). Addition of EPO to this system induces mature erythropoiesis. We hypothesised that physiologic concentrations of cytokines - stem cell factor (SCF) / EPO - and a hypoxia (H)/normoxia (N) schedule to mimic BM oxygen gradients would enhance erythropoiesis. CBMNCs were seeded (4x106 cells/scaffold) in 3D serum-free cultures supplemented with 10ng/mL SCF (D0-D28), and 100mU/mL EPO (D7-D28), with medium exchange every 3D. Three conditions were compared: N (20%), H (5%) and 2-step oxygenation HN (H D0-D7 and N thereafter). Erythroid maturation was monitored weekly by flow cytometry (CD45/CD71/CD235a) both in situ (i.e., in scaffolds) and in supernatant (S/N) cells. D0-7 H was more efficient in early induction of CD235a in the absence of exogenous EPO (H 13% vs N 8% CD45loCD71+CD235alo cells, p<0.05). This maturation profile was also observed in D10 S/N cells, in which CD45loCD71+CD235a+ cells were proportionately more in H (30%) and HN (27%) than in N (16%, p<0.05). By D14, N and HN stimulated the appearance of CD45-CD71+CD235a+ cells, whereas H maintained the CD45loCD71+CD235a-/lo phenotype. By D21, a CD45-CD71+CD235a+ mature population was clearly distinguished in all conditions, most notably in N (16%) and HN (21%) vs H (9%). At D28, more mature CD45-CD71loCD235a+ cells were observed in normoxia conditions, N 3% and HN 4%, vs H 0.3%. A renewed population of erythroid progenitors was also evident at this time (H 62%, N 51% and HN 46% CD45loCD71lo/+CD235a- cells). In order to assess the impact of H and N on erythroid gene transcription, we evaluated erythroid signatures by qRT-PCR. GATA-1 expression was detected from D7, highest for H at D14 (p<0.05), and decreased thereafter. GATA-2 expression was up-regulated only at D28, in particular in N (p<0.05), and correlated with emerging erythroid progenitors identified at this stage. At D14, EPOR expression was maximal, especially in HN (p<0.05), simultaneous with high pSTAT5 levels, suggesting activation of EPOR signalling. Also at D14, H upregulated γ-globin (p<0.05). By Western Blot, only H and HN still produced γ-globin whereas β-globin expression was clearly detected in all conditions by D28. In situ production of cytokines was evaluated by cytometric bead array in the exhausted media. IL-6, G-CSF, GM-CSF, IL-1, TNF-α and IL-17 were detected at higher concentrations during the first 7 days, declining to undetectable thereafter. IL-21 was not detected at any point. IL-3 was detected from D13, with highest expression in H (p<0.05, D22). VEGF was also expressed after D7, highest in H (p<0.05, D16 & D19), concurrent with HIF-1α up-regulation observed at D7 and D14. TNF-α was produced with variable intensity from D4. These data suggested that D7-D14 was a crucial period for culture dynamics, in particular for H and HN, with up-regulation of erythroid transcription factors, EPOR signalling, and endogenous cytokine production. BFU-E and CFU-E also dominated the first 14 days of culture. Scanning electron microscopy at D17 and D25 revealed niche-like structures in situ, which expressed STRO-1, osteopontin and vimentin at D19 by confocal immunofluorescent microscopy, indicative of an endogenous stromal cell microenvironment. CD68+ cells were also detected at D19 in proximity to CD71+ cells suggesting formation of erythroblastic islands. In this 3D ex vivo biomimicry using near-physiologic cytokine and oxygen conditions, H induced initial erythroid commitment and established an early erythroid progenitor population. N was required at later maturational stages and enhanced the γ-globin to β-globin switch. We identified D7-D14 as a crucial timeframe in this system wherein endogenous cytokine production as well as up-regulation of GATA-1, EPOR and HIF-1α was observed. We propose that a combined HN schedule in this 3D BM biomimicy may enable a more robust and physiologic culture platform to study normal and abnormal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 288-288
Author(s):  
Suhita Ray ◽  
Linda Chee ◽  
Nicholas T. Woods ◽  
Kyle J Hewitt
Keyword(s):  
Steady State ◽  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Interacting Proteins ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Capping Protein ◽  
Sam Domain ◽  
Stress Erythropoiesis

Abstract Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p&lt;0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors. To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. Disclosures No relevant conflicts of interest to declare.

the γ Isoform of the Glucocorticoid Receptor Is Ontogenetically Activated and Predicts Poor Ex-Vivo Expansion of Erythroid Cells From Adult Blood.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 642-642
Author(s):  
Giovanni Migliaccio ◽  
Massimo Sanchez ◽  
Francesca Masiello ◽  
Valentina Tirelli ◽  
Lilian Varricchio ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Optimal Growth ◽  
Human Albumin ◽  
Red Cells ◽  
Erythroid Cells ◽  
Erythroid Progenitors

Abstract Abstract 642 Ex-vivo generated erythroblasts (EBs) represent alternative transfusion products. Adult blood (AB) contains numbers of progenitor cells comparable to those present in cord blood (CB) (106 vs 1.8×106 CD34pos cells in average AB and CB donations) but generates lower numbers of erythroblasts (EBs) (∼4.8×108 vs 6.6×1010, respectively) and, in spite of its numerous advantages, is not considered suitable for ex-vivo EB production. To assess the potential of AB to generate EBs ex-vivo, the growth factors [stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO)] and optimal concentration and addition schedule of dexamethasone (DXM) and estradiol (ES) sustaining maximal EB amplification from AB mononuclear cells (MNC) were defined using media with serum previously defined as human erythroid massive amplification culture (HEMAser). Adult MNC stimulated with SCF and IL-3 in combination with EPO generated low numbers (fold increase ∼2) of EBs at all stages of maturation. Concentration response studies conducted on MNC from 10 different donors, indicated that the further addition to the cultures of DXM and ES (both at 10-6 M) increased (∼6-12-fold) the numbers of EBs generated. Delayed addition and withdrawal experiments indicated that DXM and ES exerted partially overlapping but non-redundant functions. DXM was indispensable to achieve maximal amplification in the first 10 days of culture while ES was required from day 10 on. To determine if variability in glucocorticoid receptor (GR) expression might affect ex vivo generation of EBs, expression of αa and γ GR isoforms (αaGR and γGR) by EBs from 10 AB and 5 CB was investigated. While EBs from all donors expressed αaGR, γGR was not expressed by EBs obtained from CB and from AB that generated high numbers of EBs ex vivo, suggesting that activation of γGR in EBs is ontogenetically activated in a subset of AB and may predicts poor expansion. Ex vivo produced EBs are megaloblastic (30 to 50 μm). EPO decreased their size from 40.1±1.4 to 11.6±0.3 μm by 96 h (p<0.01). Although still macrocytic (adult normocytic red cells are 8 μm), these cells are smaller than fetal red cells (12.5 μm) and therefore suitable for clinical use. Inclusion of bovine components in HEMAser precludes its use for clinical purposes. Therefore, optimal growth factor and hormone combinations identified in HEMAser were used to formulate a medium composed of pharmaceutical grade human albumin, human albumin-based-lipid liposomes and iron-saturated recombinant human-tranferrin (HEMAdef). HEMAdef sustained EB amplification as efficiently as HEMAser from CB MNC and 10-fold higher than HEMAser from AB MNC. Moreover, the numbers of EBs generated in HEMAdef by adult MNC were similar to those generated by CB MNC (750×106 vs 500×106 per 106 MNC from AB and CB, respectively). Assuming that MNC contain 102-103 EB progenitors (CD34pos cells represent 0.1% of MNC and erythroid progenitors represent 10% of CD34pos cells), it was calculated that the generation of 750×106 EBs from the progenitors present in 106 adult MNC required 19-23 divisions, a number below the theoretical Hayflick's limit for somatic cell divisions of 35. These results indicate that at least a subset of AB donors is suitable to produce ex-vivo erythroid cells for transfusion and that it should be possible, by optimizing HEMAdef components, to further increase the number of EBs that can be generated ex-vivo from AB. Disclosures: No relevant conflicts of interest to declare.

4-1BBL-Expressing aAPCs Attenuate IL-15-Induced NK Cell Expansion and Cytokine Production In Vitro but Induce NK Cell-Mediated Gvhd In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2536-2536
Author(s):  
Christian M. Capitini ◽  
Joanna L. Meadors ◽  
Monica M. Cho ◽  
Rimas J. Orentas ◽  
Crystal L. Mackall ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Biology ◽  
Cytokine Production ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Adoptive Cell Therapy ◽  
Receptor Expression ◽  
The Impact ◽  
Cells Cultured

Abstract Abstract 2536 Methods to expand natural killer (NK) cells ex vivo for adoptive cell therapy are being explored to improve outcomes after allogeneic blood and marrow transplant (alloBMT). Artificial antigen presenting cells (aAPCs) can present cytokines and/or co-stimulatory molecules that can potentially improve expansion and activity. 4-1BBL (CD137L) has demonstrated mixed results on murine and human NK cells, but the impact on murine NK cell biology after alloBMT has not been explored. NK cells were harvested from either C57BL/6 (B6) or CB6F1 spleens and cultured ex vivo with a recombinant interleukin (IL)-15/IL-15 receptor alpha (Ra) complex in the presence or absence of a CD137L+ aAPC. Because IL-15 is typically presented in trans by IL-15Ra, the complex was utilized to potently increase agonist bioactivity. NK cells cultured with IL-15/IL-15Ra alone showed a peak of 20-fold expansion, but this expansion was decreased with the addition of CD137L+ aAPCs if the ratio of aAPC to NK cells was greater than 1:1. In the presence of IL-15/IL-15Ra, the impact of CD137L+ aAPCs on expression of the inhibitory receptors, Ly49C+I and activating receptor Ly49H was variable and strain dependent, with increased expression in B6 NK cells, but decreased expression in CB6F1 NK cells. The expression of major histocompatibility complex (MHC) class I was not affected in NK cells from either strain by the presence of CD137L+ aAPCs. The production of gamma interferon and tumor necrosis factor-a was robust in NK cells expanded by IL-15/IL-15Ra alone, but attenuated with the addition of CD137L+ aAPCs. Animal experiments showed that administration of NK cells expanded ex vivo with IL-15/IL-15Ra alone was well tolerated after T cell depleted MHC-mismatched alloBMT (CB6F1–>B6), but surprisingly the addition of CD137L+ aAPCs to cultures caused NK cells to induce GVHD-associated weight loss. In summary, IL-15/IL-15Ra expanded murine NK cells demonstrate increased cytokine production and do not cause toxicity when infused after alloBMT. The presence of CD137L+ aAPCs attenuated cytokine production and increased Ly49 receptor expression in NK cells from B6 mice. Remarkably, NK cells expanded by IL-15/IL-15Ra in the presence of CD137L+ aAPCs demonstrate increased propensity to cause GVHD. Ongoing studies are exploring the anti-tumor efficacy of IL-15/IL-15Ra expanded murine NK cells cultured in the presence and absence of CD137L. Disclosures: No relevant conflicts of interest to declare.

Transcriptome- Based Analysis of EPO Dependent Cfu-e to Erythroblast Development

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3188-3188
Author(s):  
Rakesh Verma ◽  
Aishwarya Narayanan ◽  
David Kuhrt ◽  
Lei Li ◽  
Su Su ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Murine Bone Marrow ◽  
Correlation Score ◽  
Insight Into

Abstract Abstract 3188 To provide molecular insight into erythroid developmental programs, including EPO- regulated aspects, we have employed transcriptome-based approaches to analyze the stage-wise development of purified murine bone marrow- derived CFUe, proerythroblasts and maturing erythroblasts. In vivo and ex vivo, these progenitors develop as KitposCD71highTer119neg; KitnegCD71highTer119neg; and KitnegCD71highTer119pos cohorts (designated as E1, E2, E3 stages, respectively). In the context of EPO- modulation, stage E1 cells exhibited ∼250 EPO- regulated target genes. In stage-E2 proerythroblasts, in contrast, 750 transcripts proved to be EPO- regulated while stage-E3 erythroblasts exhibited only select EPO- modulated genes (<50). At E1 and E2 stages, EPO- regulated targets included overlapping yet distinct sets, and this was reflected in functional sets of GO terms. Major EPO- targets at stage E1 included cell cycle and cell biogenesis genes, while in E2 proerythroblasts, negative regulators of protein kinases (and kinase activity) constituted a major EPO/EPOR target set. As E1 CFUe transitioned to E2 proerythroblasts, an unexpected transient narrowing in the complexity of overall expressed genes was exhibited. Stage- modulated global sets of transcripts included myeloid cell differentiation factors, cell number homeostasis factors and heme biosynthetic processes. As E2 proerythroblasts transitioned to E3 erythroblasts, functional GO sets were associated predominantly with dynamics in organelle and cellular compartments. In addition, HomoloGene and Connectivity Mapping approaches were applied to compare transcriptomes of stage E1, E2 and E3 murine bone marrow erythroid progenitors with four recently studied stages of human erythroid progenitor cell development (here, termed H1, H2, H3 and H4). High correlation of stage E1 m-CFUe with not only human H1 CFUe but also H2 proerythroblasts was observed (0.59 and 0.57 correlations). Stage E2 murine proerythroblasts best corresponded to H2 human proerythroblasts (0.36 correlation score), while E3 murine erythroblasts aligned closely with human H4 late stage erythroblasts (0.58 correlation score). These latter analyses provide novel transcriptome- based comparisons, and transcript specific insight, into conserved vs distinct features of murine and human erythroid development at CFUe to maturing erythroblast stages. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Loss of Liver Kinase B1 Accelerates Maturation Time in the Developing Erythrocyte

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 936-936
Author(s):  
Zollie White III ◽  
Katie Freeman ◽  
Lorrie L Delehanty ◽  
Adam N Goldfarb
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Transferrin Saturation ◽  
Flow Cytometric Analysis ◽  
Conditional Knockout ◽  
Erythroid Progenitors ◽  
Human Cd34 ◽  
Iron Deprivation ◽  
Liver Kinase B1 ◽  
The Impact

Abstract In the US, iron-restricted anemia contributes greatly to morbidity. The erythroid iron deprivation response, characterized by a pathway in which erythropoiesis is suppressed during iron restriction, underlies this anemia. In preliminary studies, liver kinase B1 (LKB1) was implicated as a potential key component in the erythroid iron deprivation response. Normal human CD34+ hematopoietic progenitor cells cultured for 3 days in erythroid medium with 100% or 10% transferrin saturations underwent immunoblot of whole cell lysates. Reproducibly, the levels of LKB1 did not change based on the transferrin saturation; however, immunofluorescence imaging showed a shift in subcellular localization when cells were subjected to low iron conditions. To assess the effects of LKB1 loss in the erythroid compartment, we used control and LKB1 conditional knockout (STK11 F/F; EpoR-Cre+) mice. In steady-state conditions, the loss of LKB1 does not confer a change in RBC count, though, there is a baseline increase in the number of reticulocytes, and a large increase in the level of serum Erythropoietin (Epo). In a model to precipitate anemia, these mice were challenged with intraperitoneal injection of phenylhydrazine (PHZ). It was found that LKB1 is dispensable for an appropriate response to this type of stress erythropoiesis. To assess the impact of LKB1 on maturation, we performed flow cytometric analysis using an ex vivo culture system of splenic erythroblasts. LKB1-deficient erythroid progenitors show increased percentages of more advanced cells as evidenced by the surface markers CD71 and Ter119 (the mouse analogue of human glycophorin A). Current studies are underway to assess if this change is due to signaling through the AMPK pathway. These studies will provide mechanistic detail of LKB1 function and activity on erythropoiesis, improving our understanding of programs involved in maturation of differentiation and lineage selection which may ultimately help to improve health outcomes and advance treatment for various types of anemias. Disclosures No relevant conflicts of interest to declare.

Probiotic supplement consumption alters cytokine production from peripheral blood mononuclear cells: a preliminary study using healthy individuals

2013 ◽  
Vol 4 (4) ◽  
pp. 313-317 ◽  
Author(s):  
N.J. Hepburn ◽  
I. Garaiova ◽  
E.A. Williams ◽  
D.R. Michael ◽  
S. Plummer
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Healthy Individuals ◽  
Probiotic Supplementation ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Preliminary Study

The objective of this study was to examine the effect of daily probiotic supplementation upon the immune profile of healthy participants by the assessment of ex vivo cytokine production. Twenty healthy adult volunteers received a multi-strain probiotic supplement consisting of two strains of Lactobacillus acidophilus (CUL60 and CUL21), Bifidobacterium lactis (CUL34) and Bifidobacterium bifidum (CUL20) and fructooligosaccharide for 12 weeks. Blood samples were collected at baseline, 6 and 12 weeks. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured ex vivo in the presence or absence of lipopolysaccharide and cytokine production was assessed. Postintervention, a significant decrease in the production of interleukin-6 and interleukin-1β was apparent when PBMCs were incubated in the presence of lipopolysaccharide, whilst a significant increase in IL-10 and transforning growth factor-β production was seen when the cells were incubated without an additional stimulus. This preliminary study demonstrates the potential of a multi-strain probiotic supplement to alter the immune response as demonstrated by changes in ex vivo cytokine production. Such results demonstrate the potential benefit of probiotic supplementation for healthy individuals and warrants further investigation.

scholarly journals Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

2021 ◽  
Vol 67 (2) ◽  
pp. 95-101
Author(s):  
Monica Vuță ◽  
Ionela-Maria Cotoi ◽  
Ion Bogdan Mănescu ◽  
Doina Ramona Manu ◽  
Minodora Dobreanu
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Gradient Centrifugation ◽  
Intracellular Cytokine Staining ◽  
Interleukin 2 ◽  
Middle Aged

Abstract Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be stimulated directly after isolation or frozen for later use. However, cryopreservation may affect cell recovery, viability and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells. Methods: PBMCs were obtained by Ficoll gradient centrifugation from heparinized peripheral blood of 6 middle-aged clinically healthy subjects. Half of these cells (labeled “Fresh”) was further processed and the other half (labeled “Cryo”) was cryopreserved at -140°C for up to 3 months. Fresh-PBMCs were activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin for 5 hours immediately after isolation while Cryo-PBMCs were identically activated after thawing and cell resting. Activated cells were fixed, permeabilized and intracellular cytokine staining was performed using Phycoerythrin (PE)-conjugated antibodies for Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), and Interferon-gamma (IFN-g). All samples were analyzed within 24 hours by flow cytometry. Results: Both Fresh and Cryo CD3+CD4+/CD3+CD4- sub-populations partially produced each of the three cytokines. A higher percentage of CD4+ T cells produced IL-2 and TNF-a and a greater percentage of CD4- T cells were found to produce IFN-g. A significantly higher percentage of Cryo-lymphocytes was shown to produce TNF-a in both CD3+CD4+ (31.4% vs 24.9%, p=0.031) and CD3+CD4- (22.7% vs 17.9%, p=0.031) subpopulations. No notable difference was found for IL-2 and IFN-g production between Fresh and Cryo T cells. Conclusion: Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

scholarly journals Interferon Induction By HbS, SERINC5 Upregulation and Reduction of HIV-1 Nef and AP2 Contribute to HIV-1 Restriction in Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-6
Author(s):  
Namita Kumari ◽  
Marina Jerebtsova ◽  
Songping Wang ◽  
Sharmin Diaz ◽  
Sergei Nekhai
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Interferon Response ◽  
Cell Disease ◽  
Restriction Factors ◽  
Peripheral Blood Mononuclear ◽  
Hiv 1

Concerted action of numerous positively acting cellular factors is essential for Human immunodeficiency virus type 1 (HIV-1) replication but in turn is challenged by anti-viral restriction factors. Previously we showed that ex vivo one round HIV-1 replication and replication of fully competent T-tropic HIV-1(IIIB) is significantly reduced in peripheral blood mononuclear cells (PBMCs) obtained from patients with Sickle Cell Disease (SCD). Further, we identified and confirmed CDKN1A (p21) and CH25H as host restriction factors expressed in SCD PBMCs that may contribute to the HIV-1 inhibition, in addition to the previously reported SAMHD1 and IKBα. Since CH25H is an interferon stimulated gene (ISG), we analyzed IRFs and interferon expression in SCD PBMCs. Higher levels of IRF7 and IFNβ mRNA were observed in SCD PBMCs compared to controls. We probed further to ascertain if hemin or sickle Hb was responsible for interferon response. We found upregulation of IFNβ in THP-1 - derived macrophages treated with lysates of HbSS RBCs or purified HbS as compared to untreated or HbA treated controls. HbSS RBCs lysates and purified HbS inhibited HIV-1 gag mRNA expression in monocyte-derived macrophages infected with HIV-1(Ba-L). Recent clinical study showed increased levels of CD4 in HIV-1 infected SCD patients in Africa. Thus we analyzed CD4 levels in HIV-1 IIIB infected SCD PBMCs, and found them to be higher compared to controls. Levels of HIV-1 nef mRNA, that controls CD4 expression was lower in HIV-1 IIIB infected SCD PBMCs. As Nef counteracts SERINC3/5 restriction factor, we analyzed its expression as well as the expression of AP2 clathrin adaptor that is required for Nef mediated internalization of CD4. AP2 expression was lower and SERINC5 expression was higher in SCD PBMCs. CONCLUSIONS: SCD PBMCs could resist HIV-1 infection because of the increased IFNβ production by macrophages exposed to HbSS or sickle cell RBCs. SCD PBMC have increased levels of SERNIC5 and lower levels of HIV-1 Nef and host AP2 expression that, culumlatively, can increased CD4 levels and lead to the overall improved immunological health of SCD patients. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 1SC1HL150685, 5U54MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures No relevant conflicts of interest to declare.

scholarly journals An Exaggerated Monocyte-Derived Cytokine Response to Candida Hyphae in Patients With Recurrent Vulvovaginal Candidiasis

2020 ◽  
Author(s):  
Diletta Rosati ◽  
Mariolina Bruno ◽  
Martin Jaeger ◽  
Bart-Jan Kullberg ◽  
Frank van de Veerdonk ◽  
...  
Keyword(s):  
Immune Response ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Adaptive Immune Response ◽  
Vulvovaginal Candidiasis ◽  
T Helper 1 ◽  
Recurrent Vulvovaginal Candidiasis ◽  
Tnf Α ◽  
Group A ◽  
Factor Α

Abstract Background Recurrent vulvovaginal candidiasis (RVVC) affects up to 8% of women. The immunopathogenesis is poorly understood but it has been suggested that RVVC might be due to dysregulated innate immune response. The aim of this study was to compare cytokine profiles in stimulated primary mononuclear cells (PBMCs) from RVVC and healthy individuals. Methods PBMCs isolated from RVVC patients (n = 24) and healthy volunteers (n = 30) were stimulated with unspecific and pathogen-specific antigens. Cytokine production was assessed after 24 hours, 48 hours, and 7 days using ELISA. Results No significant differences in cytokine production were found in T helper 1 (Th1), Th2, and Th17 immunity in response to both unspecific and pathogen-specific stimulations. Tumor necrosis factor-α (TNF-α) production in response to C. albicans hyphae was significantly higher in patients than controls and within the patient group, a significant positive correlation was found between interleukin-1β (IL-1β) and both TNF-α and IL-6. Both IL-1β/IL-1Ra and TNF-α/IL-10 ratios in Candida hyphae-stimulated PBMCs were significantly higher in patients than controls. Conclusions Women affected by RVVC showed increased monocytes-derived cytokine production, which might contribute to an exaggerated vaginal immune response to Candida hyphae. RVVC patients show no defective Th-dependent adaptive immune response upon Candida stimulation.

scholarly journals Intranasal Niosomal In Situ Gel as a Promising Approach for Enhancing Flibanserin Bioavailability and Brain Delivery: In Vitro Optimization and Ex Vivo/In Vivo Evaluation

Pharmaceutics ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 485 ◽  
Author(s):  
Usama A. Fahmy ◽  
Shaimaa M. Badr-Eldin ◽  
Osama A. A. Ahmed ◽  
Hibah M. Aldawsari ◽  
Singkome Tima ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Oral Treatment ◽  
Drug Dissolution ◽  
Nasal Administration ◽  
Brain Delivery ◽  
Drug Bioavailability ◽  
In Vivo Evaluation ◽  
In Situ Gel ◽  
The Impact

Flibanserin (FLB) is a multifunctional serotonergic agent that was recently approved by the FDA for the oral treatment of premenopausal women with hypoactive sexual desire disorder. FLB is a centrally acting drug that has a low oral bioavailability of 33% owing to its exposure to the hepatic first-pass effect, as well as its pH-dependent solubility, which could be an obstacle hindering the drug dissolution and absorption via mucosal barriers. Thus, this work aimed at overcoming the aforementioned drawbacks and promoting the nose-to-brain delivery of FLB via the formulation of an intra-nasal in situ niosomal gel. The Box–Behnken design was employed to study the impact of Span® 85 concentration (X1), hydration time (X2), and pH of the hydrating buffer (X3) on the vesicle size and drug entrapment. The optimized formulation exhibited a spherical shape with a vesicular size of 46.35 nm and entrapment efficiency of 92.48%. The optimized FLB niosomes integrated into gellan gum-based in situ gel exhibited enhanced ex vivo permeation and improved plasma and brain concentrations after nasal administration in rats compared to raw FLB. These findings highlight the capability of the proposed intra-nasal FLB niosomal in situ gel to boost the drug bioavailability and to promote its direct delivery to the brain.

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