Truncated Pneumolysin from Streptococcus pneumoniae as a TLR4-Antagonizing New Drug for Chronic Inflammatory Conditions.

Microbial proteins have recently been found to have more benefits in clinical disease treatment because of their better-developed strategy and properties than traditional medicine. In this study, we investigated the effectiveness of a truncated peptide synthesized from the C-terminal sequence of pneumolysin, i.e., C70PLY4, in Streptococcus pneumoniae, in treating chronic inflammatory conditions. It has been shown that C70PLY4 significantly blocks the transendothelial migration of neutrophils and attenuates the formation of atherosclerotic plaque and the secretion of soluble forms of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in high-fat-diet/streptozotocin-induced inflammatory rats. The mechanism and the docking simulation analysis further indicated that C70PLY4 might serve as a Toll-like receptor 4 (TLR4) antagonist by competing for the binding site of MD2, an indispensable protein for lipopolysaccharide (LPS)-TLR4 interaction signaling, on the TLR4 structure. Moreover, compared to the full-length PLY, C70PLY4 seems to have no cytotoxicity in human vascular endothelial cells. Our study elucidated a possible therapeutic efficacy of C70PLY4 in reducing chronic inflammatory conditions and clarified the underlying mechanism. Thus, our findings identify a new drug candidate that, by blocking TLR4 activity, could be an effective treatment for patients with chronic inflammatory diseases.

Heptapeptide HP3 acts as a potent inhibitor of experimental imiquimod­induced murine psoriasis and impedes the trans­endothelial migration of mononuclear cells.

During the progression of psoriatic lesions, abundant cellular infiltration of myeloid cells, such as macrophages and activated dendritic cells, occurs in the skin and the infiltrating cells interact with naive lymphoid cells to generate a T helper (Th)1 and Th17 environment. Therapies to treat psoriasis include phototherapy, non­steroidal and steroidal drugs, as well as antibodies to block tumor necrosis factor­α, interleukin (IL)­17­A and IL­12/IL­23, which all focus on decreasing the proinflammatory hallmark of psoriasis. The present study obtained the heptapeptide HP3 derived from phage display technology that blocks mononuclear cell adhesion to endothelial cells and inhibits trans­endothelial migration in vitro. The activity of the heptapeptide in a murine model of psoriasis was also assessed, which indicated that early administration inhibited the development of psoriatic lesions. Therefore, the results suggested that HP3 may serve as a potential therapeutic target for psoriasis.

Endocan regulates acute lung inflammation through control of leukocyte diapedesis.

Acute respiratory distress syndrome is a severe form of respiratory failure, occurring in up to 20% of patients admitted to the intensive care unit with sepsis. Dysregulated leukocyte diapedesis is a major contributor to acute respiratory distress syndrome. Endocan is a circulating proteoglycan that binds to the leukocyte integrin leukocyte functional antigen-1 and blocks its interaction with its endothelial ligand, ICAM-1. The objective of this study was to evaluate the role of endocan in the control of acute lung inflammation. In vitro, endocan inhibited human leukocyte transendothelial migration as well as ICAM-1-dependent migration but had a very mild effect on ICAM-1-dependent adhesion. Endocan also acted as an inhibitor of transendothelial migration of mouse leukocytes. The effect of systemic administration of recombinant human endocan was assessed in a model of acute lung inflammation in BALB/c mice. Treatment with endocan 1 h after intratracheal LPS challenge reduced the alveolar inflammatory response, diminished histological features of acute lung injury, and improved respiratory function. These results highlight the anti-inflammatory role of human endocan and its protective effect against acute lung injury.NEW & NOTEWORTHY We show here that endocan inhibits ICAM-1-dependent human leukocyte transendothelial migration and ICAM-1-dependent adhesion. We also found that in BALB/c mice with tracheal LPS-induced acute lung injury treatment with recombinant human endocan reduces lung inflammation, notably through reduction of neutrophilic recruitment, and restores normal lung function. These results confirm the hypothesis that human endocan may have a protective effect against acute lung inflammation.

QobuR - A new in vitro human corneal epithelial model for preclinical drug screening.

A new in vitro human corneal epithelial model (QobuR) obtained from normal limbal tissue has been developed to study ocular irritancy of different ophthalmic compounded drugs. Phenotypical characterization and trans-epithelial electrical resistance (TEER) of QobuR revealed essential similarities compared with a native human cornea, displaying functional markers and TEER values near 1500 Ωcm2 at day 7th of cellular differentiation. Using this model, ocular irritancy and barrier integrity alterations were evaluated using MTT reaction and variations in TEER. We found that some of the Non-Irritant products evaluated still damage the corneal epithelial integrity and current protocols for ocular irritancy should therefore include a barrier integrity evaluation. Moreover, in order to comprehensively evaluate corneal permeability of the active ingredients, we propose the use of QobuR as an all-in-one alternative method for evaluating ocular irritancy, barrier disruptions and permeability rates of topically applied ocular drugs to improve current in vitro drug testing procedures.

Cenicriviroc inhibits trans-endothelial passage of monocytes and is associated with impaired E-selectin expression.

Incidences of cardiovascular diseases (CVD) are high among virologically suppressed HIV-infected individuals. Monocyte activation and trafficking are key mechanisms in the evolution of CVD. We studied the ability of cenicriviroc (CVC), a dual C-C chemokine receptor type 2 (CCR2) and CCR5 antagonist, to influence the migration of monocytes from HIV-infected individuals on antiretroviral therapy (ART). Monocytes were derived from 23 ART-suppressed HIV-infected and 16 HIV-uninfected donors. In a trans-endothelial migration model, monocytes, and human aortic endothelial cells (HAoECs) were exposed to cenicriviroc and migrated monocytes, quantified. Expression of CCR2 and CCR5 on monocytes and adhesion molecules (E-selectin, ICAM-1, VCAM-1, PECAM-1, and CD99) on HAoECs were measured. The single antagonists, BMS-22 (CCR2), and maraviroc (CCR5), served as controls. When both HAoECs and monocytes together were exposed to the antagonists, cenicriviroc led to a greater decrease in monocyte migration compared to BMS-22 or vehicle in both HIV-infected and HIV-uninfected groups (P < 0.05), with maraviroc having no inhibitory effect. Cenicriviroc treatment of HAoECs alone decreased monocyte migration in the HIV-infected group when compared to vehicle (P < 0.01). Inhibition of migration was not evident when monocytes alone were exposed to cenicriviroc, BMS-22 or maraviroc. Incubation of HAoECs with cenicriviroc decreased E-selectin expression (P = 0.045) but had limited effects on the other adhesion molecules. Cenicriviroc inhibits monocyte trans-endothelial migration more effectively than single chemokine receptor blockade, which may be mediated via disruption of monocyte-endothelial tethering through reduced E-selectin expression. Cenicriviroc should be considered as a therapeutic intervention to reduce detrimental monocyte trafficking.

DynaMiTES - A dynamic cell culture platform for in vitro drug testing PART 2 - Ocular DynaMiTES for drug absorption studies of the anterior eye.

In the present study, a formerly designed Dynamic Micro Tissue Engineering System (DynaMiTES) was applied with our prevalidated human hemicornea (HC) construct to obtain a test platform for improved absorption studies of the anterior eye (Ocular DynaMiTES). First, the cultivation procedure of the classic HC was slightly adapted to the novel DynaMiTES design. The obtained inverted HC was then compared to classic HC regarding cell morphology using light and scanning electron microscopy, cell viability using MTT dye reaction and epithelial barrier properties observing transepithelial electrical resistance and apparent permeation coefficient of sodium fluorescein. These tested cell criteria were similar. In addition, the effects of four different flow rates on the same cell characteristics were investigated using the DynaMiTES. Because no harmful potential of flow was found, dynamic absorption studies of sodium fluorescein with and without 0.005%, 0.01% and 0.02% benzalkonium chloride were performed compared to the common static test procedure. In this proof-of-concept study, the dynamic test conditions showed different results than the static test conditions with a better prediction of in vivo data. Thus, we propose that our DynaMiTES platform provides great opportunities for the improvement of common in vitro drug testing procedures.

Cytosolic Phospholipase A2α Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection.

Pulmonary infection by Streptococcus pneumoniae is characterized by a robust alveolar infiltration of neutrophils (polymorphonuclear cells [PMNs]) that can promote systemic spread of the infection if not resolved. We previously showed that 12-lipoxygenase (12-LOX), which is required to generate the PMN chemoattractant hepoxilin A3 (HXA3) from arachidonic acid (AA), promotes acute pulmonary inflammation and systemic infection after lung challenge with S. pneumoniae As phospholipase A2 (PLA2) promotes the release of AA, we investigated the role of PLA2 in local and systemic disease during S. pneumoniae infection. The group IVA cytosolic isoform of PLA2 (cPLA2α) was activated upon S. pneumoniae infection of cultured lung epithelial cells and was critical for AA release from membrane phospholipids. Pharmacological inhibition of this enzyme blocked S. pneumoniae-induced PMN transepithelial migration in vitro Genetic ablation of the cPLA2 isoform cPLA2α dramatically reduced lung inflammation in mice upon high-dose pulmonary challenge with S. pneumoniae The cPLA2α-deficient mice also suffered no bacteremia and survived a pulmonary challenge that was lethal to wild-type mice. Our data suggest that cPLA2α plays a crucial role in eliciting pulmonary inflammation during pneumococcal infection and is required for lethal systemic infection following S. pneumoniae lung challenge.

Inhibitory effect of trans-caryophyllene (TC) on leukocyte-endothelial attachment.

trans-Caryophyllene (TC) is a major component found in the essential oils of many spices and foods/medicinal plants. It is a natural sesquiterpene and has been the subject of numerous studies. However, the effects of TC on vascular inflammation remain unknown. In this study, we reported that TC treatment in human umbilical vein endothelial cells (HUVECs) prevented attachment of monocytic leukemia cell line THP-1 cells to endothelial cells. In addition, in vivo results indicate that TC inhibited macrophage infiltration to the aortic surface and reduced total serum levels of cholesterol and triglycerides. Importantly, administration of TC could inhibit the induction of vascular cell adhesion molecule-1 (VCAM-1) both in vitro and in vivo. Notably, our data indicate that the inhibitory effects of TC on the expression of VCAM-1 are mediated by the JAK2/STAT1/IRF-1 pathway. TC is a specific agonist of the type 2 cannabinoid receptor (CB2R). Importantly, we further verified that the inhibitory effects of TC on the expression of IRF-1 and VCAM-1 are dependent on activation of CB2R. Inhibition of CB2R by either specific inhibitors or RNA interference abolished the inhibitory effects of TC on the expression of IRF-1 and VCAM-1. Our results suggest that TC might have a capacity to suppress the development of atherosclerosis.

Barrier protective effects of 2,4,6-trihydroxy-3-geranyl acetophenone on lipopolysaccharides-stimulated inflammatory responses in human umbilical vein endothelial cells.

PHARMOCOLOGICAL RELEVANCE: 2,4,6-trihydroxy-3-geranyl acetophenone (tHGA), is a phloroglucinol compound found naturally in Melicope ptelefolia. Melicope ptelefolia has been used traditionally for centuries as natural remedy for wound infections and inflammatory diseases. AIM OF THE STUDY: Endothelial barrier dysfunction is a pathological hallmark of many diseases and can be caused by lipopolysaccharides (LPS) stimulation. Therefore, this study aims to investigate the possible barrier protective effects of tHGA upon LPS-stimulated inflammatory responses in human umbilical vein endothelial cells (HUVECs). MATERIALS AND METHODS: HUVECs were pretreated with tHGA prior to LPS stimulation, where inflammatory parameters including permeability, monocyte adhesion and migration, and release of pro-inflammatory mediators were examined. Additionally, the effect of tHGA on F-actin rearrangement and adhesion protein expression of LPS-stimulated HUVECs was evaluated. RESULTS: It was found that pretreatment with tHGA inhibited monocyte adhesion and transendothelial migration, reduced endothelial hyperpermeability and secretion of prostaglandin E2 (PGE2). Additionally, tHGA inhibited cytoskeletal rearrangement and adhesion protein expression on LPS-stimulated HUVECs. CONCLUSION: As the regulation of endothelial barrier dysfunction can be one of the therapeutic strategies to improve the outcome of inflammation, tHGA may be able to preserve vascular barrier integrity of endothelial cells following LPS-stimulated dysfunction, thereby endorsing its potential usefulness in vascular inflammatory diseases.

Antithrombotic Activity of a New Hypoglycemic Compound Limiglidole in Mouse Model of Cell Thrombosis.

Antithrombotic activity of hypoglycemic compound limiglidole that exhibits antiplatelet activity 2-fold exceeded activity of antiplatelet agent acetylsalicylic acid in the mouse model of systemic collagen-epinephrine thrombosis. Limiglidole signifi cantly reduced the relative and mean area of blood clots in the sections of mouse lungs.

Selenoglycoproteins attenuate adhesion of tumor cells to the brain microvascular endothelium via a process involving NF-κB activation.

Selenium-containing compounds and selenized yeast have anticancer properties. In order to address possible mechanisms involved in these effects, selenoglycoproteins (SGPs) were extracted from selenium-enriched yeast at pH 4.0 and 6.5 (the fractions are called SGP40 and SGP65, respectively), followed by evaluation of their impact on the interactions of lung and breast tumor cells with human brain microvascular endothelial cells (HBMECs). Extracted SGPs, especially SGP40, significantly inhibited adhesion of tumor cells to HBMECs and their transendothelial migration. Because the active components of SGPs are unknown, small selenium-containing compounds [leucyl-valyl-selenomethionyl-arginine (LVSe-MR) and methylselenoadenosine (M-Se-A)], which are normally present in selenized yeast, were introduced as additional treatment groups. Treatment of HBMECs with SGP40, LVSe-MR and M-Se-A induced changes in gene signatures, which suggested a central involvement of nuclear factor (NF)-κB-dependent pathway. These observations were confirmed in the subsequent analysis of NF-κB DNA binding activity, quantitative measurements of the expression of selected genes and proteins, and tumor cell adhesion assay with a specific NF-κB inhibitor as the additional treatment factor. These findings indicate that specific organic selenium-containing compounds have the ability to inhibit tumor cell adhesion to brain endothelial cells via down-regulation of NF-κB. SGPs appear to be more effective than small selenium-containing compounds, suggesting the role of not only selenium but also the glycoprotein component in the observed protective impact.

The α-tocopherol form of vitamin E reverses age-associated susceptibility to streptococcus pneumoniae lung infection by modulating pulmonary neutrophil recruitment.

Streptococcus pneumoniae infections are an important cause of morbidity and mortality in older patients. Uncontrolled neutrophil-driven pulmonary inflammation exacerbates this disease. To test whether the α-tocopherol (α-Toc) form of vitamin E, a regulator of immunity, can modulate neutrophil responses as a preventive strategy to mitigate the age-associated decline in resistance to S. pneumoniae, young (4 mo) and old (22-24 mo) C57BL/6 mice were fed a diet containing 30-PPM (control) or 500-PPM (supplemented) α-Toc for 4 wk and intratracheally infected with S. pneumoniae. Aged mice fed a control diet were exquisitely more susceptible to S. pneumoniae than young mice. At 2 d postinfection, aged mice suffered 1000-fold higher pulmonary bacterial burden, 2.2-fold higher levels of neutrophil recruitment to the lung, and a 2.25-fold higher rate of lethal septicemia. Strikingly, α-Toc supplementation of aged mice resulted in a 1000-fold lower bacterial lung burden and full control of infection. This α-Toc-induced resistance to pneumococcal challenge was associated with a 2-fold fewer pulmonary neutrophils, a level comparable to S. pneumoniae-challenged, conventionally fed young mice. α-Toc directly inhibited neutrophil egress across epithelial cell monolayers in vitro in response to pneumococci or hepoxilin-A3, an eicosanoid required for pneumococcus-elicited neutrophil trans-epithelial migration. α-Toc altered expression of multiple epithelial and neutrophil adhesion molecules involved in migration, including CD55, CD47, CD18/CD11b, and ICAM-1. These findings suggest that α-Toc enhances resistance of aged mice to bacterial pneumonia by modulating the innate immune response, a finding that has potential clinical significance in combating infection in aged individuals through nutritional intervention.

An inflammation loop orchestrated by S100A9 and calprotectin is critical for development of arthritis.

OBJECTIVE: The S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity. METHODS: In this study, we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion. RESULTS: Treatment with anti-S100A9 antibodies improved the clinical score by 50%, diminished immune cell infiltration, reduced inflammatory cytokines, both in serum and in the joints, and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFα, IL-1ß and IL-6, and of chemokines like MIP-1α and MCP-1. CONCLUSION: The effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively, our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore, S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed.

Dietary curcumin inhibits atherosclerosis by affecting the expression of genes involved in leukocyte adhesion and transendothelial migration.

SCOPE: The aim of the study was to examine the atheroprotective effect of dietary curcumin in a mouse model of atherosclerosis and to identify its cellular and molecular targets at the vascular level. METHODS AND RESULTS: ApoE(-/-) mice were fed with curcumin at 0.2% (wt/wt) in diet for 4 months. This supplementation reduced the extent of atherosclerotic lesion by 26% and induced changes in expression of genes implicated in cell adhesion and transendothelial migration or cytoskeleton organization, as revealed by a transcriptomic analysis in the aorta. Expression profile of these genes suggests reduction in both leukocyte adhesion and transendothelial migration. In agreement with this hypothesis, we observed a reduction (-37%) in macrophage infiltration in the plaque, as measured by immunohistochemistry, and, in vitro, a lower adhesion of monocytes to TNF-α-stimulated endothelial cells (-32%) after exposure to a nutritionally achievable concentration of curcumin. These changes in gene expression could be related to the observed increased expression of IκB protein and decrease of TNF-α-induced NF-κB/DNA binding and NF-κB-transcriptional activity upon exposure to curcumin. CONCLUSION: Our findings pointed out that the antiatherogenic effect of curcumin could be linked to its effect on gene networks and cell functions related to leukocyte adhesion and transendothelial migration via NF-κB-dependent pathways.

Effects of astragaloside IV on eosinophil activation induced by house dust mite allergen.

Astragaloside IV (AS-IV) has been noted for its reduction of eosinophilic airway inflammation in a murine model of chronic asthma. To gain a better understanding of the mechanisms involved in this anti-inflammatory phenomenon, the effect of AS-IV on human blood eosinophils was studied in vitro. Eosinophils were isolated from the blood of patients with mild atopic asthma, preincubated with AS-IV for 1 h and stimulated in the presence or absence of the house dust mite allergen Dermatophagoides pteronyssinus (Der p) 1 for 4 h. The survival of the eosinophils at 48 h was investigated using trypan blue and the surface expression of CC chemokine receptor 3 (CCR3) and intercellular adhesion molecule-1 (ICAM-1) by the eosinophils was analyzed using flow cytometry. The secretion of cytokines in the supernatants and the chemotaxis of the eosinophils were measured by ELISA and the transwell system, respectively. Der p 1 was found to prolong the survival of the eosinophils. Similarly, the expression of CCR3 and ICAM-1, secretion of interleukin (IL)-1ß, IL-5, tumor necrosis factor (TNF)-α and the granulocyte macrophage colony stimulating factor (GM-CSF) and transmigration of the eosinophils were increased in the presence of Der p 1. However, these inductive effects on the eosinophils were significantly inhibited by AS-IV (50 µg/ml). These findings suggest that AS-IV modulates eosinophil activation and trafficking in response to Der p 1 and may therefore be a useful therapeutic option in eosinophilic asthma.

Inhibitory effects of luteolin on transendothelial migration of monocytes and formation of lipid-laden macrophages.

OBJECTIVE: Because of an initial activation of proinflammatory cytokines that facilitates leukocyte transmigration, atherosclerosis is a chronic inflammatory disease and its severity is accelerated by the occurrence of complex interactions of oxidatively modified low-density lipoprotein (LDL) with monocyte-derived macrophages. METHODS: The present study investigated whether luteolin suppresses adheren junction-associated monocyte transmigration and platelet-derived growth factor-BB-mediated foam cell formation. The involvement of monocyte integrins and macrophage scavenger receptors (SRs) also was determined. RESULTS: Luteolin, non-toxic at 1 to 20 µmol/L, blocked the monocyte-endothelium interactions by inhibiting the cytokine-associated monocyte induction of integrin-ß2. Luteolin retarded the transendothelial migration of monocytes by firmly localizing the occludin present in paracellular endothelial junctions and by blunting the monocyte activity of matrix-degrading matrix metalloproteinase-9. Treatment with luteolin showed inhibitory effects on oxidized LDL-triggered foam cell formation by decreasing SR-A and SR-B1 induction in THP-1 cell-derived macrophages, which was confirmed by Oil red O and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate staining. Furthermore, luteolin attenuated the oxidized LDL-induced macrophage secretion of platelet-derived growth factor-BB, entailing the induction of SR-A and SR-B1. These results demonstrate that luteolin encumbered monocyte cytokine-instigated endothelial transmigration and oxidized LDL-elicited macrophage foam cell formation. CONCLUSION: Luteolin may qualify as an antiatherogenic agent in LDL systems, which may have implications for strategies attenuating monocyte/macrophage dysfunction-related atherosclerosis.

Imatinib decreases endometrial stromal cell transmesothial migration and proliferation in the extracellular matrix of modeled peritoneum.

OBJECTIVE: To characterize imatinib's effect on endometrial stromal cell (ESC) attachment, proliferation, and invasion in modeled peritoneum. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): Twelve normally cycling women. INTERVENTION(S): Imatinib treatment in ESCs from women without endometriosis. MAIN OUTCOME MEASURE(S): Rate of ESC attachment, proliferation, and invasion. RESULT(S): Imatinib treatment at 10 µM had no effect on ESC attachment. Treatment with 0.5 µM, 2 µM, and 10 µM of imatinib reduced ESC proliferation by 30%, 72%, and 76%, respectively. The 0.1 µM dose of imatinib had no effect on proliferation. Treatment with 5 µM and 10 µM of imatinib reduced ESC invasion by 30% and 73%, respectively. The 2 µM dose had no effect on invasion. CONCLUSION(S): Imatinib treatment reduces ESC proliferation and invasion in modeled peritoneum without altering attachment. Imatinib may have a therapeutic role in endometriosis treatment.