MTA ELTE Immunology Research Group

Budapest, Hungary

MTA ELTE Immunology Research Group

Budapest, Hungary

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Orgovan N.,Eötvös Loránd University | Orgovan N.,Hungarian Academy of Sciences | Salanki R.,Hungarian Academy of Sciences | Salanki R.,University of Pannonia | And 6 more authors.
Biosensors and Bioelectronics | Year: 2014

Adhesion and spreading of primary monocytes isolated from human blood were monitored utilizing optical waveguide lightmode spectroscopy (OWLS); a highly sensitive label-free biosensor technique using evanescent optical waves generated at a biocompatible surface. Appropriate development on a custom built setup enabled the OWLS cuvette to be operated as a 1.5ml mini-incubator, controlling both temperature and CO2 levels. The incubator-equipped OWLS is readily applicable for delicate and long-term studies on sensitive primary cells, demonstrated here through monitoring the serum dependence of the adhesion and spreading of human monocytes. Moreover, the custom-built setup enables the simultaneous monitoring of the position and overall width of the OWLS resonant peaks. This unique feature makes it possible to distinguish the refractive index variations induced by the adsorption of secreted material from refractive index changes provoked by cellular spreading. A definite attachment and spreading activity was observed on the substratum (glassy silica-titania), when the serum level of the culturing medium was 0.0-0.01%. Increasing serum concentration resulted in a steep fall in monocyte surface adhesion and spreading. 1.0% serum level practically abolished all spreading activity measured by OWLS, and the number of attached cells was significantly decreased, too. Serum addition to fully spread cells provoked a reduction in the cell-substratum contact area, clearly detectable by the biosensor. Cell spreading was inhibited by pre-coating the sensor surface with considerable amounts of serum proteins. These findings suggest that monocyte spreading is inhibited by the adsorption of serum biomolecules to the substratum, rather than by soluble factors present in the serum. All of these results were obtained completely noninvasively with real time monitoring; demonstrating the capabilities of OWLS to sensitively monitor the adhesion properties of immune cells isolated from human blood. The current study is, therefore, a significant step towards the application of label-free optical biosensors in medical diagnostics. © 2013 Elsevier B.V.


PubMed | Hungarian Academy of Sciences, University of Pannonia, Eötvös Loránd University and MTA ELTE Immunology Research Group
Type: | Journal: Scientific reports | Year: 2016

Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1-2 nanoliters from a thin (~100m) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1m positioning precision, adaptive cell targeting and below 1nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved.


Prechl J.,Diagnosticum Zrt | Prechl J.,MTA ELTE Immunology Research Group | Czirjak L.,University of Pécs
F1000Research | Year: 2015

Systemic lupus erythematosus (SLE) is a heterogeneous multifactorial systemic autoimmune disease affecting several organs. SLE can start relatively early in life and results in impaired quality of life and shortened life expectancy because of a gradual disease progression leading to cardiovascular, renal and neoplastic disease. The basic mechanisms of the pathogenesis of the disease still remain to be clarified. It is clear that complement proteins play a key and complex role in the development of SLE. Complement component C1q has been known to be a fundamental component of lupus development, but most explanations focus on its role in apoptotic debris removal. Importantly, C1q was recently found to play a key role in the maintenance of vascular endothelial integrity. We suggest that apoptotic products, endothelial cells and extracellular matrix components, which display negatively charged moieties, compete for binding to molecules of the innate humoral immune response, like C1q. Genetic or acquired factors leading to an increased load of apoptotic cell debris and decrease or absence of C1q therefore interfere with the regulation of endothelial permeability and integrity. Furthermore, we suggest that lupus is the net result of an imbalance between the two functions of immune clearance and vascular endothelial integrity maintenance, an imbalance triggered and sustained by autoimmunity, which skews C1q consumption by IgG-mediated complement classical pathway activation on autoantigens. In this triangle of innate clearance, autoimmunity and endothelial integrity, C1q plays a central role. Hence, we interpret the pathogenesis of lupus by identifying three key components, namely innate immune clearance, autoimmunity and endothelial integrity and we establish a link between these components based on the protective role that innate clearance molecules play in endothelial renewal. By including the vasoprotective role of C1q in the interpretation of SLE development we attempt to provide novel explanations for the symptoms, organ damage, diagnostic and therapeutic difficulties of the disease. © 2015 Prechl J and Czirják L.


PubMed | Eötvös Loránd University, Clinic Center, Hungarian Academy of Sciences, University of Florence and 2 more.
Type: Journal Article | Journal: PloS one | Year: 2016

Our study tested the hypothesis that immunoglobulins differ in their ability to activate the nuclear factor-B pathway mediated cellular responses. These responses are modulated by several properties of the immune complex, including the ratio of antibody isotypes binding to antigen. Immunoassays allow the measurement of antigen specific antibodies belonging to distinct immunoglobulin classes and subclasses but not the net biological effect of the combination of these antibodies. We set out to develop a biosensor that is suitable for the detection and characterization of antigen specific serum antibodies. We genetically modified the monocytoid U937 cell line carrying Fc receptors with a plasmid encoding NF-B promoter-driven GFP. This clone, U937-NF-B, was characterized with respect to FcR expression and response to solid-phase immunoglobulins. Human IgG3, IgG4 and IgG1 induced GFP production in a time- and dose-dependent manner, in this order of efficacy, while IgG2 triggered no activation at the concentrations tested. IgA elicited no response alone but showed significant synergism with IgG3 and IgG4. We confirmed the importance of activation via FcRI by direct stimulation with monoclonal antibody and by competition assays. We used citrullinated peptides and serum from rheumatoid arthritis patients to generate immune complexes and to study the activation of U937-NF-B, observing again a synergistic effect between IgG and IgA. Our results show that immunoglobulins have distinct pro-inflammatory potential, and that U937-NF-B is suitable for the estimation of biological effects of immune-complexes, offering insight into monocyte activation and pathogenesis of antibody mediated diseases.


Herbath M.,Eötvös Loránd University | Szekeres Z.,MTA ELTE Immunology Research Group | Kovesdi D.,Eötvös Loránd University | Papp K.,MTA ELTE Immunology Research Group | And 3 more authors.
Immunology Letters | Year: 2014

CpG oligodeoxynucleotides (CpG) are widely studied as promising adjuvants in vaccines against a range of diseases including infection, cancer or allergy. Conjugating antigen to CpG has been shown to potentiate the adjuvant effect via enhancing antigen uptake and danger signaling by the very same cell. In the present study, using biotinylated CpG and streptavidin as a model system, we demonstrate that CpG motif containing free and antigen-conjugated oligonucleotides do not compete in terms of cell activation via TLR9, but do compete for cellular uptake. Antigen-conjugated CpG enhances cellular association and uptake of the antigen by antigen-presenting cells (APC) and T cells. Free CpG efficiently competes with antigen-CpG conjugates in BMDC and T cells, but shows weak or no competition in B cells that have higher TLR9 expression. Vaccination with antigen-conjugated CpG or with a mixture of antigen and CpG elevates the level of antigen-specific antibodies but co-administration of CpG-antigen conjugates and free CpG adversely effects immunogenicity. These observations may help optimize CpG-based vaccine formulation. © 2014 Elsevier B.V.


Herbath M.,MTA ELTE Immunology Research Group | Papp K.,Eötvös Loránd University | Erdei A.,Eötvös Loránd University | Prechl J.,MTA ELTE Immunology Research Group
Journal of Immunology Research | Year: 2015

Natural and synthetic nucleic acids are known to exert immunomodulatory properties. Notably, nucleic acids are known to modulate immune function via several different pathways and various cell types, necessitating a complex interpretation of their effects. In this study we set out to compare the effects of a CpG motif containing oligodeoxynucleotide (ODN) with those of a control and an inhibitory non-CpG ODN during cognate B cell-T cell interactions. We employed an antigen presentation system using splenocytes from TCR transgenic DO11.10 mice and the ovalbumin peptide recognized by the TCR as model antigen. We followed early activation events by measuring CD69 expression, late activation by MHC class II expression, cell division and antibody production of switched, and nonswitched isotypes. We found that both of the tested non-CpG ODN exerted significant immunomodulatory effects on early T cell and on late B cell activation events. Importantly, a synergism between non-CpG effects and T cell help acting on B cells was observed, resulting in enhanced IgG production following cognate T cell-B cell interactions. We propose that non-CpG ODN may perform as better adjuvants when a strong antigen-independent immune activation, elicited by CpG ODNs, is undesirable. © 2015 Melinda Herbáth et al.


PubMed | Eötvös Loránd University and MTA ELTE Immunology Research Group
Type: | Journal: Journal of immunology research | Year: 2015

Natural and synthetic nucleic acids are known to exert immunomodulatory properties. Notably, nucleic acids are known to modulate immune function via several different pathways and various cell types, necessitating a complex interpretation of their effects. In this study we set out to compare the effects of a CpG motif containing oligodeoxynucleotide (ODN) with those of a control and an inhibitory non-CpG ODN during cognate B cell-T cell interactions. We employed an antigen presentation system using splenocytes from TCR transgenic DO11.10 mice and the ovalbumin peptide recognized by the TCR as model antigen. We followed early activation events by measuring CD69 expression, late activation by MHC class II expression, cell division and antibody production of switched, and nonswitched isotypes. We found that both of the tested non-CpG ODN exerted significant immunomodulatory effects on early T cell and on late B cell activation events. Importantly, a synergism between non-CpG effects and T cell help acting on B cells was observed, resulting in enhanced IgG production following cognate T cell-B cell interactions. We propose that non-CpG ODN may perform as better adjuvants when a strong antigen-independent immune activation, elicited by CpG ODNs, is undesirable.


PubMed | Eötvös Loránd University, MTA ELTE Immunology Research Group and Debrecen University
Type: Journal Article | Journal: Immunology letters | Year: 2015

The role of complement in the regulation of T cell immunity has been highlighted recently by several groups. We were prompted to reinvestigate the role of complement receptor type 1 (CR1, CD35) [corrected] in human T cells based on our earlier data showing that activated human T cells produce C3 (Torok et al. (2012) [48]) and also by results demonstrating that engagement of Membrane Cofactor Protein (MCP, CD46) induces a switch of anti-CD35-activated [corrected] helper T cells into regulatory T cells (Kemper et al. (2003) [17]). We demonstrate here that co-ligation of CD46 and CD35, [corrected] the two C3b-binding structures present on activated CD4+ human T cells significantly enhances CD25 expression, elevates granzyme B production and synergistically augments cell proliferation. The role of CR1 in the development of the Treg phenotype was further confirmed by demonstrating that its engagement enhances IL-10 production and reduces IFN release by the activated CD4+ T cells in the presence of excess IL-2. The functional in vivo relevance of our findings was highlighted by the immunohistochemical staining of tonsils, revealing the presence of CD4/CD35 [corrected] double positive lymphocytes mainly in the inter-follicular regions where direct contact between CD4+ T cells and B lymphocytes occurs. Regarding the in vivo relevance of the complement-dependent generation of regulatory T cells in secondary lymphoid organs we propose a scenario shown in the figure. The depicted process involves the sequential binding of locally produced C3 fragments to CD46 and CD35 [corrected] expressed on activated T cells, which - in the presence of excess IL-2 - leads to the development of Treg cells.


Kremlitzka M.,MTA ELTE Immunology Research Group | Macsik-Valent B.,Eötvös Loránd University | Erdei A.,Eötvös Loránd University | Erdei A.,MTA ELTE Immunology Research Group
Cellular and Molecular Life Sciences | Year: 2015

B cells are efficiently activated by CpG oligodeoxynucleotides (ODNs) to produce pro-inflammatory cytokines and antibody (Ab). Here, we describe a so far unidentified, spleen tyrosine kinase (Syk)-dependent pathway, which is indispensable for CpG-induced human B cell activation. We show that triggering of B cells by CpG results in Syk and src kinase phosphorylation, proliferation, as well as cytokine and Ab production independent of the BCR. Notably, all these functions are abrogated when Syk is inhibited. We demonstrate that CpG-induced Syk activation originates from the cell surface in a TLR9-dependent manner. While inhibition of Syk does not influence the uptake of CpG ODNs, activation of the kinase is a prerequisite for the delivery of CpG into TLR9-containing endolysosomes and for the CpG-induced up-regulation of TLR9 expression. Our results reveal an alternative, Syk-dependent pathway of CpG-induced B cell stimulation, which is initiated at the plasma membrane and seems to be an upstream requirement for endosomal TLR9-driven B cell proliferation and differentiation. © 2014 Springer Basel.


PubMed | University of Pécs and MTA ELTE Immunology Research Group
Type: | Journal: F1000Research | Year: 2015

Systemic lupus erythematosus (SLE) is a heterogeneous multifactorial systemic autoimmune disease affecting several organs. SLE can start relatively early in life and results in impaired quality of life and shortened life expectancy because of a gradual disease progression leading to cardiovascular, renal and neoplastic disease. The basic mechanisms of the pathogenesis of the disease still remain to be clarified. It is clear that complement proteins play a key and complex role in the development of SLE. Complement component C1q has been known to be a fundamental component of lupus development, but most explanations focus on its role in apoptotic debris removal. Importantly, C1q was recently found to play a key role in the maintenance of vascular endothelial integrity. We suggest that apoptotic products, endothelial cells and extracellular matrix components, which display negatively charged moieties, compete for binding to molecules of the innate humoral immune response, like C1q. Genetic or acquired factors leading to an increased load of apoptotic cell debris and decrease or absence of C1q therefore interfere with the regulation of endothelial permeability and integrity. Furthermore, we suggest that lupus is the net result of an imbalance between the two functions of immune clearance and vascular endothelial integrity maintenance, an imbalance triggered and sustained by autoimmunity, which skews C1q consumption by IgG-mediated complement classical pathway activation on autoantigens. In this triangle of innate clearance, autoimmunity and endothelial integrity, C1q plays a central role. Hence, we interpret the pathogenesis of lupus by identifying three key components, namely innate immune clearance, autoimmunity and endothelial integrity and we establish a link between these components based on the protective role that innate clearance molecules play in endothelial renewal. By including the vasoprotective role of C1q in the interpretation of SLE development we attempt to provide novel explanations for the symptoms, organ damage, diagnostic and therapeutic difficulties of the disease.

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