Defective Induction of COX-2 Expression by Psoriatic Fibroblasts Promotes Pro-inflammatory Activation of Macrophages.

Fibroblasts play an important role as members of the innate immune system through the secretion of COX-2-derived inflammatory mediators such as prostaglandin E2 (PGE2). However, it has been described that dermal fibroblasts behave like mesenchymal stem cells reducing lymphocyte recruitment and dendritic cell activation through PGE2 release. As the role of fibroblasts in psoriasis remains poorly characterized, in the present study we have evaluated the possible influence of PGE2 derived from dermal fibroblasts as modulator of the immune response in psoriatic skin. Our results indicate that under inflammatory conditions, psoriatic fibroblasts showed defective induction of COX-2, which resulted in diminished production of PGE2, in contrast to healthy fibroblasts. This phenotype correlated with deficient c-Jun N-terminal kinase (JNK) activation, in accordance with the hypothesis that alterations in members of the JNK pathway are associated with psoriasis. Furthermore, conditioned medium from psoriatic fibroblasts promoted the polarization of monocytic cells toward a pro-inflammatory profile, effect that was mimicked in healthy fibroblasts after pre-incubation with indomethacin. These results are consistent with a prominent role of dermal fibroblasts in the regulation of inflammatory response through the participation of COX-derived metabolites. This resolutive behavior seems to be defective in psoriatic fibroblasts, offering a possible explanation for the chronification of the disease and for the exacerbation triggered by nonsteroidal anti-inflammatory drugs (NSAIDS) such as indomethacin.

Adenosine A2A and A2B Receptors Differentially Modulate Keratinocyte Proliferation: Possible Deregulation in Psoriatic Epidermis.

Adenosine is a potent regulator of inflammation and immunity, but the role of adenosine receptors in keratinocytes remains controversial. We determined that in addition to A2B receptors, human epidermal keratinocytes also express A2A receptors, although to a lower extent. Through the use of selective adenosine receptor agonists and antagonists, we showed that physiological concentrations of adenosine activate A2B receptors in normal human keratinocytes, inducing cell cycle arrest through the increase of intracellular calcium but not through cAMP signaling. In contrast, the selective activation of A2A receptors by CGS-21680 induces keratinocyte proliferation via p38-mitogen-activated protein kinase activation. Adenosine and selective A2A and A2B agonists presented anti-inflammatory profiles independent of adenosine receptors but mediated by membrane phosphatase activation. Finally, keratinocyte exposure to diverse inflammatory cytokines altered adenosine receptor expression by reducing A2B and increasing A2A, a pattern also observed in psoriatic epidermis. Because increased epidermal turnover and inflammatory response are characteristics of psoriatic disease, further studies are needed to assess the role and consequences of the altered adenosine receptor expression in lesional and nonlesional psoriatic keratinocytes.

Pharmacological Interventions to Ameliorate Neuropathological Symptoms in a Mouse Model of Lafora Disease.

Lafora disease (LD, OMIM 254780) is a rare fatal neurodegenerative disorder that usually occurs during childhood with generalized tonic-clonic seizures, myoclonus, absences, drop attacks, or visual seizures. Unfortunately, at present, available treatments are only palliatives and no curative drugs are available yet. The hallmark of the disease is the accumulation of insoluble polyglucosan inclusions, called Lafora bodies (LBs), within the neurons but also in heart, muscle, and liver cells. Mouse models lacking functional EPM2A or EPM2B genes (the two major loci related to the disease) recapitulate the Lafora disease phenotype: they accumulate polyglucosan inclusions, show signs of neurodegeneration, and have a dysregulation of protein clearance and endoplasmic reticulum stress response. In this study, we have subjected a mouse model of LD (Epm2b-/-) to different pharmacological interventions aimed to alleviate protein clearance and endoplasmic reticulum stress. We have used two chemical chaperones, trehalose and 4-phenylbutyric acid. In addition, we have used metformin, an activator of AMP-activated protein kinase (AMPK), as it has a recognized neuroprotective role in other neurodegenerative diseases. Here, we show that treatment with 4-phenylbutyric acid or metformin decreases the accumulation of Lafora bodies and polyubiquitin protein aggregates in the brain of treated animals. 4-Phenylbutyric acid and metformin also diminish neurodegeneration (measured in terms of neuronal loss and reactive gliosis) and ameliorate neuropsychological tests of Epm2b-/- mice. As these compounds have good safety records and are already approved for clinical uses on different neurological pathologies, we think that the translation of our results to the clinical practice could be straightforward.

NF-κB and STAT3 inhibition as a therapeutic strategy in psoriasis: in vitro and in vivo effects of BTH.

Benzo[b]thiophen-2-yl-3-bromo-5-hydroxy-5H-furan-2-one (BTH) is a simple and interesting synthetic derivative of petrosaspongiolide M, a natural compound isolated from a sea sponge with demonstrated potent anti-inflammatory activity through inhibition of the NF-κB signaling pathway. In the present study, we report the in vitro and in vivo pharmacological effect of BTH on some parameters related to the innate and adaptive response in the pathogenesis of psoriasis. BTH inhibited the release of some of the key psoriatic cytokines such as tumor necrosis factor α, IL-8, IL-6, and CCL27 through the downregulation of NF-κB in normal human keratinocytes. Moreover, it impaired signal transducers and activators of transcription 3 (STAT3) phosphorylation and translocation to the nucleus, which resulted in decreased keratinocyte proliferation. These results were confirmed in vivo in two murine models of psoriasis: the epidermal hyperplasia induced by 12-O-tetradecanoylphorbol-13-acetate and the imiquimod-induced skin inflammation model. In both cases, topical administration of BTH prevented skin infiltration and hyperplasia through suppression of NF-κB and STAT3 phosphorylation. Our results confirm the pivotal role of both transcriptional factors in skin inflammation, as occurs in psoriasis, and highlight the potential of small molecules as therapeutic agents for the treatment of this skin disease, with BTH being a potential candidate for future drug research.

Potential antipsoriatic effect of chondroitin sulfate through inhibition of NF-κB and STAT3 in human keratinocytes.

Chondroitin sulfate (CS) is a natural glycosaminoglycan, formed by the 1-3 linkage of d-glucuronic acid to N-acetylgalactosamine, present in the extracellular matrix. It is used as a slow acting disease modifying agent in the treatment of osteoarthritis, and part of its beneficial effects are due to its antiinflammatory properties that result from an inhibitory effect on NF-κB signaling pathway. This ability raises the hypothesis that CS might be effective in other chronic inflammatory processes such as psoriasis, in which a deregulation of NF-κB is a key feature. In addition, psoriasis is characterized by an upregulation of STAT3 signaling pathway that is related to the epidermal hyperplasia. In the present study we report the pharmacological modulation of the NF-κB and STAT3 signaling pathways by CS in normal human keratinocytes. CS inhibited NF-κB activation and the release of some of the key psoriatic cytokines such as TNFα, IL-8, IL-6 and CCL27. Moreover, it impaired STAT3 translocation to the nucleus and significantly reduced STAT3 transcriptional activity by a mechanism that was independent from STAT3 phosphorylation. Our results confirm the interest of CS as a candidate for future drug research in the therapeutics of psoriasis given the need of more effective and safer oral medications for these patients.

Toward the discovery of new agents able to inhibit the expression of microsomal prostaglandin E synthase-1 enzyme as promising tools in drug development.

In our recent studies, we focused our attention on the synthesis of several gamma-hydroxybutenolides designed on the basis of petrosaspongiolide M 1 (PM) structure that has been recognized to potently inhibit the inflammatory process through the selective PLA(2) enzyme inhibition. By means of a combination of computational methods and efficient synthetic strategies, we generated small collections of PM modified analogs to identify new potent PLA(2) inhibitors, suitable for clinical development. In the course of the biological screening of our compounds, we discovered a potent and selective inhibitor of mPGES-1 expression, the benzothiophene gamma-hydroxybutenolide 2, which so far represents the only product, together with resveratrol, able to reduce PGE(2) production through the selective downregulation of mPGES-1 enzyme. In consideration that microsomal prostaglandin E synthase 1 (mPGES-1) is one of the most strategic target involved both in inflammation and in carcinogenesis processes, we decided to explore the biological effects of some structural changes of the gamma-hydroxybutenolide 2, hoping to improve its biological profile. This optimization process led to the identification of three strictly correlated compounds 14g, 16g, and 18 with higher inhibitory potency on PGE(2) production on mouse macrophage cell line RAW264.7 through the selective modulation of mPGES-1 enzyme expression.

Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host.

Type-IIA secreted phospholipase A(2) (sPLA(2)-IIA) has been proposed to play a role in the development of inflammatory diseases. It has been shown to release arachidonic acid, the precursor of proinflammatory eicosanoids, to hydrolyze phospholipids of pulmonary surfactant, and to bind to specific receptors located on cell surface membranes. However, the most established biological role of sPLA(2)-IIA is related to its potent bactericidal property in particular toward Gram-positive bacteria. This enzyme is present in animal and human biological fluids at concentrations sufficient to kill bacteria. Human recombinant sPLA(2)-IIA is able to kill Gram-positive bacteria at concentrations as low as 1.1 ng/ml. This remarkable property is due to the unique preference of sPLA(2)-IIA for anionic phospholipids such as phosphatidylglycerol, the main phospholipid component of bacterial membranes. Much higher concentrations of sPLA(2)-IIA are required for its action on host cell membranes and surfactant both of which are mainly composed by phosphatidylcholine, a poor substrate for sPLA(2)-IIA. Transgenic mice over-expressing human sPLA(2)-IIA are resistant to infection by Staphylococcus aureus, Escherichia coli, and Bacillus anthracis, the etiological agent of anthrax. Conversely, certain bacteria, such as B. anthracis, E. coli and Bordetella pertussis are able to inhibit sPLA(2)-IIA expression by host cells, thus highlighting a mechanism by which these bacteria can subvert the host immune system. Intranasal instillation of recombinant sPLA(2)-IIA protects mice from mortality caused by pulmonary anthrax. Interestingly, this protective effect was obtained even with B. anthracis strains that down-regulate the expression of endogenous sPLA(2)-IIA, indicating that instilled sPLA(2)-IIA can overcome the subversive action of B. anthracis. We conclude that sPLA(2)-IIA is an efficient endogenous antibiotic of the host and can play a role in host defense against pathogenic bacteria. It can be used as a therapeutic agent in adjunct with current therapy to treat bacteria resistant to multiple antibiotics.

Anthrax lethal toxin down-regulates type-IIA secreted phospholipase A(2) expression through MAPK/NF-kappaB inactivation.

Bacillus anthracis, the etiological agent of anthrax, produces lethal toxin (LT) that displays a metallo-proteolytic activity toward the N-terminus of the MAPK-kinases. We have previously shown that secreted type-IIA phospholipase A(2) (sPLA(2)-IIA) exhibits potent anthracidal activity. In vitro expression of sPLA(2)-IIA in guinea pig alveolar macrophages (AMs), the major source of this enzyme in lung tissues, is inhibited by LT. Here, we examined the mechanisms involved in sPLA(2)-IIA inhibition by LT. We first showed that chemical inhibitors of p38 and ERK MAPKs reduced sPLA(2)-IIA expression in AMs indicating that these kinases play a role in sPLA(2)-IIA expression. LT inhibited IL-1beta-induced p38 phosphorylation as well as sPLA(2)-IIA promoter activity in CHO cells. Inhibition of sPLA(2)-IIA promoter activity was mimicked by co-transfection with dominant negative construct of p38 (DN-p38) and reversed by the active form of p38-MAPK (AC-p38). Both LT and DN-p38 decreased IL-1beta-induced NF-kappaB luciferase activity. This contrasted with the effect of AC-p38, which enhanced this activity. However, neither LT nor specific p-38 inhibitor interfered with LPS-induced IkappaBalpha degradation or NF-kappaB nuclear translocation in AMs. Subcutaneous administration of LT to guinea pig before LPS challenge reduced sPLA(2)-IIA levels in broncho-alveolar lavages and ears. We conclude that sPLA(2)-IIA expression is induced via a sequential MAPK-NF-kappaB activation and that LT inhibits this expression likely by interfering with the transactivation of NF-kappaB in the nucleus. This inhibition, which is operating both in vitro and in vivo, may represent a mechanism by which B. anthracis subvert host defense.

Anti-inflammatory and analgesic activity of a novel inhibitor of microsomal prostaglandin E synthase-1 expression.

In a previous study, we reported a new gamma-hydroxybutenolide derivative, 4-benzo[b]thiophen-2-yl-3-bromo-5-hydroxy-5H-furan-2-one (BTH), as inhibitor of microsomal prostaglandin E synthase-1 (mPGES-1) expression in lypopolysaccharide (LPS) stimulated RAW 264.7 and TPH-1 cells, without affecting cyclooxygenase-2 (COX-2). In this study, we evaluated the in vivo effect of BTH on some acute and chronic inflammatory animal models in relation to its inhibitory profile on mPGES-1 expression. In the zymosan-induced mouse air pouch model, BTH produced a dose-dependent inhibition of prostaglandin E(2) (PGE(2)) production and mPGES-1 protein expression in pouch exudates without any effect on COX-2 protein expression. This behavior was confirmed in the chronic model of collagen-induced arthritis, where administration of BTH (5 mg/kg) clearly reduced PGE(2) and mPGES-1 expression in joint tissues, whereas COX-2 was unaffected. These effects were accompanied by the suppression of clinical and histopathological manifestations of disease such as the loss of proteoglycan, and the destruction of surface cartilage. Other enzymes participating in the metabolism of arachidonic acid, such as prostaglandin I(2) synthase, tromboxane A(2) synthase or 5-lipoxygenase were unaffected by this compound. The acetic acid-induced hyperalgesia model in LPS-sensitized mice showed a dose-dependent analgesic effect of BTH, exerting an ED(50) value of 6.2 mg/kg. Our data suggest that inhibition of mPGES-1 protein expression in acute and chronic inflammatory models by BTH, could provide a potential therapeutic target and a pharmacological tool to discern the role of the inducible enzymes COX-2 and mPGES-1 in inflammatory pathologies.

Development of a second generation of inhibitors of microsomal prostaglandin E synthase 1 expression bearing the gamma-hydroxybutenolide scaffold.

Petrosaspongiolide M (PM), a marine sesterterpene metabolite bearing the gamma-hydroxybutenolide scaffold and displaying a potent inhibitory activity toward PLA(2) enzyme, was selected by us as an attractive target in order to explore its mechanism of action at molecular level. In the course of our investigations we decided to synthetically modify the parent compound to clarify the structural determinants responsible for the activity; in fact, very recently, our research group reported the synthesis and the pharmacological properties of a first collection of PM analogues generated by Ludi approach. The synthesized compounds showed a poor or moderate activity toward PLA(2) enzymes, nevertheless we discovered a potent and selective modulator of the expression of microsomal prostaglandin E synthase 1 (mPGES-1), an enzyme highly involved in the inflammatory response, which represents an interesting target for the development of a new class of anti-inflammatory agents. In this paper we report the synthesis of a further collection of nine analogues, having the same scaffold of PM, the gamma-hydroxybutenolide, and bearing, as side chain, more complex aromatic portions, in substitution of the sesterterpene moiety. Their pharmacological behavior against PLA(2) enzymes as well as to modulate the expression of inducible cyclooxygenase 2 (COX-2) and mPGES-1 enzymes is also described.

Avarol inhibits TNF-alpha generation and NF-kappaB activation in human cells and in animal models.

Avarol is a marine sesquiterpenoid hydroquinone with interesting pharmacological properties including anti-inflammatory and antipsoriatic effects. In the present study we evaluated the pharmacological effect of avarol on some inflammatory parameters related to the pathogenesis of psoriasis. Avarol inhibited tumor necrosis factor-alpha (TNF-alpha) generation in stimulated human monocytes (IC(50) 1 microM) and TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB)-DNA binding in keratinocytes. In the mouse air pouch model, administration of avarol produced a dose-dependent reduction of TNF-alpha generation (ED(50) 9.2 nmol/pouch) as well as of interleukin (IL)-1beta, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) levels in pouch exudates. In the psoriasis-like model of 12-O-tetradecanoylphorbol-acetate-induced mouse epidermal hyperplasia, topical administration of avarol (0.6-1.2 micromol/site) reduced edema, myeloperoxidase activity, IL-1beta, IL-2 and eicosanoid levels in skin. Histopathological study confirmed the inhibition of epidermal hyperplasia as well as leukocyte infiltration. The reduction of cutaneous TNF-alpha by avarol was also detected by immunohistochemical analysis. Avarol was also capable of suppressing in vivo NF-kappaB nuclear translocation, determined in mouse skin. Our results suggested that antipsoriatic properties of avarol previously described could be mediated in part by the downregulation of several inflammatory biomarkers, such as TNF-alpha and NF-kappaB in psoriatic skin.

Synthesis and pharmacological evaluation of a selected library of new potential anti-inflammatory agents bearing the gamma-hydroxybutenolide scaffold: a new class of inhibitors of prostanoid production through the selective modulation of microsomal prostaglandin E synthase-1 expression.

As a part of our drug discovery effort, recently we clarified the molecular basis of phospholipase A2 (PLA2) inactivation by petrosaspongiolide M (PM), an interesting metabolite belonging to a marine sesterterpene family, containing in its structural architecture a gamma-hydroxybutenolide moiety and showing potent anti-inflammatory activity. In the attempt to expand structural diversity as well as to simplify crucial synthetic features of the parent compound, we decided to develop a selected library based on the densely functionalized gamma-hydroxybutenolide scaffold. The synthesized products were tested for their ability to inhibit PLA2 enzymes as well as to modulate the expression of inducible cyclooxygenase 2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1), two key enzymes highly involved in the inflammatory event, in order to discover new promising anti-inflammatory agents with better pharmacological profiles. This led us to the discovery of a promising inhibitor (4e) of prostanoid production acting by in vitro and in vivo selective modulation of microsomal prostaglandin E synthase 1 expression.

Synthesis and evaluation of diverse thio avarol derivatives as potential UVB photoprotective candidates.

Semisynthesis of 13 new thio avarol derivatives (4-16) and in vitro evaluation on the photodamage response induced by UVB irradiation are described. Their ability to inhibit NF-kappaB activation and TNF-alpha generation in HaCaT cells as well as their antioxidant capacity in human neutrophils has also been studied. Among them we have identified two monophenyl thio avarol derivatives (4-5) lacking cytotoxicity which can be considered promising UVB photoprotective agents through the potent inhibition of NF-kappaB activation with a mild antioxidant pharmacological profile.

Marine sponge metabolites for the control of inflammatory diseases.

Marine organisms are a rich source of bioactive metabolites. A number of potential anti-inflammatory compounds have been isolated from marine invertebrates that exhibit phospholipase A2 inhibitory activity. A wide range of marine compounds have been investigated for their anti-inflammatory properties. Cacospongionolide B and petrosaspongiolide M are representative examples of anti-inflammatory compounds in experimental models of acute or chronic inflammation. The mechanisms of action of these compounds include phospholipase A, inhibition as well as the control of nuclear factor-B activation and inflammatory gene expression. Although many marine compounds exhibit interesting anti-inflammatory properties, few have entered clinical trials. The future development of this class of compounds as anti-inflammatory drugs requires the introduction of novel molecular targets of therapeutic relevance in addition to biotechnological approaches for the production of these molecules.

Identification of avarol derivatives as potential antipsoriatic drugs using an in vitro model for keratinocyte growth and differentiation.

Avarol, a marine sesquiterpenoid hydroquinone, and 14 avarol derivatives have shown interesting anti-inflammatory properties in previous studies. In this study, avarol and derivatives were evaluated in high-throughput keratinocyte culture models using cytokeratin 10 and SKALP/Elafin expression as markers for respectively normal and psoriatic differentiation. Avarol and five of its derivatives (5, 10, 13, 14 and 15) were selected for further study. Only 10, 13, 14 and 15 were able to inhibit keratinocyte cell growth. Changes in expression levels of 22 genes were assessed by quantitative real time PCR (qPCR). From these genes, TNFalpha mRNA levels showed the strongest changes. For compound 13, 15 and dithranol (used as a model antipsoriatic drug), a dose-dependent downregulation of TNFalpha mRNA was found. The changes in TNFalpha mRNA were confirmed at the protein level for compound 13. Additionally, this compound was able to reduce also IL-8 and COX-2 mRNA levels and this effect was correlated with a reduction in COX-2 protein expression. The mechanism of action of this compound involves at least the inhibition of NF-kappaB-DNA binding activity. In conclusion, our high-throughput screening models in combination with quantitative assessment of changes in gene expression profiles identified the avarol derivative 13, a benzylamine derivative of avarol at the 4' position of benzoquinone ring, as an interesting anti-psoriatic drug candidate that inhibits keratinocyte cell growth and TNFalpha and COX-2 expression.

In vivo protective role of human group IIa phospholipase A2 against experimental anthrax.

Anthrax is an acute disease caused by Bacillus anthracis. Some animal species are relatively resistant to anthrax infection. This trait has been correlated to the extent of the local inflammatory reaction, suggesting innate immunity to be the first line of defense against B. anthracis infection in nonimmunized hosts. Group IIA secreted phospholipase A2 (sPLA2-IIA) is produced in particular by macrophages and possesses potent antibacterial activity especially against Gram-positive bacteria. We have previously shown in vitro that sPLA2-IIA kills both germinated B. anthracis spores and encapsulated bacilli. Here we show that sPLA2-IIA plays in vivo a protective role against experimental anthrax. Transgenic mice expressing human sPLA2-IIA are resistant to B. anthracis infection. In addition, in vivo administration of recombinant human sPLA2-IIA protects mice against B. anthracis infection. The protective effect was observed both with a highly virulent encapsulated nontoxinogenic strain and a wild-type encapsulated toxinogenic strain, showing that toxemia did not hinder the sPLA2-IIA-afforded protection. sPLA2-IIA, a natural component of the immune system, may thus be considered a novel therapeutic agent to be used in adjunct with current therapy for treating anthrax. Its anthracidal activity would be effective even against strains resistant to multiple antibiotics.

Neisseria meningitidis pili induce type-IIA phospholipase A2 expression in alveolar macrophages.

Induction of type-IIA secreted phospholipase A2 (sPLA2-IIA) expression by bacterial components other than lipopolysaccharide has not been previously investigated. Here, we show that exposure of alveolar macrophages (AM) to Neisseria meningitidis or its lipooligosaccharide (LOS) induced sPLA2-IIA synthesis. However, N. meningitidis mutant devoid of LOS did not abolish this effect. In addition, a pili-defective mutant exhibited significantly lower capacity to stimulate sPLA2-IIA synthesis than the wild-type strain. Moreover, pili isolated from a LOS-defective strain induced sPLA2-IIA expression and nuclear factor kappa B (NF-kappaB) activation. These data suggest that pili are potent inducers of sPLA2-IIA expression by AM, through a NF-kappaB-dependent process.

Protection against 2,4,6-trinitrobenzenesulphonic acid-induced colonic inflammation in mice by the marine products bolinaquinone and petrosaspongiolide M.

Proinflammatory mediators, namely eicosanoids, reactive oxygen and nitrogen species and cytokines, are clearly involved in the pathogenesis of intestinal bowel disease. bolinaquinone (BQ) and petrosaspongiolide M (PT), two marine products with potent anti-inflammatory action, have been shown to control the production of mediators in acute and chronic inflammatory processes. Hence, we have tested here the hypothesis that BQ and PT could ameliorate inflammation and oxidative stress parameters in 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis in Balb/c mice. BQ and PT were given orally in doses of 10 or 20mg/kg/day. Treatment of the animals with BQ or PT at the highest dose significantly protected against TNBS-induced inflammation, as assessed by a reduced colonic weight/length ratio and histological scoring. Neutrophilic infiltration, interleukin (IL)-1beta and prostaglandin E(2) (PGE(2)) levels, as well as cyclooxygenase-2 (COX-2) protein expression were inhibited by both compounds. Colonic nitrite and nitrate levels and protein expression of inducible nitric oxide synthase (iNOS) were also lower in the treated groups in comparison to the TNBS control. BQ and PT reduced nitrotyrosine immunodetection and colonic superoxide anion production. Neither compound inhibited the expression of the protective protein heme oxygenase-1 (HO-1), although they reduced the extension of apoptosis. Our study also indicated that PT could interfere with the translocation of p65 into the nucleus, a key step in nuclear factor-kappaB (NF-kappaB) activation. Altogether, the results suggest that BQ and PT can have potential protective actions in intestinal inflammatory diseases.

Potential antipsoriatic avarol derivatives as antioxidants and inhibitors of PGE(2) generation and proliferation in the HaCaT cell line.

The synthesis and structure-activity relationships for a series of 14 new avarol derivatives as antioxidants and inhibitors of cell proliferation and PGE(2) generation in human keratinocytes are described. Compound 6 (thiosalicylic derivative) was the most potent inhibitor of superoxide generation in human neutrophils and also potently inhibited PGE(2) generation in the human keratinocyte HaCaT cell line. Compound 7(3'-methylaminoavarone) presented the best antiproliferative profile, by the inhibition of (3)H-thymidine incorporation in HaCaT cells, with potency similar to the reference compound anthralin. None of the avarol derivatives showed any sign of cytotoxicity measured as LDH release in treated keratinocytes. The potency and pharmacological profile of derivatives are also discussed.

High bactericidal efficiency of type iia phospholipase A2 against Bacillus anthracis and inhibition of its secretion by the lethal toxin.

There is a considerable body of evidence supporting the role of secretory type II-A phospholipase A(2) (sPLA(2)-IIA) as an effector of the innate immune response. This enzyme also exhibits bactericidal activity especially toward Gram-positive bacteria. In this study we examined the ability of sPLA(2)-IIA to kill Bacillus anthracis, the etiological agent of anthrax. Our results show that both germinated B. anthracis spores and encapsulated bacilli were sensitive to the bactericidal activity of recombinant sPLA(2)-IIA in vitro. In contrast, nongerminated spores were resistant. This bactericidal effect was correlated to the ability of sPLA(2)-IIA to hydrolyze bacterial membrane phospholipids. Guinea pig alveolar macrophages, the major source of sPLA(2)-IIA in an experimental model of acute lung injury, released enough sPLA(2)-IIA to kill extracellular B. anthracis. The production of sPLA(2)-IIA was significantly inhibited by B. anthracis lethal toxin. Human bronchoalveolar lavage fluids from acute respiratory distress syndrome patients are known to contain sPLA(2)-IIA; bactericidal activity against B. anthracis was detected in a high percentage of these samples. This anthracidal activity was correlated to the levels of sPLA(2)-IIA and was abolished by an sPLA(2)-IIA inhibitor. These results suggest that sPLA(2)-IIA may play a role in innate host defense against B. anthracis infection and that lethal toxin may help the bacteria to escape from the bactericidal action of sPLA(2)-IIA by inhibiting the production of this enzyme.