Prophylactic Treatment With Simvastatin Modulates the Immune Response and Increases Animal Survival Following Lethal Sepsis Infection

Chronic use of statins may have anti-inflammatory action, promoting immunomodulation and survival in patients with sepsis. This study aimed to analyze the effects of pretreatment with simvastatin in lethal sepsis induced by cecal ligation and puncture (CLP). Male Swiss mice received prophylactic treatment with simvastatin or pyrogen-free water orally in a single daily dose for 30 days. After this period, the CLP was performed. Naïve and Sham groups were performed as non-infected controls. Animal survival was monitored for 60 h after the CLP. Half of mice were euthanized after 12 h to analyze colony-forming units (CFUs); hematological parameters; production of IL-10, IL-12, IL-6, TNF-α, IFN-γ, and MCP-1; cell counts on peritoneum, bronchoalveolar lavage (BAL), bone marrow, spleen, and mesenteric lymph node; immunephenotyping of T cells and antigen presenting cells and production of hydrogen peroxide (H2O2). Simvastatin induced an increase in survival and a decrease in the CFU count on peritoneum and on BAL cells number, especially lymphocytes. There was an increase in the platelets and lymphocytes number in the Simvastatin group when compared to the CLP group. Simvastatin induced a greater activation and proliferation of CD4+ T cells, as well as an increase in IL-6 and MCP-1 production, in chemotaxis to the peritoneum and in H2O2 secretion at this site. These data suggest that simvastatin has an impact on the survival of animals, as well as immunomodulatory effects in sepsis induced by CLP in mice

Comprehensive molecular landscape of cetuximab resistance in head and neck cancer cell lines

Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed by Western blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC50 compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC.This work was supported by Barretos Cancer Hospital and the Public Ministry of Labor Campinas (Research, Prevention, and Education of Occupational Cancer) in Campinas, Brazil, CAPESDFATD (88887.137283/2017-00). INFG is the recipient of a FAPESP Ph.D. fellowship (2017/22305-9)

INIBIÇÃO DA INFECÇÃO in vitro DE MACRÓFAGOS POR Leishmania amazonensis POR EXTRATO E FRAÇÕES DE Chenopodium ambrosioides L.

A utilização de espécies vegetais, como Chenopodium ambrosioides L., para o tratamento da leishmaniose na terapêutica tradicional tem despertado interesse na busca de novos compostos mais eficazes e menos tóxicos. Nosso grupo demonstrou as atividades imunoestimuladora e anti-Leishmania in vivo do extrato bruto hidroalcoólico (EBH) de C. ambrosioides e efeito anti-promastigota in vitro do EBH e das suas frações. Neste trabalho, avaliou-se a atividade anti-Leishmania do EBH e suas frações acetato de etila (FAc) e clorofórmica (FCHCl3) em macrófagos infectados in vitro por Leishmania amazonensis. Foram realizados dois modelos: “proflático” e “terapêutico”. No primeiro, macrófagos peritoneais de camundongos Swiss  foram tratados com EBH, FAc ou FCHCl3 nas concentrações de 62,5µg/mL, 125µg/mL e 250µg/mL e, após 4 horas, infectados com formas promastigotas do parasito na razão de 1:10 por 24 horas. No segundo, os macrófagos foram infectados com promastigotas (1:10) e, após 4 horas, tratados com EBH, FAc ou FCHCl3 por 24 horas. Foram então realizados a quantifcação das amastigotas fagocitadas e o cálculo das taxas de infecção. No modelo “proflático”, apenas os macrófagos expostos ao EBH nas maiores concentrações apresentaram  taxas de  infecção  inferiores ao controle negativo. Entretanto, no modelo “terapêutico”, as  três concentrações de EBH e também da FAc reduziram a infecção de macrófagos em relação ao controle negativo, sendo a maior concentração do EBH mais efetiva inclusive que o controle positivo.  Em conclusão, o EBH de folhas de C. ambrosioides e a sua FAc possuem efeito terapêutico anti-Leishmania na infecção in vitro de macrófagos.Descritores: Leishmaniose; Leishmania amazonensis; Chenopodium ambrosioides.AbstractInhibit  of  in  vitro  macrophage  infection  by  Leishmania  amazonensis  by  extract  and  fractions  from Chenopodium ambrosioides L. The use of plant species such as Chenopodium ambrosioides L. for the treatment of leishmaniasis in traditional medicine has aroused interest in fnding new, more effective and less toxic compounds. Our group demonstrated the immunostimulatory and in vivo anti-Leishmania activities of the crude hydroalcoholic extract (HCE) from C. ambrosioides L. and the in vitro anti-promastigote effect of the HCE and its fractions. In this study, we evaluated the anti-Leishmania activity of the HCE and its fractions ethyl acetate (FAc) and chloroform (FCHCl3) in macrophages infected in vitro with Leishmania amazonensis. Two models, “prophylactic” and “therapeutic”, were performed. In the frst, Swiss mice peritoneal macrophages were treated with CHE, FAc or FCHCl3 in concentrations of 62,5μg/mL, 125μg/mL and 250μg/mL and, after 4 hours, infected with promastigote forms in the ratio of 1:10 for 24 hours. In the second model, the macrophages were infected with promastigotes (1:10)  and,  after 4 hours,  treated with HCE, FAc or FCHCl3 for 24 hours. Quantifcation of phagocytosed amastigotes and calculation  of  infection  rates were  then  perfomed.  In  the  “prophylactic” model,  only macrophages  exposed  to  the  highest concentrations of HCE presented  infection  rates  lower  than  the negative  control. However,  in  the  “therapeutic” model,  the three concentrations of both the HCE and FAc reduced the infection of macrophages compared to the negative control, with the highest concentration of HCE being even more effective than the positive control. In conclusion, the HCE from leaves of Chenopodium ambrosioides and its FAc have an anti-Leishmania therapeutic effect on the in vitro macrophages infection.Descriptors: Leishmaniasis. Leishmania amazonensis. Chenopodium ambrosioides

EFEITO DO TRATAMENTO COM NIMESULIDA SOBRE A INFLAMAÇÃO GRANULOMATOSA EM CAMUNDONGOS

O granuloma induzido por corpo estranho é um modelo experimental de infamação subcrônica que permite a avaliação do efeito de fármacos sobre o extravasamento vascular (edema) e sobre o infltrado celular. A Nimesulida é um anti-infamatório não esteroidal (AINE) que age por vários mecanismos e é largamente utilizada por sua boa tolerabilidade e comodidade de administração. O objetivo deste trabalho foi verifcar o efeito da Nimesulida sobre o modelo de granuloma induzido por corpo estranho. Camundongos Swiss machos (n=6/grupo) receberam implantes subcutâneos de algodão previamente pesados e esterilizados. O grupo Nimesulida foi tratado com o fármaco na dose de 5mg/kg a cada 12 horas, por via oral, por seis dias. Os animais do grupo Controle receberam igual volume de água estéril e apirogênica por via oral, nos mesmos intervalos e pelo mesmo período. Um dia após o período de tratamento, os animais foram sacrifcados, os implantes de algodão retirados e obtido o peso úmido. Os implantes foram pressionados contra lâminas, posteriormente coradas e utilizadas para a contagem diferencial de células. Após dessecagem por 48 horas em estufa, foi obtido o peso seco. Houve diminuição signifcativa do peso úmido do implante e signifcativa alteração do padrão do infltrado celular do granuloma, com diminuição de neutróflos e aumento de macrófagos nos animais tratados com Nimesulida, que apresentaram um padrão tissular de infamação mais tardia se comparados aos animais do grupo Controle. Assim, o tratamento com Nimesulida modula a resposta infamatória no modelo de granuloma por corpo estranho, podendo nesses casos ser utilizada como controle positivo.Descritores: Inflamação; Granuloma; Nimesulida.Abstract:  The cotton pellet granuloma is a subchronic infammation experimental model that allows to assessing the effect of drugs on the vascular leakage (edema) and on the cell infltrate. Nimesulide is a NSAID that acts by several mechanisms and is widely used for its good tolerability and convenience of administration. This experiment aim was to verify the effect of Nimesulide on cotton pellet granuloma. Swiss male mice separated in two groups (n=6/group) received subcutaneous implants of cotton previously weighed and sterilized and began orally treatment with Nimesulide 5mg/kg at interval of  12 hours for six days. Control group animals received an equal volume of sterile and pyrogen-free water orally at the same interval and for the same period. One day after the treatment, the animals were sacrifced and had the implants removed. The wet weight was obtained and the implants pressed against glass slides, then stained and used for differential cell count. There was a signifcant decrease at the wet weigh and a signifcant change in the pattern of granuloma cellular infltrate, with replacement of neutrophils by macrophages in animal treated with Nimesulide. This group showed a more chronic tissue quality compared to the Control group. Then, treatment with Nimesulide is able to modulate the infammation in foreign body granuloma model and can be used as positive control.Descriptors: Infammation; Granuloma; Nimesulide

Rarity of monodominance in hyperdiverse Amazonian forests.

Tropical forests are known for their high diversity. Yet, forest patches do occur in the tropics where a single tree species is dominant. Such "monodominant" forests are known from all of the main tropical regions. For Amazonia, we sampled the occurrence of monodominance in a massive, basin-wide database of forest-inventory plots from the Amazon Tree Diversity Network (ATDN). Utilizing a simple defining metric of at least half of the trees ≥ 10 cm diameter belonging to one species, we found only a few occurrences of monodominance in Amazonia, and the phenomenon was not significantly linked to previously hypothesized life history traits such wood density, seed mass, ectomycorrhizal associations, or Rhizobium nodulation. In our analysis, coppicing (the formation of sprouts at the base of the tree or on roots) was the only trait significantly linked to monodominance. While at specific locales coppicing or ectomycorrhizal associations may confer a considerable advantage to a tree species and lead to its monodominance, very few species have these traits. Mining of the ATDN dataset suggests that monodominance is quite rare in Amazonia, and may be linked primarily to edaphic factors

Candida Infections and Therapeutic Strategies: Mechanisms of Action for Traditional and Alternative Agents

The Candida genus comprises opportunistic fungi that can become pathogenic when the immune system of the host fails. Candida albicans is the most important and prevalent species. Polyenes, fluoropyrimidines, echinocandins, and azoles are used as commercial antifungal agents to treat candidiasis. However, the presence of intrinsic and developed resistance against azole antifungals has been extensively documented among several Candida species. The advent of original and re-emergence of classical fungal diseases have occurred as a consequence of the development of the antifungal resistance phenomenon. In this way, the development of new satisfactory therapy for fungal diseases persists as a major challenge of present-day medicine. The design of original drugs from traditional medicines provides new promises in the modern clinic. The urgent need includes the development of alternative drugs that are more efficient and tolerant than those traditional already in use. The identification of new substances with potential antifungal effect at low concentrations or in combination is also a possibility. The present review briefly examines the infections caused by Candida species and focuses on the mechanisms of action associated with the traditional agents used to treat those infections, as well as the current understanding of the molecular basis of resistance development in these fungal species. In addition, this review describes some of the promising alternative molecules and/or substances that could be used as anticandidal agents, their mechanisms of action, and their use in combination with traditional drugs