Comparative evaluation of plant extract effects on peritoneal, medullary and J774 cells G8 macrophages / Avaliação comparativa do efeito de extratos de plantas em macrófagos peritoneais, medulares e da linhagem J774. G8

The use of medicinal plants as raw material for extracts production and pure substances isolation and subsequence development of new drugs represents a constantly growing area. However, some stages are indispensable before pharmacologically evaluating natural products such as medicines. Toxicity tests in mammalian cells are essential to initiate new drugs development or verify the substance's biocompatibility. Thus, we verified the toxicity of crude extracts and fractions with different polarities obtained from the leaves and stems of eight plant species. The toxic effect was evaluated on macrophages obtained from the bone marrow and peritoneal cavity of a Swiss webster mouse and J774 macrophages. G8 cell lineage. These macrophages were cultured in a 96-well plate, and the compounds were added at a concentration of 100 µg/mL for 24 hours. After this time, the supernatant was removed. The toxicity was evaluated for lactate dehydrogenase (LDH) release assay and the resazurin assay, which uses an indicator dye to measure oxidation-reduction reactions. The results showed a difference in the percentage of toxicity when comparing the same extract in different types of macrophages. This outcome indicates that these cells from different origins may exhibit different responses when exposed to the same natural compounds.

A utilização de plantas medicinais como matéria-prima para a produção de extratos e isolamento de substâncias puras para o desenvolvimento de novos fármacos representa uma área em constante crescimento. No entanto, existem processos a serem realizados antes de avaliar farmacologicamente produtos naturais como medicamentos. Os testes de toxicidade em células de mamíferos são fundamentais para iniciar o desenvolvimento de novas drogas ou verificar a biocompatibilidade de substâncias. Assim, verificamos a toxicidade de extratos brutos e frações com diferentes polaridades obtidos de folhas e caule de oito espécies de planta. Para comparar o efeito tóxico, os testes foram realizados em macrófagos obtidos da medula óssea e cavidade do peritônio de camundongo Swiss webster, bem como no macrófago da linhagem celular J774.G8. Esses macrófagos foram cultivados em placa de 96 poços e os compostos adicionados na concentração de 100 µg/mL por 24 horas. Após esse período o sobrenadante foi removido. A toxicidade foi avaliada pelos ensaios de detecção da enzima lactato-desidrogenase (LDH) e pelo ensaio de resazurina, que usa um corante indicador para medir as reações de oxidação-redução. Os resultados mostraram uma diferença na porcentagem de toxicidade quando comparamos o mesmo extrato em diferentes tipos de macrófagos. Este resultado indica que essas células de várias origens podem exibir respostas distintas quando expostas aos mesmos compostos naturais.

Characterization of macrophage polarization in lesions of dogs inoculated with Leishmania (Leishmania) infantum (BH401) strain

The aim of this work was to describe the anatomical pathology of dogs experimentally infected with Leishmania (Leishmania) infantum strain MCAN / BR / 2002 / BH401, a Brazilian form of L. infantum isolated from a symptomatic dog from an endemic area. For this, five beagles (three months old and both sexes) composed the experimental group. Markers of macrophage subpopulations M1 and M2 (related to resistance and susceptibility to visceral leishmaniasis) and the tissue cytokine transforming growth factor beta 1 (TGF-ß1) (one of the main cytokines related to the fibrosis process and anti-inflammatory action) were evaluated in livers, lungs and kidneys. The BH 401 L. infantum strain induced classical lesions of the visceral disease where all evaluated organs showed a chronic inflammatory reaction and tissue parasitism associated with a higher expression of CD163 and TGF-ß1 markers, might be related to the progression of the disease. In this work it was possible to conclude that the BH 401 strain reproduces canine visceral leishmaniasis that occurs naturally.(AU)

Molecular docking studies and evaluation of the antiretroviral activity and cytotoxicity of the species Lafoensia pacari Saint-Hilaire / Estudos de docking molecular e avaliação da atividade antirretroviral e citotoxicidade da espécie Lafoensia pacari Saint-Hilaire

Interest in antiviral plant species has grown exponentially and some have been reported to have anti-HIV properties. This research aims to perform the bio-guided phytochemical fractionation by antiretroviral activity of Lafoensia pacari stem barks. This in vitro experimental study involved the preparation of plant material, obtention of ethanolic extract, fractionation, purification, identification and quantification of fractions, acid-base extraction, nuclear magnetic resonance, HIV-1 RT inhibition test and molecular docking studies. From the bio-guided fractionation by the antiretroviral activity there was a higher activity in the acetanolic subfractions, highlighting the acetate subfraction - neutrals with 60.98% of RT inhibition and ellagic acid with 88.61% of RT inhibition and absence of cytotoxicity. The macrophage lineage cytotoxicity assay showed that the chloroform fraction was more toxic than the acetate fraction. The analysis of the J-resolved spectrum in the aromatic region showed a singlet at 7.48 and 6.93 ppm which was identified as ellagic acid and gallic acid, respectively. The 5TIQ enzyme obtained better affinity parameter with the ellagic acid ligand, which was confirmed by the HSQC-1H-13C spectra. Gallic acid was also favorable to form interaction with the 5TIQ enzyme, being confirmed through the HSQC-1H-13C spectrum. From the PreADMET evaluation it was found that ellagic acid is a promising molecule for its RT inhibition activity and pharmacokinetic and toxicity parameters.

O interesse por espécies vegetais com ação antiviral tem crescido exponencialmente e algumas tem sido relatadas como possuídoras de propriedades anti-HIV. Essa pesquisa tem como objetivo realizar o fracionamento fitoquímico biodirecionado pela atividade antirretroviral das cascas do caule da espécie Lafoensia pacari. Trata-se de um estudo experimental in vitro e a metodologia envolve preparo do material vegetal, obtenção do extrato etanólico, fracionamento, purificação, identificação e quantificação das frações, extração ácido-base, ressonância magnética nuclear, teste de inibição da TR do HIV-1 e estudos de docking molecular. A partir do fracionamento biodirecionado pela atividade antirretroviral verificou-se uma maior atividade nas subfrações acetanólica. Com destaque para a subfração acetanólica neutros com 60,98% de inibição de TR e o ácido elágico com 88,61% de inibição de TR e ausência de citotoxicidade. Verificou-se com o teste de citotoxicidade em linhagem de macrófagos que a fração clorofórmica foi mais tóxica que a fração acetanólica. A análise do espectro J-resolvido na região aromática apresentou um simpleto em 7.48 e 6.93 ppm que foram identificados como ácido elágico e ácido gálico respectivamente. A enzima 5TIQ obteve melhor parâmetro de afinidade com o ligante ácido elágico que foi confirmado pelos espectros HSQC-1H-13C. O ácido gálico mostrou-se também favorável a formar interação com a enzima 5TIQ, sendo confirmado através do espectro HSQC-1H-13C. Através da avaliação do PreADMET verificou-se que o ácido elágico é uma molécula promissora pela sua atividade de inibição da TR e parâmetros farmacocinéticos e de toxicidade.

Immunomodulatory effects of Blaps rynchopetera extract

Purpose: To explore the potential immunomodulatory effects of total extract and different polar parts from Blaps rynchopetera Fairmaire. Methods: Phagocytic activity was evaluated by neutral red assay, and the effect of the immune function was investigated by normal and immunocompromised mice models. Results: In vitro, total extract, as well as chloroform, ethyl acetate, n-butanol and water fractions could individually enhance the phagocytic ability of mouse peritoneal macrophages; in addition, chloroform and ethyl acetate fractions had an increasing tendency when combined stimulation with lipopolysaccharide (LPS). In vivo, ethyl acetate fraction (EAF) could enhance the immune organ index, increase the serum hemolysin level and peripheral blood immune cells of immunocompromised mice, while for normal mice, the effect was inconspicuous. Conclusions: Blaps rynchopetera extracts had noteworthy immunomodulatory effect, especially for individuals with immune disorders.

Innate immunity in chicken lines developed by EMBRAPA Suínos e Aves: antimicrobial activity of macrophages and serum

The objective of this study was to compare the antimicrobial activity of macrophages and serum in laying hen (MM, CC, and CCc) and broiler chicken lineages (TT and LL). Macrophages were evaluated for phagocytic and antimicrobial activity. Microbicidal serum activity was evaluated by the resistance test for serum and the agar test. The results showed that phagocytic activity was higher in males of the MM strain, with 13% of macrophages presenting phagocytosis, while the other lineages studied, and even female MM, presented a rate of 6% of phagocytic cells. However, antimicrobial activity in macrophages from males of CCc lineage and females of TT lineage were higher, eliminating more than 30% of the Salmonella enterica inoculum, while in the other strains, the results were similar, with inoculum reduction below 30%. In the serum resistance assay, female laying lines presented higher antibacterial activity than female broiler lines. In the trials to evaluate the microbicide activity of the serum, females of both broiler and laying lineages presented higher performance when compared with males of the same lineage. Females of laying hen lines (MM and CC) present a greater antibacterium activity than males. These results can contribute to a better understanding of the immune response in broiler chicken and laying hen lineages, to aid development of lineages of birds more resistant to pathogens.

Evaluation of the immunomodulatory activity of thalidomide on tumor-associated macrophages in the 4T1 murine metastatic breast cancer model / [Avaliação do efeito imunomodulador da talidomida em macrófagos associados a tumores no modelo murino de câncer de mama metastático 4T1]

The present work evaluated the immunomodulatory effect of thalidomide (Thal) at different doses on tumor-associated macrophages (TAMs) using a mouse model of human breast cancer. Mice were inoculated with 4T1 cells in the left flank and treated with Thal once a day at concentrations of 50, 100, and 150mg/kg body weight from the 5th day until the 28th day of tumor inoculation. The tumors were sized, proliferation index and TAMs count were evaluated in primary tumors and metastatic lungs. In addition, the metastasis rate was evaluated in the lungs. Thal at 150mg/kg significantly decreased tumor growth, proliferation index, and TAMs infiltration in primary tumors. Conversely, a higher number of TAMs and lower proliferation index were observed in metastatic lungs in mice treated with 150mg/kg of Thal. Furthermore, Thal at 150mg/kg significantly decreased the metastatic nodules in the lungs. Our findings demonstrated that Thal treatment considerably decreased the primary tumor and lung metastasis in mice associated with different TAM infiltration effects in these sites.(AU)

No presente trabalho, foi avaliado o efeito imunomodulador de diferentes doses de talidomida em macrófagos associados ao tumor (TAMs), em um modelo murino de câncer de mama. Camundongos foram inoculados com células 4T1, na região do flanco esquerdo, e tratados com talidomida, uma vez ao dia, nas doses de 50, 100 e 150mg/k, por massa corporal, do quinto dia ao 28º dia de inoculação tumoral. Os tumores foram medidos, o índice de proliferação celular e a contagem de TAMs foram avaliados nos tumores primários e nos pulmões com metástases. Além disso, a taxa de metástases pulmonares também foi avaliada. A talidomida na dose de 150mg/kg diminuiu significativamente o crescimento tumoral, o índice de proliferação celular e a infiltração de TAMs nos tumores primários. Por outro lado, maior número de TAMs e menor índice de proliferação celular foram observados nos pulmões metastáticos, em camundongos tratados com 150mg/kg de talidomida. Ademais, a talidomida na dose de 150mg/kg diminuiu significativamente os nódulos metastáticos nos pulmões. Os resultados demonstraram que o tratamento com talidomida diminuiu o crescimento tumoral e as metástases pulmonares em camundongos, associado com diferentes efeitos na infiltração de TAMs nesses locais.(AU)

Effect of IL-33 on pyroptosis of macrophages in mice with sepsis via NF-κB/p38 MAPK signaling pathway

ABSTRACT Purpose To demonstrate the effect of IL-33 on the macrophage pyroptosis in mice with sepsis through the NF-kB/p38 MAPK signal pathway. Methods In total, 24 C57BL/6 mice were divided into the sham operation group (sham) and the cecal ligation and puncture group (CLP). After CLP, 24 IL-33-/- mice were divided into the IL-33-/- group and the IL-33-/- intervention group. The latter group was intraperitoneally injected with IL-33. Mouse mortality was observed after CLP. Macrophage apoptosis in peritoneal lavage fluid was detected by flow cytometry. Serum inflammatory factor level was detected by ELISA. Apoptotic protein expression and NF-κB/p38 MAKP signaling pathway protein expression were detected by qRT-PCR and Western blot. Results Knocking out IL-33 significantly reduced the mortality of CLP mice, as well as the mRNA expression of IL-33 and the levels of serum inflammatory factors, including IL-33, IL-1β, and IL-18. It also reduced the rate of macrophage apoptosis and the expression of the apoptotic protein caspase-1 p10; increased the expression of IκBα; and reduced the protein expression of NF-κB and p38 MAPK. These effects were reversed after exogenous injection of IL-33. Conclusions IL-33 can increase the level of macrophage pyroptosis in mice with sepsis (by activating the NF-kB/p38MAPK signal pathway) and the mortality of these mice.

Primary culture macrophages from fish as potential experimental model in toxicity studies with multiwalled carbon nanotubes

Multiwalled carbon nanotube (MWCNT) has been broadly used in several sectors of society. This material when exposed to the environment might reach the aquatic animals and cause toxic effects. Here, it was evaluated the MWCNTs toxicity in melanomacrophages primary culture that was submitted to 1 µ gm L-1 MWCNTs for 24 hours. After exposition to MWCNT, 48 and 59% liver and spleen melanomacrophages were healthy, respectively. The control group presented 85% viability. Phagocytosis activity of melanomacrophages was observed by presence of black inclusions in cytoplasm. The findings indicate MWCNT was cytotoxic to melanomacrophages, where its release and effect into aquatic environment must be more studied. Finally, the melanomacrophages present large potential as experimental model for evaluation of carbon-based nanomaterial toxicity.

Primary culture macrophages from fish as potential experimental model in toxicity studies with multiwalled carbon nanotubes

Multiwalled carbon nanotube (MWCNT) has been broadly used in several sectors of society. This material when exposed to the environment might reach the aquatic animals and cause toxic effects. Here, it was evaluated the MWCNTs toxicity in melanomacrophages primary culture that was submitted to 1 µ gm L-1 MWCNTs for 24 hours. After exposition to MWCNT, 48 and 59% liver and spleen melanomacrophages were healthy, respectively. The control group presented 85% viability. Phagocytosis activity of melanomacrophages was observed by presence of black inclusions in cytoplasm. The findings indicate MWCNT was cytotoxic to melanomacrophages, where its release and effect into aquatic environment must be more studied. Finally, the melanomacrophages present large potential as experimental model for evaluation of carbon-based nanomaterial toxicity.(AU)

Utrophin, MHC and M1/M2 macrophages in GRMD dogs / Utrofina, MHC e macrófagos M1 e M2 em cães DMGR

Muscular dystrophies are hereditary diseases that lead to progressive degeneration of the skeletal musculature. Golden Retriever dogs are used as animal models because they show a hereditary muscle disease similar to muscular dystrophy in humans. Aims: To evaluate the immunostaining of M1 (CD68) and M2 (CD163) macrophages, MHC I, MHC II and, utrophin in muscles of Golden Retriever dogs affected by muscular dystrophy (GRMD). Methods: Samples from 17 male dogs affected by GRMD were divided into GI - dystrophic dogs up to one year of age; and GII - dystrophic dogs over one-year-old. Results: Immunostaining of CD163 was higher than CD68 in both GI and GII. CD68 showed no variation between groups of dystrophic animals. MHC class I immunostaining was most evident in the biceps femoris and triceps brachialis. MHC class II was expressed mildly in four dystrophic muscle types in GI and GII. Utrophin immunostaining was higher in GII. Conclusion: M2 macrophages were one of the main mononuclear inflammatory cells found in dystrophic muscles. The number of M2 in muscles of dogs with GRMD increases with age, linking this cell subtype to permanent muscle damage.

As distrofias musculares são doenças hereditárias que levam à degeneração progressiva da musculatura esquelética. Cães Golden Retriever são usados como modelos animais, pois desenvolvem uma doença muscular hereditária semelhante à distrofia muscular em humanos. Objetivos: Avaliar a imunomarcação dos macrófagos M1 (CD68) e M2 (CD163), MHC I, MHC II e utrofina nos músculos de cães Golden Retriever afetados pela distrofia muscular (DMGR). Métodos: Amostras de 17 cães machos afetados por DMGR foram divididas em cães distróficos GI - até um ano de idade; e GII - cães distróficos com mais de um ano de idade. Resultados: a imunomarcação de CD163 foi maior que CD68 nos grupos GI e GII. CD68 não mostrou variação entre os grupos de animais distróficos. A imunomarcação de MHC classe I foi mais evidente no bíceps femoral e tríceps braquial. O MHC classe II foi expresso discretamente nos quatro tipos de músculo distrófico no GI e GII. A imunomarcação de utrofina foi maior no GII. Conclusão: Os macrófagos M2 foram uma das principais células inflamatórias mononucleares encontradas nos músculos distróficos. O número de M2 nos músculos de cães com DMGR aumentou com a idade, ligando esse subtipo de célula a danos musculares permanentes.

Utrophin, MHC and M1/M2 macrophages in GRMD dogs / Utrofina, MHC e macrófagos M1 e M2 em cães DMGR

Muscular dystrophies are hereditary diseases that lead to progressive degeneration of the skeletal musculature. Golden Retriever dogs are used as animal models because they show a hereditary muscle disease similar to muscular dystrophy in humans. Aims: To evaluate the immunostaining of M1 (CD68) and M2 (CD163) macrophages, MHC I, MHC II and, utrophin in muscles of Golden Retriever dogs affected by muscular dystrophy (GRMD). Methods: Samples from 17 male dogs affected by GRMD were divided into GI - dystrophic dogs up to one year of age; and GII - dystrophic dogs over one-year-old. Results: Immunostaining of CD163 was higher than CD68 in both GI and GII. CD68 showed no variation between groups of dystrophic animals. MHC class I immunostaining was most evident in the biceps femoris and triceps brachialis. MHC class II was expressed mildly in four dystrophic muscle types in GI and GII. Utrophin immunostaining was higher in GII. Conclusion: M2 macrophages were one of the main mononuclear inflammatory cells found in dystrophic muscles. The number of M2 in muscles of dogs with GRMD increases with age, linking this cell subtype to permanent muscle damage.(AU)

As distrofias musculares são doenças hereditárias que levam à degeneração progressiva da musculatura esquelética. Cães Golden Retriever são usados como modelos animais, pois desenvolvem uma doença muscular hereditária semelhante à distrofia muscular em humanos. Objetivos: Avaliar a imunomarcação dos macrófagos M1 (CD68) e M2 (CD163), MHC I, MHC II e utrofina nos músculos de cães Golden Retriever afetados pela distrofia muscular (DMGR). Métodos: Amostras de 17 cães machos afetados por DMGR foram divididas em cães distróficos GI - até um ano de idade; e GII - cães distróficos com mais de um ano de idade. Resultados: a imunomarcação de CD163 foi maior que CD68 nos grupos GI e GII. CD68 não mostrou variação entre os grupos de animais distróficos. A imunomarcação de MHC classe I foi mais evidente no bíceps femoral e tríceps braquial. O MHC classe II foi expresso discretamente nos quatro tipos de músculo distrófico no GI e GII. A imunomarcação de utrofina foi maior no GII. Conclusão: Os macrófagos M2 foram uma das principais células inflamatórias mononucleares encontradas nos músculos distróficos. O número de M2 nos músculos de cães com DMGR aumentou com a idade, ligando esse subtipo de célula a danos musculares permanentes.(AU)

Apamin-induced alterations in J774 1. 6 macrophage metabolism / [Alterações induzidas por apamina no metabolismo de macrófagos J774 1. 6]

Among the immune system cells, macrophages have an important role. Apamin, a bee venom constituent, is important in the defense of these insects. Thus, we aimed to evaluate the metabolism of J774 1.6 macrophage cell line when exposed to isolated and purified apamin, using cytotoxicity tests by MTT reduction and analysis by flow cytometry (apoptosis / necrosis, production of reactive oxygen species (ROS), membranous lipoperoxidation (LPO), electrical potential of the mitochondrial membrane (mMP) and DNA fragmentation). None of the tested concentrations (10 to 100µg/mL) were cytotoxic according to MTT reductions. Apoptosis rates decreased at concentrations of 2.5, 5.0, and 10.0µg/mL (P<0.05), while necrosis rates increased (P<0.05). However, rates of healthy cells at the highest tested concentration (10µg/mL) did not differ from control (P>0.05). Apamin did not alter ROS, LPO, or DNA fragmentation. Therefore, all analyzed concentrations (1.25 to 10µg/mL) decreased mMP. Such decrease in apoptosis might be due to a suppression of mitochondrial pro-apoptotic messengers, as this peptide causes no oxidative stress, lipid peroxidation, and DNA damage. Highly sensitive techniques are majorly important for proper interpretation of cellular toxicity mechanisms, combined with routine laboratory methods.(AU)

Das células do sistema imunológico, macrófagos desempenham um papel fundamental. Apamina, constituinte do veneno de abelhas, é importante na defesa destas. Objetivou-se avaliar o metabolismo da linhagem de macrófagos J774 1.6 expostos à apamina isolada e purificada, avaliando-se citotoxicidade por redução de MTT e análise por citometria de fluxo (apoptose / necrose, produção de espécies reativas de oxigênio (EROs), lipoperoxidação membranosa (LPO), potencial elétrico da membrana mitocondrial (MMP) e fragmentação do DNA). Nenhuma concentração testada (10 a 100µg / mL) foi citotóxica. As taxas de apoptose diminuíram nas concentrações 2,5, 5,0 e 10,0µg / mL (P<0,05), enquanto as de necrose aumentaram (P<0,05). Entretanto, as taxas de células saudáveis na maior concentração testada (10µg / mL) não diferiram do controle (P>0,05). A apamina não alterou as ERO, a LPO nem a fragmentação do DNA. Portanto, todas as concentrações analisadas (1,25 a 10µg / mL) diminuíram a mMP. Tal diminuição na apoptose pode ser por uma supressão de mensageiros pró-apoptóticos mitocondriais, já que este peptídeo não causa estresse oxidativo, peroxidação lipídica nem dano ao DNA. Técnicas altamente sensíveis são importantes para adequada interpretação dos mecanismos de citotoxicidade.(AU)

Evaluation of cell damage and modulation of cytokines TNF-, IL-6 and IL-10 in macrophages exposed to PpIX-mediated photodynamic therapy / Avaliação do dano celular e da modulação das citocinas TNF-alpha, IL-6 e IL-10 em macrófagos expostos à terapia fotodinâmica mediada pela PpIX

Little is known regarding whether photodynamic therapy (PDT)-induced cell death can substantially compromise macrophages (M), which are important cells in PDT-induced immune responses. Here, parameters of PDT-mediated M cytotoxicity and cytokine production in response to protoporphyrin IX (PpIX) were evaluated. Peritoneal M from BALB/c mice were stimulated in vitro with PDT, light, PpIX, or lipopolysaccharide (LPS). After that, cell viability, lipid peroxidation, Nitric Oxide (NO), DNA damage, TNF-, IL-6 and IL-10 were evaluated. Short PDT exposure reduced cell viability by 1030%. There was a two-fold increase in NO and DNA degradation, despite the non-increase in lipoperoxidation. PDT increased TNF- and IL-10, particularly in the presence of LPS, and decreased the production of IL-6 to 10-fold. PDT causes cellular stress, induces NO radicals and leads to DNA degradation, generating a cytotoxic microenvironment. Furthermore, PDT modulates pro- and anti-inflammatory cytokines in M(AU)

Pouco se sabe se a morte celular induzida pela terapia fotodinâmica (PDT) compromete os macrófagos (M), envolvidos nas respostas imunes induzidas pela PDT. Neste estudo, foram avaliados parâmetros de citotoxicidade dos M mediada pela PDT e a produção de citocinas, frente à protoporfirina IX (PpIX). M peritoneais de camundongos BALB/c foram estimulados in vitro com PDT, luz, PpIX ou lipopolissacarídeo (LPS). Após isto, a viabilidade celular (VC), a lipoperoxidação, os níveis de óxido nítrico (NO), de DNA degradado, de TNF-, IL-6 e IL-10 foram avaliados. A exposição curta à PDT reduziu a VC em 10-30%. Os níveis de NO e de DNA degradado duplicaram, sem aumento da lipoperoxidação. Houve aumento de TNF- e IL-10, sendo maior na presença de LPS. Já a produção de IL-6 reduziu em dez vezes. A PDT induz estresse celular, gera radicais NO e causa dano ao DNA, tornando o microambiente citotóxico. Ainda, modula citocinas pró e anti-inflamatórias em M(AU)

Healing effect of andiroba-based emulsion in cutaneous wound healing via modulation of inflammation and transforming growth factor beta 3

Purpose:To evaluate the effects and mechanisms of andiroba-based emulsion (ABE) topical treatment on full-thickness cutaneous wounds in rats.Methods:The wounds were harvested on days 3, 7, 15, and 20 post-surgery. Wound contraction rate, quantitative immunohistochemistry [macrophages, myofibroblasts, capillaries, collagens (col) I and III, transforming growth factor β3β (TGFβ3)], and tensile strength were assessed.Results:Treated wounds were smaller, contracted earlier and had increased angiogenesis, fewer CD68+ and M2 macrophages on days 7 and 15, but higher on day 20. Myofibroblasts appeared on days 3 to 7 in untreated wounds and on days 7 to 15 in treated wounds. TGFβ3 levels were higher in the treated wounds, less dense collagen fibers, lower col I/III ratios and a higher tensile strength.Conclusion:These results demonstrate the important anti-inflammatory role of treatment and the associated modulation of macrophages, myofibroblasts, and TGFβ3 levels. Collagen fibers in the treated wounds were more organized and less dense, similar to unwounded skin, which likely contributed to the higher tensile strength.(AU)

Salmonella typhimurium vaccine to control a brazilian Salmonella heidelberg strain in broiler chickens / Vacina de Salmonella typhimurium para controlar cepa brasileira de Salmonella heidelberg em frangos de corte

Foodborne Salmonella infections in humans, which results from the consumption of contaminated poultry meat and eggs, are a major public health concern. Vaccination of animals against Salmonella is one strategy to prevent these infections and reduce the risks to public health. Live attenuated Salmonella enterica vaccines can confer protection against salmonellosis by inducing both cell-mediated and mucosal immune responses. This study assessed a live, attenuated Salmonella enterica Typhimurium (ST) vaccine in broiler chickens against a heterologous challenge with Salmonella Heidelberg (SH) by evaluating bacterial quantification, immune cells infiltration, and cytokine gene expression in the cecum. The treatments were: T1, non-vaccinated, non-challenged; T2, non-vaccinated, SH-challenged; T3, ST-vaccinated and SH-challenged. At 28 days of age, the ST-vaccinated group had significantly recovered reduction of SH in the crop (P<0,01) and cecum (P = 0,021) compared to the non-vaccinated SH-challenged group, with no significant changes (P˃0,05) in macrophages, T CD4+, or T CD8+ cells dynamics during the same period. Aerosol vaccination on the first day promoted greater interleukin-12 expression in the liver (P<0,05) and interleukin-10 expression and T CD8+ cells in the ileum 16 hours after housing. After prime-boosted oral immunization on the 13th day, the vaccinated group had greater expression of macrophages and T CD4+ cells in the liver (P<0,05) than the control group. Two doses of a live ST-attenuated vaccine promoted a partial cross-protective effect against SH strain UFPR1 challenge in broilers.

Infecções por Salmonella transmitidas por alimentos como consumo de carne de frango e ovos contaminados em seres humanos constituem um importante problema de saúde pública. A vacinação de animais contra Salmonella é uma estratégia para prevenir essas infecções e reduzir o risco para a saúde pública. As vacinas vivas atenuadas de Salmonella enterica podem conferir proteção contra a salmonelose, induzindo respostas imunológicas mediadas por células e em mucosas. Este estudo avaliou uma vacina viva e atenuada de Salmonella enterica Typhimurium (ST) em frangos de corte contra um desafio heterólogo com Salmonella Heidelberg (SH), avaliando a quantificação de Salmonella, infiltração de células imunes e a expressão de genes de citocinas no ceco. Os tratamentos foram: T1, não vacinado, não desafiado; T2, não vacinado, desafiado com SH; T3, ST-vacinado, desafiado com SH. Aos 28 dias de idade, o grupo vacinado com ST apresentou significativa redução de SH no papo (P<0,01) e no ceco (P = 0,021) comparado ao grupo T2-não vacinado SH-desafiado, sem alterações significativas na dinâmica celular de macrófagos, T CD4+ ou T CD8+ (P˃0,05) durante o mesmo período. A vacinação por aerossol no primeiro dia promoveu maior expressão de IL-12 no fígado (P<0,05), maior expressão de IL-10 e células T CD8+ no íleo, 16 horas após o alojamento. Após o reforço de imunização oral ao 13º dia, o grupo vacinado apresentou maior expressão de macrófagos e células T CD4+ no fígado (P<0,05) do que o grupo controle. Duas doses de uma vacina viva atenuada de ST promoveram um efeito de proteção cruzada parcial contra o desafio da cepa de Salmonella Heidelberg cepa UFPR1 em frangos de corte.

Salmonella typhimurium vaccine to control a brazilian Salmonella heidelberg strain in broiler chickens / Vacina de Salmonella typhimurium para controlar cepa brasileira de Salmonella heidelberg em frangos de corte

Foodborne Salmonella infections in humans, which results from the consumption of contaminated poultry meat and eggs, are a major public health concern. Vaccination of animals against Salmonella is one strategy to prevent these infections and reduce the risks to public health. Live attenuated Salmonella enterica vaccines can confer protection against salmonellosis by inducing both cell-mediated and mucosal immune responses. This study assessed a live, attenuated Salmonella enterica Typhimurium (ST) vaccine in broiler chickens against a heterologous challenge with Salmonella Heidelberg (SH) by evaluating bacterial quantification, immune cells infiltration, and cytokine gene expression in the cecum. The treatments were: T1, non-vaccinated, non-challenged; T2, non-vaccinated, SH-challenged; T3, ST-vaccinated and SH-challenged. At 28 days of age, the ST-vaccinated group had significantly recovered reduction of SH in the crop (P<0,01) and cecum (P = 0,021) compared to the non-vaccinated SH-challenged group, with no significant changes (P˃0,05) in macrophages, T CD4+, or T CD8+ cells dynamics during the same period. Aerosol vaccination on the first day promoted greater interleukin-12 expression in the liver (P<0,05) and interleukin-10 expression and T CD8+ cells in the ileum 16 hours after housing. After prime-boosted oral immunization on the 13th day, the vaccinated group had greater expression of macrophages and T CD4+ cells in the liver (P<0,05) than the control group. Two doses of a live ST-attenuated vaccine promoted a partial cross-protective effect against SH strain UFPR1 challenge in broilers.(AU)

Infecções por Salmonella transmitidas por alimentos como consumo de carne de frango e ovos contaminados em seres humanos constituem um importante problema de saúde pública. A vacinação de animais contra Salmonella é uma estratégia para prevenir essas infecções e reduzir o risco para a saúde pública. As vacinas vivas atenuadas de Salmonella enterica podem conferir proteção contra a salmonelose, induzindo respostas imunológicas mediadas por células e em mucosas. Este estudo avaliou uma vacina viva e atenuada de Salmonella enterica Typhimurium (ST) em frangos de corte contra um desafio heterólogo com Salmonella Heidelberg (SH), avaliando a quantificação de Salmonella, infiltração de células imunes e a expressão de genes de citocinas no ceco. Os tratamentos foram: T1, não vacinado, não desafiado; T2, não vacinado, desafiado com SH; T3, ST-vacinado, desafiado com SH. Aos 28 dias de idade, o grupo vacinado com ST apresentou significativa redução de SH no papo (P<0,01) e no ceco (P = 0,021) comparado ao grupo T2-não vacinado SH-desafiado, sem alterações significativas na dinâmica celular de macrófagos, T CD4+ ou T CD8+ (P˃0,05) durante o mesmo período. A vacinação por aerossol no primeiro dia promoveu maior expressão de IL-12 no fígado (P<0,05), maior expressão de IL-10 e células T CD8+ no íleo, 16 horas após o alojamento. Após o reforço de imunização oral ao 13º dia, o grupo vacinado apresentou maior expressão de macrófagos e células T CD4+ no fígado (P<0,05) do que o grupo controle. Duas doses de uma vacina viva atenuada de ST promoveram um efeito de proteção cruzada parcial contra o desafio da cepa de Salmonella Heidelberg cepa UFPR1 em frangos de corte.(AU)

Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis

Background: Lipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells. Methods: In the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.) amazonensis) and during in vivo infection using BALB/c mice. Results: The aforementioned inhibitors were deleterious to promastigote and amastigote forms of the L. (L.) amazonensis and were non-toxic to peritoneal macrophages from BALB/c mice. L. (L.) amazonensis-infected BALB/c mice treated with the inhibitor BEL presented decreased lesion size and skin parasitism; however, BEL treatment induced hepatotoxicity in BALB/c mice. Conclusions: Results presented herein suggested that PLA2 inhibitors altered L. (L.) amazonensis viability. In spite of liver toxicity, treatment with BEL was the most selective compound in vitro, as well in vivo, resulting in lower skin parasitism in the infected mice. These findings corroborate the role of PLA2 in parasite virulence and maintenance in vertebrate hosts, and suggest that molecules structurally related to BEL should be considered when planning compounds against Leishmania sp.(AU)

Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis

Background: Lipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells. Methods: In the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.) amazonensis) and during in vivo infection using BALB/c mice. Results: The aforementioned inhibitors were deleterious to promastigote and amastigote forms of the L. (L.) amazonensis and were non-toxic to peritoneal macrophages from BALB/c mice. L. (L.) amazonensis-infected BALB/c mice treated with the inhibitor BEL presented decreased lesion size and skin parasitism; however, BEL treatment induced hepatotoxicity in BALB/c mice. Conclusions: Results presented herein suggested that PLA2 inhibitors altered L. (L.) amazonensis viability. In spite of liver toxicity, treatment with BEL was the most selective compound in vitro, as well in vivo, resulting in lower skin parasitism in the infected mice. These findings corroborate the role of PLA2 in parasite virulence and maintenance in vertebrate hosts, and suggest that molecules structurally related to BEL should be considered when planning compounds against Leishmania sp.

T lymphocytes and macrophages in the intestinal tissues of dogs infected with Leishmania infantum / Linfócitos T e macrófagos nos tecidos intestinais de cães infectados com Leishmania infantum

This study was about a semi-quantitative analysis of T lymphocytes (CD4+ and CD8+, FoxP3+ regulatory T cells), and macrophages in the gut wall of dogs naturally infected with Leishmania infantum. Thirteen dogs were divided into three groups: group 1 (G1, n=5), dogs with canine visceral leishmaniasis (CVL) and infected with L. infantum amastigotes in the intestine; group 2 (G2, n=5), dogs with CVL but without intestinal amastigotes; and group 3 (G3, n=3), uninfected dogs (control group). There was no significant difference (p ≥ 0.05) on CD4+ and Treg cell numbers among the groups, whereas the levels of CD8+ T cells and macrophages were significantly higher in dogs from G1 group than in G2 and G3 (p ≤ 0.05), especially in intestinal segments with high parasite burden. Parasite burden correlated positively with levels of CD8+ T cells and macrophages (p ≤ 0.05), but was inversely correlated to levels of CD4+ T lymphocytes and FoxP3+ Treg cells. In conclusion, in the intestine of dogs with CVL, the increase of CD8+ T cells and macrophages population associated with high parasite burdens, but no changes of CD4+ T cells and FoxP3+ Treg cells suggest a possible immunoregulation by the parasite not dependent on Treg cells.(AU)

Este estudo foi uma análise semi-quantitativa de linfócitos T (CD4+, CD8+ e regulatórios - Treg FoxP3+) e macrófagos na parede intestinal de cães naturalmente infectados com Leishmania infantum. Treze cães foram divididos em três grupos: grupo 1 (G1, n=5) continha cães com leishmaniose visceral canina (LVC) e com amastigotas intestinais; grupo 2 (G2, n=5) continha cães com LVC, mas sem amastigotas intestinais e o grupo 3 (G3, n=3) continha cães não infectados (grupo controle). Verificou-se que não houve diferença significativa (p ≤ 0.05) no número de células CD4+ e de Treg entre os grupos, mas o número de células T CD8+ e macrófagos foi significativamente superior nos cães do grupo G1 em relação ao G2 e ao G3 (p ≤ 0,05), especialmente nos segmentos intestinais com altas cargas parasitárias. As altas cargas parasitarias correlacionaram positivamente com os números de CD8+ e macrófagos (p ≤ 0,05), mas negativamente com as células CD4+ e Treg. Em conclusão, no intestino dos cães com LVC, o aumento das populações de células T CD8+ e de macrófagos associado a altas cargas parasitárias, mas nenhuma alteração de células T CD4+ e células Treg FoxP3+ sugerem uma possível imunorregulação pelo parasita não dependente de células Treg.(AU)

Dexamethasone Regulates Macrophage and Cd4+Cd25+ Cell Numbers in the Chicken Spleen

Dexamethasone (DEX) is a corticoid hormone that is experimentally used to mimic the effects of increased levels of endogenous corticosterone observed during the stress response. Currently, stress is considered one of the major predisposing factors for diseases in the poultry industry. The aim of this study was to analyze the effects of DEX and/or of a 20-fold coccidial vaccine dose on leukocyte phenotypes in the spleen and cecal tonsils of chickens. Twenty specific-pathogen-free (SPF) Leghorn chickens were divided into four groups: a non-treated group (NT), a DEX-treated group (Dex), a vaccinated group (V) and a DEX-treated+vaccinated group (Dex+V). On experimental day (ED) 42, each bird in the vaccinated groups received a anti-coccidial vaccine. DEX was injected in the birds of the Dex and Dex+V groups (0.9 mg/kg) onED42 and ED45. The immunophenotyping was performed by flow cytometry analysis of splenocytes and cecal tonsils cells onED48. DEX treatment per se was unable to change CD4+CD8+, CD4+CD8+ and CD4-CD8+ populations with TCRgd or CD28 in the spleen, or macrophages and T lymphocytes in the cecal tonsils. V group birds presented higher numbers of splenic macrophages compared with those measured in the Dex+V group. The number of CD4+CD25+ cells in the spleen of birds of the V group was higher than those measured in the other experimental groups. Our data suggest that CD4+CD25+ cells and macrophages might be influenced by DEX treatment in spleen, but not in the cecal tonsils of chickens inoculated with Eimeria.