scholarly journals Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue

2021 ◽  
Vol 12 ◽  
Author(s):  
Chloe M. Doyle ◽  
Erica E. Vine ◽  
Kirstie M. Bertram ◽  
Heeva Baharlou ◽  
Jake W. Rhodes ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Human Tissue ◽  
Digestive Enzyme ◽  
Extraction Process ◽  
Mononuclear Phagocytes ◽  
Careful Selection ◽  
Functional Assays ◽  
Intestinal Immune System ◽  
Novel Method ◽  
Selection Of

The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin+ and langerin-), newly described DC3, monocytes, Mf1, Mf2, Mf3 and Mf4. We also present a novel method to account for autofluorescent signal from tissue macrophages. Finally, we demonstrate that these methods can successfully be used to sort functional, immature intestinal DCs that can be used for functional assays such as cytokine production assays.

Afterword

2020 ◽  
pp. 680-683
Author(s):  
Janice L. Waldron ◽  
Stephanie Horsley ◽  
Kari K. Veblen
Keyword(s):  
Social Media ◽  
Media Use ◽  
Careful Selection ◽  
Social Media Use ◽  
Pedagogical Tools ◽  
The Future ◽  
User Data ◽  
Selection Of ◽  
Data Surface

We all feel the implications of the force of social media—for good and for ill—in our lives and in our professional world. At the time of this writing, Facebook continues with its struggle to “clean up its act” as more revelations surrounding breaches of trust and hacked user data surface in the news and various countries attempt to hold Facebook to account. Despite this, social media use continues to grow exponentially, and the potential for responsible, ethical, and transparent social media to transform the ways in which we interact with and learn from each other increase with it. As we wait to see what the future holds for social media in society, we are reminded once again that it is the careful selection of pedagogical tools such as social media, as well the guided awareness of the challenges and benefits of those tools, that remains constant, even as tools may change, disappear, or fall out of fashion.

scholarly journals Evaluation of the Expression of CCR5 and CX3CR1 Receptors and Correlation with the Functionality of T Cells in Women infected with ZIKV during Pregnancy

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 191
Author(s):  
Débora Familiar-Macedo ◽  
Iury Amancio Paiva ◽  
Jessica Badolato-Corrêa da Silva ◽  
Fabiana Rabe de Carvalho ◽  
Helver Gonçalves Dias ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Clinical Outcome ◽  
Cell Responses ◽  
Functional Assays ◽  
Asymptomatic Children ◽  
Cd107a Expression ◽  
Congenital Zika Syndrome ◽  
Ifn Γ

There have been reports of neurological abnormalities associated with the Zika virus (ZIKV), such as congenital Zika syndrome (CZS) in children born to mothers infected during pregnancy. We investigated how the immune response to ZIKV during pregnancy is primed and conduct a thorough evaluation of the inflammatory and cytotoxic profiles as well as the expression of CCR5 and CX3CR1. We compared the reactivity of T cells to ZIKV peptides in convalescent mothers infected during pregnancy. The child’s clinical outcome (i.e., born with or without CZS) was taken to be the variable. The cells were stimulated in vitro with ZIKV peptides and evaluated using the ELISPOT and flow cytometry assays. After in vitro stimulation with ZIKV peptides, we observed a tendency toward a higher Interferon gamma (IFN-γ)-producing T cell responses in mothers who had asymptomatic children and a higher CD107a expression in T cells in mothers who had children with CZS. We found a higher frequency of T cells expressing CD107a+ and co-expressing CX3CR1+CCR5+, which is much clearer in the T cells of mothers who had CZS children. We suggest that this differential profile influenced the clinical outcome of babies. These data need to be further investigated, including the evaluation of other ZIKV peptides and markers and functional assays.

A novel method for the design and selection of neutron-attenuating media

2021 ◽  
Vol 160 ◽  
pp. 108368
Author(s):  
Abdullah G. Weiss ◽  
Moiz I. Butt ◽  
Pavel V. Tsvetkov ◽  
Mark L. Kimber ◽  
Sean M. McDeavitt
Keyword(s):  
Novel Method ◽  
Selection Of

scholarly journals 588 Targeting GITR enhances human tumour-infiltrating T cell functionality in mismatch repair proficient primary colorectal carcinoma and liver metastases

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A623-A623
Author(s):  
Yannick Rakké ◽  
Lucia Campos Carrascosa ◽  
Adriaan van Beek ◽  
Valeska de Ruiter ◽  
Michael Doukas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Colorectal Carcinoma ◽  
Liver Metastases ◽  
Mismatch Repair ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
Human Tumour ◽  
Functional Assays

BackgroundImmune checkpoint blockade (ICB; e.g. anti-PD-1/-CTLA-4) has been proven to be clinically effective in mismatch repair deficient (dMMR) colorectal carcinoma (CRC). Yet, the majority of patients carry mismatch repair proficient (pMMR) CRC, especially those with liver metastasis, and do not respond to ICB. Here, we studied the effect of immune checkpoint stimulation via GITR targeting on human tumour-infiltrating lymphocyte (TIL) functionality in pMMR primary CRC and liver metastases (CRLM).MethodsHuman TIL were isolated from freshly resected pMMR tumours of patients with primary CRC (stage 1–3) or liver metastases (table 1). GITR expression on TIL was determined using flow cytometry and compared to leukocytes isolated from blood (PBMC) and tumour-free surrounding tissues (tumour-free colon/liver, resp. TFC and TFL). Ex vivo functional assays were used to assess TIL expansion, activation and cytokine/cytotoxic mediator secretion upon CD3/CD28 bead activation and co-stimulation using an antibody-crosslinked recombinant trimeric GITR ligand (GITRL).ResultsGITR was overexpressed on TIL when compared to other stimulatory immune checkpoints (4-1BB, OX40). GITR expression was enhanced on CD4+ and CD8+ TIL compared to PBMC and TFC or TFL compartments in both primary CRC and CRLM. Among CD4+ TIL, GITR was increasingly expressed on CD45RA± FoxP3- helper T (Th), CD45RA- FoxP3int activated helper T (aTh), and CD45RA- FoxP3hi activated regulatory T cells (aTreg), respectively. Within CD8+ TIL, GITR expression was higher on TOX+ PD1Hi and putatively tumour-reactive CD103+ CD39+ TIL.1 Impaired effector cytokine production upon ex vivo PMA/ionomycin stimulation was observed in CD4+ and CD8+ GITR-expressing TIL, hinting to functional exhaustion of the target population. However, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers and proinflammatory cytokine secretion in a dose-dependent manner (figure 1). Treg depletion did not fully abrogate the stimulatory effect of GITR ligation on CD4+ and CD8+ T cell expansion, demonstrating that the stimulatory effect was partly exerted via direct targeting GITR on effector T cells. Importantly, GITR-ligation also enhanced expansion of purified CD8+CD39+ TIL. Dual treatment with GITR ligand and nivolumab (anti-PD-1) further enhanced CD8+ TIL responses compared to GITR ligand monotherapy, whereas nivolumab alone did not show any effect.Abstract 588 Table 1Patient characteristicsPatient characteristics of patients included for FACS analysis and/or functional assays. † Pathologic staging was performed according to the AJCC 8th edition criteriaAbstract 588 Figure 1GITR ligation enhances CD4+ and CD8+ TIL expansionTIL were isolated from CRC or CRLM and cultured upon CD3/CD28 activation with or without GITRL (0.1–1.0 ug/mL) for 8 days. TIL numbers were acquired by flow cytometry and normalized to counting beads. Indicated is fold change relative to ctrl-treated TIL (n=10).ConclusionsAgonistic targeting of GITR enhances ex vivo human TIL functionality in pMMR CRC and might therefore be a promising approach for novel mono- or combinatorial immunotherapies in primary CRC and CRLM.AcknowledgementsN/ATrial RegistrationN/AEthics ApprovalThe study was approved by the medical ethics committee of the Erasmus Medical Center (MEC-2012-331).ConsentN/AReferenceDuhen T, Duhen R, Montler R, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9(1):2724. doi: 10.1038/s41467-018-05072-0.

scholarly journals Tuning of a Linear-Quadratic Stabilization System for an Anti-Aircraft Missile

Aerospace ◽  
2021 ◽  
Vol 8 (2) ◽  
pp. 48
Author(s):  
Witold Bużantowicz
Keyword(s):  
Analytical Solutions ◽  
Feedback Loop ◽  
Linear Quadratic Regulator ◽  
Added Value ◽  
Tuning Parameter ◽  
Stabilization System ◽  
Linear Quadratic ◽  
Quadratic Stabilization ◽  
Novel Method ◽  
Selection Of

A description is given of an application of a linear-quadratic regulator (LQR) for stabilizing the characteristics of an anti-aircraft missile, and an analytical method of selecting the weighting elements of the gain matrix in feedback loop is proposed. A novel method of LQR tuning via a single parameter ς was proposed and tested. The article supplements and develops the topics addressed in the author’s previous work. Its added value includes the observation that the solutions obtained are symmetric pairs, and that the tuning parameter ς proposed for the designed linear-quadratic regulator enables the selection of suitable parameters for the airframe stabilizing loop for the majority of the analytical solutions of the considered Riccati equation.

Catacomb culture wagons of the Eurasian steppes

Antiquity ◽  
2014 ◽  
Vol 88 (340) ◽  
pp. 378-394 ◽  
Author(s):  
N.I. Shishlina ◽  
D.S. Kovalev ◽  
E.R. Ibragimova
Keyword(s):  
Stable Isotope ◽  
Isotope Analysis ◽  
Burial Mound ◽  
Careful Selection ◽  
Southern Russia ◽  
The Subject ◽  
The Individual ◽  
Wheeled Vehicles ◽  
Selection Of ◽  
Catacomb Culture

The origin and development of wheeled vehicles continues to fascinate today no less than when Stuart Piggott (1974) first wrote about the subject inAntiquity40 years ago. A growing number of examples from the steppes of southern Russia and Ukraine are providing new insights into the design and construction of these complex artefacts. A recent example from the Ulan IV burial mound illustrates the techniques employed and the mastery of materials, with careful selection of the kinds of wood used for the wheels, axles and other elements. Stable isotope analysis of the individual interred in this grave showed that he had travelled widely, emphasising the mobility of steppe populations.

A novel method for predicting variant selection of {101¯2} twins in pure hafnium

2021 ◽  
pp. 130738
Author(s):  
Yingdong Zhang ◽  
Geping Li ◽  
Fusen Yuan ◽  
Fuzhou Han ◽  
Muhammad Ali ◽  
...  
Keyword(s):  
Variant Selection ◽  
Novel Method ◽  
Selection Of

scholarly journals The Effects of Landscape Elements on the Breeding Sites of Bloodsucking Midge

2021 ◽  
Vol 13 (5) ◽  
pp. 2863
Author(s):  
Kaowen Grace Chang ◽  
Hungju Chien
Keyword(s):  
Ground Surface ◽  
Preventive Strategies ◽  
Careful Selection ◽  
Public Concern ◽  
Breeding Sites ◽  
Algae Growth ◽  
Potential Habitats ◽  
Landscape Factors ◽  
The Relationship ◽  
Selection Of

Forcipomyia taiwana, a bloodsucking midge that is one of the most irritating biting pests in Taiwan, has raised widespread public concern. However, we have little information about the extent to which landscape factors affect their potential habitats. As a result, landscape professionals do not have enough information to implement preventive strategies to control midges. The purpose of this study is to investigate the relationship between landscaping and algae growth for larval breeding sites of Forcipomyia taiwana. The intent is to determine the environmental strategies that make the planned landscape unsuitable for midges to breed. GIS based on data collected from 16 constructed landscape sites (317,187 m2 in total) was utilized to spatially examine the relationship between the occurrence of the algae for midge breeding sites and the ground surface types and planting characteristics in each landscape. The results revealed that the potential midge habitats can be controlled through careful selection of the ground surface, the improvement of the site drainage, and choosing plants with the appropriate characteristics. Apart from choosing the appropriate type of paving surface, the integrity of the paving installation and the coverage of the ecological surface also influence prevention efficacy.

WEIGHT GAINS IN THE FIRST FOUR WEEKS OF INFANCY: A COMPARISON OF THREE DIETS

PEDIATRICS ◽  
1956 ◽  
Vol 18 (3) ◽  
pp. 362-368
Author(s):  
Ruth T. Gross ◽  
Lincoln E. Moses
Keyword(s):  
Human Milk ◽  
Statistical Methods ◽  
Full Term ◽  
Clinical Judgement ◽  
Careful Selection ◽  
Term Infants ◽  
Weight Gains ◽  
The Many ◽  
Selection Of

Four hundred seven healthy, full-term infants were divided into three groups and fed, respectively, a formula of evaporated milk and water with 5% carbohydrate; human milk; and a special modified evaporated milk designed to simulate human milk. No other foods were added to the diet. A comparison of the three groups was made, based on weight gains from birth to the end of the first 4 weeks. The conclusions refer only to weight gains; no attempt was made to determine the superiority of any particular diet. The data show no significant differences in the 4-week weight gains among the three groups of infants, although sensitive statistical methods could be validly applied to the problem. These methods are explained. The authors wish to emphasize the many variables which must be taken into account in a study of this sort; the necessity for careful selection of valid statistical methods; the importance of critical clinical judgement in the evaluation of the results.

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