Andrea Balogh

Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies.

Placental Protein 13 (PP13) – A Placental Immunoregulatory Galectin Protecting Pregnancy

Galectins are glycan-binding proteins that regulate innate and adaptive immune responses, and some confer maternal-fetal immune tolerance in eutherian mammals. A chromosome 19 cluster of galectins has emerged in anthropoid primates, species with deep placentation and long gestation. Three of the five human cluster galectins are solely expressed in the placenta, where they may confer additional immunoregulatory functions to enable deep placentation. One of these is galectin-13, also known as Placental Protein 13 (PP13). It has a “jelly-roll” fold, carbohydrate-recognition domain and sugar-binding preference resembling other mammalian galectins. PP13 is predominantly expressed by the syncytiotrophoblast and released from the placenta into the maternal circulation. Its ability to induce apoptosis of activated T cells in vitro, and to divert and kill T cells as well as macrophages in the maternal decidua in situ, suggests important immune functions. Indeed, mutations in the promoter and an exon of LGALS13 presumably leading to altered or non-functional protein expression are associated with a higher frequency of preeclampsia and other obstetrical syndromes, which involve immune dysregulation. Moreover, decreased placental expression of PP13 and its low concentrations in first trimester maternal sera are associated with elevated risk of preeclampsia. Indeed, PP13 turned to be a good early biomarker to assess maternal risk for the subsequent development of pregnancy complications caused by impaired placentation. Due to the ischemic placental stress in preterm preeclampsia, there is increased trophoblastic shedding of PP13 immunopositive microvesicles starting in the second trimester, which leads to high maternal blood PP13 concentrations. Our meta-analysis suggests that this phenomenon may enable the potential use of PP13 in directing patient management near to or at the time of delivery. Recent findings on the beneficial effects of PP13 on decreasing blood pressure due to vasodilatation in pregnant animals suggest its therapeutic potential in preeclampsia.

Some new faces of membrane microdomains: A complex confocal fluorescence, differential polarization, and FCS imaging study on live immune cells

Lipid rafts are cholesterol‐ and glycosphingolipid‐rich plasma membrane microdomains, which control signal transduction, cellular contacts, pathogen recognition, and internalization processes. Their stability/lifetime, heterogeneity remained still controversial, mostly due to the high diversity of raft markers and cellular models. The correspondence of the rafts of living cells to liquid ordered (Lo) domains of model membranes and the effect of modulating rafts on the structural dynamics of their bulk membrane environment are also yet unresolved questions. Spatial overlap of various lipid and protein raft markers on live cells was studied by confocal laser scanning microscopy, while fluorescence polarization of DiIC18(3) and Bodipy‐phosphatidylcholine was imaged with differential polarization CLSM (DP‐CLSM). Mobility of the diI probe under different conditions was assessed by fluorescence correlation spectroscopic (FCS). GM1 gangliosides highly colocalized with GPI‐linked protein markers of rafts and a new anti‐cholesterol antibody (AC8) in various immune cells. On the same cells, albeit not fully excluded from rafts, diI colocalized much less with raft markers of both lipid and protein nature, suggesting the Lo membrane regions are not equivalents to lipid rafts. The DP‐CLSM technique was capable of imaging probe orientation and heterogeneity of polarization in the plasma membrane of live cells, reflecting differences in lipid order/packing. This property—in accordance with diI mobility assessed by FCS—was sensitive to modulation of rafts either through their lipids or proteins. Our complex imaging analysis demonstrated that two lipid probes—GM1 and a new anti‐cholesterol antibody—equivocally label the membrane rafts on a variety of cell types, while some raft‐associated proteins (MHC‐II, CD48, CD59, or CD90) do not colocalize with each other. This indicates the compositional heterogeneity of rafts. Usefulness of the DP‐CLSM technique in imaging immune cell surface, in terms of lipid order/packing heterogeneities, was also shown together with its sensitivity to monitor biological modulation of lipid rafts.

Exploiting fluorescence for multiplex immunoassays on protein microarrays

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.

Differential detergent sensitivity of extracellular vesicle subpopulations.

Extracellular vesicles (including exosomes, microvesicles and apoptotic bodies) are currently attracting rapidly increasing attention from various fields of biology due to their ability to carry complex information and act as autocrine, paracrine and even endocrine intercellular messengers. In the present study we investigated the sensitivity of size-based subpopulations of extracellular vesicles to different concentrations of detergents including sodium dodecyl sulphate, Triton X-100, Tween 20 and deoxycholate. We determined the required detergent concentration that lysed each of the vesicle subpopulations secreted by Jurkat, THP-1, MiaPaCa and U937 human cell lines. We characterized the vesicles by tunable resistive pulse sensing, flow cytometry and transmission electron microscopy. Microvesicles and apoptotic bodies were found to be more sensitive to detergent lysis than exosomes. Furthermore, we found evidence that sodium dodecyl sulphate and Triton X-100 were more effective in vesicle lysis at low concentrations than deoxycholate or Tween 20. Taken together, our data suggest that a combination of differential detergent lysis with tunable resistive pulse sensing or flow cytometry may prove useful for simple and fast differentiation between exosomes and other extracellular vesicle subpopulations as well as between vesicular and non-vesicular structures.

Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions.

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely related 3β-hydroxyl sterols, and they reacted with human lipoproteins VLDL, LDL, and HDL. They bound, usually with low avidity, to live human or murine lymphocyte and monocyte-macrophage cell lines, which was enhanced substantially by a moderate papain digestion of the cell surface, removing some protruding extracellular protein domains. Cell-bound ACHAs strongly colocalized with markers of cholesterol-rich lipid rafts and caveolae at the cell surface and intracellularly with markers of the endoplasmic reticulum and Golgi complex. These data suggest that these IgG ACHAs may serve as probes of clustered cholesterol (e.g., different lipid rafts) in live cells and thus may also have immunomodulatory potential.

Cytometry of raft and caveola membrane microdomains: from flow and imaging techniques to high throughput screening assays.

The evolutionarily developed microdomain structure of biological membranes has gained more and more attention in the past decade. The caveolin‐free “membrane rafts,” the caveolin‐expressing rafts (caveolae), as well as other membrane microdomains seem to play an essential role in controlling and coordinating cell‐surface molecular recognition, internalization/endocytosis of the bound molecules or pathogenic organisms and in regulation of transmembrane signal transduction processes. Therefore, in many research fields (e.g. neurobiology and immunology), there is an ongoing need to understand the nature of these microdomains and to quantitatively characterize their lipid and protein composition under various physiological and pathological conditions. Flow and image cytometry offer many sophisticated and routine tools to study these questions. In this review, we give an overview of the past efforts to detect and characterize these membrane microdomains by the use of classical cytometric technologies, and finally we will discuss the results and perspectives of a new line of raft cytometry, the “high throughput screening assays of membrane microdomains,” based on “lipidomic” and “proteomic” approaches.

Galectins: Double-edged Swords in the Cross-roads of Pregnancy Complications and Female Reproductive Tract Inflammation and Neoplasia.

Galectins are an evolutionarily ancient and widely expressed family of lectins that have unique glycan-binding characteristics. They are pleiotropic regulators of key biological processes, such as cell growth, proliferation, differentiation, apoptosis, signal transduction, and pre-mRNA splicing, as well as homo- and heterotypic cell-cell and cell-extracellular matrix interactions. Galectins are also pivotal in immune responses since they regulate host-pathogen interactions, innate and adaptive immune responses, acute and chronic inflammation, and immune tolerance. Some galectins are also central to the regulation of angiogenesis, cell migration and invasion. Expression and functional data provide convincing evidence that, due to these functions, galectins play key roles in shared and unique pathways of normal embryonic and placental development as well as oncodevelopmental processes in tumorigenesis. Therefore, galectins may sometimes act as double-edged swords since they have beneficial but also harmful effects for the organism. Recent advances facilitate the use of galectins as biomarkers in obstetrical syndromes and in various malignancies, and their therapeutic applications are also under investigation. This review provides a general overview of galectins and a focused review of this lectin subfamily in the context of inflammation, infection and tumors of the female reproductive tract as well as in normal pregnancies and those complicated by the great obstetrical syndromes.

CD3ζ-Chain Expression of Human T Lymphocytes Is Regulated by TNF via Src-like Adaptor Protein-Dependent Proteasomal Degradation

Decreased expression of the TCR ζ-chain has been reported in several autoimmune, inflammatory, and malignant diseases, suggesting that ζ-chain downregulation is common at sites of chronic inflammation. Although ζ-chain is critically important in T lymphocyte activation, the mechanism of the decreased ζ-chain expression is less clear. Src-like adaptor protein (SLAP) is a master regulator of T cell activation; previous data have reported that SLAP regulates immunoreceptor signaling. We have examined the mechanism and the functional consequences of CD3 ζ-chain downregulation. TNF treatment of human T lymphocytes (15–40 ng/ml) selectively downregulates CD3 ζ-chain expression in a dose-dependent manner (p < 0.05) and decreases activation-induced IL-2 expression (p < 0.01). Although blocking of the lysosomal compartment fails to restore TNF-induced CD3 ζ-chain downregulation, inhibition of the proteasome prevented the effect of TNF. Both SLAP expression and the colocalization of SLAP with CD3 ζ-chain was enhanced by TNF treatment (p < 0.05 and p < 0.01, respectively), whereas TNF-induced ζ-chain downregulation was inhibited by gene silencing of SLAP with small interfering RNA. SLAP levels of the CD4+ T lymphocytes isolated from patients with rheumatoid arthritis were more than 2-fold higher than that of the healthy donors’ (p < 0.05); moreover, TNF treatment did not alter the SLAP expression of the CD4+ cells of anti-TNF therapy-treated patients. Our present data suggest that TNF modulates T cell activation during inflammatory processes by regulating the amount of CD3 ζ-chain expression via a SLAP-dependent mechanism. These data provide evidence for SLAP-dependent regulation of CD3 ζ-chain in the fine control of TCR signaling.

New cholesterol-specific antibodies remodel HIV-1 target cells’ surface and inhibit their in vitro virus production

The importance of membrane rafts in HIV-1 infection is still in the focus of interest. Here, we report that new monoclonal anticholesterol IgG antibodies (ACHAs), recognizing clustered membrane cholesterol (e.g., in lipid rafts), rearrange the lateral molecular organization of HIV-1 receptors and coreceptors in the plasma membrane of HIV-1 permissive human T-cells and macrophages. This remodeling is accompanied with a substantial inhibition of their infection and HIV-1 production in vitro. ACHAs promote the association of CXCR4 with both CD4 and lipid rafts, consistent with the decreased lateral mobility of CXCR4, while Fab fragments of ACHAs do not show these effects. ACHAs do not directly mask the extracellular domains of either CD4 or CXCR4 nor do they affect CXCR4 internalization. No significant inhibition of HIV production is seen when the virus is preincubated with the antibodies prior to infection. Thus, we propose that the observed inhibition is mainly due to the membrane remodeling induced by cholesterol-specific antibodies on the target cells. This, in turn, may prevent the proper spatio-temporal juxtaposition of HIV-1 glycoproteins with CD4 and chemokine receptors, thus negatively interfering with virus attachment/entry.