13 research outputs found
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The Role of the Selenoprotein Glutathione Peroxidase-1 in T Cell Activation and Differentiation
Glutathione peroxidase-1 (GPx-1) is an antioxidant enzyme that plays an important role in reducing cellular oxidative stress. The expression of GPX1 has previously been shown to be upregulated upon T cell activation in vitro and CD4+ T cells that lack GPx-1 have also been shown to preferentially differentiate into the TH1 effector subtype. These observations suggest that the activity of GPx-1 may play a role in T cell activation and differentiation. In order to determine the relationship between GPx-1 expression and T cell activation, we used a combination of in vitro and in vivo experiments to correlate GPx-1 expression with the activation marker CD44 and to address whether inhibition of GPx-1 impacts the differentiation of induced regulatory T cells. Gene expression analysis done on naïve CD4+ T cells stimulated in vitro reveals that the number of GPX1 transcripts is reduced in cells cultured in the presence of recombinant PD-L1 when compared to cells cultured in the presence of control human Ig-Fc. In contrast to these data, flow cytometry data does not reveal any significant differences between the overall abundance of intracellular GPx-1 over the course of 72 hours when comparing CD4+ T cells cultured in the presence and absence of recombinant PD-L1. Using in vivo immunization and tumor models, we demonstrate that the overall expression of GPx-1 is higher both in CD44+ T cells derived from the draining lymph node and tumor infiltrating lymphocytes when compared to CD44- T cells from each microenvironment. To assess the role of GPx-1 during activation and differentiation of naïve CD4+ T cells in vitro, we used a small molecule inhibitor of GPx-1, mercaptosuccinate (MS). Our data reveal that the addition of MS reduces the overall number of CD44+ T cells and that the presence of MS increases the oxidative state of CD4+ T cells in a dose dependent manner. These data support the role of GPx-1 as an important cytosolic and mitochondrial antioxidant enzyme. Our data also reveal that the presence of MS in cell culture media induces the expression of the transcription factor Forkhead box P3 (FoxP3) in a dose-dependent manner that is independent of the presence of TGF-β. Overall, these data suggest that GPx-1 is an important antioxidant enzyme that may play a role in regulating T cell activation and differentiation. Additionally, we provide preliminary evidence to support the idea that the redox tone of the cell may be important for fate determination. Further work is needed to assess the functionality and durability of these FoxP3 expressing T cells, and to address whether or not these cells exhibit the typical characteristics of induced regulatory T cells
Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins
Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDPglucose: glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins
Transfected HEK293 Cells Expressing Functional Recombinant Intercellular Adhesion Molecule 1 (ICAM-1) – A Receptor Associated with Severe Plasmodium falciparum Malaria
Intercellular adhesion molecule 1 (ICAM-1) is a membrane-bound glycoprotein expressed on endothelial cells and cells of the immune system. Human ICAM-1 mediates adhesion and migration of leucocytes, and is implicated in inflammatory pathologies, autoimmune diseases and in many cancer processes. Additionally, ICAM-1 acts as receptor for pathogens like human rhinovirus and Plasmodium falciparum malaria parasites. A group of related P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains, the DBLβ, mediates ICAM-1 binding of P. falciparum-infected erythrocytes. This ICAM‑1-binding phenotype has been suggested to be involved in the development of cerebral malaria. However, more studies identifying cross-reactive antibody and ICAM-1-binding epitopes and the establishment of a clinical link between DBLβ expression and e.g. cerebral malaria are needed before the DBLβ domains can be put forward as vaccine candidates and go into clinical trials. Such studies require availability of functional recombinant ICAM-1 in large quantities. In this study, we compared recombinant ICAM-1 expressed in HEK293 and COS-7 cells with mouse myeloma NS0 ICAM-1 purchased from a commercial vendor in terms of protein purity, yield, fold, ability to bind DBLβ, and relative cost. We present a HEK293 cell-based, high-yield expression and purification scheme for producing inexpensive, functional ICAM‑1. ICAM-1 expressed in HEK293 is applicable to malaria research and can also be useful in other research fields
Binding of the malaria PfEMP1 antigen DBLβ3_D4 to ICAM-1-Fc.
<p>Concentration-dependent binding of recombinant <i>P. falciparum</i> 3D7 PFD1235w DBLβ3_D4 to ICAM-1-Fc<sub>HEK239,</sub> ICAM-1-Fc<sub>COS-7</sub> and ICAM-1-Fc<sub>NS0</sub> (R&D Systems) by ELISA. The binding of DBLβ3_D4 to ICAM-1-Fc<sub>HEK239</sub> was repeated in three independent experiments (mean and standard deviation shown) while the assay using ICAM-1-Fc<sub>COS-7</sub> and ICAM-1-Fc<sub>NS0</sub> (R&D Systems) was done one time each.</p
Comparison of ICAM-1-Fc by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
<p>SDS-PAGE gel electrophoresis of 5 µg of ICAM-1-Fc expressed in HEK293 cells, COS-7 cells or in mouse myeloma NS0 (R&D Systems) cells. 5 µl protein marker (M) was loaded onto the gel. Samples were reduced using DTT (+) or non-reduced (−). Arrows indicate ICAM-1-Fc bands.</p
Purification of ICAM1-Fc expressed by HEK293 cells.
<p>Data from exp. #1 is shown here as an example of purification of ICAM-1-Fc<sub>HEK293</sub>. (<b>A</b>) Dot blot showing 2 µl of cell supernatant at day of harvest, 2 µl diafiltrated supernatant (column input) and 2 µl column run-through. 1.8 µg and two ten-fold dilutions hereof of the eluted ICAM-1-Fc was dotted onto the membrane. ICAM-1-Fc was detected using HRP-conjugated anti-human IgG antibody. (<b>B</b>) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis of 5 µl protein marker (lane 1), 10 µl column input (lane 2), 10 µl column run-through (lane 3) and 9 µg eluted ICAM-1-Fc (lane 4+5). Samples were reduced using DTT (+) or non-reduced (−). Arrows indicate ICAM-1-Fc bands.</p
Reactivity of monoclonal ICAM antibodies.
<p>The reactivity of seven anti-human ICAM-1 specific monoclonal antibodies (clones 15.2, RR1/1, 84H10, LB2, BBIG-I1, 8.4A6 and My13) against ICAM-1-Fc expressed in HEK293, COS-7 or mouse myeloma NS0 (R&D Systems) cells were tested using ELISA. One CD36 specific monoclonal antibody (clone FA6.152) was included as negative control. Data shown are the mean reactivity (three independent experiments) of the antibodies to ICAM-1. Errors indicate S.D.</p
Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins
Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDPglucose: glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins
Shedding of Hepatitis C Virus in Semen of Human Immunodeficiency Virus-Infected Men.
Background.  The epidemic of sexually transmitted hepatitis C virus (HCV) infection among human immunodeficiency virus (HIV)-infected men who have sex with men (MSM) has been documented for over a decade. Despite this, there is no consensus as to the risk factors for sexual acquisition of HCV in these men. Methods.  We obtained paired semen and blood samples at 2-week intervals from HIV-infected MSM with recent and chronic HCV infection and quantified HCV in semen. Results.  Hepatitis C virus was quantified in 59 semen specimens from 33 men. Hepatitis C virus was shed in 16 (27%) of semen specimens from 11 (33%) of the men. Median HCV viral load (VL) in semen was 1.49 log10 IU/mL. Hepatitis C virus VL in blood was significantly higher at the time of HCV shedding in semen than when HCV shedding in semen was not detected (P = .002). Furthermore, there was a significant correlation between the HCV VL in blood and semen overall (rs = 0.41; P = .001), and in the subgroup with recent HCV infection (rs = 0.37; P = .02), but not in the subgroup with chronic HCV infection (rs = 0.34; P = .1). Conclusions.  One third of HIV-infected MSM coinfected with HCV shed HCV into their semen. Based on the HCV VL in semen in this study, an average ejaculate would deliver up to 6630 IU of virus into the rectum of the receptive partner. Therefore, our data strongly support that condoms should be used during anal intercourse among MSM to prevent transmission of HCV