scholarly journals Extracellular vesicles released by the human gut symbiont Bacteroides thetaiotaomicron in the mouse intestine are enriched in a selected range of proteins that influence host cell physiology and metabolism

2020 ◽  
Author(s):  
Regis Stentz ◽  
Udo Wegmann ◽  
Maria Guirro ◽  
Will Bryant ◽  
Avani Ranjit ◽  
...  
Keyword(s):  
Host Cell ◽  
Extracellular Vesicles ◽  
Cell Function ◽  
Bile Salt Hydrolase ◽  
Cell Physiology ◽  
Bacteroides Thetaiotaomicron ◽  
Human Gut ◽  
The Impact

Abstract It is becoming increasingly clear that bacterial extracellular vesicles (BEVs) produced by members of the intestinal microbiota can contribute to microbe-host cell interactions that impact on host health. A major unresolved question is the nature of the cargo packaged into these BEVs and how they can impact on host cell function. Here we have analysed the proteome of BEVs produced by the major human gut symbiont Bacteroides thetaiotaomicron in both in vitro cultures using defined and complex medias, and in vivo in fed or fasted animals to determine the impact of nutrient stress on the BEV proteome, and to identify proteins specifically enriched in BEVs produced in vivo. In contrast to BEVs produced in vitro where limiting nutrient provision resulted in an increase in a large fraction of proteins, the protein content of BEVs extracted from fasted versus fed mice was less affected with similar numbers of proteins showing increased and decreased abundance. We identified 102 proteins exclusively enriched in BEVs in vivo of which the majority (66/102) were enriched independently of their expression in the parent cells implicating the existence of an active mechanism to drive the selection of a group of proteins for their secretion into BEVs within the intestine. Amongst these abundantly expressed proteins in BEVs in vivo were a bile salt hydrolase and a dipeptidyl peptidase IV that were characterised further and shown to be active and able to degrade host-derived substrates with defined roles in metabolism. Collectively these findings provide additional evidence for the role of BEVs in microbiota-host interactions with their contents playing key roles in the maintenance of intestinal homeostasis, and host metabolism.

scholarly journals Intestinal IgA Regulates Expression of a Fructan Polysaccharide Utilization Locus in Colonizing Gut Commensal Bacteroides thetaiotaomicron

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Payal Joglekar ◽  
Hua Ding ◽  
Pablo Canales-Herrerias ◽  
Pankaj Jay Pasricha ◽  
Justin L. Sonnenburg ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Ex Vivo ◽  
Microbial Colonization ◽  
Bacteroides Thetaiotaomicron ◽  
Content Type ◽  
Microbial Utilization ◽  
Polysaccharide Utilization Locus ◽  
The Impact

ABSTRACT Gut-derived immunoglobulin A (IgA) is the most abundant antibody secreted in the gut that shapes gut microbiota composition and functionality. However, most of the microbial antigens targeted by gut IgA remain unknown, and the functional effects of IgA targeting these antigens are currently understudied. This study provides a framework for identifying and characterizing gut microbiota antigens targeted by gut IgA. We developed a small intestinal ex vivo culture assay to harvest lamina propria IgA from gnotobiotic mice, with the aim of identifying antigenic targets in a model human gut commensal, Bacteroides thetaiotaomicron VPI-5482. Colonization by B. thetaiotaomicron induced a microbe-specific IgA response that was reactive against diverse antigens, including capsular polysaccharides, lipopolysaccharides, and proteins. IgA against microbial protein antigens targeted membrane and secreted proteins with diverse functionalities, including an IgA specific against proteins of the polysaccharide utilization locus (PUL) that are necessary for utilization of fructan, which is an important dietary polysaccharide. Further analyses demonstrated that the presence of dietary fructan increased the production of fructan PUL-specific IgA, which then downregulated the expression of fructan PUL in B. thetaiotaomicron, both in vivo and in vitro. Since the expression of fructan PUL has been associated with the ability of B. thetaiotaomicron to colonize the gut in the presence of dietary fructans, our work suggests a novel role for gut IgA in regulating microbial colonization by modulating their metabolism. IMPORTANCE Given the significant impact that gut microbes have on our health, it is essential to identify key host and environmental factors that shape this diverse community. While many studies have highlighted the impact of diet on gut microbiota, little is known about how the host regulates this critical diet-microbiota interaction. In our present study, we discovered that gut IgA targeted a protein complex involved in the utilization of an important dietary polysaccharide: fructan. While the presence of dietary fructans was previously thought to allow unrestricted growth of fructan-utilizing bacteria, our work shows that gut IgA, by targeting proteins responsible for fructan utilization, provides the host with tools that can restrict the microbial utilization of such polysaccharides, thereby controlling their growth.

scholarly journals Methods to Investigate miRNA Function: Focus on Platelet Reactivity

2020 ◽  
Author(s):  
Alix Garcia ◽  
Sylvie Dunoyer-Geindre ◽  
Richard J. Fish ◽  
Marguerite Neerman-Arbez ◽  
Jean-Luc Reny ◽  
...  
Keyword(s):  
Cell Function ◽  
Platelet Reactivity ◽  
Cell Physiology ◽  
Plasma Samples ◽  
Small Noncoding Rnas ◽  
Cellular Processes ◽  
Extraction And Purification ◽  
Large Panel

AbstractMicroRNAs (miRNAs) are small noncoding RNAs modulating protein production. They are key players in regulation of cell function and are considered as biomarkers in several diseases. The identification of the proteins they regulate, and their impact on cell physiology, may delineate their role as diagnostic or prognostic markers and identify new therapeutic strategies. During the last 3 decades, development of a large panel of techniques has given rise to multiple models dedicated to the study of miRNAs. Since plasma samples are easily accessible, circulating miRNAs can be studied in clinical trials. To quantify miRNAs in numerous plasma samples, the choice of extraction and purification techniques, as well as normalization procedures, are important for comparisons of miRNA levels in populations and over time. Recent advances in bioinformatics provide tools to identify putative miRNAs targets that can then be validated with dedicated assays. In vitro and in vivo approaches aim to functionally validate candidate miRNAs from correlations and to understand their impact on cellular processes. This review describes the advantages and pitfalls of the available techniques for translational research to study miRNAs with a focus on their role in regulating platelet reactivity.

scholarly journals IMMU-46. EXAMINATION OF THE EFFECTS OF DEXAMETHASONE ON THE EFFICACY OF IMMUNOTHERAPY IN GLIOMA USING GENE-MEDIATED CYTOTOXIC IMMUNOTHERAPY

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi129-vi129
Author(s):  
Marilin Koch ◽  
Mykola Zdioruk ◽  
M Oskar Nowicki ◽  
Estuardo Aguilar ◽  
Laura Aguilar ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Immune Cell ◽  
Symptomatic Treatment ◽  
Tumor Cell Death ◽  
The Impact

Abstract RATIONALE Dexamethasone is frequently used in symptomatic treatment of glioma patients, although it is known to cause immune suppression. Checkpoint inhibitor immunotherapies have not yet been successful in glioma treatments. Gene-mediated cytotoxic immunotherapy (GMCI) is an immunotherapeutic approach that uses aglatimagene besadenovec with an anti-herpetic prodrug to induce immunogenic tumor cell death and immune cell attraction to the tumor site with potent CD8 T cell activation. GMCI is currently in clinical trials for solid tumors including glioblastoma, where it showed encouraging survival results in a Phase 2 study that did not limit the use of dexamethasone. However, the effects of dexamethasone on its efficacy have not been explored. METHODS We investigated the effects of dexamethasone on GMCI in vitro using cytotoxicity and T-cell-killing assays in glioblastoma cell lines. The impact of dexamethasone in vivo was assessed in an orthotopic syngeneic murine glioblastoma model. RESULTS Cyotoxicity assays showed that Dexamethasone has a slight impact on GMCI in vitro. In contrast, we observed a highly significant effect in T-cell-functional assays in which killing was greatly impaired. Immune cell response assays revealed a reduced T-cell proliferation after co-culture with supernatant from dexamethasone or combination treated glioblastoma cells in contrast to GMCI alone. In a murine model, the combination of GMCI and dexamethasone resulted in a significant reduction in median symptom-free survival (29d) in comparison to GMCI alone (39.5d) (P = 0.0184). CONCLUSION Our data suggest that high doses of dexamethasone may negatively impact the efficacy of immunotherapy for glioma, which may be a consequence of impaired T cell function. These results support the idea that there is a need in identifying possible alternatives to dexamethasone to maximize the effectiveness of immunostimulatory therapies such as GMCI.

scholarly journals Lipopolysaccharide administration alters extracellular vesicles in human lung-cancer cells and mice

2020 ◽  
Author(s):  
Leandra B. Jones ◽  
Sanjay Kumar ◽  
Courtnee’ R. Bell ◽  
Brennetta J. Crenshaw ◽  
Mamie T. Coats ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Bacterial Infection ◽  
Cell Line ◽  
Extracellular Vesicles ◽  
A549 Cells ◽  
Human Lung Cancer ◽  
Diagnostic Tools ◽  
The Impact

AbstractExtracellular vesicles (EVs) play a fundamental role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. In this study, we explored the in vitro impact of bacterial lipopolysaccharide administration on a cell line that represents a target for bacterial infection in the host. Administration of lipopolysaccharide at varying concentrations to this A549 cell line caused only modest changes in cell death, but EV numbers were significantly changed. After treatment with the highest concentration of lipopolysaccharide, EVs derived from A549 cells packaged significantly less interleukin-6 and lysosomal-associated membrane protein 1. We also examined the impact of lipopolysaccharide administration on exosome biogenesis and cargo composition in BALB/c mice. Serum-isolated EVs from lipopolysaccharide-treated mice showed significantly increased lysosomal-associated membrane protein 1 and toll-like receptor 4 levels compared with EVs from control mice. In summary, this study demonstrated that EV numbers and cargo were altered using these in vitro and in vivo models of bacterial infection.

scholarly journals In vitro Characterization of Insulin−Producing β-Cell Spheroids

2021 ◽  
Vol 8 ◽  
Author(s):  
Yonela Ntamo ◽  
Ebrahim Samodien ◽  
Joleen Burger ◽  
Nolan Muller ◽  
Christo J. F. Muller ◽  
...  
Keyword(s):  
Glucose Utilization ◽  
Cell Function ◽  
3D Culture ◽  
Glucose Sensing ◽  
Blue Staining ◽  
Cell Physiology ◽  
Β Cell ◽  
3D Spheroids

Over the years, immortalized rodent β-cell lines such as RIN, HIT, MIN, βTC, and INS-1 have been used to investigate pancreatic β-cell physiology using conventional two-dimensional (2D) culture techniques. However, physical and physiological limitations inherent to 2D cell culture necessitates confirmatory follow up studies using sentient animals. Three-dimensional (3D) culture models are gaining popularity for their recapitulation of key features of in vivo organ physiology, and thus could pose as potential surrogates for animal experiments. In this study, we aimed to develop and characterize a rat insulinoma INS-1 3D spheroid model to compare with 2D monolayers of the same cell line. Ultrastructural verification was done by transmission electron microscopy and toluidine blue staining, which showed that both 2D monolayers and 3D spheroids contained highly granulated cells with ultrastructural features synonymous with mature pancreatic β-cells, with increased prominence of these features observed in 3D spheroids. Viability, as assessed by cellular ATP quantification, size profiling and glucose utilization, showed that our spheroids remained viable for the experimental period of 30 days, compared to the limiting 5-day passage period of INS-1 monolayers. In fact, increasing ATP content together with spheroid size was observed over time, without adverse changes in glucose utilization. Additionally, β-cell function, assessed by determining insulin and amylin secretion, showed that the 3D spheroids retained glucose sensing and insulin secretory capability, that was more acute when compared to 2D monolayer cultures. Thus, we were able to successfully demonstrate that our in vitro INS-1 β-cell 3D spheroid model exhibits in vivo tissue-like structural features with extended viability and lifespan. This offers enhanced predictive capacity of the model in the study of metabolic disease, β-cell pathophysiology and the potential treatment thereof.

scholarly journals IAP inhibitors enhance co-stimulation to promote tumor immunity

2010 ◽  
Vol 207 (10) ◽  
pp. 2195-2206 ◽  
Author(s):  
Michael Dougan ◽  
Stephanie Dougan ◽  
Joanna Slisz ◽  
Brant Firestone ◽  
Matthew Vanneman ◽  
...  
Keyword(s):  
T Cell ◽  
Cytotoxic Activity ◽  
Small Molecule ◽  
Cell Function ◽  
Tumor Vaccine ◽  
Nuclear Factor Κb ◽  
Cell Responses ◽  
The Impact

The inhibitor of apoptosis proteins (IAPs) have recently been shown to modulate nuclear factor κB (NF-κB) signaling downstream of tumor necrosis factor (TNF) family receptors, positioning them as essential survival factors in several cancer cell lines, as indicated by the cytotoxic activity of several novel small molecule IAP antagonists. In addition to roles in cancer, increasing evidence suggests that IAPs have an important function in immunity; however, the impact of IAP antagonists on antitumor immune responses is unknown. In this study, we examine the consequences of IAP antagonism on T cell function in vitro and in the context of a tumor vaccine in vivo. We find that IAP antagonists can augment human and mouse T cell responses to physiologically relevant stimuli. The activity of IAP antagonists depends on the activation of NF-κB2 signaling, a mechanism paralleling that responsible for the cytotoxic activity in cancer cells. We further show that IAP antagonists can augment both prophylactic and therapeutic antitumor vaccines in vivo. These findings indicate an important role for the IAPs in regulating T cell–dependent responses and suggest that targeting IAPs using small molecule antagonists may be a strategy for developing novel immunomodulating therapies against cancer.

scholarly journals Effects of long-term in vitro exposure to phosphodiesterase type-3 inhibitors on follicle and oocyte development

Reproduction ◽  
2005 ◽  
Vol 130 (2) ◽  
pp. 177-186 ◽  
Author(s):  
D Nogueira ◽  
R Cortvrindt ◽  
B Everaerdt ◽  
J Smitz
Keyword(s):  
Somatic Cell ◽  
Cell Function ◽  
Oocyte Growth ◽  
Phosphodiesterase Type ◽  
Type 3 ◽  
The Impact ◽  
Phosphodiesterase Type 3

Germinal vesicle (GV)-stage oocytes retrieved from antral follicles undergo nuclear maturation in vitro, which typically occurs prior to cytoplasmic maturation. Short-term culture with meiotic inhibitors has been applied to arrest oocytes at the GV stage aiming to synchronize nuclear and ooplasmic maturity. However, the results obtained are still far from the in vivo situation. In order to acquire competence, immature oocytes may require meiotic arrest in vitro for a more extended period. The phosphodiesterase type 3-inhibitor (PDE3-I) is a potent meiotic arrester. The effects of a prolonged culture with PDE3-I on oocyte quality prior to and after reversal from the inhibition are not known. This study tested the impact of long-term in vitro exposure of two PDE3-Is, org9935 and cilostamide, on oocytes using a mouse follicle culture model. The results showed that PDE3-I (maximum of 10 μM) during a 12-day culture of follicle-enclosed oocytes did not alter somatic cell proliferation, differentiation or follicle survival. In addition, the steroid production profile was not significantly modified by a 12-day exposure to PDE3-I. The recombinant human chorionic gonadotrophin/recombinant human epidermal growth factor stimulus induced a characteristic normal progesterone peak of luteinization and normal mucification of the cumulus cells, while the enclosed oocyte remained blocked at the GV stage. In vitro maturation of denuded or cumulus-enclosed oocytes derived from org9935- or cilostamide-exposed follicles progressed through meiosis and formed morphologically normal meiotic spindles with chromosomes properly aligned at the equator. In conclusion, long-term culture with PDE3-I was harmless to somatic cell function, differentiation, oocyte growth and maturation. Our results suggested that PDE3-I can be applied when extended oocyte culture is required to improve ooplasmic maturation.

scholarly journals Novel approaches for analysing gut microbes and dietary polyphenols: challenges and opportunities

Microbiology ◽  
2010 ◽  
Vol 156 (11) ◽  
pp. 3224-3231 ◽  
Author(s):  
R. A. Kemperman ◽  
S. Bolca ◽  
L. C. Roger ◽  
E. E. Vaughan
Keyword(s):  
Gut Microbiota ◽  
Health Effects ◽  
Fruit And Vegetables ◽  
Human Gut ◽  
Microbial Composition ◽  
Dietary Polyphenols ◽  
Host Health ◽  
The Impact

Polyphenols, ubiquitously present in the food we consume, may modify the gut microbial composition and/or activity, and moreover, may be converted by the colonic microbiota to bioactive compounds that influence host health. The polyphenol content of fruit and vegetables and derived products is implicated in some of the health benefits bestowed on eating fruit and vegetables. Elucidating the mechanisms behind polyphenol metabolism is an important step in understanding their health effects. Yet, this is no trivial assignment due to the diversity encountered in both polyphenols and the gut microbial composition, which is further confounded by the interactions with the host. Only a limited number of studies have investigated the impact of dietary polyphenols on the complex human gut microbiota and these were mainly focused on single polyphenol molecules and selected bacterial populations. Our knowledge of gut microbial genes and pathways for polyphenol bioconversion and interactions is poor. Application of specific in vitro or in vivo models mimicking the human gut environment is required to analyse these diverse interactions. A particular benefit can now be gained from next-generation analytical tools such as metagenomics and metatranscriptomics allowing a wider, more holistic approach to the analysis of polyphenol metabolism. Understanding the polyphenol–gut microbiota interactions and gut microbial bioconversion capacity will facilitate studies on bioavailability of polyphenols in the host, provide more insight into the health effects of polyphenols and potentially open avenues for modulation of polyphenol bioactivity for host health.

scholarly journals Mesenchymal Stromal Cells From Emphysematous Donors and Their Extracellular Vesicles Are Unable to Reverse Cardiorespiratory Dysfunction in Experimental Severe Emphysema

2021 ◽  
Vol 9 ◽  
Author(s):  
Mariana A. Antunes ◽  
Cassia L. Braga ◽  
Tainá B. Oliveira ◽  
Jamil Z. Kitoko ◽  
Ligia L. Castro ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Therapeutic Potential ◽  
Chronic Obstructive ◽  
Severe Emphysema ◽  
Arginase 1 ◽  
The Impact

Although bone marrow-derived mesenchymal stromal cells (BM-MSCs) from patients with chronic obstructive pulmonary disease (COPD) appear to be phenotypically and functionally similar to BM-MSCs from healthy sources in vitro, the impact of COPD on MSC metabolism and mitochondrial function has not been evaluated. In this study, we aimed to comparatively characterize MSCs from healthy and emphysematous donors (H-MSCs and E-MSCs) in vitro and to assess the therapeutic potential of these MSCs and their extracellular vesicles (H-EVs and E-EVs) in an in vivo model of severe emphysema. For this purpose, C57BL/6 mice received intratracheal porcine pancreatic elastase once weekly for 4 weeks to induce emphysema; control animals received saline under the same protocol. Twenty-four hours after the last instillation, animals received saline, H-MSCs, E-MSCs, H-EVs, or E-EVs intravenously. In vitro characterization demonstrated that E-MSCs present downregulation of anti-inflammatory (TSG-6, VEGF, TGF-β, and HGF) and anti-oxidant (CAT, SOD, Nrf2, and GSH) genes, and their EVs had larger median diameter and lower average concentration. Compared with H-MSC, E-MSC mitochondria also exhibited a higher respiration rate, were morphologically elongated, expressed less dynamin-related protein-1, and produced more superoxide. When co-cultured with alveolar macrophages, both H-MSCs and E-MSCs induced an increase in iNOS and arginase-1 levels, but only H-MSCs and their EVs were able to enhance IL-10 levels. In vivo, emphysematous mice treated with E-MSCs or E-EVs demonstrated no amelioration in cardiorespiratory dysfunction. On the other hand, H-EVs, but not H-MSCs, were able to reduce the neutrophil count, the mean linear intercept, and IL-1β and TGF-β levels in lung tissue, as well as reduce pulmonary arterial hypertension and increase the right ventricular area in a murine model of elastase-induced severe emphysema. In conclusion, E-MSCs and E-EVs were unable to reverse cardiorespiratory dysfunction, whereas H-EVs administration was associated with a reduction in cardiovascular and respiratory damage in experimental severe emphysema.

Enforced expression of MLL-AF4 fusion in cord blood CD34+ cells enhances the hematopoietic repopulating cell function and clonogenic potential but is not sufficient to initiate leukemia

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4746-4758 ◽  
Author(s):  
Rosa Montes ◽  
Verónica Ayllón ◽  
Ivan Gutierrez-Aranda ◽  
Isidro Prat ◽  
M. Carmen Hernández-Lamas ◽  
...  
Keyword(s):  
Cord Blood ◽  
Cell Function ◽  
Lymphoblastic Leukemia ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Dismal Prognosis ◽  
Clonogenic Potential ◽  
The Impact

Abstract Infant acute lymphoblastic leukemia harboring the fusion mixed-lineage leukemia (MLL)-AF4 is associated with a dismal prognosis and very brief latency. Our limited understanding of transformation by MLL-AF4 is reflected in murine models, which do not accurately recapitulate the human disease. Human models for MLL-AF4 disease do not exist. Hematopoietic stem or progenitor cells (HSPCs) represent probable targets for transformation. Here, we explored in vitro and in vivo the impact of the enforced expression of MLL-AF4 in human cord blood-derived CD34+ HSPCs. Intrabone marrow transplantation into NOD/SCID-IL2Rγ−/− mice revealed an enhanced multilineage hematopoietic engraftment, efficiency, and homing to other hematopoietic sites on enforced expression of MLL-AF4. Lentiviral transduction of MLL-AF4 into CD34+ HSPCs increased the in vitro clonogenic potential of CD34+ progenitors and promoted their proliferation. Consequently, cell cycle and apoptosis analyses suggest that MLL-AF4 conveys a selective proliferation coupled to a survival advantage, which correlates with changes in the expression of genes involved in apoptosis, sensing DNA damage and DNA repair. However, MLL-AF4 expression was insufficient to initiate leukemogenesis on its own, indicating that either additional hits (or reciprocal AF4-MLL product) may be required to initiate ALL or that cord blood-derived CD34+ HSPCs are not the appropriate cellular target for MLL-AF4-mediated ALL.

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