scholarly journals Impact of individual-level factors on Ex vivo mycobacterial growth inhibition: Associations of immune cell phenotype, cytomegalovirus-specific response and sex with immunity following BCG vaccination in humans

Tuberculosis ◽  
2019 ◽  
Vol 119 ◽  
pp. 101876
Author(s):  
Satria A. Prabowo ◽  
Steven G. Smith ◽  
Karin Seifert ◽  
Helen A. Fletcher
Keyword(s):  
Growth Inhibition ◽  
Immune Cell ◽  
Ex Vivo ◽  
Cell Phenotype ◽  
Specific Response ◽  
Individual Level ◽  
Bcg Vaccination ◽  
Mycobacterial Growth

scholarly journals Using ex vivo culture to assess dynamic phenotype changes in human prostate macrophages following exposure to therapeutic drugs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Clovis Boibessot ◽  
France-Hélène Joncas ◽  
Aerin Park ◽  
Zohra Berrehail ◽  
Jean-François Pelletier ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Cell ◽  
Ex Vivo ◽  
Cell Phenotype ◽  
Gleason Grade ◽  
Tumor Resistance ◽  
Grade Group ◽  
In Vivo Models ◽  
Novel Associations

AbstractWithin the prostate tumor microenvironment (TME) there are complex multi-faceted and dynamic communication occurring between cancer cells and immune cells. Macrophages are key cells which infiltrate and surround tumor cells and are recognized to significantly contribute to tumor resistance and metastases. Our understanding of their function in the TME is commonly based on in vitro and in vivo models, with limited research to confirm these model observations in human prostates. Macrophage infiltration was evaluated within the TME of human prostates after 72 h culture of fresh biopsies samples in the presence of control or enzalutamide. In addition to immunohistochemistry, an optimized protocol for multi-parametric evaluation of cellular surface markers was developed using flow cytometry. Flow cytometry parameters were compared to clinicopathological features. Immunohistochemistry staining for 19 patients with paired samples suggested enzalutamide increased the expression of CD163 relative to CD68 staining. Techniques to validate these results using flow cytometry of dissociated biopsies after 72 h of culture are described. In a second cohort of patients with Gleason grade group ≥ 3 prostate cancer, global macrophage expression of CD163 was unchanged with enzalutamide treatment. However, exploratory analyses of our results using multi-parametric flow cytometry for multiple immunosuppressive macrophage markers suggest subgroup changes as well as novel associations between circulating biomarkers like the neutrophil to lymphocyte ratio (NLR) and immune cell phenotype composition in the prostate TME. Further, we observed an association between B7–H3 expressing tumor-associated macrophages and the presence of intraductal carcinoma. The use of flow cytometry to evaluate ex vivo cultured prostate biopsies fills an important gap in our ability to understand the immune cell composition of the prostate TME. Our results highlight novel associations for further investigation.

scholarly journals Ex vivo mycobacterial growth inhibition assay (MGIA) for tuberculosis vaccine testing - a protocol for mouse splenocytes

2015 ◽  
Author(s):  
Andrea Andrea Zelmer ◽  
Rachel Tanner ◽  
Elena Stylianou ◽  
Sheldon Morris ◽  
Angelo Izzo ◽  
...  
Keyword(s):  
Immune Response ◽  
Clinical Trials ◽  
Growth Inhibition ◽  
Ex Vivo ◽  
Inhibition Assay ◽  
Growth Inhibition Assay ◽  
Mouse Splenocytes ◽  
Vaccine Testing ◽  
Mycobacterial Growth ◽  
Detailed Protocol

The testing of vaccines for tuberculosis is costly and time-consuming, and dependent on preclinical animal challenge models and clinical trials. We have recently developed a mycobacterial growth inhibition assay (MGIA) to test vaccine efficacy ex vivo. This assay measures the summative effect of the host immune response and may serve as a novel tool to facilitate vaccine testing. It has generated much interest recently, and to facilitate technology transfer and reproducibility between laboratories, we here describe a detailed protocol for an ex vivo MGIA in mouse splenocytes.

scholarly journals Adaption of the ex vivo mycobacterial growth inhibition assay for use with murine lung cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hannah Painter ◽  
Satria A. Prabowo ◽  
Felipe Cia ◽  
Lisa Stockdale ◽  
Rachel Tanner ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Ex Vivo ◽  
Lung Cells ◽  
Inhibition Assay ◽  
Growth Inhibition Assay ◽  
Murine Lung ◽  
Mycobacterial Growth

scholarly journals Polyfunctional CD4 T-cells correlate with in vitro mycobacterial growth inhibition following Mycobacterium bovis BCG-vaccination of infants

Vaccine ◽  
2016 ◽  
Vol 34 (44) ◽  
pp. 5298-5305 ◽  
Author(s):  
Steven G. Smith ◽  
Andrea Zelmer ◽  
Rose Blitz ◽  
Helen A. Fletcher ◽  
Hazel M. Dockrell
Keyword(s):  
T Cells ◽  
Growth Inhibition ◽  
Mycobacterium Bovis ◽  
Cd4 T Cells ◽  
Mycobacterium Bovis Bcg ◽  
Bcg Vaccination ◽  
Mycobacterial Growth

scholarly journals The influence of the gut microbiome on BCG-induced trained immunity

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Martin Stražar ◽  
Vera P. Mourits ◽  
Valerie A. C. M. Koeken ◽  
L. Charlotte J. de Bree ◽  
Simone J. C. F. M. Moorlag ◽  
...  
Keyword(s):  
Immune Responses ◽  
Gut Microbiome ◽  
De Novo ◽  
Ex Vivo ◽  
Specific Response ◽  
Dependent Manner ◽  
De Novo Genome Assembly ◽  
Bcg Vaccination ◽  
Vaccine Responses ◽  
Phenylalanine Metabolism

Abstract Background The bacillus Calmette-Guérin (BCG) vaccine protects against tuberculosis and heterologous infections but elicits high inter-individual variation in specific and nonspecific, or trained, immune responses. While the gut microbiome is increasingly recognized as an important modulator of vaccine responses and immunity in general, its potential role in BCG-induced protection is largely unknown. Results Stool and blood were collected from 321 healthy adults before BCG vaccination, followed by blood sampling after 2 weeks and 3 months. Metagenomics based on de novo genome assembly reveals 43 immunomodulatory taxa. The nonspecific, trained immune response is detected by altered production of cytokines IL-6, IL-1β, and TNF-α upon ex vivo blood restimulation with Staphylococcus aureus and negatively correlates with abundance of Roseburia. The specific response, measured by IFN-γ production upon Mycobacterium tuberculosis stimulation, is associated positively with Ruminococcus and Eggerthella lenta. The identified immunomodulatory taxa also have the strongest effects on circulating metabolites, with Roseburia affecting phenylalanine metabolism. This is corroborated by abundances of relevant enzymes, suggesting alternate phenylalanine metabolism modules are activated in a Roseburia species-dependent manner. Conclusions Variability in cytokine production after BCG vaccination is associated with the abundance of microbial genomes, which in turn affect or produce metabolites in circulation. Roseburia is found to alter both trained immune responses and phenylalanine metabolism, revealing microbes and microbial products that may alter BCG-induced immunity. Together, our findings contribute to the understanding of specific and trained immune responses after BCG vaccination.

scholarly journals Inflamm-Aging Is Associated with Lower Plasma PTX3 Concentrations and an Impaired Capacity of PBMCs to Express hTERT following LPS Stimulation

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Aaron L. Slusher ◽  
Tiffany M. Zúñiga ◽  
Edmund O. Acevedo
Keyword(s):  
Gene Expression ◽  
Young Adults ◽  
Telomere Length ◽  
Immune Cell ◽  
Ex Vivo ◽  
Middle Aged ◽  
Human Telomerase Reverse Transcriptase ◽  
Negatively Associated ◽  
Htert Gene ◽  
Age Related

Age-related elevations in proinflammatory cytokines, known as inflamm-aging, are associated with shorter immune cell telomere lengths. Purpose. This study examined the relationship of plasma PTX3 concentrations, a biomarker of appropriate immune function, with telomere length in 15 middle-aged (40-64 years) and 15 young adults (20-31 years). In addition, PBMCs were isolated from middle-aged and young adults to examine their capacity to express a key mechanistic component of telomere length maintenance, human telomerase reverse transcriptase (hTERT), following ex vivo cellular stimulation. Methods. Plasma PTX3 and inflammatory cytokines (i.e., IL-6, IL-10, TGF-β, and TNF-α), PBMC telomere lengths, and PBMC hTERT gene expression and inflammatory protein secretion following exposure to LPS, PTX3, and PTX3+LPS were measured. Results. Aging was accompanied by the accumulation of centrally located visceral adipose tissue, without changes in body weight and BMI, and alterations in the systemic inflammatory milieu (decreased plasma PTX3 and TGF-β; increased TNF-α (p≤0.050)). In addition, shorter telomere lengths in middle-aged compared to young adults (p=0.011) were negatively associated with age, body fat percentages, and plasma TNF-α (r=−0.404, p=0.027; r=−0.427, p=0.019; and r=−0.323, p=0.041, respectively). Finally, the capacity of PBMCs to increase hTERT gene expression following ex vivo stimulation was impaired in middle-aged compared to young adults (p=0.033) and negatively associated with telomere lengths (r=0.353, p=0.028). Conclusions. Proinflammation and the impaired hTERT gene expression capacity of PBMCs may contribute to age-related telomere attrition and disease.

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