scholarly journals Macrophage Plasticity and Function in the Lung Tumour Microenvironment Revealed in 3D Heterotypic Spheroid and Explant Models

Biomedicines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 302
Author(s):  
Lauren Evans ◽  
Kate Milward ◽  
Richard Attanoos ◽  
Aled Clayton ◽  
Rachel Errington ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Tumour Microenvironment ◽  
Therapeutic Interventions ◽  
Lung Tumour ◽  
Cell Responses ◽  
Dual Analysis ◽  
Macrophage Plasticity ◽  
And Function ◽  
Functional Analyses

In non-small cell lung cancer (NSCLC), stroma-resident and tumour-infiltrating macrophages may facilitate an immunosuppressive tumour microenvironment (TME) and hamper immunotherapeutic responses. Analysis of tumour-associated macrophage (TAM) plasticity in NSCLC is largely lacking. We established a novel, multi-marker, dual analysis approach for assessing monocyte-derived macrophage (Mf polarisation and M1/M2 phenotypic plasticity. We developed a flow cytometry-based, two-marker analysis (CD64 and CD206) of CD14+ cells. The phenotype and immune function of in vitro-induced TAMs was studied in a heterotypic spheroid and tumour-derived explant model of NSCLC. Heterotypic spheroids and NSCLC explants skewed Mfs from an M1- (CD206loCD64hi) to M2-like (CD206hiCD64lo) phenotype. Lipopolysaccharide (LPS) and IFNg treatment reversed M2-like Mf polarisation, indicating the plasticity of Mfs. Importantly, antigen-specific CD8+ T cell responses were reduced in the presence of tumour explant-conditioned Mfs, but not spheroid-conditioned Mfs, suggesting explants are likely a more relevant model of the immune TME than cell line-derived spheroids. Our data indicates the importance of multi-marker, functional analyses within Mf subsets and the advantages of the ex vivo NSCLC explant model in immunomodulation studies. We highlight the plasticity of the M1/M2 phenotype using the explant model and provide a tool for studying therapeutic interventions designed to reprogram M2-like Mf-induced immunosuppression.

In vitro, In vivo and Ex vivo Models for Peripheral Nerve Injury and Regeneration

2021 ◽  
Vol 19 ◽  
Author(s):  
Andrew Li ◽  
Clifford Pereira ◽  
Elise Eleanor Hill ◽  
Olivia Vukcevich ◽  
Aijun Wang
Keyword(s):  
Peripheral Nerve ◽  
Peripheral Nerve Injury ◽  
Ex Vivo ◽  
Peripheral Nerve Regeneration ◽  
Therapeutic Interventions ◽  
Peripheral Nerve Injuries ◽  
Traumatic Injuries ◽  
And Function

: Peripheral nerve injuries (PNI) frequently occur secondary to traumatic injuries. Recovery from these injuries can be expectedly poor, especially in proximal injuries. In order to study and improve peripheral nerve regeneration, scientists rely on peripheral nerve models to identify and test therapeutic interventions. In this review, we discuss the best described and most commonly used peripheral nerve models that scientists have and continue to use to study peripheral nerve physiology and function.

scholarly journals hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function

2022 ◽  
Author(s):  
Homa Majd ◽  
Ryan M Samuel ◽  
Jonathan T Ramirez ◽  
Ali Kalantari ◽  
Kevin Barber ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Xenograft Model ◽  
Developmental Relationships ◽  
Drug Candidates ◽  
Effective Strategies ◽  
Wide Range ◽  
Functional Features ◽  
And Function

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.  

scholarly journals BHLHE40 Regulates IL-10 and IFN-γ Production in T Cells but Does Not Interfere With Human Type 1 Regulatory T Cell Differentiation

2021 ◽  
Vol 12 ◽  
Author(s):  
Molly Javier Uyeda ◽  
Robert A. Freeborn ◽  
Brandon Cieniewicz ◽  
Rosa Romano ◽  
Ping (Pauline) Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
Ex Vivo ◽  
Surface Expression ◽  
Surface Molecules ◽  
Ifn Γ ◽  
And Function ◽  
Tr1 Cells

Type 1 regulatory T (Tr1) cells are subset of peripherally induced antigen-specific regulatory T cells. IL-10 signaling has been shown to be indispensable for polarization and function of Tr1 cells. However, the transcriptional machinery underlying human Tr1 cell differentiation and function is not yet elucidated. To this end, we performed RNA sequencing on ex vivo human CD49b+LAG3+ Tr1 cells. We identified the transcription factor, BHLHE40, to be highly expressed in Tr1 cells. Even though Tr1 cells characteristically produce high levels of IL-10, we found that BHLHE40 represses IL-10 and increases IFN-γ secretion in naïve CD4+ T cells. Through CRISPR/Cas9-mediated knockout, we determined that IL10 significantly increased in the sgBHLHE40-edited cells and BHLHE40 is dispensable for naïve CD4+ T cells to differentiate into Tr1 cells in vitro. Interestingly, BHLHE40 overexpression induces the surface expression of CD49b and LAG3, co-expressed surface molecules attributed to Tr1 cells, but promotes IFN-γ production. Our findings uncover a novel mechanism whereby BHLHE40 acts as a regulator of IL-10 and IFN-γ in human CD4+ T cells.

scholarly journals Comparing the Effects of the mTOR Inhibitors Azithromycin and Rapamycin on In Vitro Expanded Regulatory T Cells

2019 ◽  
Vol 28 (12) ◽  
pp. 1603-1613 ◽  
Author(s):  
Marcus Bergström ◽  
Malin Müller ◽  
Marie Karlsson ◽  
Hanne Scholz ◽  
Nils Tore Vethe ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Mtor Inhibitors ◽  
Allogeneic Transplantation ◽  
Suppressive Effect ◽  
And Function ◽  
Treg Phenotype ◽  
Suppression Assay

Adoptive transfer of autologous polyclonal regulatory T cells (Tregs) is a promising option for reducing graft rejection in allogeneic transplantation. To gain therapeutic levels of Tregs there is a need to expand obtained cells ex vivo, usually in the presence of the mTOR inhibitor Rapamycin due to its ability to suppress proliferation of non-Treg T cells, thus promoting a purer Treg yield. Azithromycin is a bacteriostatic macrolide with mTOR inhibitory activity that has been shown to exert immunomodulatory effects on several types of immune cells. In this study we investigated the effects of Azithromycin, compared with Rapamycin, on Treg phenotype, growth, and function when expanding bulk, naïve, and memory Tregs. Furthermore, the intracellular concentration of Rapamycin in CD4+ T cells as well as in the culture medium was measured for up to 48 h after supplemented. Treg phenotype was assessed by flow cytometry and Treg function was measured as inhibition of responder T-cell expansion in a suppression assay. The concentration of Rapamycin was quantified with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Azithromycin and Rapamycin both promoted a FoxP3-positive Treg phenotype in bulk Tregs, while Rapamycin also increased FoxP3 and FoxP3+Helios positivity in naïve and memory Tregs. Furthermore, Rapamycin inhibited the expansion of naïve Tregs, but also increased their suppressive effect. Rapamycin was quickly degraded in 37°C medium, yet was retained intracellularly. While both compounds may benefit expansion of FoxP3+ Tregs in vitro, further studies elucidating the effects of Azithromycin treatment on Tregs are needed to determine its potential use.

scholarly journals MPST but not CSE is the primary regulator of hydrogen sulfide production and function in the coronary artery

2016 ◽  
Vol 310 (1) ◽  
pp. H71-H79 ◽  
Author(s):  
Maggie M. Kuo ◽  
Dae Hee Kim ◽  
Sandeep Jandu ◽  
Yehudit Bergman ◽  
Siqi Tan ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Coronary Artery ◽  
Ex Vivo ◽  
Human Coronary Artery ◽  
Sodium Hydrosulfide ◽  
Physiological Relevance ◽  
Hydrogen Sulfide Production ◽  
And Function

Hydrogen sulfide (H2S) has emerged as an important gasotransmitter in the vasculature. In this study, we tested the hypothesis that H2S contributes to coronary vasoregulation and evaluated the physiological relevance of two sources of H2S, namely, cystathionine-γ-lyase (CSE) and 3-mercaptypyruvate sulfertransferase (MPST). MPST was detected in human coronary artery endothelial cells as well as rat and mouse coronary artery; CSE was not detected in the coronary vasculature. Rat coronary artery homogenates produced H2S through the MPST pathway but not the CSE pathway in vitro. In vivo coronary vasorelaxation response was similar in CSE knockout mice, wild-type mice (WT), and WT mice treated with the CSE inhibitor propargylglycine, suggesting that CSE-produced H2S does not have a significant role in coronary vasoregulation in vivo. Ex vivo, the MPST substrate 3-mercaptopyruvate (3-MP) and H2S donor sodium hydrosulfide (NaHS) elicited similar coronary vasoreactivity responses. Pyruvate did not have any effects on vasoreactivity. The vasoactive effect of H2S appeared to be nitric oxide (NO) dependent: H2S induced coronary vasoconstriction in the presence of NO and vasorelaxation in its absence. Maximal endothelial-dependent relaxation was intact after 3-MP and NaHS induced an increase in preconstriction tone, suggesting that endothelial NO synthase activity was not significantly inhibited. In vitro, H2S reacted with NO, which may, in part explain the vasoconstrictive effects of 3-MP and NaHS. Taken together, these data show that MPST rather than CSE generates H2S in coronary artery, mediating its effects through direct modulation of NO. This has important implications for H2S-based therapy in healthy and diseased coronary arteries.

scholarly journals Talin is required for integrin-mediated platelet function in hemostasis and thrombosis

2007 ◽  
Vol 204 (13) ◽  
pp. 3103-3111 ◽  
Author(s):  
Brian G. Petrich ◽  
Patrizia Marchese ◽  
Zaverio M. Ruggeri ◽  
Saskia Spiess ◽  
Rachel A.M. Weichert ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Ex Vivo ◽  
Focal Adhesions ◽  
Β1 Integrin ◽  
Normal Morphology ◽  
And Function ◽  
Specific Deletion

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin αIIbβ3-mediated platelet aggregation and β1 integrin–mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet β1 and β3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian α2β1 and αIIbβ3 integrins in vivo.

scholarly journals Energy metabolism manipulates the fate and function of tumour myeloid-derived suppressor cells

2019 ◽  
Vol 122 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Cong Hu ◽  
Bo Pang ◽  
Guangzhu Lin ◽  
Yu Zhen ◽  
Huanfa Yi
Keyword(s):  
Metabolic Pathways ◽  
Immune Surveillance ◽  
Suppressor Cells ◽  
Tumour Microenvironment ◽  
Metabolic Energy ◽  
Myeloid Derived Suppressor Cells ◽  
Cell Responses ◽  
And Function ◽  
Promote Tumour Development

AbstractIn recent years, a large number of studies have been carried out in the field of immune metabolism, highlighting the role of metabolic energy reprogramming in altering the function of immune cells. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells generated during a large array of pathological conditions, such as cancer, inflammation, and infection, and show remarkable ability to suppress T-cell responses. These cells can also change their metabolic pathways in response to various pathogen-derived or inflammatory signals. In this review, we focus on the roles of glucose, fatty acid (FA), and amino acid (AA) metabolism in the differentiation and function of MDSCs in the tumour microenvironment, highlighting their potential as targets to inhibit tumour growth and enhance tumour immune surveillance by the host. We further highlight the remaining gaps in knowledge concerning the mechanisms determining the plasticity of MDSCs in different environments and their specific responses in the tumour environment. Therefore, this review should motivate further research in the field of metabolomics to identify the metabolic pathways driving the enhancement of MDSCs in order to effectively target their ability to promote tumour development and progression.

scholarly journals In vitro and in vivo activities of flavonoids – apigenin, baicalin, chrysin, scutellarin – in regulation of hypertension – a review for their possible effects in pregnancy-induced hypertension

2019 ◽  
Vol 65 (1) ◽  
pp. 55-70 ◽  
Author(s):  
Marcin Ożarowski ◽  
Radosław Kujawski ◽  
Przemysław Ł. Mikołajczak ◽  
Karolina Wielgus ◽  
Andrzej Klejewski ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Biological Activities ◽  
Wide Spectrum ◽  
Chemical Compounds ◽  
New Drugs ◽  
Future Application ◽  
Mediators Of Inflammation ◽  
And Function

Summary Flavonoids and their conjugates are the most important group of natural chemical compounds in drug discovery and development. The search for pharmacological activity and new mechanisms of activity of these chemical compounds, which may inhibit mediators of inflammation and influence the structure and function of endothelial cells, can be an interesting pharmacological strategy for the prevention and adjunctive treatments of hypertension, especially induced by pregnancy. Because cardiovascular diseases have multi-factorial pathogenesis these natural chemical compounds with wide spectrum of biological activities are the most interesting source of new drugs. Extracts from one of the most popular plant used in Traditional Chinese Medicine, Scutellaria baicalensis Georgi could be a very interesting source of flavonoids because of its exact content in quercetin, apigenin, chrysin and scutellarin as well as in baicalin. These flavonoids exert vasoprotective properties and many activities such as: anti-oxidative via several pathways, anti-in-flammatory, anti-ischaemic, cardioprotective and anti-hypertensive. However, there is lack of summaries of results of studies in context of potential and future application of flavonoids with determined composition and activity. Our review aims to provide a literature survey of in vitro, in vivo and ex vivo pharmacological studies of selected flavonoids (apigenin, chrysin and scutellarin, baicalin) in various models of hypertension carried out in 2008–2018.

scholarly journals Increased Activation and Oligoclonality of Peripheral CD8+ T Cells in the Chronic Human Helminth Infection Alveolar Echinococcosis

2002 ◽  
Vol 70 (3) ◽  
pp. 1168-1174 ◽  
Author(s):  
Burkhard J. Manfras ◽  
Stefan Reuter ◽  
Thomas Wendland ◽  
Peter Kern
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Alveolar Echinococcosis ◽  
Ex Vivo ◽  
Active Role ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
And Function

ABSTRACT Alveolar echinococcosis (AE) in humans is a chronic disease characterized by slowly expanding liver lesions. Cellular immunity restricts the spreading of the extracellular pathogen, but functional contributions of CD4+ and CD8+ T cells are not defined. Here we studied ex vivo the phenotype and function of circulating T-cell subsets in AE patients by means of flow cytometry, T-cell receptor spectratyping, and lymphocyte proliferation. AE patients with parasitic lesions displayed a significant increase of activation of predominantly CD8+ T cells compared to healthy controls and AE patients without lesions. In vitro, proliferative T-cell responses to polyclonal stimulation with recall antigens and Echinococcus multilocularis vesicular fluid antigen were sustained during chronic persisting infection in all AE patients. Only in AE patients with parasitic lesions did T-cell receptor spectratyping reveal increased oligoclonality of CD8+ but not CD4+ T cells, suggesting a persistent antigenic drive for CD8+ T cells with subsequent proliferation of selected clonotypes. Thus, our data provide strong evidence for an active role of CD8+ T cells in AE.

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