scholarly journals Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

2020 ◽  
Vol 117 (44) ◽  
pp. 27329-27338
Author(s):  
Eugene Joeh ◽  
Timothy O’Leary ◽  
Weichao Li ◽  
Richard Hawkins ◽  
Jonathan R. Hung ◽  
...  
Keyword(s):  
Immune Responses ◽  
Hepatic Stellate Cells ◽  
Mononuclear Cells ◽  
Three Dimensional ◽  
Stellate Cells ◽  
Live Cells ◽  
Quantitative Mass Spectrometry ◽  
Peripheral Blood Mononuclear ◽  
Galectin 3 ◽  
Labeling Method

Galectin-3 is a glycan-binding protein (GBP) that binds β-galactoside glycan structures to orchestrate a variety of important biological events, including the activation of hepatic stellate cells and regulation of immune responses. While the requisite glycan epitopes needed to bind galectin-3 have long been elucidated, the cellular glycoproteins that bear these glycan signatures remain unknown. Given the importance of the three-dimensional (3D) arrangement of glycans in dictating GBP interactions, strategies that allow the identification of GBP receptors in live cells, where the native glycan presentation and glycoprotein expression are preserved, have significant advantages over static and artificial systems. Here we describe the integration of a proximity labeling method and quantitative mass spectrometry to map the glycan and glycoprotein interactors for galectin-3 in live human hepatic stellate cells and peripheral blood mononuclear cells. Understanding the identity of the glycoproteins and defining the structures of the glycans will empower efforts to design and develop selective therapeutics to mitigate galectin-3–mediated biological events.

scholarly journals Mapping glycan-mediated galectin-3 interactions by live cell proximity labeling

2020 ◽  
Author(s):  
Eugene Joeh ◽  
Timothy O’Leary ◽  
Weichao Li ◽  
Richard Hawkins ◽  
Jonathan R. Hung ◽  
...  
Keyword(s):  
Hepatic Fibrosis ◽  
Hepatic Stellate Cells ◽  
Cell Activation ◽  
Weak Interactions ◽  
Stellate Cell ◽  
Three Dimensional ◽  
Stellate Cells ◽  
Living Cells ◽  
Live Cells ◽  
Galectin 3

AbstractGalectin-3 is a glycan-binding protein (GBP) that binds β-galactoside glycan structures to orchestrate a variety of important biological events, including the activation of hepatic stellate cells to cause hepatic fibrosis. While the requisite glycan epitopes needed to bind galectin-3 have long been elucidated, the cellular glycoproteins that bear these glycan signatures remain unknown. Given the importance of the three-dimensional arrangement of glycans in dictating GBP interactions, strategies that allow the identification of GBP receptors in live cells, where the native glycan presentation and glycoprotein expression are preserved, possess significant advantages over static and artificial systems. Here, we describe the integration of a proximity labeling method and quantitative mass spectrometry to map the glycan and glycoprotein interactors for galectin-3 in live hepatic stellate cells. Understanding the identity of the glycoproteins and defining the structures of the glycans required for galectin-3 mediated hepatic stellate cell activation will empower efforts to design and develop selective therapeutics to mitigate hepatic fibrosis.SignificanceBecause of the weak interactions between individual glycan-binding proteins (GBP), such as galectin-3, and glycans, strategies that allow the direct interrogation of these interactions in living cells remain limited. Thus, the glycan and glycoprotein ligands that are physiologically relevant for galectin-3 binding are insufficiently described. Here, we used a proximity labeling approach that catalytically tags interactors for galectin-3 and identified its pertinent glycan and glycoprotein counter-receptors in live hepatic stellate cells. This study demonstrates that proximity labeling is a powerful tool for mapping GBP complexes in living cells, and when coupled with chemical inhibitors, it can discriminate between protein-protein and protein-glycan interactions.Graphical Abstract

scholarly journals Chemerin Overexpression in the Liver Protects against Inflammation in Experimental Non-Alcoholic Steatohepatitis

Biomedicines ◽  
2022 ◽  
Vol 10 (1) ◽  
pp. 132
Author(s):  
Rebekka Pohl ◽  
Susanne Feder ◽  
Elisabeth M. Haberl ◽  
Lisa Rein-Fischboeck ◽  
Thomas S. Weiss ◽  
...  
Keyword(s):  
Hepatic Stellate Cells ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Stellate Cells ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Alcoholic Steatohepatitis ◽  
Huh7 Cells ◽  
Chemokine Ligand ◽  
Underlying Mechanisms

Non-alcoholic steatohepatitis (NASH) is marked by macrophage infiltration and inflammation. Chemerin is a chemoattractant protein and is abundant in hepatocytes. The aim of this study was to gain insight into the role of hepatocyte-produced prochemerin in NASH. Therefore, mice were infected with adeno-associated virus 8 to direct hepatic overexpression of prochemerin in a methionine–choline deficient dietary model of NASH. At the end of the study, hepatic and serum chemerin were higher in the chemerin-expressing mice. These animals had less hepatic oxidative stress, F4/80 and CC-chemokine ligand 2 (CCL2) protein, and mRNA levels of inflammatory genes than the respective control animals. In order to identify the underlying mechanisms, prochemerin was expressed in hepatocytes and the hepatic stellate cells, LX-2. Here, chemerin had no effect on cell viability, production of inflammatory, or pro-fibrotic factors. Notably, cultivation of human peripheral blood mononuclear cells (PBMCs) in the supernatant of Huh7 cells overexpressing chemerin reduced CCL2, interleukin-6, and osteopontin levels in cell media. CCL2 was also low in RAW264.7 cells exposed to Hepa1–6 cell produced chemerin. In summary, the current study showed that prochemerin overexpression had little effect on hepatocytes and hepatic stellate cells. Of note, hepatocyte-produced chemerin deactivated PBMCs and protected against inflammation in experimental NASH.

Traditional Acupuncture Increases the Content of Beta-Endorphin in Immune Cells and Influences Mitogen Induced Proliferation

1991 ◽  
Vol 19 (02) ◽  
pp. 101-104 ◽  
Author(s):  
Mauro Bianchi ◽  
Edda Jotti ◽  
Paola Sacerdote ◽  
Alberto E. Panerai
Keyword(s):  
Immune Responses ◽  
T Lymphocyte ◽  
Peripheral Blood Mononuclear Cells ◽  
Immune Cells ◽  
Lymphocyte Proliferation ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Beta Endorphin ◽  
T Lymphocyte Proliferation ◽  
Blood Mononuclear Cells

We measured beta-endorphin concentrations in peripheral blood mononuclear cells and mitogen-induced T-lymphocyte proliferation in patient who underwent treatment with traditional acupuncture. Traditional acupuncture increased both the concentrations of the opioid in the immune cells and lymphocyte proliferation. Our data are consistent with the hypothesis that traditional acupuncture modulates immune responses in man.

scholarly journals A preliminary study to evaluate the immune responses induced by immunization of dogs with inactivated Ehrlichia canis organisms

2005 ◽  
Vol 72 (2) ◽  
Author(s):  
Sunita Mahan ◽  
P.J. Kelly ◽  
S.M. Mahan
Keyword(s):  
Immune Responses ◽  
Western Blotting ◽  
Lymphocyte Proliferation ◽  
Mononuclear Cells ◽  
Antibody Responses ◽  
Ehrlichia Canis ◽  
Intracellular Pathogen ◽  
Peripheral Blood Mononuclear ◽  
Canine Monocytic Ehrlichiosis ◽  
Antibody Titres

Ehrlichia canis is an intracellular pathogen that causes canine monocytic ehrlichiosis. Although the role of antibody responses cannot be discounted, control of this intracellular pathogen is expected to be by cell mediated immune responses. The immune responses in dogs immunized with inactivated E. canis organisms in combination with Quil A were evaluated. Immunization provoked strong humoral and cellular immune responses, which were demonstrable by Western blotting and lymphocyte proliferation assays. By Western blotting antibodies to several immunodominant E. canis proteins were detected in serum from immunized dogs and antibody titres increased after each immunization. The complement of immunogenic proteins recognized by the antisera were similar to those recognized in serum from infected dogs. Upon challenge with live E. canis, rapid anamnestic humoral responses were detected in the serum of immunized dogs and primary antibody responses were detected in the serum from control dogs. Following immunization, a lymphocyte proliferative response (cellular immunity) was detected in peripheral blood mononuclear cells (PBMNs) of immunized dogs upon stimulation with E. canis antigens. These responses were absent from non-immunized control dogs until after infection with live E. canis, when antigen specific-lymphocyte proliferation responses were also detected in the PBMNs of the control dogs. It can be thus concluded that immunization against canine monocytic ehrlichiosis may be feasible. However, the immunization regimen needs to be optimized and a detailed investigation needs to be done to determine if this regimen can prevent development of acute and chronic disease.

scholarly journals Cytokine Profiles and Cell Proliferation Responses to Truncated ORF2 Protein in Iranian Patients Recovered from Hepatitis E Infection

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Reza Taherkhani ◽  
Fatemeh Farshadpour ◽  
Manoochehr Makvandi ◽  
Hamid Rajabi Memari ◽  
Ali Reza Samarbafzadeh ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Immune Responses ◽  
Mononuclear Cells ◽  
Hepatitis E ◽  
Control Group ◽  
Peripheral Blood Mononuclear ◽  
Orf2 Protein ◽  
Cellular Immune ◽  
Iranian Patients

Background.The aim of this study was to evaluatehepatitis E virus(HEV) specific cellular immune responses to truncated ORF2 protein in Iranian patients recovered from HEV infection. Information about HEV-specific immune responses could be useful in finding an effective way for development of HEV vaccine.Methods.A truncated form of HEV ORF2 protein containing amino acids 112-608 was used to stimulate peripheral blood mononuclear cells (PBMCs) separated from HEV-recovered and control groups. Finally, the levels of four cytokines, IFN-γELISPOT, and cell proliferative responses following stimulation with the truncated ORF2 protein were assessed in the both groups.Results.The truncated ORF2 protein was able to induce IFN-γELISPOT and cell proliferation responses and to produce significant amounts of IFN-γand IL-12 cytokines, but low amounts of IL-10 and IL-4 cytokinesin vitro. These responses were significantly higher in the recovered group compared to the control group. These results indicate the antigenic nature of the truncated ORF2 protein and production of T helper type 1 cytokines.Conclusion.The truncated ORF2 protein can effectively induce significant cellular immune responsesand can be introduced as a potential vaccine candidate. However, further studies are required to evaluate this proteinin vivo.

Clinical significance of insulin peptide-specific interferon-γ-related immune responses in ketosis-prone type 2 diabetes

2021 ◽  
Author(s):  
Atsushi Satomura ◽  
Yoichi Oikawa ◽  
Akifumi Haisa ◽  
Seiya Suzuki ◽  
Shunpei Nakanishi ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Immune Responses ◽  
Elispot Assay ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Β Cell ◽  
Peripheral Blood Mononuclear ◽  
Β Cell Function ◽  
Ifn Γ

Abstract Context Unprovoked A−β+ ketosis-prone type 2 diabetes (KPD) is characterized by the sudden onset of diabetic ketosis/ketoacidosis (DK/DKA) without precipitating factors, negative anti-islet autoantibodies (“A−”), and preservation of β-cell function (“β+”) after recovery from DKA. Although this phenotype often appears with acute hyperglycemia and DK/DKA just like acute-onset type 1 diabetes (AT1D), the involvement of anti-islet immune responses remains unknown. Objective We sought to clarify the immunological role of insulin-associated molecules in unprovoked A−β+ KPD. Methods In this cross-sectional study, blood samples from 75 participants (42 with AT1D and 33 with KPD) were evaluated for interferon (IFN)-γ-secreting peripheral blood mononuclear cells (PBMCs) reactive to four insulin B-chain amino acid 9–23-related peptides (B:9–23rPep) using an enzyme-linked immunospot (ELISpot) assay. Results Overall, 36.4% (12/33) of KPD participants showed positive IFN-γ ELISpot assay results; the positivity rate in KPD was similar to that in AT1D (38.1%; 16/42) and significantly higher than the previously reported rate in type 2 diabetes (8%; 2/25; P < 0.0167). Moreover, B:9–23rPep-specific IFN-γ-producing PBMC frequency was negatively correlated with age and ad lib serum C-peptide levels in all KPD participants and positively correlated with HbA1c level in KPD participants with positive IFN-γ ELISpot results. Conclusions These findings suggest the involvement of B:9–23rPep-specific IFN-γ-related immunoreactivity in the pathophysiology of some unprovoked A−β+ KPD. Moreover, increased immunoreactivity may reflect transiently decreased β-cell function and increased disease activity at the onset of DK/DKA, thereby playing a key role in DK/DKA development in this KPD phenotype.

scholarly journals Th1/Th2 Cytokine Profile in Patients Coinfected with HIV and Leishmania in Brazil

2011 ◽  
Vol 18 (10) ◽  
pp. 1765-1769 ◽  
Author(s):  
Maria Zilma Andrade Rodrigues ◽  
Maria Fernanda Rios Grassi ◽  
Sanjay Mehta ◽  
Xing-Quan Zhang ◽  
Luana Leandro Gois ◽  
...  
Keyword(s):  
Immune Responses ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Content Type ◽  
Th2 Cytokine ◽  
Cytokine Levels ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

ABSTRACTTo evaluate the effects of HIV on immune responses in cutaneous leishmaniasis (CL), we quantified cytokine levels from plasma and stimulated peripheral blood mononuclear cells (PBMCs) from individuals infected with HIV and/or CL. Gamma interferon (IFN-γ) and interleukin 13 (IL-13) levels and the ratio of IFN-γ to IL-10 produced in response to stimulation with solubleLeishmaniaantigens were significantly lower in HIV-Leishmania-coinfected patients than in CL-monoinfected patients.

scholarly journals Humoral and Cellular Immune Responses to Trypanosoma cruzi-Derived Paraflagellar Rod Proteins in Patients with Chagas' Disease

2003 ◽  
Vol 71 (6) ◽  
pp. 3165-3171 ◽  
Author(s):  
Vladimir Michailowsky ◽  
Keith Luhrs ◽  
Manoel Otávio C. Rocha ◽  
David Fouts ◽  
Ricardo T. Gazzinelli ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Immune Responses ◽  
Chagas Disease ◽  
Mononuclear Cells ◽  
Clinical Symptoms ◽  
Cellular Responses ◽  
Peripheral Blood Mononuclear ◽  
Cellular Immune Responses ◽  
Ifn Γ ◽  
Cellular Immune

ABSTRACT Sera and peripheral blood mononuclear cells (PBMC) from patients displaying different clinical symptoms as well as from normal uninfected individuals (NI) were used to evaluate the humoral and cellular responses of Chagas' disease patients to Trypanosoma cruzi-derived paraflagellar rod proteins (PFR). Our results show that sera from both asymptomatic Chagas' disease patients (ACP) and cardiac Chagas' disease patients (CCP) have higher levels of antibodies to PFR than sera from NI. Immunoglobulin G1 (IgG1) and IgG3 were the main Ig isotypes that recognized PFR. We also tested three recombinant forms of PFR, named rPAR-1, rPAR-2, and rPAR-3, by Western blot analysis. Sera from seven out of eight patients with Chagas' disease recognized one of the three rPAR forms. Sera from 75, 50, and 37.5% of Chagas' disease patients tested recognized rPAR-3, rPAR-2, and rPAR-1, respectively. PFR induced proliferation of 100 and 70% of PBMC from ACP and CCP, respectively. Further, stimulation of cells from Chagas' disease patients with PFR enhanced the frequencies of both small and large CD4+ CD25+ and CD4+ CD69+ lymphocytes, as well as that of small CD8+ CD25+ lymphocytes. Finally, we evaluated the ability of PFR to elicit the production of gamma interferon (IFN-γ) by PBMC from patients with Chagas' disease. Fifty percent of the PBMC from ACP as well as CCP produced IFN-γ upon stimulation with PFR. PFR enhanced the percentages of IFN-γ-producing cells in both CD3+ and CD3− populations. Within the T-cell population, large CD4+ T lymphocytes were the main source of IFN-γ.

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