scholarly journals Differential Uptake of Murine and Human Exosomes by Normal and Inflamed Peripheral Tissues and Brain

2022 ◽  
Author(s):  
William A Banks ◽  
Priyanka Sharma ◽  
K. M. Hansen ◽  
Nils Ludwig ◽  
T. L. Whiteside
Keyword(s):  
Path Analysis ◽  
Target Cells ◽  
Multiple Time ◽  
Tissue Uptake ◽  
Heat Map ◽  
Uptake Inhibition ◽  
Peripheral Tissues ◽  
Human T Cells ◽  
Wheatgerm Agglutinin ◽  
Map Analysis

Abstract Background: Exosomes function as an intercellular communication system conveying messages from donor to target cells in nearby or distant tissues. Many aspects of exosome trafficking remain unresolved, however. Here, we investigated uptake of ten radiolabeled murine or human exosomes of various cellular origins by the liver, kidney, spleen, and lung of male CD-1 mice. Methods: We radioactively labeled 10 exosomes from mouse or human cancerous or non-cancerous lines, injected them intravenously into male CD-1 mice, and studied their tissue uptake. We examined the ability of wheatgerm agglutinin (WGA), mannose-6 phosphate (M6P), and inflammation induced by lipopolysaccharide (LPS) to modulate uptake. We measured uptake rate using multiple-time regression analysis and used heat mapping and path analysis to correlate tissue and exosomal influences on uptake. Results: Except for the uptake of SCCVII exosomes by kidney, all exosomes were taken up by all tissues, although the uptake levels varied broadly among exosomes and tissues. The liver/serum uptake ratio for exosomes from primary human T-cells was the highest at 4,500 mL/g. Species of origin (mouse vs human) or source (cancerous vs noncancerous cells) did not influence tissue uptake. The uptake of some exosomes was altered by WGA and LPS but not by M6P, except for uptake inhibition of J774A.1 exosomes by liver, suggesting use of the M6P receptor. WGA or LPS treatments enhanced uptake of exosomes by brain and lung but inhibited uptake by liver and spleen. Response to LPS was not, however, predictive of response to WGA. No evidence for a universal binding site controlling exosome uptake was obtained. Applying path analysis and heat map analysis to the data, including our published results for brain, we found that exosome uptake patterns for lung and brain responded similarly to WGA or to LPS, whereas those for liver and spleen clustered together. In path analysis, the 10 exosomes clustered into distinct groups, suggesting that their bindings sites are similarly clustered. Conclusions: Uptake of exosomes by peripheral tissues is differentially regulated by both exosomes and target tissues and is dependent on the number and types of mutually interactive binding sites.

Abstract P492: Putative Protein Biomarkers For Reductive Stress Cardiomyopathy

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Rajasekaran Namakkal-Soorappan ◽  
Cynthia L David ◽  
Krishna Parsawar
Keyword(s):  
Protein Quality ◽  
Redox Homeostasis ◽  
Differentially Expressed ◽  
Related Factor ◽  
Protein Biomarkers ◽  
Heat Map ◽  
Reductive Stress ◽  
Tandem Mass Tag ◽  
Map Analysis ◽  
Dose Dependent

Background: Nuclear factor erythroid 2-related factor 2 (NRF2) signaling is vital for redox homeostasis. We reported that transgenic mice expressing constitutively active Nrf2 (CaNrf2) exhibit reductive stress (RS). Here in, we identified novel protein signatures reacting to RS-induced cardiomyopathy. Methods: Tandem Mass Tag (TMT) proteomic analysis was performed in the heart tissues of Ca-Nrf2-transgenic (TG-low & TG-high) and non-transgenic (NTg) mice at 6 months of age (N= 4/group). Differentially expressed proteins (DEPs) were then identified using Scaffold. Validated the key DEPs using immunoblotting. PANTHER and STRING analysis were used to identify potential targets and their interactions. Results: A total of 1105 proteins were extracted from 24369 spectra. Of note, 226 and 261 proteins were differentially expressed in TG-L and TG-H vs. NTg hearts indicating a unique proteome signature for RS. Heat map analysis revealed a clear distinction between the TG-L and TG-H due to the dose-dependent effects of transgene/RS. Majority of the DEPs that are significantly altered in RS mice found to involve in stress related pathways such as antioxidants, NADPH, protein quality control (PQC), etc. Interestingly, some of these proteins were redox modified at their cysteine residues under chronic RS setting. Conclusions: TMT based proteomic analyses revealed unique proteome signatures for RS. The cysteine modifications in multiple proteins likely to cause pathological alterations via impaired PQC mechanisms. Molecular studies related to RS-mediated redox modifications in structural and functional cardiac proteome are underway.

scholarly journals As2O3 Enhances Retroviral Reverse Transcription and Counteracts Ref1 Antiviral Activity

2003 ◽  
Vol 77 (5) ◽  
pp. 3167-3180 ◽  
Author(s):  
Lionel Berthoux ◽  
Greg J. Towers ◽  
Cagan Gurer ◽  
Paolo Salomoni ◽  
Pier Paolo Pandolfi ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Leukemia Virus ◽  
Target Cells ◽  
Virion Assembly ◽  
Human T Cells ◽  
Pml Bodies ◽  
Steady State Levels ◽  
Nuclear Structures ◽  
Apoptosis Inducing Agents ◽  
Hiv 1

ABSTRACT Potent drugs such as cyclosporine have provided effective probes of signal transduction pathways and, as well, of human immunodeficiency virus type 1 (HIV-1) replication mechanisms. Recently, it was reported that As2O3, a drug used to treat acute promyelocytic leukemia (PML), stimulates HIV-1 replication. We found that As2O3 accelerates the kinetics of a spreading HIV-1 infection in human T cells and increases the number of cells bearing HIV-1 provirus after a single round of infection. The stimulatory effect occurred after membrane fusion and resulted in increased steady-state levels of newly synthesized viral cDNA. Stimulation was independent of HIV-1 env and most viral accessory genes, and As2O3 had no detectable effects on viral expression postintegration or virion assembly. Murine leukemia virus (MLV) transduction was enhanced by As2O3 to the same extent as HIV-1 transduction, but As2O3 had no additional effect on Fv1 restriction. In contrast, As2O3 largely overcame the specific block to N-tropic MLV reverse transcription posed by human Ref1. As2O3 disrupts PML bodies, nuclear structures named for a major component, the PML protein. We observed no changes in PML bodies in response to HIV-1 infection. Experiments with PML-null target cells indicated that PML has no effect on HIV-1 infectivity and is dispensable for the stimulatory effect of As2O3. As2O3 caused cell death in uninfected cells at the same concentrations which stimulate HIV-1 replication. Among four additional apoptosis-inducing agents, a boost in HIV-1 infectivity was observed only with carbonyl cyanide m-chlorophenylhydrazone, a compound which, like As2O3, disrupts the mitochondrial transmembrane potential. In summary, As2O3 stimulates retroviral reverse transcription, perhaps via effects on mitochondria, and provides a useful tool for characterizing Ref1.

Peripheral Alloantigen Drives Early Dysfunction and Eventual Exhaustion of CTL Following Delayed Donor Leukocyte Infusions.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2346-2346
Author(s):  
Barry R Flutter ◽  
Farnaz Fallah-Arani ◽  
Clare Bennett ◽  
Janani Sivakumaran ◽  
Gordon J Freeman ◽  
...  
Keyword(s):  
T Cell ◽  
Significant Risk ◽  
T Cell Response ◽  
Target Cells ◽  
Peripheral Tissues ◽  
Hematopoietic System ◽  
Cd8 Cells ◽  
Ifn Γ

Abstract T cell immunotherapies for cancer should ideally generate high levels of anti-tumor activity, with minimal host injury and permit the prolonged survival of functional memory/effector cells to prevent tumor recurrence. Following allogeneic stem cell transplantation, delayed donor leukocyte infusion (DLI) is one strategy employed to induce graft-versus-leukemia (GVL) responses while limiting the risk of host injury in terms of graft-versus-host disease. However, patients remain at significant risk of relapse following DLI and murine models of delayed DLI indicate that this results from the eventual loss of functional, alloreactive cytotoxic T lymphocytes (CTL) [Mapara et al. Transplantation 2003]. We hypothesised that the loss of functional CTL is driven by persistent stimulation of donor CD8 cells by alloantigen expressed by peripheral tissues. In order to follow and characterise an alloreactive CD8 response under conditions in which alloantigen was present or absent in peripheral tissues, we employed a model in which either parental B6 (H2b) or B6 x DBA-2 F1 (BDF1, H2dxb) mice were lethally irradiated and reconstituted with a mixture of B6 and BDF1 T cell depleted bone marrow. 8-10 weeks later congenic CD45.1 B6 splenocytes were transferred into the established mixed chimeras. This allowed us to test the importance of peripheral antigen in the loss of alloreactive CTL responses, since alloantigen was either restricted to the hematopoietic system (B6 +BDF1 → B6) or was ubiquitously expressed (B6 +BDF1 → BDF1). Following transfer of CD45.1 B6 splenocytes, the ensuing alloantigenspecific T cell response in both groups led to the elimination of alloantigen-positive (BDF1-derived) hematopoietic elements. Thereafter, alloreactive CD8 cells resided in an environment in which peripheral alloantigen was present (PA+) or absent (PA-). We observed similar kinetics of initial CD45.1+ CD8 cell proliferation and expansion and similar acquisition of a CD44highCD62Llow phenotype. However, by day 60, there were striking differences in the phenotype and function of transferred CD8 cells. In PA- hosts, CD45.1+ CD8 cells killed allogeneic target cells effectively both in vitro and in vivo, underwent rapid proliferation in a mixed leukocyte reaction and produced the effector cytokine, IFN-γ. In contrast CD45.1+ CD8 cells from PA+ hosts had little or no cytotoxic activity, did not proliferate to alloantigen and were IFN-γlow. Moreover, CD45.1+ CD8 cells from PA+ hosts displayed high levels of the co-inhibitory receptor PD-1, low levels of the IL-7Rα chain and responded poorly to IL-7 and IL-15 in vitro, a phenotype typical of the ‘exhaustion’ signature observed in CTL following chronic antigen exposure. In comparison, CD45.1+ CD8 cells from PA- hosts expressed significantly lower levels of PD-1, higher levels of IL-7Rα and demonstrated better responsiveness to IL-7 and IL-15 in vitro. In vitro PD-1 or PD-L1 blockade restored IFN-γ generation to CD45.1+ CD8 cells from PA+ hosts, suggesting that the PD-1 pathway may play a functional role in driving exhaustion of these cells. Importantly we observed no loss of long-term alloreactive CD4 responses in either PA+ or PA- hosts. This finding is consistent with a model in which peripheral alloantigen drives exhaustion since the majority of cells expressing Class II alloantigens in PA+ and PA- hosts would be restricted to the hematopoietic system and thus, would have been cleared in the initial alloresponse. The full exhausted phenotype of alloreactive CD8 cells described above was not seen until at least 30 days after transfer to PA+ hosts. However, as early as day 14, CTL primed in PA+ hosts produced less IFN-γ in comparison to those primed in PA-hosts, even though they were still equivalent in terms of their cytotoxicity. Furthermore, when CD8 cells primed in PA+ hosts were transferred to secondary antigen-free hosts, they still displayed reduced ‘fitness’ compared to CTL originally primed in PA- hosts. These data show that peripheral alloantigen qualitatively affects donor CTL function during priming and drives their eventual exhaustion. Additionally they suggest that blockade of co-inhibitory signals may have potential in restoring function to such cells as has been demonstrated in models of chronic infection.

scholarly journals Functional subclasses of T-lymphocytes bearing different Ly antigens. I. The generation of functionally distinct T-cell subclasses is a differentiative process independent of antigen.

1975 ◽  
Vol 141 (6) ◽  
pp. 1376-1389 ◽  
Author(s):  
H Cantor ◽  
E A Boyse
Keyword(s):  
T Cell ◽  
Lymphoid Cells ◽  
Target Cells ◽  
Transitional Form ◽  
Peripheral Tissues ◽  
Killer Activity ◽  
Immunologic Function ◽  
Helper Activity ◽  
Early Effects ◽  
Cytotoxic Function

Ly alloantigens coded by two unlinked genetic loci (Ly-1 and Ly-2/Ly-3) are expressed on lymphoid cells undergoing thymus-dependent differentiation. Peripheral Thy-1+ cells from C57BL/6 mice can be divided into three subclasses on the basis of differential expression of Ly-1, Ly-2, and Ly-3; about 50% express all three Ly antigens (Ly -123+), about 33% only Ly-1 (Ly-1+), and about 6-8% Ly-2 and Ly-3 (Ly-23+). Cells of the Ly-123+ subclasses are the first peripheral Thy-1+ cells to appear in ontogeny, and are reduced in the periphery shortly after adult thymectomy. In contrast, Ly-1+ and Ly-23+ subclasses appear later in the peripheral tissues than do Ly-123+ cells, and are resistant to the early effects of adult thymectomymperiheral lymphoid populations depleted of Ly-1+ cells and Ly-123+ cells (and thereby enriched for Ly-23+ cells) were incapable of developing significant helper activity to SRBC but generated substantial levels of cytotoxic activity to allogeneic target cells. The same lymphoid populations, depleted of Ly-23+ cells and Ly-123+ cells (and thereby enriched for Ly-1+ cells), produced substantial helper responses but were unable to generate appreciable levels of killer activity. These experiments imply that commitment of T cells to participate exclusively in either helper or cytotoxic function is a differentiative process that takes place before they encounter antigen, and is accompanied by exclusion of different Ly groups, Lu-23 or Ly-1 respectively, from TL+Ly-123+ T-cell precursors. It is yet to be decided whether the TL-phase by Ly-123+ subclass is a transitional form or a separately differentiated subclass with a discrete immunologic function.

scholarly journals Variability and trends of climate extremes indices from the observed and downscaled GCMs data over 1950–2020 period in Chattogram City, Bangladesh

2021 ◽  
Author(s):  
Lia Pervin ◽  
Sabbir Mostafa Khan
Keyword(s):  
Regional Climate ◽  
Climate Extremes ◽  
Climate Indices ◽  
Heat Map ◽  
Maximum Rainfall ◽  
The Sustainable Development ◽  
Slope Test ◽  
Daily Data ◽  
Increasing Trend ◽  
Map Analysis

Abstract This study was intended to evaluate the variability and trends of climate extremes by incorporating daily data from Chattogram station and from the high-resolution Coordinated Regional Climate Downscaling Experiment (CORDEX) for two different time series. Here, we also focused on evaluating the performance of the selected RCMs (CanESM2, CSIRO, and GFDL from CORDEX) using Taylor diagrams and heat map analysis. Twenty-two extreme climate indices from ETCCDI were computed for 1950–1989 and 1990–2020 periods. Mann–Kendall and Sen's slope test were performed to estimate the trends from the indices from both station and RCMs data. Highly significant increasing trend for the warm days and warm nights’ frequencies were found, whereas, the frequency of cold days and cold nights indicated significantly decreasing trend. On the other hand, mild increasing trend in 1-day and 5-day maximum rainfall was detected. Also, the average annual precipitation has increased by 6% from the 1950–1989 to 1990–2020 period. During the last three decades, the region has experienced more heavier rainfall in the monsoon but increased water stress in the dry season. The two-fold effects of climate change on the local hydrology revealed by this study need to be addressed properly for the sustainable development of this region.

scholarly journals Intraocular Viral Communities Associated With Post-fever Retinitis

2021 ◽  
Vol 8 ◽  
Author(s):  
Kotakonda Arunasri ◽  
Gumpili Sai Prashanthi ◽  
Mudit Tyagi ◽  
Rajeev R. Pappuru ◽  
Sisinthy Shivaji
Keyword(s):  
Control Group ◽  
Ocular Infection ◽  
Vitreous Fluid ◽  
Heat Map ◽  
Barr Virus ◽  
Ocular Infections ◽  
Viral Communities ◽  
Epstein Barr ◽  
Human Viruses ◽  
Map Analysis

The virome of ocular fluids is naive. The results of this study highlight the virome in the vitreous fluid of the eye of individuals without any ocular infection and compare it with the virome of the vitreous fluid of individuals with retinitis. A total of 1,016,037 viral reads were generated from 25 vitreous fluid samples comprising control and post-fever retinitis (PFR) samples. The top 10 viral families in the vitreous fluids comprised of Myoviridae, Siphoviridae, Phycodnaviridae, Herpesviridae, Poxviridae, Iridoviridae, Podoviridae, Retroviridae, Baculoviridae, and Flaviviridae. Principal coordinate analysis and heat map analysis clearly discriminated the virome of the vitreous fluid of the controls from that of the PFR virome. The abundance of 10 viral genera increased significantly in the vitreous fluid virome of the post-fever retinitis group compared with the control group. Genus Lymphocryptovirus, comprising the human pathogen Epstein-Barr virus (EBV) that is also implicated in ocular infections was significantly abundant in eight out of the nine vitreous fluid viromes of post-fever retinitis group samples compared with the control viromes. Human viruses, such as Hepacivirus, Circovirus, and Kobuvirus, were also significantly increased in abundance in the vitreous fluid viromes of post-fever retinitis group samples compared with the control viromes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analysis and the network analysis depicted an increase in the immune response by the host in the post-fever retinitis group compared with the control group. All together, the results of the study indicate changes in the virome in the vitreous fluid of patients with the post-fever retinitis group compared to the control group.

scholarly journals A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity

2021 ◽  
Vol 7 (11) ◽  
pp. 222
Author(s):  
Claudia Coronnello ◽  
Rosalia Busà ◽  
Luca Cicero ◽  
Albert Comelli ◽  
Ester Badami
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Photon Emission ◽  
Target Cells ◽  
Multiple Time ◽  
Chromium Release Assay ◽  
Cellular Division ◽  
High Background ◽  
Chromium Release ◽  
Time Point

The cytotoxic activity of T cells and Natural Killer cells is usually measured with the chromium release assay (CRA), which involves the use of 51Chromium (51Cr), a radioactive substance dangerous to the operator and expensive to handle and dismiss. The accuracy of the measurements depends on how well the target cells incorporate 51Cr during labelling which, in turn, depends on cellular division. Due to bystander metabolism, the target cells spontaneously release 51Cr, producing a high background noise. Alternative radioactive-free methods have been developed. Here, we compare a bioluminescence (BLI)-based and a carboxyfluorescein succinimidyl ester (CFSE)-based cytotoxicity assay to the standard radioactive CRA. In the first assay, the target cells stably express the enzyme luciferase, and vitality is measured by photon emission upon the addition of the substrate d-luciferin. In the second one, the target cells are labelled with CFSE, and the signal is detected by Flow Cytometry. We used these two protocols to measure cytotoxicity induced by treatment with NK cells. The cytotoxicity of NK cells was determined by adding increasing doses of human NK cells. The results obtained with the BLI method were consistent with those obtained with the CRA- or CFSE-based assays 4 hours after adding the NK cells. Most importantly, with the BLI assay, the kinetic of NK cells’ killing was thoroughly traced with multiple time point measurements, in contrast with the single time point measurement the other two methods allow, which unveiled additional information on NK cell killing pathways.

Application of unsupervised clustering algorithm and heat-map analysis for selection of lactic acid bacteria isolated from dairy samples based on desired probiotic properties

LWT ◽  
2020 ◽  
Vol 118 ◽  
pp. 108839 ◽  
Author(s):  
Yousef Nami ◽  
Bahman Panahi ◽  
Hossein Mohammadzadeh Jalaly ◽  
Reza Vaseghi Bakhshayesh ◽  
Mohammad Amin Hejazi
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Clustering Algorithm ◽  
Unsupervised Clustering ◽  
Probiotic Properties ◽  
Heat Map ◽  
Map Analysis ◽  
Selection Of

scholarly journals Transport of Extracellular Vesicles across the Blood-Brain Barrier: Brain Pharmacokinetics and Effects of Inflammation

2020 ◽  
Vol 21 (12) ◽  
pp. 4407 ◽  
Author(s):  
William A. Banks ◽  
Priyanka Sharma ◽  
Kristin M. Bullock ◽  
Kim M. Hansen ◽  
Nils Ludwig ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Surface Proteins ◽  
Brain Barrier ◽  
Multiple Time ◽  
Blood Brain ◽  
Cancerous Cell ◽  
Wheatgerm Agglutinin ◽  
Mannose 6 Phosphate

Extracellular vesicles can cross the blood–brain barrier (BBB), but little is known about passage. Here, we used multiple-time regression analysis to examine the ability of 10 exosome populations derived from mouse, human, cancerous, and non-cancerous cell lines to cross the BBB. All crossed the BBB, but rates varied over 10-fold. Lipopolysaccharide (LPS), an activator of the innate immune system, enhanced uptake independently of BBB disruption for six exosomes and decreased uptake for one. Wheatgerm agglutinin (WGA) modulated transport of five exosome populations, suggesting passage by adsorptive transcytosis. Mannose 6-phosphate inhibited uptake of J774A.1, demonstrating that its BBB transporter is the mannose 6-phosphate receptor. Uptake rates, patterns, and effects of LPS or WGA were not predicted by exosome source (mouse vs. human) or cancer status of the cell lines. The cell surface proteins CD46, AVβ6, AVβ3, and ICAM-1 were variably expressed but not predictive of transport rate nor responses to LPS or WGA. A brain-to-blood efflux mechanism variably affected CNS retention and explains how CNS-derived exosomes enter blood. In summary, all exosomes tested here readily crossed the BBB, but at varying rates and by a variety of vesicular-mediated mechanisms involving specific transporters, adsorptive transcytosis, and a brain-to-blood efflux system.

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