Polarized monocyte response to cytokine stimulation

Background  

Mononuclear phagocytes (MPs) stand at the crossroads between the induction of acute inflammation to recruit and activate immune effector cells and the downmodulation of the inflammatory process to contain collateral damage. This decision is extensively modulated by the cytokine microenvironment, which includes a broad array of cytokines whose direct effect on MPs remains largely unexplored. Therefore, we tested whether polarized responses of MPs to pathogens are related to the influence of selected cytokines or represent a mandatory molecular switch through which most cytokines operate.     

A comparative study of protein synthesis in <Emphasis Type="Italic">in vitro</Emphasis> systems: from the prokaryotic reconstituted to the eukaryotic extract-based

Background  

Cell-free protein synthesis is not only a rapid and high throughput technology to obtain proteins from their genes, but also provides an in vitro platform to study protein translation and folding. A detailed comparison of in vitro protein synthesis in different cell-free systems may provide insights to their biological differences and guidelines for their applications.     

A systematic approach for testing expression of human full-length proteins in cell-free expression systems

Background  

The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems.     

Biotinylated-sortase self-cleavage purification (BISOP) method for cell-free produced proteins

Background  

Technology used for the purification of recombinant proteins is a key issue for the biochemical and structural analyses of proteins. In general, affinity tags, such as glutathione-S-transferase or six-histidines, are used to purify recombinant proteins. Since such affinity tags often interfere negatively with the structural and functional analyses of proteins, they are usually removed by treatment with proteases. Previously, Dr. H. Mao reported self-cleavage purification of a target protein by fusing the sortase protein to its N-terminal end, and subsequently obtained tag-free recombinant protein following expression in Escherichia coli. This method, however, is yet to be applied to the cell-free based protein production.     

RNA-directed cell-free synthesis of the galactose enzymes of Escherichia coli

Summary Conditions are described for the RNA-directed cell-free synthesis of the three galactose enzymes of Escherichia coli. Together with the DNA-directed synthesis described previously, this system permits the measurement of the three gene-products encoded by the gal operon as active enzymes synthesized in vitro in response to either gal-DNA or gal-RNA. The yield of enzyme is proportional to the amount of RNA added. Thus, the RNA-directed enzyme synthesis can serve as an assay system for functional mRNA. This test has been employed to determine the kinetics of synthesis and degradation of functional gal mRNA under the conditions of cell-free enzyme synthesis. The functional half-life of gal mRNA in this system is 6–7 minutes and is higher than expected from in vivo measurements.In contrast to the DNA-directed cell-free synthesis, the RNA-directed synthesis of the galactose enzymes is neither stimulated by cyclic adenosine-3:5-monophosphate nor by inducer.     

Fluorescent 3-hydroxy-4-pyridinone hexadentate iron chelators: intracellular distribution and the relevance to antimycobacterial properties

Abstract  

We report the synthesis and characterization of a fluorescent iron chelator (4), shown to be effective in inhibiting the growth of Mycobacterium avium in macrophages, together with the synthesis and characterization of two unsuccessful analogues selected to facilitate identification of the molecular properties responsible for the antimicrobial activity. Partition of the chelators in liposomes was investigated and the compounds were assessed with respect to uptake by macrophages, responsiveness to iron overload/iron deprivation and intracellular distribution by flow cytometry and confocal microscopy. The synthesis of the hexadentate chelators is based on a tetrahedral structure to which three bidentate 3-hydroxy-4-pyridinone chelating units are linked via amide bonds. The structure is synthetically versatile, allowing further addition of functional groups such as fluorophores. Here, we analyse the non-functionalized hexadentate unit (3) and the corresponding rhodamine B (4) and fluorescein (5) labelled chelators. The iron(III) stability constant was determined for 3 and the values log β = 34.4 and pFe³⁺ = 29.8 indicate an affinity for iron of the same order of magnitude as that of mycobacteria siderophores. Fluorescence properties in the presence of liposomes show that 4 strongly interacts with the lipid phase, whereas 5 does not. Such different behaviour may explain their distinct intracellular localization as revealed by confocal microscopy. The flow cytometry and confocal microscopy studies indicate that 4 is readily engulfed by macrophages and targeted to cytosol and vesicles of the endolysosomal continuum, whereas 5 is differentially distributed and only partially colocalizes with 4 after prolonged incubation. Differential distribution of the compounds is likely to account for their different efficacy against mycobacteria.     

Drosophila photoreceptor cells exploited for the production of eukaryotic membrane proteins: receptors, transporters and channels

Background

Membrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies.

Principal Findings

We describe the homologous and heterologous expression of 10 different targets from the three major MP classes - G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2–0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter - both involved in synaptic transmission - showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies.

Significance

We demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of eukaryotic MPs that have so far not been accessible to biochemical and biophysical studies.     

A simple and high-sensitivity method for analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis

Background  

Ubiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is also implicated in several critical cellular processes. Although Arabidopsis genome research has estimated more than 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrate a novel, simple and high-sensitive method for in vitro analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis and luminescent detection.     

Elevated Procoagulant Endothelial and Tissue Factor Expressing Microparticles in Women with Recurrent Pregnancy Loss

Background

15% of reproducing couples suffer from pregnancy loss(PL) and recurs in 2-3%. One of the most frequently hypothesized causes of unexplained PL refers to a defective maternal haemostatic response leading to uteroplacental thrombosis. Hereditary thrombophilia and antiphospholipid antibodies have been extensively described as risk factors for PL in women with unknown aetiology. Recently, a new marker has emerged: the cell-derived procoagulant circulating microparticles(MPs) which have been reported to have a major role in many thrombosis complicated diseases. This study aims to analyze the significance of procoagulant MPs in women suffering from unexplained recurrent pregnancy loss(RPL), and characterize their cellular origin.

Method and Findings

115 women with RPL were analyzed for common thrombophilia markers and different cell derived MPs-total annexinV, platelet(CD41a), endothelial(CD146,CD62e), leukocyte(CD45), erythrocyte(CD235a) and tissue factor(CD142)(TF) expressing MPs and were compared with 20 healthy non-pregnant women. Methodology for MP analysis was standardized by participating in the “Vascular Biology Scientific and Standardization Committee workshop”.

Results

Total annexinV, TF and endothelial MPs were found significantly increased(p<0.05, 95% confidence interval) in women with RPL. The procoagulant activity of MPs measured by STA-PPL clotting time assay was found in correspondence with annexinV MP levels, wherein the clot time was shortened in samples with increased MP levels. Differences in platelet, leukocyte and erythrocyte derived MPs were not significant. Thirty seven of 115 women were found to carry any of the acquired or hereditary thrombophilia markers. No significant differences were seen in the MP profile of women with and without thrombophilia marker.

Conclusion

The presence of elevated endothelial, TF and phosphatidylserine expressing MPs at a distance (at least 3 months) from the PL suggests a continued chronic endothelial damage/activation which may get exaggerated at the onset of pregnancy. The data suggests that MPs may contribute to uteroplacental thrombosis and are associated with the pathogenesis of RPL.     

Isolation and characterization of microparticles in sputum from cystic fibrosis patients

Background

Microparticles (MPs) are membrane vesicles released during cell activation and apoptosis. MPs have different biological effects depending on the cell from they originate. Cystic fibrosis (CF) lung disease is characterized by massive neutrophil granulocyte influx in the airways, their activation and eventually apoptosis. We investigated on the presence and phenotype of MPs in the sputum, a rich non-invasive source of inflammation biomarkers, of acute and stable CF adult patients.

Methods

Spontaneous sputum, obtained from 21 CF patients (10 acute and 11 stable) and 7 patients with primary ciliary dyskinesia (PCD), was liquefied with Sputasol. MPs were counted, visualized by electron microscopy, and identified in the supernatants of treated sputum by cytofluorimetry and immunolabelling for leukocyte (CD11a), granulocyte (CD66b), and monocyte-macrophage (CD11b) antigens.

Results

Electron microscopy revealed that sputum MPs were in the 100-500 nm range and did not contain bacteria, confirming microbiological tests. CF sputa contained higher number of MPs in comparison with PCD sputa. Levels of CD11a⁺-and CD66b⁺-, but not CD11b⁺-MPs were significantly higher in CF than in PCD, without differences between acute and stable patients.

Conclusions

In summary, MPs are detectable in sputa obtained from CF patients and are predominantly of granulocyte origin. This novel isolation method for MPs from sputum opens a new opportunity for the study of lung pathology in CF.     

Cell-free production of VDAC directly into liposomes for integration with biomimetic membrane systems

The mitochondrial voltage-dependent anion channel (VDAC) is a pivotal protein since it provides the major transport pathway between the cytosol and the mitochondrial intermembrane space and it is implicated in cell apoptosis by functioning as a gatekeeper for the trafficking of mitochondrial death molecules. VDAC is a beta-barrel channel with a large conductance, and we use it as a model transport protein for the design of biomimetic systems. To overcome the limitations of classical overexpression methods for producing and purifying membrane proteins (MPs) we describe here the use of an optimized cell-free system. In a one-step reaction VDAC is obtained directly integrated into liposomes and purified by ultracentrifugation. We then combine proteoliposomes with different bilayers models in order to validate VDAC insertion and functionality. This VDAC biomimetic model is the first example validating the use of a cell-free expression system for production of MPs into liposomes and tethered bilayers as a toolbox to build a wide range of biomimetic devices.     

TNFα expressed on the surface of microparticles modulates endothelial cell fate in rheumatoid arthritis

Background

Rheumatoid arthritis (RA) is associated with a high prevalence of atherosclerosis. Recently increased levels of microparticles (MPs) have been reported in patients with RA. MPs could represent a link between autoimmunity and endothelial dysfunction by expressing tumor necrosis factor alpha (TNFα), a key cytokine involved in the pathogenesis of RA, altering endothelial apoptosis and autophagy. The aim of this study was to investigate TNFα expression on MPs and its relationship with endothelial cell fate.

Methods

MPs were purified from peripheral blood from 20 healthy controls (HC) and from 20 patients with RA, before (time (T)0) and after (T4) 4-month treatment with etanercept (ETA). Surface expression of TNFα was performed by flow cytometry analysis. EA.hy926 cells, an immortalized endothelial cell line, were treated with RA-MPs purified at T0 and at T4 and also, with RA-MPs in vitro treated with ETA. Apoptosis and autophagy were then evaluated.

Results

RA-MPs purified at T0 expressed TNFα on their surface and this expression significantly decreased at T4. Moreover, at T0 RA-MPs, significantly increased both apoptosis and autophagy levels on endothelial cells, in a dose-dependent manner. RA-MPs did not significantly change these parameters after 4 months of in vivo treatment with ETA.

Conclusions

Our data demonstrate that MPs isolated from patients with RA exert a pathological effect on endothelial cells by TNFα expressed on their surface. In vivo and in vitro treatment with ETA modulates this effect, suggesting anti-TNF therapy protects against endothelial damage in patients with RA.

     

Proteomic analysis of microparticles isolated from malaria positive blood samples

Background

Malaria continues to be a great public health concern due to the significant mortality and morbidity associated with the disease especially in developing countries. Microparticles (MPs), also called plasma membrane derived extracellular vesicles (PMEVs) are subcellular structures that are generated when they bud off the plasma membrane. They can be found in healthy individuals but the numbers tend to increase in pathological conditions including malaria. Although, various studies have been carried out on the protein content of specific cellular derived MPs, there seems to be paucity of information on the protein content of circulating MPs in malaria and their association with the various signs and symptoms of the disease. The aim of this study was therefore to carry out proteomic analyses of MPs isolated from malaria positive samples and compare them with proteins of MPs from malaria parasite culture supernatant and healthy controls in order to ascertain the role of MPs in malaria infection.

Methods

Plasma samples were obtained from forty-three (43) malaria diagnosed patients (cases) and ten (10) healthy individuals (controls). Malaria parasite culture supernatant was obtained from our laboratory and MPs were isolated from them and confirmed using flow cytometry. 2D LC-MS was done to obtain their protein content. Resultant data were analyzed using SPSS Ver. 21.0 statistical software, Kruskal Wallis test and Spearman’s correlation coefficient r.

Results

In all, 1806 proteins were isolated from the samples. The MPs from malaria positive samples recorded 1729 proteins, those from culture supernatant were 333 while the control samples recorded 234 proteins. The mean number of proteins in MPs of malaria positive samples was significantly higher than that in the control samples. Significantly, higher quantities of haemoglobin subunits were seen in MPs from malaria samples and culture supernatant compared to control samples.

Conclusion

A great number of proteins were observed to be carried in the microparticles (MPs) from malaria samples and culture supernatant compared to controls. The greater loss of haemoglobin from erythrocytes via MPs from malaria patients could serve as the initiation and progression of anaemia in P.falciparum infection. Also while some proteins were upregulated in circulating MPs in malaria samples, others were down regulated.

     

A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells

Synthetic biology is an emerging field that aims at constructing artificial biological systems by combining engineering and molecular biology approaches. One of the most ambitious research line concerns the so-called semi-synthetic minimal cells, which are liposome-based system capable of synthesizing the lipids within the liposome surface. This goal can be reached by reconstituting membrane proteins within liposomes and allow them to synthesize lipids. This approach, that can be defined as biochemical, was already reported by us (Schmidli et al. J. Am. Chem. Soc. 113, 8127-8130, 1991). In more advanced models, however, a full reconstruction of the biochemical pathway requires (1) the synthesis of functional membrane enzymes inside liposomes, and (2) the local synthesis of lipids as catalyzed by the in situ synthesized enzymes. Here we show the synthesis and the activity - inside liposomes - of two membrane proteins involved in phospholipids biosynthesis pathway. The proteins, sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT), have been synthesized by using a totally reconstructed cell-free system (PURE system) encapsulated in liposomes. The activities of internally synthesized GPAT and LPAAT were confirmed by detecting the produced lysophosphatidic acid and phosphatidic acid, respectively. Through this procedure, we have implemented the first phase of a design aimed at synthesizing phospholipid membrane from liposome within from within — which corresponds to the autopoietic growth mechanism.     

Characterization of Microparticles after Hepatic Ischemia-Reperfusion Injury

Background

Hepatic ischemia-reperfusion (I/R) is a well-studied model of liver injury and has demonstrated a biphasic injury followed by recovery and regeneration. Microparticles (MPs) are a developing field of study and these small membrane bound vesicles have been shown to have effector function in other physiologic and pathologic states. This study was designed to quantify the levels of MPs from various cell origins–platelets, neutrophils, and endolethial cells–following hepatic ischemia-reperfusion injury.

Methods

A murine model was used with mice undergoing 90 minutes of partial hepatic ischemia followed by various times of reperfusion. Following reperfusion, plasma samples were taken and MPs of various cell origins were labeled and levels were measured using flow cytometry. Additionally, cell specific MPs were further assessed by Annexin V, which stains for the presence of phosphatidylserine, a cell surface marker linked to apoptosis. Statistical analysis was performed using one-way analysis of variance with subsequent Student-Newman-Keuls test with data presented as the mean and standard error of the mean.

Results

MPs from varying sources show an increase in circulating levels following hepatic I/R injury. However, the timing of the appearance of different MP subtypes differs for each cell type. Platelet and neutrophil-derived MP levels demonstrated an acute elevation following injury whereas endothelial-derived MP levels demonstrated a delayed elevation.

Conclusion

This is the first study to characterize circulating levels of cell-specific MPs after hepatic I/R injury and suggests that MPs derived from platelets and neutrophils serve as markers of inflammatory injury and may be active participants in this process. In contrast, MPs derived from endothelial cells increase after the injury response during the reparative phase and may be important in angiogenesis that occurs in the regenerating liver.     

Comparison of BCG,MPL and cationic liposome adjuvant systems in leishmanial antigen vaccine formulations against murine visceral leishmaniasis

Background  

The development of an effective vaccine against visceral leishmaniasis (VL) caused by Leishmania donovani is an essential aim for controlling the disease. Use of the right adjuvant is of fundamental importance in vaccine formulations for generation of effective cell-mediated immune response. Earlier we reported the protective efficacy of cationic liposome-associated L. donovani promastigote antigens (LAg) against experimental VL. The aim of the present study was to compare the effectiveness of two very promising adjuvants, Bacille Calmette-Guerin (BCG) and Monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) with cationic liposomes, in combination with LAg, to confer protection against murine VL.     

The relationship between disease activity, sleep, psychiatric distress and pain sensitivity in rheumatoid arthritis: a cross-sectional study

Introduction

Cell stimulation leads to the shedding of phosphatidylserine (PS)-rich microparticles (MPs). Because autoimmune diseases (AIDs) are characterized by cell activation, we investigated level of circulating MPs as a possible biomarker in primary Sjögren's syndrome (pSS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).

Methods

We measured plasma levels of total, platelet and leukocyte MPs by prothrombinase capture assay and flow cytometry in 43 patients with pSS, 20 with SLE and 24 with RA and in 44 healthy controls (HCs). Secretory phospholipase A2 (sPLA2) activity was assessed by fluorometry. Soluble CD40 ligand (sCD40L) and soluble P-selectin (sCD62P), reflecting platelet activation, were measured by ELISA.

Results

Patients with pSS showed increased plasma level of total MPs (mean ± SEM 8.49 ± 1.14 nM PS equivalent (Eq), P < 0.0001), as did patients with RA (7.23 ± 1.05 n PS Eq, P = 0.004) and SLE (7.3 ± 1.25 nM PS Eq, P = 0.0004), as compared with HCs (4.13 ± 0.2 nM PS Eq). Patients with AIDs all showed increased level of platelet MPs (P < 0.0001), but only those with pSS showed increased level of leukocyte MPs (P < 0.0001). Results by capture assay and flow cytometry were correlated. In patients with high disease activity according to extra-glandular complications (pSS), DAS28 (RA) or SLEDAI (SLE) compared with low-activity patients, the MP level was only slightly increased in comparison with those having a low disease activity. Platelet MP level was inversely correlated with anti-DNA antibody level in SLE (r = -0.65; P = 0.003) and serum β2 microglobulin level in pSS (r = -0.37; P < 0.03). The levels of total and platelet MPs were inversely correlated with sPLA2 activity (r = -0.37, P = 0.0007; r = -0.36, P = 0.002, respectively). sCD40L and sCD62P concentrations were significantly higher in pSS than in HC (P ≤ 0.006).

Conclusions

Plasma MP level is elevated in pSS, as well as in SLE and RA, and could be used as a biomarker reflecting systemic cell activation. Level of leukocyte-derived MPs is increased in pSS only. The MP level is low in case of more severe AID, probably because of high secretory phospholipase A2 (sPLA2) activity, which leads to consumption of MPs. Increase of platelet-derived MPs, sCD40L and sCD62P, highlights platelet activation in pSS.