Mitogen activated protein kinases SakA(HOG1) and MpkC collaborate for Aspergillus fumigatus virulence.

Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen-activated protein kinases of the high-osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild-type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.

The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence.

Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ΔsitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitA strain is more sensitive to several metals and ions, such as MnCl2, CaCl2, and LiCl, but it is more resistant to ZnSO4. The ΔsitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.

Interleukin 1 receptor-driven neutrophil recruitment accounts to MyD88-dependent pulmonary clearance of legionella pneumophila infection in vivo.

Legionella pneumophila, the etiological agent of Legionnaires' disease, triggers activation of multiple innate immune pathways that lead to the restriction of bacterial replication in vivo. Despite the critical role for MyD88 in infection clearance, the receptors and mechanisms responsible for MyD88-mediated pulmonary bacterial clearance are still unclear. Here, we used flagellin mutants of L. pneumophila, which bypass the NAIP5/NLRC4-mediated restriction of bacterial replication, to assess the receptors involved in MyD88-mediated pulmonary bacterial clearance. By systematically comparing pulmonary clearance of L. pneumophila in C57BL/6 MyD88(-/-), TLR2(-/-), TLR3(-/-), TLR4(-/-), TLR9(-/-), IL-1R(-/-), and IL-18(-/-) mice, we found that, while the knockout of a single Toll-like receptor or interleukin 18 resulted only in minor impairment of bacterial clearance, deficiency in the interleukin 1 (IL-1) receptor led to a significant impairment. IL-1/MyD88-mediated pulmonary bacterial clearance occurs via processes involving the recruitment of neutrophils. Collectively, our data contribute to the understanding of the effector mechanisms involved in MyD88-mediated pulmonary bacterial clearance.

Identification and functional characterization of K(+) transporters encoded by Legionella pneumophila kup genes.

Legionnaires' disease is an emerging, severe, pneumonia-like illness caused by the Gram-negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L. pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L. pneumophila genes with high similarity to the Escherichia coli K(+) transporters. These three genes were expressed by L. pneumophila and have been designated kupA, kupB and kupC. Investigation using the L. pneumophila kup mutants revealed that kupA is involved in K(+) acquisition during axenic growth. The kupA mutants replicated efficiently in rich axenic media, but poorly in a chemically defined medium. The kupA mutants were defective in the recruitment of polyubiquitinated proteins to the Legionella-containing vacuole that is formed in macrophages and displayed an intracellular multiplication defect during the replication in Acanthamoeba castellanii and in mouse macrophages. We found that bafilomycin treatment of macrophages was able to rescue the growth defects of kupA mutants, but itdid not influence the replication of wild-type bacteria. The defects identified in kupA mutants of L. pneumophila were complemented by the expression E. coli trkD/Kup gene in trans, a bona fide K(+) transporter encoded by E. coli. Collectively, our data indicate that KupA is a functional K(+) transporter expressed by L. pneumophila that facilitates the bacterial replication intracellularly and in nutrient-limited conditions.

Activation of NLRC4 by flagellated bacteria triggers caspase-1-dependent and -independent responses to restrict Legionella pneumophila replication in macrophages and in vivo.

Although NLRC4/IPAF activation by flagellin has been extensively investigated, the downstream signaling pathways and the mechanisms responsible for infection clearance remain unclear. In this study, we used mice deficient for the inflammasome components in addition to wild-type (WT) Legionella pneumophila or bacteria deficient for flagellin (flaA) or motility (fliI) to assess the pathways responsible for NLRC4-dependent growth restriction in vivo and ex vivo. By comparing infections with WT L. pneumophila, fliI, and flaA, we found that flagellin and motility are important for the colonization of the protozoan host Acanthamoeba castellanii. However, in macrophages and mammalian lungs, flagellin expression abrogated bacterial replication. The flagellin-mediated growth restriction was dependent on NLRC4, and although it was recently demonstrated that NLRC4 is able to recognize bacteria independent of flagellin, we found that the NLRC4-dependent restriction of L. pneumophila multiplication was fully dependent on flagellin. By examining infected caspase-1(-/-) mice and macrophages with flaA, fliI, and WT L. pneumophila, we could detect greater replication of flaA, which suggests that caspase-1 only partially accounted for flagellin-dependent growth restriction. Conversely, WT L. pneumophila multiplied better in macrophages and mice deficient for NLRC4 compared with that in macrophages and mice deficient for caspase-1, supporting the existence of a novel caspase-1-independent response downstream of NLRC4. This response operated early after macrophage infection and accounted for the restriction of bacterial replication within bacteria-containing vacuoles. Collectively, our data indicate that flagellin is required for NLRC4-dependent responses to L. pneumophila and that NLRC4 triggers caspase-1-dependent and -independent responses for bacterial growth restriction in macrophages and in vivo.

Pomegranate Extract Potentiates the Anti-Demineralizing, Anti-Biofilm, and Anti-Inflammatory Actions of Non-Alcoholic Mouthwash When Associated with Sodium-Fluoride Trimetaphosphate.

This study investigated the anti-caries and anti-inflammatory effects of mouthwash formulations containing Punica granatum (pomegranate) peel extract (PPE), sodium-trimetaphosphate, and low concentrations of fluoride. PPE was characterized using high-performance liquid chromatography (ellagic acid and punicalagin). Total phenolics were quantified among formulations, and their stability was analyzed for 28 days. The formulation effects were evaluated as follows: (1) inorganic component concentration and reduced demineralization on bovine enamel blocks subjected to pH cycling; (2) anti-biofilm effect on dual-biofilms of Streptococcus mutans ATCC 25175 and Candida albicans ATCC 10231 treated for 1 and 10 min, respectively; and (3) cytotoxicity and production of inflammatory mediators (interleukin-6 and tumor necrosis factor-alpha). The formulation containing 3% PPE, 0.3% sodium-trimetaphosphate, and 225 ppm of fluoride resulted in a 34.5% surface hardness loss; a 13% (treated for 1 min) and 36% (treated for 10 min) biofilm reduction in S. mutans; a 26% (1 min) and 36% (10 min) biofilm reduction in C. albicans; absence of cytotoxicity; and anti-inflammatory activity confirmed by decreased interleukin-6 production in mouse macrophages. Thus, our results provide a promising prospect for the development of an alcohol-free commercial dental product with the health benefits of P. granatum that have been recognized for a millennium.

Artepillin C Reduces Allergic Airway Inflammation by Induction of Monocytic Myeloid-Derived Suppressor Cells.

Propolis is a natural product produced by bees that is primarily used in complementary and alternative medicine and has anti-inflammatory, antibacterial, antiviral, and antitumoral biological properties. Some studies have reported the beneficial effects of propolis in models of allergic asthma. In a previous study, our group showed that green propolis treatment reduced airway inflammation and mucus secretion in an ovalbumin (OVA)-induced asthma model and resulted in increased regulatory T cells (Treg) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) frequencies in the lungs, two leukocyte populations that have immunosuppressive functions. In this study, we evaluated the anti-inflammatory effects of artepillin C (ArtC), the major compound of green propolis, in the context of allergic airway inflammation. Our results show that ArtC induces in vitro differentiation of Treg cells and monocytic MDSC (M-MDSC). Furthermore, in an OVA-induced asthma model, ArtC treatment reduced pulmonary inflammation, eosinophil influx to the airways, mucus and IL-5 secretion along with increased frequency of M-MDSC, but not Treg cells, in the lungs. Using an adoptive transfer model, we confirmed that the effect of ArtC in the reduction in airway inflammation was dependent on M-MDSC. Altogether, our data show that ArtC exhibits an anti-inflammatory effect and might be an adjuvant therapy for allergic asthma.

Incorporation of indomethacin into a mesoporous silica nanoparticle enhances the anti-inflammatory effect Indomethacin into a mesoporous silica.

PURPOSE: We evaluated the analgesic, anti-inflammatory and toxicological effects of indomethacin incorporated into mesoporous silica nanoparticles (IND+NP). METHODS: Nociception was evaluated by the formalin assay. The anti-inflammatory potential was assessed by cell migration and paw edema assays, modulation of nitric oxide and cytokines (IL-6, IL-10 and TNF-α) by macrophages production. Toxicity was evaluated in peritoneal macrophages and by the locomotion assay and assessment of gastric injuries, presence of occult blood and hepatic and renal markers. RESULTS: IND+NP reduced nociception during phases 1 by 53% and 2 by 79% of the formalin assay and the influx of peritoneal cells by 94%, indicating an analgesic and anti-inflammatory effect more efficiently than indomethacin alone. Indomethacin, but not IND+NP, caused macroscopic gastric injuries, the presence of fecal occult blood, and an increase of ALT levels. In the paw edema assay, IND+NP reduced edema by 21%. IND+NP has no effect on the LPS-induced production of nitric oxide, IL-6, IL-10 and TNF-α on no cytotoxic concentrations. CONCLUSIONS: The incorporation of indomethacin into mesoporous silica nanoparticles effectively increased the activity of the drug observed in the formalin and cell migration assays and prevented the gastric and hepatic damage associated with its use.

Green propolis increases myeloid suppressor cells and CD4+Foxp3+ cells and reduces Th2 inflammation in the lungs after allergen exposure.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis is a natural product produced by honeybees used as a medicine at least to 300 BC. In the last decades, several studies showed biological and pharmacological properties of propolis, witch scientifically explains the empirical use for centuries. The anti-inflammatory activity of propolis with the purpose to reduce Th2 inflammation has been evaluated in allergic asthma. However, it remains to be determined how propolis negatively regulates the immune response after allergen re-exposure. AIM OF THE STUDY: We hypothesized that the anti-inflammatory activity of propolis is dependent on the induction of myeloid derived suppressor cells (MDSC) and regulatory T cells. MATERIALS AND METHODS: To assess this hypothesis, we used an ovalbumin-induced asthma model to evaluate the effect of EPP-AF® dry extract from Brazilian green propolis. RESULTS: Propolis treatment decreased pulmonary inflammation and mucus production as well as eosinophils and IL-5 in the broncoalveolar lavage. Propolis enhanced also in vitro differentiation and in vivo frequency of lung MDSC and CD4+Foxp3+ regulatory T cells. CONCLUSIONS: Together these results confirm the immunomodulatory potential of propolis during sensitization and challenge with allergen. In addition, the collecting findings show, for the first time, that propolis increases the frequency of MDSC and CD4+Foxp3+ regulatory T cells in the lungs, and suggest that it could be use as target for development of new immunotherapy or adjuvant immunotherapy for asthma.

Erratum to "Mitogen activated protein kinases (MAPK) and protein phosphatases are involved in Aspergillus fumigatus adhesion and biofilm formation" [Cell Surf. 1 (2018) 43-56].

[This corrects the article DOI: 10.1016/j.tcsw.2018.03.002.].

Aspectos da abordagem terapêutica sobre candidíase vulvovaginal / Aspects of the therapeutic approach to vulvovaginal candidiasis / Aspectos del abordaje terapéutico de la candidiasis vulvovaginal

A candidíase vulvovaginal, é uma infecção da vulva e vagina causada por vários tipos de Candida spp. Essa patologia afeta 75% de todas as mulheres pelo menos uma vez durante a vida, ocorrendo com mais frequência durante a idade fértil. A transmissão dessa infeção fúngica ocorre por meio de contato com mucosas e secreções em pele de portadores ou doentes, contato sexual, água contaminada e transmissão vertical. Alguns outros sintomas característicos mais vistos em casos de CVV, são lesões brancas, cremosas e planas, sendo mais intensos no período pré-menstrual, quando a acidez vaginal aumenta. numerosos antifúngicos estão disponíveis no mercado, os quais são encontrados para administração oral na forma de comprimidos ou, para uso tópico, na forma de cremes, loções, comprimidos vaginais, supositórios e tampões revestidos. O objetivo geral do trabalho foi analisar através da revisão de literatura, tratamentos convencionais e alternativos para abordagem terapêutica da Candidíase Vulvovaginal contextuando a mesma, utilizando definições, dados epidemiológicos e sua sintomatologia frente à sociedade. O presente trabalho é uma revisão integrativa, que teve a coleta de dados realizada de março de 2021 a outubro de 2021 nas bases de dados Lilacs, Scielo, Google acadêmico, A busca resultou em 902 artigos, dos quais 14 atenderam ao critério de inclusão. A busca por tratamentos frente a candidíase vulvovaginal tem se mostrado ampla de acordo com os artigos selecionadas. Concluímos que a patologia candidíase vulvovaginal, vem apresentando resistência em algumas abordagens terapêuticas, assim como algumas mulheres não aderem há algum tipo de tratamento, devido à falta de conhecimento sobre a patologia.

Vulvovaginal candidiasis is an infection of the vulva and vagina caused by various types of Candida spp. This condition affects 75% of all women at least once in their lifetime, occurring more frequently during their childbearing years. The transmission of this fungal infection occurs through contact with mucous membranes and secretions on the skin of patients or patients, sexual contact, contaminated water and vertical transmission. Some other characteristic symptoms more seen in cases of VVC are white, creamy and flat lesions, being more intense in the premenstrual period, when the vaginal acidity increases. numerous antifungals are available on the market which are available for oral administration in tablet form or, for topical use, in the form of creams, lotions, vaginal tablets, suppositories and coated tampons. The general objective of the work was to analyze, through a literature review, conventional and alternative treatments for the therapeutic approach of Vulvovaginal Candidiasis in its context, using definitions, epidemiological data and its symptoms in society. The present work is an integrative review, which had data collection carried out from March 2021 to October 2021 in the Lilacs, Scielo, Google academic databases. The search resulted in 902 articles, of which 14 met the inclusion criteria. The search for treatments against vulvovaginal candidiasis has been shown to be wide according to the selected articles. We conclude that the vulvovaginal candidiasis pathology has been showing resistance in some therapeutic approaches, as well as some women do not adhere to any type of treatment, due to lack of knowledge about the pathology.

La candidiasis vulvovaginal es una infección de la vulva y la vagina cau- sada por diversos tipos de Candida spp. Esta afección afecta al 75% de las mujeres al menos una vez en la vida, siendo más frecuente durante la edad fértil. La transmisión de esta infección fúngica se produce por contacto con mucosas y secreciones de la piel de pacientes o enfermos, contacto sexual, agua contaminada y transmisión vertical. Otros síntomas característicos más observados en los casos de CVV son las lesiones blancas, cremosas y planas, siendo más intensas en el período premenstrual, cuando aumenta la acidez vaginal. Existen en el mercado numerosos antifúngicos disponibles para adminis- tración oral en forma de comprimidos o, para uso tópico, en forma de cremas, lociones, comprimidos vaginales, supositorios y tampones recubiertos. El objetivo general del tra- bajo fue analizar, a través de una revisión bibliográfica, los tratamientos convencionales y alternativos para el abordaje terapéutico de la Candidiasis Vulvovaginal en su contexto, utilizando definiciones, datos epidemiológicos y su sintomatología en la sociedad. El pre- sente trabajo es una revisión integradora, que tuvo recolección de datos realizada de marzo de 2021 a octubre de 2021 en las bases de datos académicas Lilacs, Scielo, Google. La búsqueda resultó en 902 artículos, de los cuales 14 cumplieron los criterios de inclu- sión. La búsqueda de tratamientos contra la candidiasis vulvovaginal se ha mostrado am- plia según los artículos seleccionados. Concluimos que la patología de la candidiasis vul- vovaginal viene mostrando resistencia en algunos abordajes terapéuticos, así como algu- nas mujeres no se adhieren a ningún tipo de tratamiento, debido al desconocimiento de la patología.

Mitogen activated protein kinases (MAPK) and protein phosphatases are involved in Aspergillus fumigatus adhesion and biofilm formation.

The main characteristic of biofilm formation is extracellular matrix (ECM) production. The cells within the biofilm are surrounded by ECM which provides structural integrity and protection. During an infection, this protection is mainly against cells of the immune system and antifungal drugs. A. fumigatus forms biofilms during static growth on a solid substratum and in chronic aspergillosis infections. It is important to understand how, and which, A. fumigatus signal transduction pathways are important for the adhesion and biofilm formation in a host during infection. Here we investigated the role of MAP kinases and protein phosphatases in biofilm formation. The loss of the MAP kinases MpkA, MpkC and SakA had an impact on the cell surface and the ECM during biofilm formation and reduced the adherence of A. fumigatus to polystyrene and fibronectin-coated plates. The phosphatase null mutants ΔsitA and ΔptcB, involved in regulation of MpkA and SakA phosphorylation, influenced cell wall carbohydrate exposure. Moreover, we characterized the A. fumigatus protein phosphatase PphA. The ΔpphA strain was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and reduced chitin, decreased conidia phagocytosis by Dictyostelium discoideum and reduced adhesion and biofilm formation. Finally, ΔpphA strain was avirulent in a murine model of invasive pulmonary aspergillosis and increased the released of tumor necrosis factor alpha (TNF-α) from bone marrow derived macrophages (BMDMs). These results show that MAP kinases and phosphatases play an important role in signaling pathways that regulate the composition of the cell wall, extracellular matrix production as well as adhesion and biofilm formation in A. fumigatus.

Pro-inflammatory Ca++-activated K+ channels are inhibited by hydroxychloroquine.

Antimalarials have demonstrated beneficial effects in Systemic Lupus Erithematosus and Rheumatoid Arthritis. However, the mechanisms and the molecular players targeted by these drugs remain obscure. Although hydroxychloroquine (HCQ) is a known ion channel inhibitor, this property has not been linked to its anti-inflammatory effects. We aimed to study whether HCQ inhibits pro-inflammatory ion channels. Electrophysiology experiments demonstrated that HCQ inhibited Ca++-activated K+ conductance in THP-1 macrophages in a dose-dependent manner. In macrophages, ATP-induced K+ efflux plays a key role in activating the NLRP3 inflammasome. ATP-induced IL-1beta secretion was controlled by the KCa1.1 inhibitor iberiotoxin. NS1619 and NS309 (KCa1.1 and KCa3.1 activators respectively) induced the secretion of IL-1beta. This effect was inhibited by HCQ and also by iberiotoxin and clotrimazol (KCa3.1 inhibitor), arguing against off-target effect. In vitro, HCQ inhibited IL-1beta and caspase 1 activation induced by ATP in a dose-dependent manner. HCQ impaired K+ efflux induced by ATP. In vivo, HCQ inhibited caspase 1-dependent ATP-induced neutrophil recruitment. Our results show that HCQ inhibits Ca++-activated K+ channels. This effect may lead to impaired inflammasome activation. These results are the basis for i) a novel anti-inflammatory mechanism for HCQ and ii) a new strategy to target pro-rheumatic Ca++-activated K+ channels.

Physicochemical characterization by AFM, FT-IR and DSC and biological assays of a promising antileishmania delivery system loaded with a natural Brazilian product.

The control and treatment of Leishmaniasis, a neglected and infectious disease affecting approximately 12 million people worldwide, are challenging. Leishmania parasites multiply intracellularly within macrophages located in deep skin and in visceral tissues, and the currently employed treatments for this disease are subject to significant drawbacks, such as resistance and toxicity. Thus, the search for new Leishmaniasis treatments is compulsory, and Ocotea duckei Vattimo, a plant-derived product from the biodiverse Brazilian flora, may be a promising new treatment for this disease. In this regard, the aim of this work was to develop and characterize a delivery system based on solid lipid nanoparticles (SLN) that contain the liposoluble lignan fraction (LF) of Ocotea duckei Vattimo, which targets the Leishmania phagolysosome of infected macrophages. LF-loaded SLNs were obtained via the hot microemulsion method, and their physical and chemical properties were comprehensively assessed using PCS, AFM, SEM, FT-IR, DSC, HPLC, kinetic drug release studies, and biological assays. The size of the developed delivery system was 218.85±14.2 nm, its zeta potential was -30 mV and its entrapment efficiency (EE%) was high (the EEs% of YAN [yangambin] and EPI-YAN [epi-yangambin] markers were 94.21±0.40% and 94.20±0.00%, respectively). Microscopy, FT-IR and DSC assays confirmed that the delivery system was nanosized and indicated a core-shell encapsulation model, which corroborated the measured kinetics of drug release. The total in vitro release rates of YAN and EPI-YAN in buffer (with sink conditions attained) were 29.6±8.3% and 34.3±8.9%, respectively, via diffusion through the cellulose acetate membrane of the SLN over a period of 4 h. After 24 h, the release rates of both markers reached approximately 45%, suggesting a sustained pattern of release. Mathematical modeling indicated that both markers, YAN and EPI-YAN, followed matrix diffusion-based release kinetics (Higuchi's model) with an estimated diffusion coefficient (D) of 1.3.10(-6) cm(2)/s. The LF-loaded SLNs were non-toxic to murine macrophages (20-80 µg mL(-1) range) and exerted a prominent anti-leishmanial effect (20 µg mL(-1)). These data suggest this new and well-characterized lipid nanoparticle delivery system safely and effectively kills Leishmania and warrants further clinical investigation.

Opposing roles of LTB4 and PGE2 in regulating the inflammasome-dependent scorpion venom-induced mortality.

Tityus serrulatus sting causes thousands of deaths annually worldwide. T. serrulatus-envenomed victims exhibit local or systemic reaction that culminates in pulmonary oedema, potentially leading to death. However, the molecular mechanisms underlying T. serrulatus venom (TsV) activity remain unknown. Here we show that TsV triggers NLRP3 inflammasome activation via K(+) efflux. Mechanistically, TsV triggers lung-resident cells to release PGE2, which induces IL-1ß production via E prostanoid receptor 2/4-cAMP-PKA-NFκB-dependent mechanisms. IL-1ß/IL-1R actions account for oedema and neutrophil recruitment to the lungs, leading to TsV-induced mortality. Inflammasome activation triggers LTB4 production and further PGE2 via IL-1ß/IL-1R signalling. Activation of LTB4-BLT1/2 pathway decreases cAMP generation, controlling TsV-induced inflammation. Exogenous administration confirms LTB4 anti-inflammatory activity and abrogates TsV-induced mortality. These results suggest that the balance between LTB4 and PGE2 determines the amount of IL-1ß inflammasome-dependent release and the outcome of envenomation. We suggest COX1/2 inhibition as an effective therapeutic intervention for scorpion envenomation.

Aspergillus fumigatus MADS-Box Transcription Factor rlmA Is Required for Regulation of the Cell Wall Integrity and Virulence.

The Cell Wall Integrity (CWI) pathway is the primary signaling cascade that controls the de novo synthesis of the fungal cell wall, and in Saccharomyces cerevisiae this event is highly dependent on the RLM1 transcription factor. Here, we investigated the function of RlmA in the fungal pathogen Aspergillus fumigatus We show that the ΔrlmA strain exhibits an altered cell wall organization in addition to defects related to vegetative growth and tolerance to cell wall-perturbing agents. A genetic analysis indicated that rlmA is positioned downstream of the pkcA and mpkA genes in the CWI pathway. As a consequence, rlmA loss-of-function leads to the altered expression of genes encoding cell wall-related proteins. RlmA positively regulates the phosphorylation of MpkA and is induced at both protein and transcriptional levels during cell wall stress. The rlmA was also involved in tolerance to oxidative damage and transcriptional regulation of genes related to oxidative stress adaptation. Moreover, the ΔrlmA strain had attenuated virulence in a neutropenic murine model of invasive pulmonary aspergillosis. Our results suggest that RlmA functions as a transcription factor in the A. fumigatus CWI pathway, acting downstream of PkcA-MpkA signaling and contributing to the virulence of this fungus.

Validation of a RP-HPLC-DAD Method for Chamomile (Matricaria recutita) Preparations and Assessment of the Marker, Apigenin-7-glucoside, Safety and Anti-Inflammatory Effect.

Chamomile is a medicinal plant, which presents several biological effects, especially the anti-inflammatory effect. One of the compounds related to this effect is apigenin, a flavonoid that is mostly found in its glycosylated form, apigenin-7-glucoside (APG), in natural sources. However, the affectivity and safety of this glycoside have not been well explored for topical application. In this context, the aim of this work was to develop and validate a reversed-phase high-performance liquid chromatography (RP-HPLC-DAD) method to quantify APG in chamomile preparations. Additionally, the safety and the anti-inflammatory potential of this flavonoid were verified. The RP-HPLC-DAD method was developed and validated with linearity at 24.0-36.0 µg/mL range (r = 0.9994). Intra- and interday precision (RSD) were 0.27-2.66% and accuracy was 98.27-101.21%. The validated method was applied in the analysis of chamomile flower heads, glycolic extract, and Kamillen cream, supporting the method application in the quality control of chamomile preparations. Furthermore, the APG safety was assessed by MTT cytotoxicity assay and mutagenic protocols and the anti-inflammatory activity was confirmed by a diminished TNF-α production showed by mice macrophages treated with APG following LPS treatment.

The Aspergillus fumigatus pkcA G579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model.

Aspergillus fumigatus is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved Cell Wall Integrity (CWI) pathway. In A. fumigatus this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that pkcA is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential A. fumigatus pkcAG579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcAG579R mutation has a reduced activation of the downstream Mitogen-Activated Protein Kinase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcAG579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The pkcAG579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the A. fumigatus cell wall during infection.

Inflammasome activation is critical to the protective immune response during chemically induced squamous cell carcinoma.

Chronic inflammation affects most stages of tumorigenesis, including initiation, promotion, malignant differentiation, invasion and metastasis. Inflammasomes have been described as involved with persistent inflammation and are known to exert both pro and antitumour effects. We evaluated the influence of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase (CASP)-1 in the antitumor immune response using a multistage model of squamous cell carcinoma (SCC) development. Absence of ASC and CASP-1 resulted in an earlier incidence and increased number of papilloma. Loss of inflammassome function in mice resulted in decreased presence of natural killer (NK), dendritic (DC), CD4(+), CD8(+) and CD45RB(+) T cells in the tumor lesions as well as in lymph nodes (LN) compared with WT mice. Increased percentage of CD4(+)CD25(+)Foxp3(+) T cells was associated with association with inflammasome loss of function. Moreover, significant differences were also found with neutrophils and macrophage infiltrating the lesions. Myeloperoxidase (MPO), but not elastase (ELA), activity oscillated among the groups during the SCC development. Levels of proinflammatory cytokines IL-1ß, IL-18, Tumor Necrosis Factor (TNF)-α and Interferon (IFN)-γ were decreased in the tumor microenvironment in the absence of inflammasome proteins. These observations suggest a link between inflammasome function and SCC tumorigenesis, indicating an important role for inflammasome activation in the control of SCC development.

The mouse as a model for pulmonary legionella infection.

Legionella pneumophila is an intracellular bacterium that was evolutionarily selected to survive in freshwater environments by infecting free-living unicellular protozoa. Once humans inhale contaminated water droplets, the bacteria reach the pulmonary alveoli where they are phagocytized by resident alveolar macrophages. Depending on host immunity and bacterial virulence genes, the infection may progress to an acute pneumonia called Legionnaires' disease, which can be fatal. Of note, an effective immune response is critical to the outcome of the human infection. These clinical observations highlight the importance of animal models of pulmonary infection for in vivo investigation of bacterial pathogenesis and host responses. In this chapter we provide detailed protocols for intranasal infection of mouse with L. pneumophila.