Release Of Proteases Research Articles

Colorectal Cancer Invasion and Atrophy of the Enteric Nervous System: Potential Feedback and Impact on Cancer Progression.

Colorectal cancer (CRC) invasion within the large intestine wall results in the replacement of normal tissue architecture by tumour mass. Cancer cells digest the extracellular matrix (ECM) by the release of proteolytic enzymes. The disintegration of matrix ground substance activates several deposited growth factors which stimulate cell proliferation. Stromal (mainly fibroblasts), immune and cancer cells dominate in this area and become involved in a network of multimodal interactions which significantly induce proliferation of colon cancer cells, inhibit their apoptosis and promote their spreading within the local tumour microenvironment. Cancer invasion destroys nerve fibres and neurons of the local enteric nervous system (ENS) and induces subsequent atrophy of the submucosal and myenteric plexuses in areas adjacent to the cancer boundary. Interestingly, the reduction of plexuses’ size is accompanied by the increased number of galanin-immunoreactive neurons and increased galanin content in parts of the colon located close to the tumour. Galanin, a neuroprotective peptide, may inhibit the extrinsic pathway of apoptosis and in this way promote cancer cell survival. The possible role of acetylcholine and some ENS neuropeptides was also discussed. Invasion of cancer cells spreads along nerve fibres with the involvement of locally-released neutrophins which promote, via their specific receptors, cancer cell proliferation and pro-survival signalling pathways. Thus, during CRC development cancer cells and neurons of the ENS release many neurotransmitters/neuropeptides which affect key cellular signalling pathways promoting cancer cell proliferation and pro-survival phenotype. The multiple interactions between ENS neurons, cancer cells and other cell types present in the colon wall increase cancer cell invasiveness and have a negative impact on the course of CRC.

SAT-138 Neutrophil Elastase Promotes Proliferative Signals in Prostate Cells Through EGFR and DDR1

Studies examining many different cancers have demonstrated that inflammation plays a critical role in tumor progression, in part through the release of proteases from stromal cells that function to either remodel the tumor microenvironment or to directly stimulate cancer cells to grow. One specific protease, neutrophil elastase (NE), has been shown to be a critical regulator of cancer growth in several mouse models. Accordingly, our laboratory demonstrated that NE, most likely from granulocytic myeloid-derived suppressor cells, potentially promotes prostate cancer progression in several different in-vivo and in-vitro models. To date, however, little is known regarding the mechanisms utilized by NE to promote tumor growth. It has been suggested that NE might cleave epidermal growth factor (EGF) or transforming growth factor-α from the cell surface to induce activation of EGFR/ERK signal transduction in an autocrine fashion. Alternatively, NE has been shown to enter into early endosomes to degrade insulin receptor substrate-I, ultimately resulting in phosphoinositol 3-kinase hyperactivity and subsequent tumor cell proliferation. Here we demonstrate that NE triggered proliferative signals in six prostate cell lines representing the spectrum of prostate cell differentiation, including normal prostatic epithelium, benign prostatic hypertrophy, and metastatic prostate cancer. Focusing on ERK signaling, we found that the stimulatory effect of NE on ERK phosphorylation was dose dependent and was abrogated by small interfering RNA induced EGFR knockdown, as well as by pretreatment of cells with irreversible EGFR inhibitor AG1478. Unlike EGF, however, NE-initiated EGFR phosphorylation was minimal. Thus, while EGFR appears to be critical for NE-induced ERK activation, perhaps it is not extensively activated directly by NE. Notably, discoidin domain receptor-1 (DDR1) was strongly expressed in normal prostate epithelium cells, but gradually decreased and had little expression in benign and metastatic prostate cancer cells sequentially. Nevertheless, similar to EGFR knockdown, silencing of DDR1 in all cell types inhibited NE mediated pERK upregulation, suggesting that DDR1 may also be important for NE-induced action. Together, our data suggest that NE, in concert with low level signals from the EGFR and DDR1, play an important role in promoting prostate cell proliferation both in normal and cancerous prostate epithelial cells.

Proteolytic Activation of ENaC Mediates Fructose‐Induced Salt‐Sensitive Hypertension

Proteolytic cleavage of the amiloride‐sensitive epithelial sodium channel (ENaC) by serine proteases such as trypsin leads to channel activation. In nephrotic syndrome, glomerular injury allows filtration of such enzymes causing ENaC‐dependent Na retention and hypertension(PMID: 23216619). Dietary fructose also causes Na retention and salt‐sensitive hypertension at least, in part, via altered hormonal signaling and metabolism in the proximal nephron. Proximal tubules normally secrete proteases into the forming urine but whether they are a source of proteases that cleave and activate ENaC in fructose‐induced salt‐sensitive hypertension is unknown. We hypothesized that dietary fructose increases expression and release of proteases from the proximal nephron, and that these enzymes activate ENaC thereby causing Na retention and salt‐sensitive hypertension. To test this hypothesis we first measured proximal nephron protease expression and urinary protease excretion in rats on either a high‐salt diet containing 4% NaCl without fructose (high salt) or one containing high salt plus 20% fructose in the drinking water (high salt/fructose) for 7 days. High salt/fructose treatment increased proximal nephron expression of trypsin I, an enzyme known to cleave and activate ENaC, by 70% (p < 0.01) compared to high salt alone. Urinary excretion of trypsin I was 8.4±1.3 arbitrary units/μg protein in rats fed high salt while it was 20.3±4.6 arbitrary units/μg protein in those on the high salt/fructose diet, 142% greater (p < 0.02). There was no difference in total urinary protein excretion between groups. Finally, we examined the effect of high salt/fructose and high salt plus 20% glucose (high salt/glucose) on blood pressure before and after oral amiloride. After 7 days, the systolic blood pressure of rats on high salt/fructose was 142±6 mm Hg while it was only 121±6 mm Hg in those on high salt/glucose (p < 0.05). After 10 days, the systolic blood pressure of rats on high salt/fructose was 148±6 mm Hg while it was only 124±5 in those on high salt/glucose (p < 0.02). Amiloride treatment for 24 hrs reduced systolic blood pressure in rats on the high salt/fructose diet from 148±6 to 134±5 mm Hg but did not decrease it in the high salt/glucose group. We conclude that proteolytic cleavage of ENaC contributes to fructose‐induced salt‐sensitive hypertension and that the source of the protease(s) is likely the proximal tubule rather than glomerular filtration.Support or Funding InformationNLHBI HL128053

15-Epi-LXA4 and 17-epi-RvD1 restore TLR9-mediated impaired neutrophil phagocytosis and accelerate resolution of lung inflammation

Timely resolution of bacterial infections critically depends on phagocytosis of invading pathogens by polymorphonuclear neutrophil granulocytes (PMNs), followed by PMN apoptosis and efferocytosis. Here we report that bacterial DNA (CpG DNA) and mitochondrial DNA impair phagocytosis and attenuate phagocytosis-induced apoptosis in human PMNs through Toll-like receptor 9 (TLR9)-mediated release of neutrophil elastase and proteinase 3 and subsequent down-regulation of the complement receptor C5aR. Consistently, CpG DNA delays pulmonary clearance of Escherichia coli in mice and suppresses PMN apoptosis, efferocytosis, and generation of proresolving lipid mediators, thereby prolonging lung inflammation evoked by E. coli Genetic deletion of TLR9 renders mice unresponsive to CpG DNA. We also show that aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and 17-epi-resolvin D1 (17-epi-RvD1) through the receptor ALX/FPR2 antagonize cues from CpG DNA, preserve C5aR expression, restore impaired phagocytosis, and redirect human PMNs to apoptosis. Treatment of mice with 15-epi-LXA4 or 17-epi-RvD1 at the peak of inflammation accelerates clearance of bacteria, blunts PMN accumulation, and promotes PMN apoptosis and efferocytosis, thereby facilitating resolution of E. coli-evoked lung injury. Collectively, these results uncover a TLR9-mediated endogenous mechanism that impairs PMN phagocytosis and prolongs inflammation, and demonstrate both endogenous and therapeutic potential for 15-epi-LXA4 and 17-epi-RvD1 to restore impaired bacterial clearance and facilitate resolution of acute lung inflammation.

Expression of mast cell in aggressive periodontitis.

Aggressive periodontitis is a progressive disease that can cause tooth loss within a short period of time. Mast cells are found in tissues with various allergic and inflammatory conditions including periodontal disease through the release of cytokines, chemokines, and proteolytic enzymes. Given the findings of some studies on the related role of mast cells, this study was conducted to determine the number of mast cells in aggressive periodontitis. This study was conducted on 30 participants divided into two groups visiting the Faculty of Dentistry of University of Medical Sciences. A total of 15 participants with aggressive periodontitis and 15 healthy participants were investigated. For counting mast cells, toluidine blue staining was used. The percentage of mast cells and demographic factors were evaluated in the two groups and the results were analyzed in SPSS using descriptive statistics and independent t-tests. A P value <0.05 was considered significant. The mean percentage of mast cells in the aggressive periodontitis group was 27.1±8.9 which was significantly higher than that of the healthy group, i.e. 12.6±5.1 (P<0.001). In general, the results of the study showed that the percentage of mast cells in the aggressive periodontitis group was more than two times higher than that of the healthy group, which is indicative of an increase in the number of mast cells in aggressive periodontitis disease.

Human neutrophils functionality under effect of an Asp49 phospholipase A2 isolated from Bothrops atrox venom.

Bothrops envenomation is associated with a cellular inflammatory response, characterized by pronounced neutrophil infiltration at the site of injury. Neutrophils act as the first line of defence, owing to their ability to migrate to the infected tissue, promoting an acute inflammatory response. At the site of inflammation, neutrophils perform defence functions such as phagocytosis, release of proteolytic enzymes, generation of reactive oxygen species (ROS), and synthesis of inflammatory mediators such as cytokines and lipid mediators. Neutrophils can also form neutrophil extracellular nets (NETs), webs composed of chromatin and granule proteins. This occurs after neutrophil activation and delivers high concentrations of anti-microbial molecules to the site of injury. This study evaluated the impact of BaTX-II, an Asp49 phospholipase A2 (PLA2) isolated from Bothrops atrox snake venom on human neutrophils in vitro. At non-toxic concentrations, BaTX-II induced hydrogen peroxide production by neutrophils, and this was reduced by wortmannin, a PI3K inhibitor. BaTX-II stimulated IL-1β, IL-8, LTB4, myeloperoxidase (MPO), and DNA content release, consistent with NET formation. This is the first study to show the triggering of relevant pro-inflammatory events by PLA2 Asp49 isolated from secretory venom.

Post-Ischaemic Immunological Response in the Brain: Targeting Microglia in Ischaemic Stroke Therapy

Microglia, the major endogenous immune cells of the central nervous system, mediate critical degenerative and regenerative responses in ischaemic stroke. Microglia become “activated”, proliferating, and undergoing changes in morphology, gene and protein expression over days and weeks post-ischaemia, with deleterious and beneficial effects. Pro-inflammatory microglia (commonly referred to as M1) exacerbate secondary neuronal injury through the release of reactive oxygen species, cytokines and proteases. In contrast, microglia may facilitate neuronal recovery via tissue and vascular remodelling, through the secretion of anti-inflammatory cytokines and growth factors (a profile often termed M2). This M1/M2 nomenclature does not fully account for the microglial heterogeneity in the ischaemic brain, with some simultaneous expression of both M1 and M2 markers at the single-cell level. Understanding and regulating microglial activation status, reducing detrimental and promoting repair behaviours, present the potential for therapeutic intervention, and open a longer window of opportunity than offered by acute neuroprotective strategies. Pharmacological modulation of microglial activation status to promote anti-inflammatory gene expression can increase neurogenesis and improve functional recovery post-stroke, based on promising preclinical data. Cell-based therapies, using preconditioned microglia, are of interest as a method of therapeutic modulation of the post-ischaemic inflammatory response. Currently, there are no clinically-approved pharmacological options targeting post-ischaemic inflammation. A major developmental challenge for clinical translation will be the selective suppression of the deleterious effects of microglial activity after stroke whilst retaining (or enhancing) the neurovascular repair and remodelling responses of microglia.

Pulsed Low-Frequency Magnetic Fields Induce Tumor Membrane Disruption and Altered Cell Viability

Tumor cells express a unique cell surface glycocalyx with upregulation of sulfated glycosaminoglycans and charged glycoproteins. Little is known about how electromagnetic fields interact with this layer, particularly with regard to harnessing unique properties for therapeutic benefit. We applied a pulsed 20-millitesla (mT) magnetic field with rate of rise (dB/dt) in the msec range to cultured tumor cells to assess whether this affects membrane integrity as measured using cytolytic assays. A 10-min exposure of A549 human lung cancer cells to sequential 50- and 385-Hz oscillating magnetic fields was sufficient to induce intracellular protease release, suggesting altered membrane integrity after the field exposure. Heparinase treatment, which digests anionic sulfated glycan polymers, before exposure rendered cells insensitive to this effect. We further examined a non-neoplastic human primary cell line (lung lymphatic endothelial cells) as a typical normal host cell from the lung cancer microenvironment and found no effect of field exposure on membrane integrity. The field exposure was also sufficient to alter proliferation of tumor cells in culture, but not that of normal lymphatic cells. Pulsed magnetic field exposure of human breast cancer cells that express a sialic-acid rich glycocalyx also induced protease release, and this was partially abrogated by sialidase pretreatment, which removes cell surface anionic sialic acid. Scanning electron microscopy showed that field exposure may induce unique membrane “rippling” along with nanoscale pores on A549 cells. These effects were caused by a short exposure to pulsed 20-mT magnetic fields, and future work may examine greater magnitude effects. The proof of concept herein points to a mechanistic basis for possible applications of pulsed magnetic fields in novel anticancer strategies.

Immunohistochemical evaluation of CD117 in mast cell of aggressive periodontitis.

Context:Aggressive periodontitis is a type of inflammatory response in periodontal tissues that is differentiated from chronic by its earlier onset, its high rate of progression, subgingival microbial composition, the difference in the host's immune response, and family history of the disease. Mast cells live in tissues and contribute to the various allergic and inflammatory conditions, including periodontal diseases, through the release of cytokines, chemokines, and proteolytic enzymes. CD117, the receptor of the stem cell factor expressed by the mast cells, is the hallmark of mast cell development. This study aimed to determine the expression level of CD117 in the mast cells of aggressive periodontitis using immunohistochemistry.Aims:This study aimed to determine the expression level of CD117 in the mast cells of aggressive periodontitis using immunohistochemistry.Subjects and Methods:The study was conducted on tissue samples from 15 patients with aggressive periodontitis and 15 patients with healthy gingival tissue. Samples were stained for CD117 markers using immunohistochemistry. After the microscopic examination of samples, all data were entered into SPSS 20 and analyzed.Statistical Analysis Used:Statistical data were analyzed using Kolmogorov–Smirnov test and independent t-test.Results:According to the results, the mean expression levels of CD117 in the aggressive periodontitis and normal gingival groups were 19.08 ± 6.9 and 18.2 ± 8.1, respectively, which were not statistically significant (P = 0.75). The Kolmogorov–Smirnov test verified the normality of distribution of CD117 expression level. Given the normal distribution of the scores, the independent t-test was used to compare the CD117 expression level in the two study groups.Conclusions:There was no significant difference in the level of CD117 expression between the control and aggressive periodontitis groups.

ATP amplifies NADPH-dependent and -independent neutrophil extracellular trap formation

Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes, including the release of proteases, phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation, or NETosis, is a specific and highly efficient process, which is induced by a variety of stimuli leading to expulsion of DNA, proteases and antimicrobial peptides to the extracellular space. However, uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here, we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly, neutrophils isolated from Panx1−/− mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF), a Panx1 channel inhibitor, decreased NETosis in wild-type neutrophils to the extent observed in Panx1−/− neutrophils. Thus, we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target.

Description and Characterization of a Novel Human Mast Cell Line for Scientific Study.

Background: Laboratory of allergic diseases 2 (LAD2) human mast cells were developed over 15 years ago and have been distributed worldwide for studying mast cell proliferation, receptor expression, mediator release/inhibition, and signaling. LAD2 cells were derived from CD34+ cells following marrow aspiration of a patient with aggressive mastocytosis with no identified mutations in KIT. Another aspiration gave rise to a second cell line which has recently been re-established (LADR). We queried whether LADR had unique properties for the preclinical study of human mast cell biology. Methods: LADR and LAD2 cells were cultured under identical conditions. Experiments examined proliferation, beta-hexosaminidase (β-hex) release, surface receptor and granular protease expression, infectivity with HIV, and gene expression. Results: LADR cells were larger and more granulated as seen with Wright–Giemsa staining and flow cytometry, with cell numbers doubling in 4 weeks, in contrast to LAD2 cells, which doubled every 2 weeks. Both LADR and LAD2 cells released granular contents following aggregation of FcεRI. LADR cells showed log-fold increases in FcεRI/CD117 and expressed CD13, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, and CD195, while LAD2 cells expressed CD33, CD34, CD63, CD117, CD133, CD193 but not CD13, CD123, CD184, or CD195. LADR tryptase expression was one-log-fold increased. LADR cell and LAD2 cell chymase expression were similar. Both cell lines could be infected with T-tropic, M-tropic, and dual tropic HIV. Following monomeric human IgE stimulation, LADR cells showed greater surface receptor and mRNA expression for CD184 and CD195. Expression arrays revealed differences in gene upregulation, especially for the suppressor of cytokine signaling (SOCS) family of genes with their role in JAK2/STAT3 signaling and cellular myelocytomatosis oncogene (c-MYC) in cell growth and regulation. Conclusions: LADR cells are thus unique in that they exhibit a slower proliferation rate, are more advanced in development, have increased FcεRI/CD117 and tryptase expression, have a different profile of gene expression, and show earlier infectivity with HIV-BAL, LAV, and TYBE when compared to LAD2 cells. This new cell line is thus a valuable addition to the few FcεRI+ human mast cell lines previously described and available for scientific inquiry.

Bafilomycin-A1 and ML9 Exert Different Lysosomal Actions to Induce Cell Death.

Bafilomycin-A1 and ML9 are lysosomotropic agents, irrespective of cell types. However, the mechanisms of lysosome targeting either bafilomycin-A1 or ML9 are unclear. The present research has been carried out by different molecular and biochemical analyses like western blot, confocal imaging and FACS studies, as well as molecular docking. Our data shows that pre-incubation of neonatal cardiomyocytes with ML9 for 4h induced cell death, whereas a longer period of time (24h) with bafilomycin-A1 was required to induce an equivalent effect. Neither changes in ROS nor ATP production is associated with such death mechanisms. Flow cytometry, LC3-II expression levels, and LC3-GFP puncta formation revealed a similar lysosomotropic effect for both compounds. We used a molecular docking approach, that predicts a stronger inhibitory activity against V-ATPase-C1 and C2 domains for bafilomycin-A1 in comparison to ML9. Bafilomycin-A1 and ML9 are lysosomotropic agents, involved in cell death events. But such death events are not associated with ATP and ROS production. Furthermore, both the drugs target lysosomes through different mechanisms. For the latter, cell death is likely due to lysosomal membrane permeabilization and release of lysosomal proteases into the cytosol.

The efficacy of platelet rich plasma in the treatment of resistant plantar Fasciitis-follow up of 12 months

Background: Platelet-rich plasma (PRP) has been used as an alternative therapy for plantar fasciitis (PF) to reduce heel pain and improve functional restoration. PRP has a role in the inflammatory, coagulation processes as well as immunity modulation. During the degranulation of platelets they release cytokines and other growth factors. These promote angiogenesis, help in tissue remodelling and wound healing. They may have a pain relieving effect depending on the release of proteases with analgesic properties. This is a study of 26 patients treated with PRP for the treatment of resistant PF. Objective: To compare the efficacy of local injection of PRP at different time intervals for the treatment of resistant PF.Materials and methods: In this study, 26 patients diagnosed with resistant Plantar Fasciitis, underwent local injection of PRP and were followed up at 3, 6, 9 and 12 months and VAS recorded. Results: In this study, the VAS showed significant improvement from the pre injection VAS to the VAS at 3 months, 6 months, 9 months and 12 months after injection. The difference in the VAS from 3 months to 6, 9 and 12 months post was statistically significant. The difference in VAS from 6 months to 9 months and 12 months post injection was statistically highly significant and very highly significant respectively. In between 9 months to 12 months post injection, there was no statistical significance. Conclusion: Platelet Rich Plasma provides good symptomatic relief in the treatment of Plantar Fasciitis with sustained gradual relief of pain and improving daily function and activity level. Platelet Rich Plasma proved to be effective modality in providing continuous and sustained relief of pain over a period of 12 months.

Complement activation on neutrophils initiates endothelial adhesion and extravasation

Neutrophils are essential to the pathogenesis of many inflammatory diseases. In the autoantibody-mediated K/BxN model of inflammatory arthritis, the alternative pathway (AP) of complement and Fc gamma receptors (FcγRs) are required for disease development while the classical pathway is dispensable. The reason for this differential requirement is unknown. We show that within minutes of K/BxN serum injection complement activation (CA) is detected on circulating neutrophils, as evidenced by cell surface C3 fragment deposition. CA requires the AP factor B and FcγRs but not C4, implying that engagement of FcγRs by autoantibody or immune complexes directly triggers AP C3 convertase assembly. The absence of C5 does not prevent CA on neutrophils but diminishes the upregulation of adhesion molecules. In vivo two-photon microscopy reveals that CA on neutrophils is critical for neutrophil extravasation and generation of C5a at the site of inflammation. C5a stimulates the release of neutrophil proteases, which contribute to the degradation of VE-cadherin, an adherens junction protein that regulates endothelial barrier integrity. C5a receptor antagonism blocks the extracellular release of neutrophil proteases, suppressing VE-cadherin degradation and neutrophil transendothelial migration in vivo. These results elucidate the AP-dependent intravascular neutrophil-endothelial interactions that initiate the inflammatory cascade in this disease model but may be generalizable to neutrophil extravasation in other inflammatory processes.

Expression, Role, and Regulation of Neutrophil Fcγ Receptors.

Neutrophils are best known for their critical role in host defense, for which they utilize multiple innate immune mechanisms, including microbe-associated pattern recognition, phagocytosis, production of reactive oxygen species, and the release of potent proteases, mediators, antimicrobials, and neutrophil extracellular traps. Beyond their well-established contribution to innate immunity, neutrophils were more recently reported to interact with various other cell types, including cells from the adaptive immune system, thereby enabling neutrophils to tune the overall immune response of the host. Neutrophils express different receptors for IgG antibodies (Fcγ receptors), which facilitate the engulfment of IgG-opsonized microbes and trigger cell activation upon cross-linking of several receptors. Indeed, FcγRs (via IgG antibodies) confer neutrophils with a key feature of the adaptive immunity: an antigen-specific cell response. This review summarizes the expression and function of FcγRs on human neutrophils in health and disease and how they are affected by polymorphisms in the FCGR loci. Additionally, we will discuss the role of neutrophils in providing help to marginal zone B cells for the production of antibodies, which in turn may trigger neutrophil effector functions when engaging FcγRs.

Roles of Growth Differentiation Factor 15 in Atherosclerosis and Coronary Artery Disease.

The majority of acute cardiovascular events (CVE) in patients are caused by occlusive thrombosis because of rupture or erosion of atherosclerotic plaques.1 Growth differentiation factor 15 (GDF‐15), a stress‐responsive member of the transforming growth factor‐β (TGF‐β) cytokine superfamily, has been shown to be a strong and independent predictor of mortality and disease progression in patients with atherosclerosis and coronary artery disease (CAD), such as acute coronary syndromes (ACS) and stable angina pectoris.2 The development of atherosclerosis is dependent upon a high‐inflammatory content, which has been shown to modulate lesion initiation, progression, and potentially devastating thrombotic complications.3 Angiogenesis plays an important role in the progression of atherosclerotic plaque and complications.4, 5, 6 Atherosclerosis and cancer arise from multiple factors and are consolidated from the very early stages of development up to the advanced forms in inflammatory processes. Uncontrolled cell proliferation and oxidative stress and angiogenesis appear to be unifying causal factors in both diseases.7 A local inflammatory state occurring in atherosclerotic lesions has been implicated in angiogenesis through activation of endothelial cells, release of chemokines, cytokines, growth factors, lipid mediators, proteases, and increase of endothelial metabolic rate. The angiogenesis allows extravasation of the plasma component, leading to future thromboembolic events.8, 9, 10, 11 GDF‐15 might be an acute phase modifier of TGF‐βRII‐dependent proinflammatory responses to atherosclerotic plaque rupture and thrombus formation12 (Figure). Although the exact biological functions of GDF‐15 are still poorly understood, it has been shown to regulate inflammatory and angiogenesis pathways (Figure). GDF‐15 exhibits differing and even opposing functions under various circumstances. For instance, GDF‐15 has proapoptosis, antiapoptosis, proangiogenesis, antiangiogenesis, proinflammatory, and anti‐inflammatory properties.12, 13 Therefore, GDF‐15 exhibits a complex pattern with beneficial and harmful functions. GDF‐15 promoter contains p53‐transcription factor binding sites that are required and sufficient for the induction of GDF‐15 expression.14 Activation of p53 is a fundamental cellular response to inflammation, oxidative stress, hypoxia, telomere erosion, and oncogene activation. The circulating levels of GDF‐15 reflect these acute and chronic inflammatory conditions linked with atherosclerosis and CAD. Open in a separate window Figure 1 Schematic overview of a vulnerable plaque in advanced atherosclerosis. Plaque formation is initiated by endothelial cell dysfunction and subsequent angiogenesis and release of proinflammatory factors mediated by GDF‐15, contributing to the progression of atherosclerotic lesions and the development of plaque rupture and thrombus formation in atherosclerotic status. CRP indicates C‐reactive protein; GDF‐15, growth differentiation factor 15; MAPK, mitogen‐activated protein kinase; M‐CSF, macrophage colony‐stimulating factor; TGF‐βRII, transforming growth factor‐βRII; TNF‐α, tumor necrosis factor‐α.

Effect of non-surgical periodontal therapy on serum and crevicular level of advanced glyacation end product in chronic periodontitis among a sample of the Egyptian population

Background: AGEs are formed under oxidative conditions and are called advanced glyco-xidation end products. Periodontitis is a multifactorial inflammatory condition. The periodontal tissue damage is caused directly by bacteria and indirectly by inflammation and immunologic response against the bacteria. This inflammatory response is characterized by generation of prolonged release of neutrophil proteolytic enzymes, pro-inflammatory mediators and reactive oxygen species (ROS). Therefore, periodontitis is strictly linked to numerous pathologies which are related to production of oxidative stress and AGEs. Methods: The study was conducted on thirty participants, they were divided into two groups; group (A) which included fifteen medically free subjects, and group (B) which included fifteen patients suffering from periodontitis. Serum and GCF samples were collected for assessment of AGEs before and after non -surgical periodontal therapy. Data were analysed by SPSS. Results: Periodontitis group showed statistically significant higher mean AGE levels than control group in both serum and GCF samples. Moreover, there was a statistically significant decrease in AGE levels in both serum and GCF after treatment.Conclusion: Crevicular level of AGEs can be used as a non-invasive diagnostic and prognostic biomarker for periodontitis.

Salicylate and Procyanidin-Rich Stem Extracts of Gaultheria procumbens L. Inhibit Pro-Inflammatory Enzymes and Suppress Pro-Inflammatory and Pro-Oxidant Functions of Human Neutrophils Ex Vivo.

Salicylate-rich plants are an attractive alternative to synthetic anti-inflammatory drugs due to a better safety profile and the advantage of complementary anti-inflammatory and antioxidant effects of the co-occurring non-salicylate phytochemicals. Here, the phytochemical value and biological effects in vitro and ex vivo of the stems of one of such plants, Gaultheria procumbens L., were evaluated. The best extrahent for effective recovery of the active stem molecules was established in comparative studies of five extracts. The UHPLC-PDA-ESI-MS3, HPLC-PDA, and UV-photometric assays revealed that the selected acetone extract (AE) accumulates a rich polyphenolic fraction (35 identified constituents; total content 427.2 mg/g dw), mainly flavanols (catechins and proanthocyanidins; 201.3 mg/g dw) and methyl salicylate glycosides (199.9 mg/g dw). The extract and its model components were effective cyclooxygenase-2, lipoxygenase, and hyaluronidase inhibitors; exhibited strong antioxidant capacity in six non-cellular in vitro models (AE and procyanidins); and also significantly and dose-dependently reduced the levels of reactive oxygen species (ROS), and the release of cytokines (IL-1β, IL-8, TNF-α) and proteinases (elastase-2, metalloproteinase-9) in human neutrophils stimulated ex vivo by lipopolysaccharide (LPS) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). The cellular safety of AE was demonstrated by flow cytometry. The results support the application of the plant in traditional medicine and encourage the use of AE for development of new therapeutic agents.

Polysaccharides based microspheres for multiple encapsulations and simultaneous release of proteases.

The work is focused on the development of microspheres based on the combination of two polysaccharides; chitosan and alginic acid with the aim to allocate, hold, release and protect environmentally sensible molecules. The microspheres were prepared using a solvent-free, low cost and scalable approach and two enzymes; trypsin and protease from Aspergillus Oryzae have been used as a model to evaluate the microspheres peculiarities. The proteins were encapsulated during the microspheres preparation. The relationship between the polysaccharides weight ratio and the morphology, stability and ability of the carrier to allocate the enzymes has been evaluated. The enzymatic activity and the release kinetics were assessed in different conditions to assess the impact of the external environment. Obtained results demonstrate the efficacy of the prepared microspheres to preserve the activity of relevant bioactive compounds which are highly relevant in food, cosmetic and pharmaceutic, but the application is limited due to their high sensibility.

Cathepsin G Activity as a New Marker for Detecting Airway Inflammation by Microscopy and Flow Cytometry.

Muco-obstructive lung diseases feature extensive bronchiectasis due to the uncontrolled release of neutrophil serine proteases into the airways. To assess if cathepsin G (CG) is a novel key player in chronic lung inflammation, we developed membrane-bound (mSAM) and soluble (sSAM) FRET reporters. The probes quantitatively revealed elevated CG activity in samples from 46 patients. For future basic science and personalized clinical applications, we developed a rapid, highly informative, and easily translatable small-molecule FRET flow cytometry assay for monitoring protease activity including cathepsin G. We demonstrated that mSAM distinguished healthy from patient cells by FRET-based flow cytometry with excellent correlation to confocal microscopy data.