scholarly journals Acute lymph node slices are a functional model system to study immunity ex vivo

2019 ◽  
Author(s):  
Maura C. Belanger ◽  
Alexander G. Ball ◽  
Megan A. Catterton ◽  
Andrew W.L. Kinman ◽  
Parastoo Anbaei ◽  
...  
Keyword(s):  
Lymph Node ◽  
Time Course ◽  
Spatial Organization ◽  
Ex Vivo ◽  
Functional Model ◽  
Repeated Measurements ◽  
Cytokine Secretion ◽  
Immune Functions ◽  
Tissue Slices ◽  
Spatial Complexity

AbstractThe lymph node is a highly organized and dynamic structure that is critical for facilitating the intercellular interactions that constitute adaptive immunity. Most ex vivo studies of the lymph node begin by reducing it to a cell suspension, thus losing the spatial organization, or fixing it, thus losing the ability to make repeated measurements. Live murine lymph node tissue slices offer the potential to retain spatial complexity and dynamic accessibility, but their viability, level of immune activation, and retention of antigen-specific functions have not been validated. Here we systematically characterized live murine lymph node slices as a platform to study immunity. Live lymph node slices maintained the expected spatial organization and cell populations while reflecting the 3D spatial complexity of the organ. Slices collected under optimized conditions were comparable to cell suspensions in terms of both 24-hr viability and inflammation. Slices responded to T cell receptor cross-linking with increased surface marker expression and cytokine secretion, in some cases more strongly than matched lymphocyte cultures. Furthermore, slices processed protein antigens, and slices from vaccinated animals responded to ex vivo challenge with antigen-specific cytokine secretion. In summary, lymph node slices provide a versatile platform to investigate immune functions in spatially organized tissue, enabling well-defined stimulation, time-course analysis, and parallel read-outs.

scholarly journals Spatially resolved microfluidic stimulation of lymphoid tissue ex vivo

The Analyst ◽  
2017 ◽  
Vol 142 (4) ◽  
pp. 649-659 ◽  
Author(s):  
Ashley E. Ross ◽  
Maura C. Belanger ◽  
Jacob F. Woodroof ◽  
Rebecca R. Pompano
Keyword(s):  
Lymph Node ◽  
Targeted Delivery ◽  
Lymphoid Tissue ◽  
Ex Vivo ◽  
Tissue Slices ◽  
Microfluidic Platform ◽  
Spatially Resolved ◽  
Local Stimulation ◽  
Stimulation Of

We present the first microfluidic platform for local stimulation of lymph node tissue slices and demonstrate targeted delivery of a model therapeutic.

scholarly journals Ex vivo infection of murine precision-cut lung tissue slices with Mycobacterium abscessus: a model to study antimycobacterial agents

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Carmen Amelia Molina-Torres ◽  
Oscar Noé Flores-Castillo ◽  
Irma Edith Carranza-Torres ◽  
Nancy Elena Guzmán-Delgado ◽  
Ezequiel Viveros-Valdez ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Time Course ◽  
Ex Vivo ◽  
Virulent Strain ◽  
Mycobacterium Abscessus ◽  
New Drugs ◽  
Histopathological Analysis ◽  
Ex Vivo Model ◽  
Tissue Slices ◽  
Precision Cut Lung Slices

Abstract Background Multidrug-resistant infections due to Mycobacterium abscessus often require complex and prolonged regimens for treatment. Here, we report the evaluation of a new ex vivo antimicrobial susceptibility testing model using organotypic cultures of murine precision-cut lung slices, an experimental model in which metabolic activity, and all the usual cell types of the organ are found while the tissue architecture and the interactions between the different cells are maintained. Methods Precision cut lung slices (PCLS) were prepared from the lungs of wild type BALB/c mice using the Krumdieck® tissue slicer. Lung tissue slices were ex vivo infected with the virulent M. abscessus strain L948. Then, we tested the antimicrobial activity of two drugs: imipenem (4, 16 and 64 μg/mL) and tigecycline (0.25, 1 and 4 μg/mL), at 12, 24 and 48 h. Afterwards, CFUs were determined plating on blood agar to measure the surviving intracellular bacteria. The viability of PCLS was assessed by Alamar Blue assay and corroborated using histopathological analysis. Results PCLS were successfully infected with a virulent strain of M. abscessus as demonstrated by CFUs and detailed histopathological analysis. The time-course infection, including tissue damage, parallels in vivo findings reported in genetically modified murine models for M. abscessus infection. Tigecycline showed a bactericidal effect at 48 h that achieved a reduction of > 4log10 CFU/mL against the intracellular mycobacteria, while imipenem showed a bacteriostatic effect. Conclusions The use of this new organotypic ex vivo model provides the opportunity to test new drugs against M. abscessus, decreasing the use of costly and tedious animal models.

scholarly journals Acute Lymph Node Slices Are a Functional Model System to Study Immunity Ex Vivo

2021 ◽  
Vol 4 (1) ◽  
pp. 128-142
Author(s):  
Maura C. Belanger ◽  
Alexander G. Ball ◽  
Megan A. Catterton ◽  
Andrew W.L. Kinman ◽  
Parastoo Anbaei ◽  
...  
Keyword(s):  
Lymph Node ◽  
Ex Vivo ◽  
Functional Model ◽  
Model System

scholarly journals A31 COMPLEX ROLE OF DIETARY FIBERS IN IBD: MICROBES MEDIATE FIBER-INDUCED INFLAMMATION

2021 ◽  
Vol 4 (Supplement_1) ◽  
pp. 148-150
Author(s):  
H Armstrong ◽  
R Valcheva ◽  
D Santer ◽  
Z Zhang ◽  
A Rieger ◽  
...  
Keyword(s):  
Immune Responses ◽  
Anaerobic Fermentation ◽  
Ex Vivo ◽  
Severe Disease ◽  
Cytokine Secretion ◽  
Dietary Fibers ◽  
Gut Health ◽  
T84 Cells ◽  
Microbial Composition ◽  
Barrier Integrity

Abstract Background Dietary fibers pass through the bowel undigested and are fermented within the intestine by microbes, typically promoting gut health. However, many IBD patients describe experiencing sensitivity to fibers. β-glucan, found on the surface of fungal cells during fungal infection, has been shown to bind to fiber receptors, such as Dectin-1, on host immune cells, resulting in a pro-inflammatory response. These fungal fibres share properties with dietary fibers. Aims As an altered gut microbial composition has been associated with IBD, we hypothesized that the loss of fiber-fermenting microbes populating the gut in IBD could lead to dietary fibers not being efficiently broken down into their beneficial biproducts (e.g. short chain fatty acids; SCFA), resulting in binding of intact fibers to pro-inflammatory host cell receptors. Methods Immune and epithelial cell lines and colonic biopsies cultured ex vivo were incubated with oligofructose or inulin (5g/L), or pre-fermented fibers (24hr anaerobic fermentation). Immune responses were measured by cytokine secretion (ELISA), and expression (qPCR). Barrier integrity was measured by transepithelial resistance (TEER). Food frequency questionnaire (FFQ) data of patient fiber consumption were correlated with gut microbes (shotgun sequencing) and immune responses to fiber in patient biopsies. Results Unfermented oligofructose induced IL-1β secretion in leukocytes (macrophage, T cell, neutrophil) and in colon biopsies from pediatric Crohn disease (CD; n=38) and ulcerative colitis (UC; n=20) patients cultured ex vivo, but not in non-IBD patients (n=21). IL-1β secretion was greater in patients with more severe disease. Pre-fermentation of oligofructose by whole-microbe intestinal washes from non-IBD patients or remission patients reduced secretion of IL-1β, while whole microbe intestinal washes from severe IBD patients were unable to ferment oligofructose or reduce cytokine secretion. Fiber effects on IL-1β secretion in biopsies positively correlated with effects on barrier integrity in T84 cells. Fiber-associated immune responses in patient biopsies cultured ex vivo (ELISA) correlated with fiber avoidance (FFQ) and gut microbiome (sequencing) in matching patient samples. Conclusions Our findings demonstrate that intolerance and avoidance of prebiotic fibers in select IBD patients is associated with the inability to ferment these fibers, leading to pro-inflammatory immune responses and intestinal barrier disruption. This highlights select disease state scenarios, in which administration of fermentable fibers should be avoided and tailored dietary interventions should be considered in IBD patients. Funding Agencies CIHRWeston Foundation

scholarly journals The long-term course of fatigue following breast cancer diagnosis

2020 ◽  
Vol 4 (1) ◽  
Author(s):  
Karin Biering ◽  
Morten Frydenberg ◽  
Helle Pappot ◽  
Niels Henrik Hjollund
Keyword(s):  
Breast Cancer ◽  
Time Course ◽  
Breast Cancer Diagnosis ◽  
Patient Characteristics ◽  
Repeated Measurements ◽  
Diagnosis And Treatment ◽  
Persistent Problem ◽  
Treatment Regimens ◽  
Long Time

Abstract Purpose Fatigue following breast cancer is a well-known problem, with both high and persistent prevalence. Previous studies suffer from lack of repeated measurements, late recruitment and short periods of follow-up. The course of fatigue from diagnosis and treatment to the long-time outcome status is unknown as well as differences in the level of fatigue between treatment regimens. The purpose of this study was to describe the long-time course of fatigue from the time of clinical suspicion of breast cancer, its dependence of patient characteristics and treatment regimens and the comparison with the course of fatigue among women with the same suspicion, but not diagnosed with breast cancer. Methods Three hundred thirty-two women referred to acute or subacute mammography was followed with questionnaires from before the mammography and up to 1500 days. Fatigue was measured by the Multidimensional Fatigue Inventory (MFI-20). The women reported their initial level of fatigue before the mammography and thus without knowledge of whether they had cancer or not. Both women with and without cancer were followed. Women with cancer were identified in the clinical database established by Danish Breast Cancer Cooperative Group (DBCG) to collect information on treatment regimen. Results Compared to fatigue scores before diagnosis, women with breast cancer reported a large increase of fatigue, especially in the first 6 months, followed by a slow decrease over time. Despite the long follow-up period, the women with breast cancer did not return to their level of fatigue at time of the mammography. Women without breast cancer, experienced a rapid decrease of fatigue after disproval of diagnosis followed by a steadier period. Conclusions Fatigue is a persistent problem in women diagnosed with breast cancer, even several years following diagnosis and treatment. The women with breast cancer were most affected by fatigue in the first 6 months after diagnosis.

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.

Author Reply: What Jealousy Can Tell Us About Theories of Emotion

2018 ◽  
Vol 10 (4) ◽  
pp. 291-292 ◽  
Author(s):  
Christine R. Harris ◽  
Mingi Chung
Keyword(s):  
Time Course ◽  
Functional Model ◽  
Author Reply ◽  
Motivational State

We clarify aspects of our Dynamic Functional Model of Jealousy in response to D’Arms (2018) and Stets (2018). Our model proposes that jealousy is an evolved motivational state that arises over threat by a rival to one’s relationship or some aspect of one’s relationship (such as attention, affection, or love). The formation or loss of relationships rarely occurs instantaneously. Therefore, we argue that jealousy, whose goal is to remove or reduce the rival threat, can occur over a longer time course than is often assumed in theories of specific emotions. We further suggest that other emotions such as grief and fear also can occur over extended periods. This raises challenges for emotion theories that assume that emotions must be short-lived.

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