Kinetic analysis of mineral formation during in vitro modeling of matrix vesicle mineralization: effect of annexin A5, phosphatidylserine, and type II collagen

Matrix vesicles (MVs) are involved in de novo mineral formation by nearly all vertebrate tissues. The driving force for MV mineralization is a nucleational core composed of three principal constituents: (i) amorphous calcium phosphate (ACP), complexed in part with phosphatidylserine (PS) to form (ii) calcium-phosphate-lipid complexes (CPLX), and (iii) annexin A5 (AnxA5), the principal lipid-dependent Ca(2+)-binding protein in MVs. We describe methods for reconstituting the nucleational core using a biomimetic approach and for analyzing the kinetics of its induction of mineral formation. The method is based on light scattering by the nascent crystallites at 340 nm and monitors mineral formation at regular intervals without disturbing the system using an automated plate reader. It yields precise replicate values that typically agree within less than 5%. As with MVs, mineral formation by the synthetic complex follows a sigmoidal pattern; following a quiescent induction period, rapid formation ensues for a limited time, followed by a distinct decline in rate that continues to slow, ultimately reaching a maximal asymptotic value. Key to quantization of mineral formation is the use of first-derivative analysis, which defines the induction time, the rate and the amount of initial mineral formation. Furthermore, using a five-parameter logistic curve-fitting algorithm, the maximal amount of mineral formation can be predicted accurately. Using these methods, we document the dramatic finding that AnxA5 synergistically activates PS-CPLX, transforming it from a very weak nucleator of mineral formation to a potent one. The methods presented should enable systematic study of the effects of numerous other factors thought to contribute to mineral formation.     

Diversity of mineral cell coverings and their formation processes: a review focused on the siliceous cell coverings

Mineral cell coverings are found in various protists. Some macroalgae accumulate calcium carbonate in the intercellular space, and some unicellular organisms use calcium carbonate or silica for the construction of loricas, scales, and frustules. Diatoms are representatives of those utilizing silica for the material of the cell covering called a frustule. The development of the frustule is initiated in a silica-deposition vesicle (SDV), which occurs just beneath the plasma membrane and, subsequently, the silicified cell covering expands its area, following the expansion of the SDV from valve face to valve mantle. Sequential valve development with whole valves is reviewed in several diatoms placed in different phylogenetic positions. Every diatom commences its valve formation from its pattern center and then develops by means of individual procedures. The results indicate that the valve development reflects the phylogeny of diatoms. In addition, recent progress in silica biomineralization is briefly reviewed, and the phylogeny of ability concerning siliceous cell covering formation is inferred. Electronic Publication     

Isotopic effects on retention times of caffeine and its metabolites 1,3,7-trimethyluric acid, theophylline, theobromine and paraxanthine

Physicochemical parameters that influence gas chromatographic separation are numerous. Consequently, isotope labelling, because it modifies physicochemical properties, can induce isotopic effects on retention time. Caffeine has been chosen to study this influence because as itself and its metabolites, it allows the preparation of different methylxanthine isotopomers and thus is one of the best models to study isotopic effects induced by stable isotope labelling. Using a caffeine molecule labelled with deuterium at different positions and rat hepatocytes to obtain metabolites, it was possible to study the influence of labelling on retention time [(14% cyanopropylphenyl)methylpolysiloxane] and to point out the role of each labelled site. It appears that isotopic effects induced by the labelling depend not only on the number of labelling atoms but also on whether this labelling is at position 1, 3 or 7 and, consequently, on the role of the labelled site on the function of the molecule.     

The effects of isomaltulose,isomalt, and isomaltulose-based oligomers on mineral absorption and retention

We carried out a balance study to examine the effects of isomaltulose, lactose, isomalt, and isomaltulose-based oligomers (IBOs) on mineral (calcium, magnesium, phosphorus, and iron) absorption and retention. Four-week-old male Wistar rats were divided into five groups of six rats each and fed a basal diet or diet the containing either 5% isomaltulose, 5% lactose, 5% isomalts or isomaltulose-based oligomers (IBOs) ad libitum for 16 d. After 1 wk, the animals were subjected to a 5-d mineral (calcium, magnesium, phosphorus, and iron) balance study. The isomalt feeding, as well as the IBOs feeding, led to significantly elevated mineral absorption and retention. On the other hand, lactose feeding, widely known to enhance calcium absorption, increased only calcium absorption and isomaltulose feeding did not affect mineral absorption or retention. The organic acids in cecum contents were increased by IBOs or isomalt feeding. Succinic and acetic acids in cecum contents were significantly increased by IBOs feeding. Similarly, succinic acetic, and i-valeric acids and total amount of organic acid in cecum content were significantly increased by isomalt feeding. Although the organic acids in cecum contents were increased by IBOs or isomalt feeding, the PH values and acidity in cecum contents were not changed by IBOs or isomalt feeding. The effect of addition of various organic acids to the mucosal fluid was examined with in vitro study using a hindgut segment. By the addition of acetic acid, and butyric acid, the mineral (calcium, magnesium, and phosphorus) uptake was increased.     

Long-term effects of induced mineral accretion on growth, survival and corallite properties of Porites cylindrica Dana

The mineral accretion technology involves the accumulation of dissolved mineral ions within the vicinity of underwater electrodes and their deposition via electrochemical processes onto the positive electrode forming a natural substrate. Branches of Porites cylindrica were reared under the presence of mineral accretion (treated nubbins), without mineral accretion (untreated), and under undisturbed (control) conditions for 6 months (mineral accretion phase). The electricity for the treated nubbins was cut off after the sixth month, and all remaining nubbins were allowed to grow for another 6 months (post mineral accretion phase). The longitudinal growth rate of the treated nubbins was relatively high during the mineral accretion phase then dropped during the post mineral accretion phase. Statistical analysis revealed longitudinal growth to be significant over time, but the significant differences between treatments lay only between the first until the second bi-monthly period (from January to May 2000) and the rest of the observation period (June to January 2001). This indicates that growth enhancement occurred only during the early stages of the mineral accretion phase. There were also significant differences in girth growth between phases and between treatments as well as a significant phase by treatment interaction again indicating that significant differences between treatments occurred only during a certain phase. In terms of overall survival, the treated nubbins fared better than the untreated nubbins. The corals in all treatments showed the same trend in corallite size and density after 1 year. Corallite sizes increased from the tip towards the base of the transplant. The reverse trend occurred with corallite density which decreased from the tip towards the base. Phenotypic alteration of the corallites (decrease in size and increase in density from the middle region of the nubbin towards the base) had occurred in the treated nubbins while they were exposed to mineral accretion. This effect disappeared, however, after 6 months. It appears, therefore, that enhancement of growth took place only during active mineral accretion, though there were positive, longer-term effects on survival.     

Observations on the in vitro formation, development and differentiation of trilaminar vesicles formed from enzymatically dispersed porcine blastocysts

The formation of trophoblastic vesicles and their use as a model for cell-to-cell interactions in the embryonic development of the mouse have been extensively studied both in vitro and in vivo . In this report, we describe the formation of trilaminar vesicles in vitro from enzymatically dispersed porcine blastocysts. Trilaminar vesicles were generated from single cell suspensions of enzymatically dispersed porcine blastocysts recovered 12 through 15 days post-breeding. Blastocysts at 10 and 11 days of development failed to form TV. No preference for self-aggregation was apparent as evident by the formation of vesicles containing cells from two different blastocysts origins. Two distinct morphological types of vesicles were formed: vesicles that displayed complete homogeneity in cell density and vesicles that had a small area of greater cell density resembling the embryonic disc. Some TV displayed localized areas of nucleated eosinophilic cells within the mesenchymal layer of the trilaminar membrane. In rare instances (2%) TV displayed contractility associated with a raised area of cells on the outer surface of the membrane. The cells comprising this contractile region were positive for Ca(++)-activated myofibriliary ATPase acitivity. Transfer of TV to the uterine horn of a gilt failed to delay a return to estrus. Transfer of TV to the uterine horn of pseudopregnant rabbits induced a localized inflammation and infiltration of leukocytes three days after transfer but no inflammation was evident at 9 or 16 days post-transfer.     

Estimation of photosynthesis and calcification rates at a fringing reef by accounting for diurnal variations and the zonation of coral reef communities on reef flat and slope: a case study for the Shiraho reef, Ishigaki Island, southwest Japan

Seven coral reef communities were defined on Shiraho fringing reef, Ishigaki Island, Japan. Net photosynthesis and calcification rates were measured by in situ incubations at 10 sites that included six of the defined communities, and which occupied most of the area on the reef flat and slope. Net photosynthesis on the reef flat was positive overall, but the reef flat acts as a source for atmospheric CO₂, because the measured calcification/photosynthesis ratio of 2.5 is greater than the critical ratio of 1.67. Net photosynthesis on the reef slope was negative. Almost all excess organic production from the reef flat is expected to be effused to the outer reef and consumed by the communities there. Therefore, the total net organic production of the whole reef system is probably almost zero and the whole reef system also acts as a source for atmospheric CO₂. Net calcification rates of the reef slope corals were much lower than those of the branching corals. The accumulation rate of the former was approximately 0.5 m kyr⁻¹ and of the latter was ~0.7–5 m kyr⁻¹. Consequently, reef slope corals could not grow fast enough to keep up with or catch up to rising sea levels during the Holocene. On the other hand, the branching corals grow fast enough to keep up with this rising sea level. Therefore, a transition between early Holocene and present-day reef communities is expected. Branching coral communities would have dominated while reef growth kept pace with sea level rise, and the reef was constructed with a branching coral framework. Then, the outside of this framework was covered and built up by reef slope corals and present-day reefs were constructed.     

The vesicle-to-micelle transition of phosphatidylcholine vesicles induced by nonionic detergents: effects of sodium chloride, sucrose and urea

The vesicle-to-micelle transition of egg phosphatidylcholine LUVs induced by octylglucoside was studied in buffers with 0-4 M sodium chloride, sucrose or urea. We used both light scattering and fluorescent probes to follow the lipid-detergent complexes in these buffers. The vesicle-to-micelle transition process was fundamentally the same in each solute. However, the detergent-to-lipid ratio required for micelle formation shifted in ways that depended on the aqueous solute. The partitioning of octylglucoside between the vesicles and the aqueous phase was primarily determined by the change in its critical micelle concentration (cmc) induced by each solute. Specifically, the cmc decreased in high salt and sucrose buffers but increased in high concentrations of urea. Cmc for two additional nonionic detergents, decyl- and dodecyl-maltoside, and three zwittergents (3-12, 3-14 and 3-16) were determined as a function of concentration for each of the solutes. In all cases NaCl and sucrose decreased the solubility of the detergents, whereas urea increased their solubilities. The effects clearly depended on acyl chain length in urea-containing solutions, but this dependence was less clear with increasing NaCl and sucrose concentrations. The contributions of these solutes to solubility and to interfacial interactions in the bilayers, pure and mixed micelles are considered.     

Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry

Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid-liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.     

Pore formation, polymerization, hemolytic and leukotoxic effects of a new Enterobacter cloacae toxin neutralized by antiserum

A new toxin of Enterobacter cloacae was purified and studied by SDS-PAGE electrophoresis with the purpose of investigating its ability to generate polymers and their molecular mass. Monomer of 13.3 kDa and structures of multimeric mass were detected. The toxin of 66 kDa was the most abundant form of toxin. This polymer and the monomer were selected to examine blood cells damage. Membrane pores caused by both toxin forms seemed to be of similar dimension (estimated in 3.6 nm by experiments with osmotic protectors) and were able to lyse erythrocytes and leukocytes. The results obtained indicate that polymerization and pore formation are involved in the molecular events that participate in the cytotoxic effects of E. cloacae toxin. Immunization of rabbits with 13.3kDa toxin generated antibody response capable of inhibiting oxidative stress as well as hemolytic and leukotoxic effects. Immunoblotting indicated that monomer and polymer reacted with antihemolysin serum. The importance of E. cloacae toxin "in vivo" was studied in animals by means of assays performed in peritoneum of rats, inoculated with the hemolytic strain (C1) and a non-hemolytic variant (C4). Both strains stimulated infiltration of leukocytes in peritoneum, but C1 caused cell death and lysis wheras assays with C4 maintained the viability of leukocytes even within 5 h after extraction of samples.     

Determination of endogenous hormones, sugars and mineral nutrition levels during the induction, initiation and differentiation stage and their effects on flower formation in olive

Olive (Olea europaea L.) is one of the most important crop plants grown in the Mediterranean region. Varying levels of hormones, sugars and mineral nutrient are thought to influence flower bud formation. The objective of this study was to evaluate the changes in endogenous sugar, mineral nutrition and hormone levels in leaf, node and fruit samples of Memecik olive during the induction, initiation and differentiation periods in on (bearing) and off (non-bearing) years. Leaf, node and fruit samples of mature 15-year-old Memecik olive were used. The samples were taken during the induction, initiation and differentiation periods of olive in on (2000) and off (2001) years. Sugar (glucose, fructose and sucrose), mineral nutrient (N, P, K, Ca, Mg, Fe, Zn, Mn and Cu) and hormone [abscisic acid (ABA), indole acetic acid (IAA), gibberellic acid (GA₃, GA₄) and zeatin (Z)] levels were determined in on and off years. Hormone and sugar levels were measured by gas chromatography and high-performance liquid chromatography. The K, Ca, Mg, Fe, Mn, Zn and Cu levels were quantified by an atomic absorption spectrophotometry. Nitrogen was determined by the Kjeldahl procedure, and P by a spectrophotometric method. The differences in any of the sugar concentrations, with the exception of fructose, were not significant in on and off years. Hormone levels, however, were significantly different in on and off years. Glucose had the highest concentrations in both years, followed by sucrose and fructose, respectively. The highest macro and micro element concentrations were found to be Ca and Fe, respectively. Thus, the results suggest that carbohydrates and mineral nutrients may not have a direct effect to induce flower initiation. However, high GA₃ level exhibited an inhibitory effect on floral formation during the induction and initiation periods. On the other hand, the high concentrations of GA₄, ABA and certain cytokinin levels may have a positive effect on flower formation in olive during the induction and initiation periods.     

Bacterial phytotoxins, syringomycin, syringostatin and syringotoxin, exert their effect on the plasma membrane H+-ATPase partly by a detergent-like action and partly by inhibition of the enzyme

Syringostatin is a newly discovered phytotoxin produced by a phytopathogenic bacterium Pseudomonas syringae pv. syringae lilac isolate. The effects of syringostatin and the similar phytotoxins, syringomycin and syringotoxin, on H⁻-ATPase activity were investigated using cultured mung bean ( Vigna radiata L. cv. Ryokuto) cells or plasma membrane vesicles isolated from mung bean hypocotyls. ³¹P-NMR analysis of cultured cells treated with syringostatin revealed that the cytoplasmic pH was decreased. When plasma membrane was prepared by a two-step method (Dextran gradient followed by a sucrose gradient). syringostatin, syringomycin and syringotoxin inhibited the H⁺-ATPase activity in a dose-dependent manner. In contrast, these toxins stimulated H⁺-ATPase activity when plasma membrane was prepared by a one-step method (sucrose gradient). While these toxins inhibited the H⁺-ATPase activity of inside-out plasma membrane vesicles, the H⁺-ATPase activity of right-side-out vesicles was stimulated. The detergent. Triton X-100, abolished this stimulatory effect of the toxins on the H⁺-ATPase of right-side-out vesicles and of one-step purified plasma membrane. The toxins also inhibited the activity of the plasma membrane H⁺-ATPase solubilized with deoxycholate and Zwittergent 3–14. Taken together, these results indicate that these toxins exert their effects partly by a detergent-like action on the plasma membrane and partly by inhibition of the enzyme.     

Preparation and characterization of iron-containing liposomes: their effect on soluble iron uptake by Caco-2 cells

The aim of this work was to study the iron uptake of Caco-2 cells incubated with five different formulations of liposomes containing iron. The vesicles were also characterized before, during, and after in vitro digestion. Caco-2 cells were incubated with digested and nondigested liposomes, and soluble iron uptake was determined. Nondigested liposomes made with chitosan (CHI) or the cationic lipid, DC-Cholesterol (DC-CHOL), generated the highest iron uptake. However, these two formulations were highly unstable under in vitro digestion, resulting in nonmeasurable iron uptake. Digested conventional liposomes composed of soybean phosphatidylcholine (SPC), hydrogentated phosphatidylcholine (HSPC), or HSPC and cholesterol (CHOL) presented the highest iron-uptake values. These liposomal formulations protected iron from oxidation and improved iron uptake from intestinal cells, compared to an aqueous solution of ferrous sulphate.     

Immobilized thylakoids in a cross-linked albumin matrix: effects of cations studied by electron microscopy, fluorescence emission, photoacoustic spectroscopy, and kinetic measurements

Immobilization of lettuce (Lactuca sativa) thylakoids has been performed by using glutaraldehyde and bovine serum albumin. Confirming previous reports, a stabilization of the O₂ evolution activity of the photosystem II (PSII) under storage and functional conditions has been observed. The present work is devoted to the role played by mono-and divalent cations, during the immobilization process itself, on the O₂ production. Four types of measurements have been employed: kinetic measurements, low temperature (77 K) fluorescence emission, photoacoustic (PA) spectroscopy, and electron microscopy observations. We show that the effect of glutaraldehyde is complex because it acts as an inhibitor, a stabilizing agent, and a cross-linking reactive. In the present studies, the thylakoids are immobilized within a polymeric insoluble albumin matrix. The highest activity yield and the best storage conditions are obtained when 0.15 mm Na⁺ (or K⁺), 1 mm Mg²⁺, and 0.1 mm Mn²⁺ are present in the resuspending media before the immobilization. Due to modifications of the ionic content during such a process, structural differences are observed on the stacking degree of thylakoids. No modification of the fluorescence and PA spectra after the immobilization are found. Furthermore, a correlation between activities and spectral changes have been shown: when the activities increase, the F₇₃₅ to F₆₉₅ ratio increases and the PA₆₇₆ to PA₄₄₀ ratio decreases.     

Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions

Initiation of atherosclerosis is characterized by accumulation of aggregates of small lipid droplets and vesicles in the extracellular matrix of the arterial intima. The droplets and vesicles have features that suggest that they are formed from modified plasma-derived low density lipoprotein (LDL) particles. A variety of hydrolytic enzymes and prooxidative agents that could lead to extracellular assembly of LDL-derived droplets and vesicles are present in the arterial intima. In fact, in vitro studies have demonstrated that extensive oxidation of LDL and treatment of LDL with either proteolytic or lipolytic enzymes will induce LDL aggregation and fusion and treatment of LDL with cholesterol esterase will cause formation of vesicles. Fusion of LDL particles proceeds faster in vitro when they are bound to components of the extracellular matrix derived from the arterial intima, such as proteoglycans, and, depending on the type of modification, the strength of binding of modified LDL to the matrix components may either increase or decrease. In the present article, we discuss molecular mechanisms that provide clues as to how aggregated lipid droplets and vesicles may be derived from modified LDL particles. We also describe how these modified forms of LDL, by means of their trapping to the extracellular matrix, may lead to extracellular lipid accumulation in the arterial intima.     

Effect of different matrices on physiological amino acids analysis by liquid chromatography: evaluation and correction of the matrix effect

Free amino acids in plasma, muscle and liver tissues were determined using the Pico-Tag method which involves HPLC and pre-column derivatization. A study of the accuracy in the determination is carried out by means of external calibration, standard-added calibration, Youden calibration and analyte recovery. It is conclusively shown that the method is affected by a proportional systematic error due to matrix effects, but not by a constant one. A new function named matrix-corrected calibration is proposed to correct for proportional bias when free analyte matrices for matrix-matched addition is unavailable.     

In vitro effects of fluor-hydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity

The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard-tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH⁻ groups and F⁻ ions. The aim of this experimental investigation was to evaluate cytotoxic, genotoxic and mutagenic effects of FHA and FA eluates on Chinese hamster V79 cells and to compare them with the effects of hydroxyapatite (HA) eluate. Cytotoxicity of the biomaterials tested was evaluated by use of the cell colony-formation assay and by direct counting of the cells in each colony. Genotoxicity was assessed by single-cell gel electrophoresis (comet assay) and mutagenicity was evaluated by the Hprt gene-mutation assay and in bacterial mutagenicity tests using Salmonella typhimurium TA100. The results show that the highest test concentrations of the biomaterials (100% and 75% eluates) induced very weak inhibition of colony growth (about 10%). On the other hand, the reduction of cell number per colony induced by these concentrations was in the range from 43% to 31%. The comet assay showed that biomaterials induced DNA breaks, which increased with increasing test concentrations in the order HA < FHA < FA. None of the biomaterials induced mutagenic effects compared with the positive control (N-methyl-N′-nitro-N-nitrosoguanidine), and DNA breakage was probably the reason for the inhibition of cell division in V79 cell colonies.     

The effect of IL-1alpha on the expression of matrix metalloproteinases, plasminogen activators, and their inhibitors in osteoblastic ROS 17/2.8 cells

Interleukin-1 (IL-1) plays key roles in altering bone matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), and the plasminogen activation system, including tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) , and plasminogen activator inhibitor type-1 (PAI-1). In this study, we examined the effect of IL-1alpha on the expression of the MMPs, TIMPs, tPA, uPA, and PAI-1 genes in osteoblasts derived from the rat osteosarcoma cell line ROS 17/2.8. The cells were cultured in alpha-minimum essential medium containing 10% fetal bovine serum with 0 or 100 U/ml of IL-1alpha for up to 14 days. The levels of MMPs, TIMPs, uPA, tPA, and PAI-1 expression were estimated by determining the mRNA levels using real-time RT-PCR and by determining protein levels using ELISA. In IL-1alpha cultures, the expression levels of MMP-1, -2, -3, -13, and -14 exceeded that of the control through day 14 of culture, and the expression of MMPs increased markedly from the proliferative to the later stages of culture. The TIMP-1, -2, and -3 expression levels increased from the initial to the proliferative stages of culture. The expression of tPA increased greatly during the proliferative stage of culture, and uPA expression increased throughout the culture period, increasing markedly from the proliferative to the later stages of culture. In contrast, PAI-1 expression decreased in the presence of IL-1alpha through day 14. These results suggest that IL-1alpha stimulate bone matrix turnover by increasing MMPs, tPA, and uPA production and decreasing PAI-1 production by osteoblasts, and incline the turnover to the resolution.     

Extracellular vesicles derived from Echinococcus granulosus hydatid cyst fluid from patients: isolation,characterization and evaluation of immunomodulatory functions on T cells

Cystic echinococcosis is a chronic and complex zoonotic disease. The mechanisms underlying the parasite’s establishment, growth and persistence are not completely understood, and are thought be modulated by a crosstalk through extracellular vesicles. Here, EVs were isolated from the hydatid cyst fluid of patients with cystic echinococcosis and protoscolex culture supernatant. Proteomic analysis of these EVs revealed several parasite- and human-derived proteins. Very few studies have performed proteomic analysis of EVs isolated from HCF and PCS. Our proteomic analysis of the EVs derived from HCF and PCS facilitated identification of 1175 proteins, wherein 1026 and 38 proteins were exclusively identified in the EVs derived from HCF (HCF-EVs) and PCS (PCS-EVs), respectively, and 111 proteins were shared in both. The results of co-culture of PCS-EVs with murine peripheral blood mononuclear cells showed that PCS-EVs significantly regulated T lymphocyte functions in a dose-dependent manner. Collectively, our results provide valuable information on parasite survival strategies and new insights into the role of these EVs in the establishment and persistence of hydatid cysts.