scholarly journals APRIL drives a co-ordinated but diverse response as a foundation for plasma cell longevity.

2021 ◽  
Author(s):  
Matthew A Care ◽  
Gina Marie Doody ◽  
Sophie J Stephenson ◽  
Reuben M Tooze
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Plasma Cell ◽  
Stress Responses ◽  
Time Course ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Regulated Gene Expression ◽  
Cell Longevity

Antibody secreting cells (ASCs) survive in niche microenvironments, but cellular responses driven by particular niche signals are incompletely defined. The TNF superfamily member APRIL provides a niche signal that can support the transition of transitory plasmablasts into long-lived plasma cells. Here we explore how APRIL helps to establish the biological programs that promote life in the niche, by studying the initial response of primary human plasmablast to APRIL. Under conditions allowing the maturation of ex vivo or in vitro generated plasmablasts, we find that APRIL drives activation of ERK, p38 and JNK. This is accompanied by a classical NFκB response. Under these conditions induction of AKT phosphorylation is also observed with similar kinetics, paralleled by FOXO1 phosphorylation and nuclear exclusion. Time course gene expression data resolve the downstream co-ordinated transcriptional response. The APRIL-signal propagates via immediate early genes and classical NFκB responsive targets to converge onto modules of MYC- and OCT2-regulated gene expression linked to cell growth, as well as leading to enhanced expression of ICAM1 and SQSTM1 associated with adhesion and metabolic/stress responses. Thus, APRIL drives a combination of multiple transcriptional programs that co-ordinate cell growth, stress response and adhesion in human ASCs, providing a broad foundation to support plasma cell longevity.

scholarly journals Generation of human long-lived plasma cells by developmentally regulated epigenetic imprinting

2021 ◽  
Vol 5 (3) ◽  
pp. e202101285
Author(s):  
Chester J Joyner ◽  
Ariel M Ley ◽  
Doan C Nguyen ◽  
Mohammad Ali ◽  
Alessia Corrado ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Epigenetic Changes ◽  
Developmentally Regulated ◽  
Apoptotic Gene ◽  
Apoptotic Genes ◽  
Distinct Morphology ◽  
Antibody Secreting Cells

Antibody secreting cells (ASCs) circulate after vaccination and infection and migrate to the BM where a subset known as long-lived plasma cells (LLPCs) persists and secrete antibodies for a lifetime. The mechanisms by which circulating ASCs become LLPCs are not well elucidated. Here, we show that human blood ASCs have distinct morphology, transcriptomes, and epigenetics compared with BM LLPCs. Compared with blood ASCs, BM LLPCs have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. LLPCs up-regulate pro-survival genes MCL1, BCL2, and BCL-XL while simultaneously down-regulating pro-apoptotic genes HRK1, CASP3, and CASP8. Consistent with reduced gene expression, the pro-apoptotic gene loci are less accessible in LLPCs. Of the pro-survival genes, only BCL2 is concordant in gene up-regulation and loci accessibility. Using a novel in vitro human BM mimetic, we show that blood ASCs undergo similar morphological and molecular changes that resemble ex vivo BM LLPCs. Overall, our study demonstrates that early-minted blood ASCs in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPCs.

scholarly journals Emerging Roles of Wisp-1 and SFRP4 in Proliferation and Differentiation Potential of Wharton's Jelly Mesenchymal Stem/Stromal Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4375-4375
Author(s):  
Aristea Batsali ◽  
Charalampos Pontikoglou ◽  
Emmanuel Agrafiotis ◽  
Elisavet Kouvidi ◽  
Irene Mavroudi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Time Course ◽  
Ex Vivo ◽  
Statistical Significance ◽  
Adipogenic Differentiation ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Proliferation And Differentiation

Abstract We have previously shown (Batsali A et al., Blood 2013:122, 1212) that ex vivo expanded human mesenchymal stem/stromal cells (MSCs) derived from the Wharton's jelly (WJ) of the umbilical cord exhibit increased proliferative capacity and reduced potential to differentiate in vitro to adipocytes and osteocytes, compared to bone marrow (BM) derived-MSCs. Provided that the WNT-pathways are involved in proliferation and differentiation of BM-MSCs, we assumed that the aforementioned findings might be attributed, at least in part, to aberrant WNT-signaling in WJ-MSCs. In support of this hypothesis, we found that gene expression of the Wnt antagonist sFRP4, a promoter of adipogenesis in human adipose tissue-derived MSCs, was significantly down-regulated in WJ-MSCs and that mRNA levels of WNT-induced secreted protein-1, (WISP-1), a regulator of osteogenesis in BM-MSCs, were also significantly reduced in WJ-MSCs. These observations imply a connection between these WNT-associated molecules and the biological properties of WJ-MSCs, which requires, however, further investigation. The present study was undertaken so as to explore the effects of WISP-1 and sFRP4 in growth and differentiation of ex-vivo expanded WJ-MSCs. MSCs were isolated from consenting healthy donors’ BM aspirates (n=5) and from the WJ of full-term neonates (n=5) after written informed consent of the family. MSCs were in vitro expanded, re-seeded for a total of 4 passages (P) and phenotypically characterized by flow cytometry at P3. WJ-MSCs were grown in the absence or presence of rhWISP-1 or rhsFRP4 and cell proliferation was assessed by a methyl-triazolyl-tetrazolium (MTT)-assay. In addition, WJ-MSCs were induced to differentiate in vitro to osteoblasts and adipocytes, in the absence or presence of rhWISP-1 or rhsFRP4 respectively. Differentiation was quantified by cytochemical stains and by the expression of adipocyte- and osteocyte-specific genes by real time RT-PCR. Relative gene expression was calculated by the ΔCt method. The expression of WISP-1 and sFRP4 by non-differentiated WJ- and BM-MSCs as well as by WJ-MSCs during osteogenesis and adipogenesis, respectively, was also evaluated by real time RT-PCR. Culture-expanded cells from both WJ and BM displayed typical morphological and immunophenotypic MSC characteristics and were able to differentiate into osteoblasts and adipocytes. In line with our previous work WISP-1 and sFRP4 mRNA were significantly decreased in WJ-MSCs, compared to BM-MSCs. To explore the role of WISP-1 in WJ-MSCs' growth we cultured cells in the presence of 50 ng/ml or 100 ng/ml rhWISP1 and assessed cell proliferation at multiple time points, throughout a 14-day culture. WISP-1 treatment did not lead to any significant effect in cell numbers. Next, we investigated the time course of WISP1 gene expression during osteoinduction. In all samples, WISP1 mRNA levels increased during osteogenesis. As compared to day0 (exposure to osteogenic medium), the increase in gene expression reached statistical significance at days 7 and 14. Furthermore, WISP-1 gene expression was significantly higher at day 14, compared to day 7. To investigate the functional effects of WISP1 on the osteoblastic differentiation of WJ-MSCs, cells were cultured for 7 days in osteogenic medium supplemented with 50ng/ml rh-WISP1. A significant increase in the expression of RUNX2 and ALP was detected, compared to non-treated cells. To investigate the impact of sFRP4 in WJ-MSC's proliferation we exposed cells to 20nM rhsFRP4 for 14 days. Live cell numbers, at various time points, were significantly reduced in treated cells. Regarding the time course of sFRP4 expression during adipogenic differentiation, sFRP4 mRNA levels increased during adipogenesis reaching statistical significance at days7 and 14, as compared to day0. In addition, sFRP4 gene expression was significantly higher at day 14 as compared to day 7. Finally, when cells underwent adipogenic differentiation in the presence of rhSFRP4, a significant increase in PPARG and CEBPA mRNA levels was detected at day 14, as compared to non-treated cells Collectively, our results suggest that WISP-1 and sFRP4 may be actively implicated in proliferation and differentiation of WJ-MSCs. The functional role of these WNT-related molecules in the biology of WJ-MSCs requires deeper understanding, in view of the growing interest for the use of WJ-MSCs in cell-based therapies. Disclosures No relevant conflicts of interest to declare.

Anti-Inflammatory Effects of Novel Standardized Platelet Rich Plasma Releasates on Knee Osteoarthritic Chondrocytes and Cartilage in vitro

2019 ◽  
Vol 20 (11) ◽  
pp. 920-933 ◽  
Author(s):  
Lucía Gato-Calvo ◽  
Tamara Hermida-Gómez ◽  
Cristina R. Romero ◽  
Elena F. Burguera ◽  
Francisco J. Blanco
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Cartilage Explants ◽  
Ex Vivo Model ◽  
Chondrocyte Viability ◽  
Platelet Concentration ◽  
The Absolute ◽  
Anti Inflammatory

Background: Platelet Rich Plasma (PRP) has recently emerged as a potential treatment for osteoarthritis (OA), but composition heterogeneity hampers comparison among studies, with the result that definite conclusions on its efficacy have not been reached. Objective: 1) To develop a novel methodology to prepare a series of standardized PRP releasates (PRP-Rs) with known absolute platelet concentrations, and 2) To evaluate the influence of this standardization parameter on the anti-inflammatory properties of these PRP-Rs in an in vitro and an ex vivo model of OA. Methods: A series of PRPs was prepared using the absolute platelet concentration as the standardization parameter. Doses of platelets ranged from 0% (platelet poor plasma, PPP) to 1.5·105 platelets/µl. PRPs were then activated with CaCl2 to obtain releasates (PRP-R). Chondrocytes were stimulated with 10% of each PRP-R in serum-free culture medium for 72 h to assess proliferation and viability. Cells were co-stimulated with interleukin (IL)-1β (5 ng/ml) and 10% of each PRP-R for 48 h to determine the effects on gene expression, secretion and intra-cellular content of common markers associated with inflammation, catabolism and oxidative stress in OA. OA cartilage explants were co-stimulated with IL-1β (5 ng/ml) and 10% of either PRP-R with 0.75·105 platelets/µl or PRP-R with 1.5·105 platelets/µl for 21 days to assess matrix inflammatory degradation. Results: Chondrocyte viability was not affected, and proliferation was dose-dependently increased. The gene expression of all pro-inflammatory mediators was significantly and dose-independently reduced, except for that of IL-1β and IL-8. Immunoblotting corroborated this effect for inducible NO synthase (NOS2). Secreted matrix metalloproteinase-13 (MMP-13) was reduced to almost basal levels by the PRP-R from PPP. Increasing platelet dosage led to progressive loss to this anti-catabolic ability. Safranin O and toluidine blue stains supported the beneficial effect of low platelet dosage on cartilage matrix preservation. Conclusion: We have developed a methodology to prepare PRP releasates using the absolute platelet concentration as the standardization parameter. Using this approach, the composition of the resulting PRP derived product is independent of the donor initial basal platelet count, thereby allowing the evaluation of its effects objectively and reproducibly. In our OA models, PRP-Rs showed antiinflammatory, anti-oxidant and anti-catabolic properties. Platelet enrichment could favor chondrocyte proliferation but is not necessary for the above effects and could even be counter-productive.

scholarly journals Cell–cell coupling and DNA methylation abnormal phenotypes in the after-hours mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Federico Tinarelli ◽  
Elena Ivanova ◽  
Ilaria Colombi ◽  
Erica Barini ◽  
Edoardo Balzani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Neuronal Networks ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Neuronal Cultures ◽  
Functional Impairments ◽  
After Hours ◽  
The Impact

Abstract Background DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. Methods In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. Results We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. Conclusions The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.

scholarly journals Plasma cells shape the mesenchymal identity of ovarian cancers through transfer of exosome-derived microRNAs

2021 ◽  
Vol 7 (9) ◽  
pp. eabb0737
Author(s):  
Zhengnan Yang ◽  
Wei Wang ◽  
Linjie Zhao ◽  
Xin Wang ◽  
Ryan C. Gimple ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Ovarian Cancer Cells ◽  
Mesenchymal Phenotype ◽  
Ovarian Cancers ◽  
Novel Approach

Ovarian cancer represents a highly lethal disease that poses a substantial burden for females, with four main molecular subtypes carrying distinct clinical outcomes. Here, we demonstrated that plasma cells, a subset of antibody-producing B cells, were enriched in the mesenchymal subtype of high-grade serous ovarian cancers (HGSCs). Plasma cell abundance correlated with the density of mesenchymal cells in clinical specimens of HGSCs. Coculture of nonmesenchymal ovarian cancer cells and plasma cells induced a mesenchymal phenotype of tumor cells in vitro and in vivo. Phenotypic switch was mediated by the transfer of plasma cell–derived exosomes containing miR-330-3p into nonmesenchymal ovarian cancer cells. Exosome-derived miR-330-3p increased expression of junctional adhesion molecule B in a noncanonical fashion. Depletion of plasma cells by bortezomib reversed the mesenchymal characteristics of ovarian cancer and inhibited in vivo tumor growth. Collectively, our work suggests targeting plasma cells may be a novel approach for ovarian cancer therapy.

scholarly journals Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 730
Author(s):  
Biji Mathew ◽  
Leianne A. Torres ◽  
Lorea Gamboa Gamboa Acha ◽  
Sophie Tran ◽  
Alice Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Extracellular Vesicles ◽  
Time Course ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Paracrine Effects

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

scholarly journals Suitability of a Progenitor Cell-Enriching Device for In Vitro Applications

Coatings ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 146
Author(s):  
Antonio Celentano ◽  
Tami Yap ◽  
Giuseppe Pantaleo ◽  
Rita Paolini ◽  
Michael McCullough ◽  
...  
Keyword(s):  
Cell Viability ◽  
Time Course ◽  
Ex Vivo ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Test ◽  
Initial Reduction ◽  
Metal Wear ◽  
Class 1 ◽  
Electron Energy Loss

Rigenera® is a novel class-1 medical device that produces micro-grafts enriched of progenitors cells without ex vivo manipulation of donor tissues. The manufacturer’s protocol has been supported for a wide variety of clinical uses in the field of regenerative medicine. This study aimed to evaluate its potential use for in vitro cell models. Human primary oral fibroblasts were cultured under standard conditions and processed through Rigenera® over a time course of up to 5 min. Cell viability was assessed using a Trypan Blue exclusion test. It is possible to process fibroblasts through Rigenera® although an initial reduction of cell viability was observed. Additionally, debris was evident in the cell suspension of the processed samples. Scanning electron microscopy (SEM) microanalysis of the debris and electron energy-loss spectroscopy confirmed the presence of metal wear possibly due to the processing conditions used in this study. Interestingly, pore sizes within Rigeneracons® grids were found to range between 250–400 μm. This is the first report assessing the suitability of Rigenera® and Rigeneracons® for in vitro applications. Whilst Rigenera® workflow was found to be amenable to laboratory uses, our results strongly suggest that further research and development is necessary to support the utilization of this technology for enrichment of micro-graft derived cells and cell sorting in vitro.

scholarly journals Maturation of Human Long-lived Plasma Cells Results in Resistance to Apoptosis by Transcriptional and Epigenetic Regulation

2021 ◽  
Author(s):  
Chester J Joyner ◽  
Ariel Ley ◽  
Doan Nguyen ◽  
Muhammad Ali ◽  
Alessia Corrado ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plasma Cells ◽  
The Novel ◽  
Epigenetic Changes ◽  
Apoptosis Pathway ◽  
Apoptotic Gene ◽  
Survival Gene ◽  
Distinct Morphology ◽  
Antibody Secreting Cells

Antibody secreting cells (ASC) circulate after vaccination and migrate to the bone marrow (BM) where a subset known as long-lived plasma cells (LLPC) persist and secrete antibodies for a lifetime. The mechanisms of how circulating ASC become LLPC are not well elucidated. Here, we show that human blood ASCs have distinct morphology, transcriptomes, and epigenetics compared to BM LLPC. LLPC acquire transcriptional and epigenetic changes in the apoptosis pathway to support their survival. Upregulation of pro-survival gene expression accompanies downregulation of pro-apoptotic gene expression in LLPC. While pro-apoptotic gene loci are less accessible, pro-survival gene loci are not always accompanied by accessibility changes. Importantly, we show similar LLPC morphological and transcriptional maturation of blood ASC in response to the novel in vitro BM mimetic. In all, our study demonstrates that blood ASC in the BM microniche must undergo morphological and molecular changes to mature into apoptotic-resistant LLPC.

scholarly journals Production of interleukin-1 by bone marrow myeloma cells

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 380-387 ◽  
Author(s):  
F Cozzolino ◽  
M Torcia ◽  
D Aldinucci ◽  
A Rubartelli ◽  
A Miliani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Interleukin 1 ◽  
Long Bone ◽  
Bone Lesions ◽  
Normal Donor ◽  
Biologic Activity ◽  
Culture Supernatants

Plasma cells isolated from bone marrow (BM) aspirates of 12 patients with multiple myeloma (MM) and nine patients with monoclonal gammopathy of undetermined significance (MGUS) were analyzed for production of cytokines with bone-resorbing activity, such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and lymphotoxin (LT). Culture supernatants of plasma cells from MM, but not from MGUS or normal donor, invariably contained high amounts of IL-1-beta and lower amounts of IL-1-alpha. With a single exception, TNF/LT biologic activity was not detected in the same supernatants. IL-6 was present in two of five supernatants tested. Normal B lymphocytes released both IL-1 and TNF/LT activities for four days after activation in vitro; however, production of these cytokines ceased at the final stage of plasma cell. Unexpectedly, the mRNA extracted from MM plasma cell hybridized with TNF- and LT- specific, as well as IL-1-specific probes, although the culture supernatants did not contain detectable TNF/LT biologic activity. When tested in the fetal rat long bone assay, MM plasma cell supernatants displayed a strong osteoclast-activating factor (OAF) activity, which was greatly reduced but not completely abolished by neutralizing anti- IL-1 antibodies. Anti-TNF or anti-LT antibodies were ineffective in the same test. We conclude that the IL-1 released in vivo by malignant plasma cells has a major role in pathogenesis of lytic bone lesions of human MM.

scholarly journals RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells

2021 ◽  
Author(s):  
Rui Fu ◽  
Kimberly Wellman ◽  
Amber Baldwin ◽  
Juilee Rege ◽  
Kathryn Walters ◽  
...  
Keyword(s):  
Gene Expression ◽  
Time Course ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ex Vivo ◽  
Human Cell Line ◽  
Rna Decay ◽  
Type I ◽  
Aldosterone Production

ABSTRACTAngiotensin II (AngII) binds to the type I angiotensin receptor in the adrenal cortex to initiate a cascade of events leading to the production of aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in our knowledge related to precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate siRNA screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. Together, these data support a model in which coordinated increases in transcription and regulated RNA decay facilitates the major transcriptomic changes required to implement a pro-steroidogenic gene expression program that is temporally restricted to prevent aldosterone overproduction.

Sign in / Sign up

Export Citation Format

Share Document