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1.
J Transl Med ; 17(1): 6, 2019 01 03.
Article in English | MEDLINE | ID: mdl-30602374

ABSTRACT

BACKGROUND: Hepatitis B virus (HBV) infections represent a global health problem and chronic hepatitis B (CHB) leads to liver cirrhosis and hepatocellular carcinoma. Thus, timely diagnosis of hepatitis B is crucial to ensure adequate treatment. We developed a powerful and rapid whole blood-based cytokine release assay assessing cellular immune responses to HBV antigens. IL-2 and IFNγ release in this assay depicts hepatitis B vaccination status. Of note, CHB goes along with elevated C5a concentrations in plasma. We aim at mimicking the proinflammatory microenvironment associated with HBV infection to enhance the diagnostic quality of our HBV specific cytokine release assay. We specifically investigated the potential of the complement factors C3a and C5a as costimulators and analyzed their potential effects on activation marker expression on T cells and antigen presenting cells. RESULTS: Whole blood from 87 healthy individuals (n = 59 hepatitis B vaccinated, n = 28 unvaccinated) was stimulated with HBV surface antigen (HBsAg) in presence or absence of C3a or C5a, respectively. Further, C3a and C5a were used in combination to investigate potential synergistic effects. IL-2 and IFNγ levels in plasma were quantified using ELISA. Complement factor C5a specifically enhances HBsAg-mediated IL-2 (690.3 ± 195.4 pg/ml vs. 789.4 ± 216.5 pg/ml) and IFNγ (146.0 ± 43.1 pg/ml vs. 336.7 ± 67.9 pg/ml) responses in whole blood. Similar cytokine levels were measured when both C3a and C5a were used. With a diagnostic specificity of 90% the IFNγ release assay reached a diagnostic sensitivity of 49.2% upon whole blood stimulation with HBsAg alone, but of 78.9% when HBsAg was combined with C3a and C5a. CONCLUSIONS: Innate signals mediated via complement pathways contribute to HBV-specific cellular immune responses. The massively improved diagnostic sensitivity of the IFNγ release assay after addition of C3a and C5a demonstrates that these effects render whole blood-based cytokine release assays even more potent as screening tools in HBV immunology and HBV vaccination studies.


Subject(s)
Complement C3a/metabolism , Complement C5a/metabolism , Hepatitis B virus/immunology , Inflammation/immunology , Inflammation/pathology , Interferon-gamma Release Tests , Adult , Antigen-Presenting Cells/metabolism , Female , Hepatitis B Surface Antigens/blood , Hepatitis B, Chronic/blood , Hepatitis B, Chronic/immunology , Hepatitis B, Chronic/virology , Humans , Interferon-gamma/blood , Interleukin-2/blood , Male , Middle Aged , Tissue Donors , Vaccination
2.
J Virol Methods ; 248: 195-201, 2017 10.
Article in English | MEDLINE | ID: mdl-28739303

ABSTRACT

BACKGROUND: Chronic hepatitis B leads to liver cirrhosis and hepatocellular carcinoma. To develop a therapeutic vaccine for chronic hepatitis B patients it is necessary to assess cellular immune responses to hepatitis B virus (HBV) antigens. We investigated the potential of toll-like receptor (TLR) 9 agonists, i.e. CpG oligonucleotides, as costimulators to increase diagnostic sensitivity and specificity of our HBV- specific cytokine release assay. METHODS: Whole blood from 80 healthy individuals (n=51 hepatitis B vaccinated, n=29 unvaccinated) was stimulated with hepatitis B surface antigen (HBsAg) or hepatitis B core antigen (HBcAg) in presence or absence of CpG oligonucleotides. IL2 and IFNγ secretion in plasma was assessed using ELISA. RESULTS: CpG oligonucleotides specifically enhanced HBsAg-mediated IL2 (276±79pg/ml vs. 320±82pg/ml) and IFNγ (77±35pg/ml vs. 401±121pg/ml) responses in whole blood. When IFNγ release was considered as readout depicting the hepatitis B vaccination status, the according assay reached a diagnostic sensitivity of 61% without, but of 76% with additional CpG oligonucleotide stimulation at a diagnostic specificity of 90%. CONCLUSIONS: We show that innate signals mediated via TLRs contribute to HBV-specific cellular immune responses. CpG oligonucleotides can be used to make whole blood based cytokine release assays even more powerful as screening tools in HBV immunology.


Subject(s)
Cytokines/blood , Hepatitis B virus/immunology , Hepatitis B, Chronic/immunology , Oligodeoxyribonucleotides/immunology , Adult , Aged , Cytokines/immunology , Female , Hepatitis B/immunology , Hepatitis B Core Antigens/immunology , Hepatitis B Surface Antigens/blood , Hepatitis B Surface Antigens/immunology , Hepatitis B Vaccines/immunology , Hepatitis B, Chronic/diagnosis , Humans , Immunity, Cellular , Interferon-gamma/blood , Interferon-gamma/immunology , Interleukin-2/blood , Interleukin-2/immunology , Lymphocyte Activation , Male , Middle Aged , Oligodeoxyribonucleotides/pharmacology , Vaccination
3.
J Transl Med ; 13: 157, 2015 May 13.
Article in English | MEDLINE | ID: mdl-25968473

ABSTRACT

BACKGROUND: Interferon gamma release assays (IGRA) have been developed to support easy and fast diagnosis of diseases like tuberculosis, and CMV in transplant patients. IGRAs focus on cellular immunity especially memory T cells and thus also allow rapid screening prior to complex flow cytometric testing. Here, we describe a novel, sensitive whole blood based cytokine release assay capable of assessing T cell responsiveness to HBV antigens in Hepatitis B patients and assessing hepatitis B vaccination status in healthy individuals. METHODS: Seventy two chronic Hepatitis B patients (CHB), 8 acute hepatitis B patients (AHB) and 80 healthy controls (HC) were tested by ELISA for IFNγ- and IL2-secretion in whole blood after challenge with synthetic peptide libraries of hepatitis B core antigen (HBcAg) or hepatitis B surface antigen (HBsAg). RESULTS: The developed IGRA test reliably differentiated between Hepatitis B patients, vaccinees and unvaccinated healthy controls. Treatment naïve and treated CHB patients showed a weaker IFNγ response to HBcAg (16 ± 5 and 35 ± 28 pg/ml, respectively) compared to the AHB group (82 ± 39 pg/ml), whereas HC remained unresponsive (6 ± 1 pg/ml). IL2 levels after HBcAg challenge were also higher in the AHB group compared to naive and treated CHB as well as HC (47 ± 21 vs. 12 ± 3, 15 ± 10 and 12 ± 9 pg/ml, respectively). HBsAg stimulation led to increased IFNγ and IL2 levels in the AHB group (33 ± 12 and 22 ± 12 pg/ml) and even higher levels in HC due to a high hepatitis B vaccination rate (41 ± 10 and 167 ± 58 pg/ml). Naive and treated CHB patients developed no or only weaker IFNγ or IL2 responses to HBsAg (5 ± 2 and 12 ± 7 pg/ml, for naive CHB, 12 ± 10 and 18 ± 15 pg/ml, for treated CHB). For HC, IL2 release after HBsAg stimulation depicted hepatitis B vaccination status with a diagnostic sensitivity and specificity of 85 % and 90 %. CONCLUSION: Our novel whole blood based cytokine release assay constitutes an easy and robust tool for screening HBV specific cellular immunity as alternative to flow cytometry or ELISPOT assays.


Subject(s)
Hepatitis B Core Antigens/blood , Hepatitis B Surface Antigens/blood , Hepatitis B virus/immunology , Hepatitis B, Chronic/blood , Hepatitis B, Chronic/immunology , Interferon-gamma Release Tests/methods , T-Lymphocytes/immunology , Adult , Case-Control Studies , Female , Hepatitis B virus/drug effects , Hepatitis B, Chronic/drug therapy , Humans , Interferon-gamma/blood , Interleukin-2/metabolism , Male , Middle Aged , Nucleotides/pharmacology , Nucleotides/therapeutic use , Peptides/immunology , ROC Curve , T-Lymphocytes/drug effects , Vaccination
4.
J Immunol Methods ; 414: 82-90, 2014 Dec 01.
Article in English | MEDLINE | ID: mdl-25450001

ABSTRACT

BACKGROUND: Interferon gamma release assays (IGRAs) are widely used to detect pathogen specific cellular immunity. Cytomegalovirus (CMV) is the foremost problematic viral infection in immunocompromised patients such as transplant or HIV infected patients. CMV antibody ELISAs are not able to predict CMV specific cellular immunity during immunosuppression. We developed a CMV specific IGRA comparing synthetic CMV peptides, native lysate and recombinant antigen. In addition, TLR agonists were tested to enhance CMV antigen immunogenicity. METHODS: 397 healthy controls (HC) were stratified according to CMV IgM and IgG serostatus and subsequently tested for IFNγ- and IL2-secretion in whole blood after challenge with synthetic, native or recombinant CMV antigens and TLR agonists by ELISA. The selected TLR agonists were lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), zymosan (Zym), polyinosinic-polycytidylic acid (Poly(I:C)), flagellin (Fla), R848, loxoribine (Lox) and bropirimine (Bro). RESULTS: Synthetic pp65 peptides elicited strong IFNγ responses in CMV seropositive, but not seronegative HC (6418 vs. 13 pg/ml). Native lysates and recombinant pp65 induced equally high IFNγ responses in seropositive (35,877 and 26,428 pg/ml) and increased background IFNγ expression in seronegative HC (43 and 1148 pg/ml). Diagnostic sensitivity and specificity with regard to anti-CMV serology reached 100% for synthetic pp65 and native CMV lysate, but 57% and 100% for recombinant pp65, respectively. TLR agonists LTA and Poly(I:C) augmented IFNγ responses after challenge with synthetic pp65 peptide, native lysate or recombinant pp65 in seropositive HC. Seronegative HC remained unaffected. IL2 production was negligible compared to IFNγ. CONCLUSION: IGRAs using synthetic CMV peptides or native lysate showed the best cytokine signal to noise ratio compared to recombinant antigen and TLR agonists LTA and Poly(I:C) constitute potential costimulating reagents.


Subject(s)
Antigens, Viral/immunology , Cytomegalovirus Infections/diagnosis , Cytomegalovirus/isolation & purification , Interferon-gamma Release Tests/methods , Phosphoproteins/immunology , Toll-Like Receptors/agonists , Viral Matrix Proteins/immunology , Adult , Biomarkers/analysis , Cytomegalovirus/immunology , Cytomegalovirus Infections/immunology , Cytomegalovirus Infections/virology , Female , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Interferon-gamma/blood , Interferon-gamma/metabolism , Interleukin-2/blood , Interleukin-2/metabolism , Male , Middle Aged , Phosphoproteins/chemical synthesis , Poly I-C/immunology , Viral Matrix Proteins/chemical synthesis
5.
PLoS One ; 9(8): e104742, 2014.
Article in English | MEDLINE | ID: mdl-25116393

ABSTRACT

BACKGROUNDS: The Loeys-Dietz syndrome (LDS) is an inherited connective tissue disorder caused by mutations in the transforming growth factor ß (TGF-ß) receptors TGFBR1 or TGFBR2. Most patients with LDS develop severe aortic aneurysms resulting in early need of surgical intervention. In order to gain further insight into the pathophysiology of the disorder, we investigated circulating outgrowth endothelial cells (OEC) from the peripheral blood of LDS patients from a cohort of 23 patients including 6 patients with novel TGF-ß receptor mutations. METHODS AND RESULTS: We performed gene expression profiling of OECs using microarray analysis followed by quantitative PCR for verification of gene expression. Compared to OECs of age- and sex-matched healthy controls, OECs isolated from three LDS patients displayed altered expression of several genes belonging to the TGF-ß pathway, especially those affecting bone morphogenic protein (BMP) signalling including BMP2, BMP4 and BMPR1A. Gene expression of BMP antagonist Gremlin-1 (GREM1) showed the most prominent up-regulation. This increase was confirmed at the protein level by immunoblotting of LDS-OECs. In immunohistochemistry, abundant Gremlin-1 protein expression could be verified in endothelial cells as well as smooth muscle cells within the arterial media. Furthermore, Gremlin-1 plasma levels of LDS patients were significantly elevated compared to healthy control subjects. CONCLUSIONS: These findings open new avenues in the understanding of the pathogenesis of Loeys-Dietz syndrome and the development of new diagnostic serological methods for early disease detection.


Subject(s)
Endothelial Cells/pathology , Intercellular Signaling Peptides and Proteins/biosynthesis , Loeys-Dietz Syndrome/physiopathology , Protein Serine-Threonine Kinases/genetics , Receptors, Transforming Growth Factor beta/genetics , Adult , Aorta/metabolism , Aorta/pathology , Bone Morphogenetic Protein 2/biosynthesis , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 4/biosynthesis , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein Receptors, Type I/biosynthesis , Bone Morphogenetic Protein Receptors, Type I/genetics , Cell Proliferation/genetics , Early Diagnosis , Female , Gene Expression Profiling , Humans , Latent TGF-beta Binding Proteins/biosynthesis , Loeys-Dietz Syndrome/diagnosis , Loeys-Dietz Syndrome/genetics , Male , Middle Aged , Muscle, Smooth, Vascular/metabolism , RNA, Messenger/biosynthesis , Receptor, Transforming Growth Factor-beta Type I , Receptor, Transforming Growth Factor-beta Type II , Young Adult
6.
J Immunol Methods ; 396(1-2): 107-15, 2013 Oct 31.
Article in English | MEDLINE | ID: mdl-23954282

ABSTRACT

BACKGROUND: Interferon gamma release assays (IGRA) have been developed to support the diagnosis of diseases like tuberculosis, which lack robust serological test systems. IGRAs focus on cellular immunity especially memory T cells and thus complement serological testing. However, the low frequency of antigen-specific memory T cells in peripheral blood limits IFNγ production to minute amounts and constitutes a major challenge for downstream test systems. We hypothesized that certain toll like receptor (TLR) agonists might enhance IFNγ production in IGRAs after antigen challenge without inducing background cytokine production. In addition, we investigated the potential use of IL2 release after TLR agonist application as another surrogate marker in cytokine release assays. METHODS: 176 healthy controls (HC) were tested for IFNγ- and IL2-secretion in whole blood in the presence of different TLR agonists with and without antigen challenge by ELISA. The selected TLR agonists were lipopolysaccharide (LPS ≙ TLR4), lipoteichoic acid (LTA ≙ TLR2), peptidoglycan (PGN ≙ TLR2), zymosan (Zym ≙ TLR2 and 6), polyinosinic-polycytidylic acid (Poly I:C ≙ TLR3), flagellin (Fla ≙ TLR5), R848 (≙TLR7 and 8), loxoribine (Lox ≙ TLR7) and bropirimine (Bro ≙ TLR7). RESULTS: TLR2 agonists LTA and PGN increased IFNγ secretion after antigen challenge nearly twofold (740 vs. 443 pg/ml for LTA and 969 vs. 469 pg/ml for PGN, respectively) without eliciting higher background expression. TLR3 agonist Poly(I:C) and TLR5 agonist Fla also induced a twofold increase in IFNγ synthesis (2.230 vs. 1.085 pg/ml for Poly(I:C) and 518 vs. 278 pg/ml for Fla, respectively), but background expression was slightly increased (114 vs. 7 pg/ml for Poly(I:C) and 47 vs. 12 pg/ml for Fla, respectively). IL2 production was not increased after antigen challenge in the presence of LTA, PGN, Poly(I:C) or Fla. The agonists LPS, Zym, R848, Lox and Bro did not raise cytokine synthesis after antigen challenge or they generated high levels of cytokines by themselves. CONCLUSION: Of all tested agonists TLR2-specific LTA and PGN met the requirements to increase IFNγ synthesis in whole blood after challenge with recall antigens without heightening basal cytokine levels alone. Thus, they constitute a potential costimulating reagent for IGRAs. IL2 did not show any potential as a surrogate marker in cytokine release assays in combination with TLR agonists.


Subject(s)
Interferon-gamma/blood , Lipopolysaccharides/metabolism , Peptidoglycan/metabolism , Teichoic Acids/metabolism , Toll-Like Receptor 2/blood , Adult , Biomarkers/blood , Cytokines/biosynthesis , Cytokines/metabolism , Female , Humans , Interferon-gamma/biosynthesis , Male , Toll-Like Receptor 2/agonists
7.
Eur J Immunol ; 43(10): 2741-9, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23817958

ABSTRACT

Human multipotent mesenchymal stromal cells (MSCs) are clinically applied to treat autoimmune diseases and graft-versus-host disease due to their immunomodulatory properties. Several molecules have been identified to mediate these effects, including constitutively expressed galectin-1. However, there are indications in the literature that MSCs exert enhanced immunosuppressive functions after interaction with an inflammatory environment. Therefore, we analyzed how inflammatory stimuli influence the expression of the galectin network in MSCs and functionally tested the relevance for the immunomodulatory effects of MSCs. We found that galectin-9 was strongly induced in MSCs upon interaction with activated PBMCs. Proinflammatory cytokines, such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), and also ligands of the Toll-like receptors (TLRs) TLR2, TLR3, and TLR4 elicited similar induction of galectin-9 in activated PBMCs. Galectin-9 was not only upregulated intracellularly, but also released by MSCs in significant amounts into the supernatant after exposure to proinflammatory stimuli. In proliferation assays, MSCs with a galectin-9 knockdown lost a significant portion of their antiproliferative effects on T cells. In conclusion, we found that unlike constitutively expressed galectin-1, galectin-9 is induced by several proinflammatory stimuli and released by MSCs. Thus, galectin-9 contributes to the inducible immunomodulatory functions of MSCs.


Subject(s)
Autoimmune Diseases/therapy , Galectins/metabolism , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/immunology , T-Lymphocytes/immunology , Autoimmune Diseases/immunology , Cell Proliferation , Cells, Cultured , Galectins/genetics , Humans , Immunosuppression Therapy , Inflammation/immunology , Interferon-gamma/metabolism , RNA, Small Interfering/genetics , Toll-Like Receptors/immunology , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation
8.
Blood ; 116(9): 1559-69, 2010 Sep 02.
Article in English | MEDLINE | ID: mdl-20505159

ABSTRACT

The matrix metalloproteinase (MMP) MT1-MMP plays pivotal roles in leukocyte physiology such as monocyte diapedesis, dendritic cell migration, and T-cell homing. MT1-MMP is a surface-anchored "master switch" proteinase that cleaves a variety of substrates including extracellular matrix components, matrix receptors, and also other MMPs. However, little is known about the mechanisms enabling intracellular trafficking and exposure of MT1-MMP on the cell surface. We now show that, in primary human macrophages, MT1-MMP-positive vesicles travel bidirectionally along microtubules, in a process regulated by KIF5B and KIF3A/KIF3B kinesins. SiRNA-induced knockdown revealed that transport by KIF5B and KIF3A/KIF3B is crucial for delivery of MT1-MMP to the cell surface and also for surface-associated functions of MT1-MMP, such as shedding of the matrix receptors CD44 and syndecan-1 or degradation of extracellular matrix at podosomes. These data show that kinesin-mediated intracellular transport of MT1-MMP is a pivotal process that allows macrophages to dynamically modify their pericellular environment. These data also identify specific kinesins as potential targets for the early manipulation of MT1-MMP activity in tissues.


Subject(s)
Extracellular Matrix/metabolism , Hyaluronan Receptors/metabolism , Kinesins/metabolism , Macrophages/metabolism , Matrix Metalloproteinase 14/metabolism , Blotting, Western , Cell Membrane/metabolism , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Kinesins/genetics , Macrophages/cytology , Matrix Metalloproteinase 14/genetics , Monocytes/cytology , Monocytes/metabolism , Plasmids , Syndecan-1/metabolism
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