Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA

Purpose: To investigate the role of thioredoxin (TRX), a novel regulator of extracellular transglutaminase 2 (TG2), in celiac patients IgA (CD IgA) mediated TG2 enzymatic activation. Methods: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs) under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA) were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. Results: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. Conclusions: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX. © 2013 Nadalutti et al.

Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals

The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.

Physical and functional characterisation of apoptotic cell-derived extracellular vesicles

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in the exposure of 'flags' at the dying cell surface and the release of attractive signals to recruit phagocytes. Together these changes ensure efficient phagocytic removal of dying cells and prevention of inflammatory and autoimmune disorders. Extracellular vesicles (EV) are released from a variety of cells (both viable and apoptotic) and they serve as a novel means of intercellular communication. They range in size: 70-100nm ('exosomes') through 100-1000nm ('microparticles') to large vesicles released from dying cells ('apoptotic bodies'). Release of apoptotic cell-derived extracellular vesicles (acdEV) of less than 1000nm is an important mechanism by which phagocytes are attracted to sites of cell death. Using a variety of approaches we characterize the release, physical characteristics and function of acdEV. Using fluorescence microscopy we demonstrate release of ICAM-3 on acdEV from dying leukocytes and, through the use of resistive pulse technology (qNano, IZON Science), we accurately size and quantitate acdEV release. The function of acdEV is revealed through the use of both horizontal chemotaxis assays (Dunn chambers) and vertical transwell migration assays (Cell-IQ, CM Technologies). These assays reveal potent chemoattractive capacity of acdEV and associated ICAM-3. Additionally we demonstrate an additional novel function of acdEV as anti-inflammatory immune-modulators. These data support an integrated approach to the physical and functional analyses of EV.

Characterisation of ICAM-3 and extracellular vesicles in the clearance of apoptotic cells

Apoptotic cell clearance by phagocytes is a vital part of programmed cell death that prevents dying cells from undergoing necrosis which may lead to inflammatory and autoimmune disorders. Apoptotic cells (AC) are removed by phagocytes, in a process that involves 'find me' and 'eat me' signals that facilitate the synapsing and engulfment of cell corpses. Extracellular vesicles (EV) are shed during apoptosis and promote phagocyte recruitment. Binding of AC is achieved by multiple ligand-receptor interactions. One interesting AC associated ligand is ICAM-3, a highly glycosylated adhesion molecule of the IgSF family, expressed on human leukocytes. On viable cells ICAM-3 participates in initiating immune responses, whereas on AC we show it attracts phagocytes through EV and aids in the binding of AC to the phagocytes. This project aims to characterize the role of ICAM-3 and EV in the clearance of AC and to identify the mechanisms that underlie their function in apoptotic cell clearance. Human B cells induced to apoptosis by UV irradiation were observed during their progression from viable to apoptotic via flow cytometry. The involvement of ICAM-3 in mediating interaction between AC and MØ was assessed. The ability of ICAM3 on EV to mediate chemoattraction was observed using chemotaxis assays. Additionally the anti-inflammatory effect was assessed using LPS-induced TNF-α production that suggested it may have anti-inflammatory effects. Future work in this project will assess the role of ICAM3 on EV from different phases of apoptosis to exert functional effects both in vitro and in vivo.

Characterization of neutrophil function in Papillon-Lefèvre syndrome

Papillon-Lefévre syndrome is a rare, inherited, autosomal-recessive disease, characterized by palmoplantar keratosis and severe prepubertal periodontitis, leading to premature loss of all teeth. Papillon-Lefévre syndrome is caused by a mutation in the cathepsin C gene, resulting in complete loss of activity and subsequent failure to activate immune response proteins. Periodontitis in Papillon-Lefévre syndrome is thought to arise from failure to eliminate periodontal pathogens as a result of cathepsin C deficiency, although mechanistic pathways remain to be elucidated. The aim of this study was to characterize comprehensively neutrophil function in Papillon-Lefévre syndrome. Peripheral blood neutrophils were isolated from 5 patients with Papillon-Lefévre syndrome, alongside matched healthy control subjects. For directional chemotactic accuracy, neutrophils were exposed to the chemoattractants MIP-1α and fMLP and tracked by real-time videomicroscopy. Reactive oxygen species generation was measured by chemiluminescence. Neutrophil extracellular trap formation was assayed fluorometrically, and proinflammatory cytokine release was measured following overnight culture of neutrophils with relevant stimuli. Neutrophil serine protease deficiencies resulted in a reduced ability of neutrophils to chemotax efficiently and an inability to generate neutrophil extracellular traps. Neutrophil extracellular trap-bound proteins were also absent in Papillon-Lefévre syndrome, and Papillon-Lefévre syndrome neutrophils released higher levels of proinflammatory cytokines in unstimulated and stimulated conditions, and plasma cytokines were elevated. Notably, neutrophil chemoattractants MIP-1α and CXCL8 were elevated in Papillon-Lefévre syndrome neutrophils, as was reactive oxygen species formation. We propose that relentless recruitment and accumulation of hyperactive/reactive neutrophils (cytokines, reactive oxygen species) with increased tissue transit times into periodontal tissues, alongside a reduced antimicrobial capacity, create a locally destructive chronic inflammatory cycle in Papillon-Lefévre syndrome.

The extracellular surface of the GLP-1 receptor is a molecular trigger for biased agonism

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems. © 2010 Elsevier Inc.

Antibacterial Activity of Extracellular Products from Cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes that can be found in freshwater and marine environments as well as in soil. These organisms produce a variety of different biologically active compounds exhibiting anti-bacterial, anti-fungal and anti-cancer properties among others. In this study, cyanobacterial isolates were screened for their ability to produce extracellular antibacterial products. Cyanobacteria were isolated from fresh water and soil samples collected in the Pembroke Pines, FL area. Twenty- seven strains of cyanobacteria were isolated belonging to the following genera: Limnothrix, Nostoc, Fischerella, Anabaena, Pseudoanabaena, Lyngbya, Leptolyngbya, Tychonema, and Calothrix. Individual strains were grown in liquid culture in laboratory conditions. Following 14-day cultivation, the culture liquid was filtered and tested for activity against the following bacteria: Escherichia coli, Bacillus megatarium, Staphylococcus aureus, and Micrococcus luteus. Among all genera of cyanobacterial strains tested, Fischerella exhibited the greatest inhibitory activity. An attempt was made to isolate the active compound from the culture liquid of the active strains. Lipophilic extracts from culture liquid were obtained from three selected Fischerella strains. The extracts proved to have varying levels of activity against the tested bacteria. Inhibitory activity from all three Fischerella strains was detected against B. megatarium and M luteus. The only strain that was active against S. aureus was Fischerella sp. 114-12 while none of the extracts showed activity against E. coli. This kind of screening has potential pharmaceutical and agricultural benefits, including possible discovery of novel antibiotics.

Estudo in silico das interações da protease NS3-NS2B de DENV-2 com o inibidor peptídico Bz-nKRR-H com finalidades terapêuticas

Dengue virus is an important patogen that causes Dengue desease in all world, and belongs to Flavivirus gender. The virus consists of enveloped RNA with a single strand positive sense, 11Kb genome. The RNA is translated into a polyprotein precursor, wich is cleaved into 3 structural proteins (C, prM e E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B e NS5). The NS3 is a multifunctional protein, that besides to promote the polyprotein precursor cleavage, also have NTPase, helicase and RTPase activity. The NS3 needs a hydrophilic segment of 40 residues from the transmembrane NS2B protein (who acts like cofator) to realize this functions. Actually, there's no vacines available on the market, and the treatment are just symptomatic. The tetrapeptide inhibitor Bz-Nle-Lys-Arg-Arg-H (Ki de 5,8-7,0 M) was showed as a potent inhibitor μ for NS3prot in Dengue virus. That is a inteligent alternative to treat the dengue desease. The present work aimed analyse the interactions of the ligand bounded to the activity site to provid a clear and depth vision of that interaction. For this purpouse, it was conducted an in silico study, by using quantum mechanical calculations based on Density Functional Theory (DFT), with Generalized Gradient approximation (GGA) to describe the effects of exchange and correlation. The interaction energy of each amino acid belonging to the binding site to the ligand was calculated the using the method of molecular fragmentation with conjugated caps (MFCC). Besides energy, we calculated the distances, types of molecular interactions and atomic groups involved. The theoretical models used were satisfactory and show a more accurate description when the dielectric constant = 20 ε and 80 was used. The results demonstrate that the interaction energy of the system reached convergence at 13.5 A. Within a radius of 13,5A the most important residues were identified. Met49, Met84 and Asp81 perform interactions of hydrogen with the ligant. The Asp79 and Asp75 residues present high energy of attraction. Arg54, Arg85 and Lys 131 perform hydrogen interactions with the ligand, however, appear in BIRD graph having high repulsion energy with the inhibitor. The data also emphasizes the importance of residue Tyr161 and the involvement of the catalytic triad composed by Asp75, His51 and Ser135

Vesicoureteral reflux and the extracellular matrix connection.

Primary vesicoureteral reflux (VUR) is a common pediatric condition due to a developmental defect in the ureterovesical junction. The prevalence of VUR among individuals with connective tissue disorders, as well as the importance of the ureter and bladder wall musculature for the anti-reflux mechanism, suggest that defects in the extracellular matrix (ECM) within the ureterovesical junction may result in VUR. This review will discuss the function of the smooth muscle and its supporting ECM microenvironment with respect to VUR, and explore the association of VUR with mutations in ECM-related genes.

The extracellular matrix contributes to mechanotransduction in uterine fibroids.

The role of the extracellular matrix (ECM) and mechanotransduction as an important signaling factor in the human uterus is just beginning to be appreciated. The ECM is not only the substance that surrounds cells, but ECM stiffness will either compress cells or stretch them resulting in signals converted into chemical changes within the cell, depending on the amount of collagen, cross-linking, and hydration, as well as other ECM components. In this review we present evidence that the stiffness of fibroid tissue has a direct effect on the growth of the tumor through the induction of fibrosis. Fibrosis has two characteristics: (1) resistance to apoptosis leading to the persistence of cells and (2) secretion of collagen and other components of the ECM such a proteoglycans by those cells leading to abundant disposition of highly cross-linked, disoriented, and often widely dispersed collagen fibrils. Fibrosis affects cell growth by mechanotransduction, the dynamic signaling system whereby mechanical forces initiate chemical signaling in cells. Data indicate that the structurally disordered and abnormally formed ECM of uterine fibroids contributes to fibroid formation and growth. An appreciation of the critical role of ECM stiffness to fibroid growth may lead to new strategies for treatment of this common disease.

Seawater carbonate chemistry and conditions of Mytilus edulis extracellular body fluids and shell composition in a pH-treatment experiment: Acid-base status, trace elements and delta11B, 2012

Mytilus edulis were cultured for 3 months under six different seawater pCO2 levels ranging from 380 to 4000 µatm. Specimen were taken from Kiel Fjord (Western Baltic Sea, Germany) which is a habitat with high and variable seawater pCO2 and related shifts in carbonate system speciation (e.g., low pH and low CaCO3 saturation state). Hemolymph (HL) and extrapallial fluid (EPF) samples were analyzed for pH and total dissolved inorganic carbon (CT) to calculate pCO2 and [HCO3]. A second experiment was conducted for 2 months with three different pCO2 levels (380, 1400 and 4000 µatm). Boron isotopes (delta11B) were investigated by LA-MC-ICP-MS (Laser Ablation-Multicollector-Inductively Coupled Plasma-Mass Spectrometry) in shell portions precipitated during experimental treatment time. Additionally, elemental ratios (B/Ca, Mg/Ca and Sr/Ca) in the EPF of specimen from the second experiment were measured via ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry). Extracellular pH was not significantly different in HL and EPF but systematically lower than ambient water pH. This is due to high extracellular pCO2 values, a prerequisite for metabolic CO2 excretion. No accumulation of extracellular [HCO3] was measured. Elemental ratios (B/Ca, Mg/Ca and Sr/Ca) in the EPF increased slightly with pH which is in accordance with increasing growth and calcification rates at higher seawater pH values. Boron isotope ratios were highly variable between different individuals but also within single shells. This corresponds to a high individual variability in fluid B/Ca ratios and may be due to high boron concentrations in the organic parts of the shell. The mean delta11B value shows no trend with pH but appears to represent internal pH (EPF) rather than ambient water pH.

Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation.

METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1β response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover.

RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1β release from macrophages, through lysosomal destabilization. IL-1β secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1β release was significantly reduced in primary macrophages from ctss(-/-) mice.

CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1β release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.

Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta

Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.

Modest elevation in BNP in asymptomatic hypertensive patients reflects sub-clinical cardiac remodeling, inflammation and extracellular matrix changes

In asymptomatic subjects B-type natriuretic peptide (BNP) is associated with adverse cardiovascular outcomes even at levels well below contemporary thresholds used for the diagnosis of heart failure. The mechanisms behind these observations are unclear. We examined the hypothesis that in an asymptomatic hypertensive population BNP would be associated with sub-clinical evidence of cardiac remodeling, inflammation and extracellular matrix (ECM) alterations. We performed transthoracic echocardiography and sampled coronary sinus (CS) and peripheral serum from patients with low (n = 14) and high BNP (n = 27). Peripheral BNP was closely associated with CS levels (r = 0.92, p<0.001). CS BNP correlated significantly with CS levels of markers of collagen type I and III turnover including: PINP (r = 0.44, p = 0.008), CITP (r = 0.35, p = 0.03) and PIIINP (r = 0.35, p = 0.001), and with CS levels of inflammatory cytokines including: TNF-α (r = 0.49, p = 0.002), IL-6 (r = 0.35, p = 0.04), and IL-8 (r = 0.54, p<0.001). The high BNP group had greater CS expression of fibro-inflammatory biomarkers including: CITP (3.8±0.7 versus 5.1±1.9, p = 0.007), TNF-α (3.2±0.5 versus 3.7±1.1, p = 003), IL-6 (1.9±1.3 versus 3.4±2.7, p = 0.02) and hsCRP (1.2±1.1 versus 2.4±1.1, p = 0.04), and greater left ventricular mass index (97±20 versus 118±26 g/m(2), p = 0.03) and left atrial volume index (18±2 versus 21±4, p = 0.008). Our data provide insight into the mechanisms behind the observed negative prognostic impact of modest elevations in BNP and suggest that in an asymptomatic hypertensive cohort a peripheral BNP measurement may be a useful marker of an early, sub-clinical pathological process characterized by cardiac remodeling, inflammation and ECM alterations.