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1.
Mem. Inst. Oswaldo Cruz ; 116: e200417, 2021. tab, graf
Article in English | LILACS | ID: biblio-1154880

ABSTRACT

BACKGROUND Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-β signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains. OBJECTIVES To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole. METHODS Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence. FINDINGS Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole. MAIN CONCLUSIONS These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.


Subject(s)
Animals , Mice , Toxoplasma/physiology , Macrophages, Peritoneal/parasitology , Nitric Oxide Synthase/metabolism , Macrophages/parasitology , Nitric Oxide/biosynthesis , Toxoplasmosis, Animal/parasitology , Macrophages/metabolism
2.
Mem. Inst. Oswaldo Cruz ; 115: e190408, 2020. graf
Article in English | LILACS | ID: biblio-1101276

ABSTRACT

BACKGROUND The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host. OBJECTIVES In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated. METHODS Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry. FINDINGS The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(β1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines. MAIN CONCLUSIONS Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.


Subject(s)
Humans , Male , Female , Adult , Young Adult , Phagocytosis/immunology , Leishmania braziliensis/immunology , Monocytes/parasitology , Leishmania infantum/immunology , Antimony/pharmacology , Nitric Oxide/biosynthesis , Antiprotozoal Agents/pharmacology , Leishmania braziliensis/drug effects , Drug Resistance , Monocytes/immunology , Leishmania infantum/drug effects , Flow Cytometry , Immunity, Innate
3.
Acta cir. bras ; 34(11): e201901102, Nov. 2019. tab, graf
Article in English | LILACS | ID: biblio-1054682

ABSTRACT

Abstract Purpose: To investigate the effect of Picroside II on testicular ischemia and reperfusion (l/R) injury and the underlying mechanism. Methods: Sprague-Dawley rats were randomly divided into 4 groups: sham operated group (Sham), Sham with Picroside II treatment group (Sham+ Pic II), l/R group (l/R) and l/R with Picroside II treatment group (I/R+ Pic II). l/R model was established by rotating the left testis 720° in a clock-wise direction for 4 hours. The histopathologic and spermatogenetic evaluation was performed. The apoptosis changes and the levels of HO-1 (heme oxygenase-1), MPO (myeloperoxidase), NOX (NADPH oxidase), SOD (superoxide dismutase), XO (xanthine oxidase) and NOS (nitric oxide synthase) were measured. Results: The seminiferous tubules were damaged in l/R rats, but Picroside II alleviated the changes induced by l/R. The increased level of apoptosis was decreased by Picroside II (P=0.01, 9.05±0.35 vs. 4.85±0.25). The activities of HO-1, MPO, NOX, XO and MDA content were increased and the SOD activity was decreased in l/R (P<0.05) and could be reversed by Picroside II (P=0.03, 405.5±7.5 vs. 304±17U/mgprot; P=0.02, 0.99±0.05 vs. 0.52±0.04 mgprot; P=0.01, 260+7 vs. 189±2 mgprot; P=0.04, 10.95+0.55 vs. 8.75+0.35 U/mgprot; P=0.045, 6.8+0.7 vs. 3.75+0.35 mgprot; P=0.04, 44.5+3.5 vs. 57.5+3.5 mgprot). Western blot showed that the expression of iNOS, nNOS and eNOS were increased in l/R (P<0.05); however, they were decreased after Picroside II treatment (P<0.05). Conclusion: Picroside II attenuated testicular I/R injury in rats mainly through suppressing apoptosis and oxidative stress through reduction of nitric oxide synthesis.


Subject(s)
Animals , Male , Testis/blood supply , Reperfusion Injury/prevention & control , Cinnamates/pharmacology , Apoptosis/drug effects , Oxidative Stress/drug effects , Iridoid Glucosides/pharmacology , Nitric Oxide/biosynthesis , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Random Allocation , Blotting, Western , Rats, Sprague-Dawley , Peroxidase/analysis , In Situ Nick-End Labeling , Heme Oxygenase-1/analysis , Malondialdehyde/analysis , NADP/analysis
4.
Braz. j. med. biol. res ; 51(5): e6693, 2018. graf
Article in English | LILACS | ID: biblio-889091

ABSTRACT

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.


Subject(s)
Animals , Male , Mice , Adenosine Triphosphate/physiology , Receptors, Purinergic/metabolism , Leydig Cells/physiology , Nitric Oxide/physiology , Arginine/administration & dosage , Arginine/metabolism , Thionucleotides/administration & dosage , Thionucleotides/metabolism , Action Potentials , Cells, Cultured , Cyclic GMP/administration & dosage , Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , Patch-Clamp Techniques , NG-Nitroarginine Methyl Ester/administration & dosage , NG-Nitroarginine Methyl Ester/metabolism , Nitric Oxide/biosynthesis
5.
J. appl. oral sci ; 25(3): 265-273, May-June 2017. tab, graf
Article in English | LILACS, BBO | ID: biblio-893620

ABSTRACT

Abstract Elderly denture wearers are commonly affected by Candida-associated denture stomatitis (DS), an inflammatory process of the oral mucosa strongly associated with Candida spp and other microorganisms, as well as local and systemic factors. The impaired immune response against pathogens is among the inherent host factors that have been also associated with the pathogenesis of DS. Mononuclear phagocytes respond to the pathogens through phagocytosis followed by the production of several substances inside the phagosomes, among them are the reactive nitrogen species (RNS). A failure in these mechanisms may contribute to the DS development. Objective The aim of this study was to investigate the influence of aging on the internalization and the production of nitric oxide (NO) by peritoneal adherent cells (PAC), in response to Candida albicans (C. albicans). Material and methods PAC obtained from young and aged mice were challenged with dead or viable C. albicans by using predetermined proportions (cells:yeast) for 30 and 120 minutes. Phagocytosis was analyzed by acridine orange dye, and NO production by the Griess reaction. Results C. albicans phagocytosis by PAC from aged mice was similar to that of young mice, although the cells from older mice cells present more internalized fungi compared with matched control. In addition, a tendency towards impaired NO production by peritoneal mononuclear phagocytes from aged mice was observed. Conclusions PAC from aged mice may capture and store many fungi, which in turn may mean that these cells are effectively unable to eliminate fungi, probably due to impaired NO production. Therefore, considering the important role of C. albicans overgrowth in the pathogenesis of DS and the aspects observed in this study, aging may favor the onset and severity of local candidosis such as DS and its systemic forms.


Subject(s)
Animals , Male , Phagocytosis/physiology , Stomatitis, Denture/metabolism , Stomatitis, Denture/microbiology , Candida albicans/pathogenicity , Aging/physiology , Cell Adhesion/physiology , Nitric Oxide/biosynthesis , Peritoneum/cytology , Reference Values , Time Factors , Candida albicans/isolation & purification , Candidiasis/microbiology , Age Factors , Mice, Inbred C57BL , Nitric Oxide/analysis
6.
Acta cir. bras ; 31(9): 586-596, Sept. 2016. tab, graf
Article in English | LILACS | ID: lil-795992

ABSTRACT

ABSTRACT PURPOSE: To evaluate the contribution of L-arginine oral or topical rout of administration in the surgical wound healing process. METHODS: L-arginine was orally or topically administrated to mice after a laparotomy model procedure. The wounds were analyzed to evaluate the granulation tissue by HE analysis, collagen deposition, iNOS and cytokines production by immunochemisyry on wound progress. Mice used in this model were healthy, immunosupressed or diabetic and all of them were treated with different concentration of L-arginine and rout of administration. RESULTS: Suggested that groups treated with L-arginine orally or topically improved wound repair when compared with non-treatad mice. L- arginine treatment stimulated TGF-β and restricted NO production leading to a mild Th1 response and collagen deposition in injured area, when it was orally administrated. Topical administration decreased IL-8 and CCR1 expression by wound cells but did not interfere with TNF-α and IL-10 production, ratifying the decrease of inflammatory response, the oral administration however, presented a higher iNOS and TGF-β expression then. L-arginine treatment also improved the improved the wound healing in immunosupressed or diabetic mice. CONCLUSION: L-arginine administrated orally or topically can be considered an important factor in the recuperation of tissues.


Subject(s)
Animals , Male , Mice , Arginine/administration & dosage , Wound Healing/drug effects , Cytokines/metabolism , Transforming Growth Factor beta/biosynthesis , Nitric Oxide Synthase Type II/biosynthesis , Surgical Wound/drug therapy , Arginine/metabolism , Wounds and Injuries/pathology , Administration, Oral , Administration, Topical , Collagen/biosynthesis , Immunocompromised Host , Diabetes Mellitus, Experimental/metabolism , Disease Models, Animal , Inflammation/metabolism , Nitric Oxide/biosynthesis
7.
Rev. Soc. Bras. Med. Trop ; 49(1): 68-73, Jan.-Feb. 2016. graf
Article in English | LILACS | ID: lil-776538

ABSTRACT

Abstract: INTRODUCTION: Leishmaniasis is a zoonotic disease caused by protozoa of the genus Leishmania . Cutaneous leishmaniasis is the most common form, with millions of new cases worldwide each year. Treatments are ineffective due to the toxicity of existing drugs and the resistance acquired by certain strains of the parasite. METHODS: We evaluated the activity of sodium nitroprusside in macrophages infected with Leishmania (Leishmania) amazonensis . Phagocytic and microbicidal activity were evaluated by phagocytosis assay and promastigote recovery, respectively, while cytokine production and nitrite levels were determined by ELISA and by the Griess method. Levels of iNOS and 3-nitrotyrosine were measured by immunocytochemistry. RESULTS: Sodium nitroprusside exhibited in vitro antileishmanial activity at both concentrations tested, reducing the number of amastigotes and recovered promastigotes in macrophages infected with L. amazonensis . At 1.5µg/mL, sodium nitroprusside stimulated levels of TNF-α and nitric oxide, but not IFN-γ. The compound also increased levels of 3-nitrotyrosine, but not expression of iNOS, suggesting that the drug acts as an exogenous source of nitric oxide. CONCLUSIONS: Sodium nitroprusside enhances microbicidal activity in Leishmania -infected macrophages by boosting nitric oxide and 3-nitrotyrosine.


Subject(s)
Animals , Tyrosine/analogs & derivatives , Trypanocidal Agents/pharmacology , Nitroprusside/pharmacology , Macrophages, Peritoneal/parasitology , Nitric Oxide/biosynthesis , Tyrosine/biosynthesis , Tyrosine/drug effects , Immunohistochemistry , Mice , Mice, Inbred BALB C
8.
Article in English | WPRIM | ID: wpr-42471

ABSTRACT

Bucillamine is used for the treatment of rheumatoid arthritis. This study investigated the protective effects of bucillamine against cisplatin-induced damage in auditory cells, the organ of Corti from postnatal rats (P2) and adult Balb/C mice. Cisplatin increases the catalytic activity of caspase-3 and caspase-8 proteases and the production of free radicals, which were significantly suppressed by pretreatment with bucillamine. Bucillamine induces the intranuclear translocation of Nrf2 and thereby increases the expression of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GSS), which further induces intracellular antioxidant glutathione (GSH), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2). However, knockdown studies of HO-1 and SOD2 suggest that the protective effect of bucillamine against cisplatin is independent of the enzymatic activity of HO-1 and SOD. Furthermore, pretreatment with bucillamine protects sensory hair cells on organ of Corti explants from cisplatin-induced cytotoxicity concomitantly with inhibition of caspase-3 activation. The auditory-brainstem-evoked response of cisplatin-injected mice shows marked increases in hearing threshold shifts, which was markedly suppressed by pretreatment with bucillamine in vivo. Taken together, bucillamine protects sensory hair cells from cisplatin through a scavenging effect on itself, as well as the induction of intracellular GSH.


Subject(s)
Animals , Antioxidants/metabolism , Apoptosis/drug effects , Caspase 3/metabolism , Caspase 8/metabolism , Cell Line , Cisplatin/toxicity , Cysteine/analogs & derivatives , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Glutathione/metabolism , Heme Oxygenase-1/genetics , Intracellular Space/metabolism , Male , Metabolic Detoxication, Phase II/genetics , Mice , NF-E2-Related Factor 2/genetics , Nitric Oxide/biosynthesis , Organ of Corti/drug effects , RNA Interference , Rats , Reactive Oxygen Species/metabolism , Superoxide Dismutase/genetics
9.
Bauru; s.n; 2015. 119 p. ilus, graf.
Thesis in Portuguese | LILACS | ID: lil-794221

ABSTRACT

Os mastócitos (MCs) estão presentes tanto no periodonto normal quanto inflamado, em diferentes quantidades e em vários locais. Nos últimos anos, a eficácia e a contribuição dos MCs em eliminar bactérias, através de sua atividade microbicida intracelular, estão se tornando cada vez mais reconhecidas. Assim, a partir de MCs murinos desafiados in vitro com o periodontopatógeno Aggregatibacter actinomycetemcomitans (ATCC 29523) por 3, 5, 10 e 24 horas, o presente estudo teve como objetivo investigar a capacidade microbicida intracelular de MCs, e comparar com a capacidade microbicida de macrófagos peritoneais murinos (MPs), considerados fagócitos profissionais, por meio da contagem das unidades formadoras de colônias. Além disso, avaliou-se a produção e liberação de mediadores microbicidas, óxido nítrico (NO) e peróxido de hidrogênio (H2O2), por meio do método colorimétrico de Griess e pela degradação de substratos fluorescentes, respectivamente. Para a análise estatística, foram utilizados os testes estatísticos ANOVA Fatorial seguido do teste de Tukey e teste de correlação de Pearson (p<0.05). Nossos resultados revelaram que os MCs foram capazes de eliminar eficientemente o periodontopatógeno, principalmente após 10h de desafio intracelular. Comparando-se a atividade microbicida dos dois tipos celulares, verificou-se, nos períodos de 3h e 5h de desafio, um menor percentual de colônias viáveis no interior de MPs, em comparação aos MCs. Inversamente, nos períodos de 10h e 24h, observaram-se menores valores percentuais de colônias intracelulares nos MCs em relação aos MPs. Além disso, a produção/liberação de NO bem como, em menor proporção, de H2O2 pelos MCs foram concordantes com a sua capacidade microbicida. Este é o primeiro estudo que demonstra a eficiente ação microbicida intracelular de MCs murinos contra Aggregatibacter actinomycetemcomitans, com produção e liberação de substâncias potencialmente bactericidas, e de forma mais eficaz que os macrófagos...


Mast cells (MCs) are present in both normal and inflamed periodontal tissues, in varying amounts and locations. Recently, MCs contribution in eliminating bacteria and its effectiveness, through its intracellular microbicidal activity, have been increasingly recognized. Thus, this study aimed to investigate the intracellular microbicide capacity of MCs, and compare it with the microbicide capacity of murine peritoneal macrophages (MPs), considered professional phagocytes, by counting the colony forming units. Both cell types were challenged in vitro with periodontopathogen Aggregatibacter actinomycetemcomitans (ATCC 29523) by 3, 5, 10 and 24 hours. Additionally, the production and release of microbicidal agents, nitric oxide (NO) and hydrogen peroxide (H2O2) were evaluated by means of colorimetric Griess method and by the degradation of fluorescent substrates, respectively. Statistical analysis was performed by ANOVA Factorial test followed by Tukey and Pearson's correlation test (p <0.05). Our results revealed that MCs are able to efficiently eliminate periodontopathogen, mainly after 10 hours of intracellular challenge. The microbicidal activity of both cell types, in 3 and 5 hours of challenge showed a lower percentage of viable colonies inside MPs, compared to MCs. Contradictorily, in 10 and 24 hours a lower percentage of intracellular colonies in MCs was observed in relation to MPs. Moreover, the production/release of NO and, in minor proportion, of H2O2 by MCs was in agreement with its microbicidal capacity. Therefore, this is the first report to describe the intracellular microicidal activity of murine MCs against Aggregatibacter actinomycetemcomitans, concerning production and release of potentially bactericidal substances, which is more effective than macrophages. These results suggest the importance of these cells in pathogenesis and defense mechanisms of biofilm-associated periodontal disease...


Subject(s)
Animals , Male , Female , Mice , Aggregatibacter actinomycetemcomitans/growth & development , Bone Marrow Cells/physiology , Mast Cells/physiology , Colony Count, Microbial , Periodontal Diseases/pathology , Nitric Oxide/biosynthesis , Hydrogen Peroxide/metabolism , Time Factors
10.
Bauru; s.n; 2015. 119 p. ilus, graf.
Thesis in Portuguese | LILACS, BBO | ID: biblio-867426

ABSTRACT

Os mastócitos (MCs) estão presentes tanto no periodonto normal quanto inflamado, em diferentes quantidades e em vários locais. Nos últimos anos, a eficácia e a contribuição dos MCs em eliminar bactérias, através de sua atividade microbicida intracelular, estão se tornando cada vez mais reconhecidas. Assim, a partir de MCs murinos desafiados in vitro com o periodontopatógeno Aggregatibacter actinomycetemcomitans (ATCC 29523) por 3, 5, 10 e 24 horas, o presente estudo teve como objetivo investigar a capacidade microbicida intracelular de MCs, e comparar com a capacidade microbicida de macrófagos peritoneais murinos (MPs), considerados fagócitos profissionais, por meio da contagem das unidades formadoras de colônias. Além disso, avaliou-se a produção e liberação de mediadores microbicidas, óxido nítrico (NO) e peróxido de hidrogênio (H2O2), por meio do método colorimétrico de Griess e pela degradação de substratos fluorescentes, respectivamente. Para a análise estatística, foram utilizados os testes estatísticos ANOVA Fatorial seguido do teste de Tukey e teste de correlação de Pearson (p<0.05). Nossos resultados revelaram que os MCs foram capazes de eliminar eficientemente o periodontopatógeno, principalmente após 10h de desafio intracelular. Comparando-se a atividade microbicida dos dois tipos celulares, verificou-se, nos períodos de 3h e 5h de desafio, um menor percentual de colônias viáveis no interior de MPs, em comparação aos MCs. Inversamente, nos períodos de 10h e 24h, observaram-se menores valores percentuais de colônias intracelulares nos MCs em relação aos MPs. Além disso, a produção/liberação de NO bem como, em menor proporção, de H2O2 pelos MCs foram concordantes com a sua capacidade microbicida. Este é o primeiro estudo que demonstra a eficiente ação microbicida intracelular de MCs murinos contra Aggregatibacter actinomycetemcomitans, com produção e liberação de substâncias potencialmente bactericidas, e de forma mais eficaz que os macrófagos...


Mast cells (MCs) are present in both normal and inflamed periodontal tissues, in varying amounts and locations. Recently, MCs contribution in eliminating bacteria and its effectiveness, through its intracellular microbicidal activity, have been increasingly recognized. Thus, this study aimed to investigate the intracellular microbicide capacity of MCs, and compare it with the microbicide capacity of murine peritoneal macrophages (MPs), considered professional phagocytes, by counting the colony forming units. Both cell types were challenged in vitro with periodontopathogen Aggregatibacter actinomycetemcomitans (ATCC 29523) by 3, 5, 10 and 24 hours. Additionally, the production and release of microbicidal agents, nitric oxide (NO) and hydrogen peroxide (H2O2) were evaluated by means of colorimetric Griess method and by the degradation of fluorescent substrates, respectively. Statistical analysis was performed by ANOVA Factorial test followed by Tukey and Pearson's correlation test (p <0.05). Our results revealed that MCs are able to efficiently eliminate periodontopathogen, mainly after 10 hours of intracellular challenge. The microbicidal activity of both cell types, in 3 and 5 hours of challenge showed a lower percentage of viable colonies inside MPs, compared to MCs. Contradictorily, in 10 and 24 hours a lower percentage of intracellular colonies in MCs was observed in relation to MPs. Moreover, the production/release of NO and, in minor proportion, of H2O2 by MCs was in agreement with its microbicidal capacity. Therefore, this is the first report to describe the intracellular microicidal activity of murine MCs against Aggregatibacter actinomycetemcomitans, concerning production and release of potentially bactericidal substances, which is more effective than macrophages. These results suggest the importance of these cells in pathogenesis and defense mechanisms of biofilm-associated periodontal disease.


Subject(s)
Animals , Male , Female , Mice , Aggregatibacter actinomycetemcomitans/growth & development , Bone Marrow Cells/physiology , Mast Cells/physiology , Colony Count, Microbial , Periodontal Diseases/pathology , Nitric Oxide/biosynthesis , Hydrogen Peroxide/metabolism , Time Factors
11.
Rev. argent. microbiol ; 46(3): 242-255, oct. 2014.
Article in Spanish | LILACS | ID: biblio-1015096

ABSTRACT

Las bacterias forman biopelículas de manera ubicua, y esta característica les otorga una flexibilidad que es resultado, en parte, de una matriz compleja construida según las exigencias de las condiciones ambientales. Aunque los estadios de la formación de las biopelículas bacterianas se conocen con detalle, para entender con profundidad la formación de las biopelículas es deseable un conocimiento mayor de los mecanismos de señalización. Las bacterias detectan cambios en la densidad de población por regulación del quórum y condiciones específicas, empleando señales como el di-GMPc y el óxido nítrico. La importancia del conocimiento de estas vías de señalización radica en que controlan una variedad de funciones, como la formación de biopelículas y la movilidad, y proporcionan a las bacterias beneficios en la colonización del hospedador, la defensa contra competidores y los cambios adversos del entorno. Por la trascendencia que revisten estos aspectos, revisamos aquí las redes de regulación y la conexión de la señalización entre quorum sensing, di-GMPc y óxido nítrico


Bacterial biofilms are ubiquitous in nature, and their flexibility is derived in part from a complex extracellular matrix that can be made-to-order to cope with environmental demand. Although common developmental stages leading to biofilm formation have been described, an in-depth knowledge of genetic and signaling is required to understand biofilm formation. Bacteria detect changes in population density by quorum sensing and particular environmental conditions, using signals such as cyclic di-GMP or nitric oxide. The significance of understanding these signaling pathways lies in that they control a broad variety of functions such as biofilm formation, and motility, providing benefits to bacteria as regards host colonization, defense against competitors, and adaptation to changing environments. Due to the importance of these features, we here review the signaling network and regulatory connections among quorum sensing, c-di-GMP and nitric oxide involving biofilm formation


Subject(s)
Biofilms/growth & development , Cyclic GMP/biosynthesis , Quorum Sensing/physiology , Nitric Oxide/biosynthesis
12.
Mem. Inst. Oswaldo Cruz ; 109(6): 767-774, 09/09/2014. tab, graf
Article in English | LILACS | ID: lil-724002

ABSTRACT

Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.


Subject(s)
Animals , Cattle , Male , Mice , Lipid Droplets/parasitology , Macrophage Activation/physiology , Macrophages, Peritoneal/parasitology , Toxoplasma/physiology , Vacuoles/parasitology , Host-Parasite Interactions , Indomethacin/pharmacology , Lipid Droplets/physiology , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Macrophages, Peritoneal/chemistry , Macrophages, Peritoneal/physiology , Macrophages, Peritoneal/ultrastructure , Nitric Oxide/biosynthesis , Primary Cell Culture , Prostaglandins E/antagonists & inhibitors , Prostaglandins E/biosynthesis , Vacuoles/physiology
13.
Mem. Inst. Oswaldo Cruz ; 109(4): 459-465, 03/07/2014. graf
Article in English | LILACS | ID: lil-716311

ABSTRACT

Nitric oxide (NO) participates in neuronal lesions in the digestive form of Chagas disease and the proximity of parasitised glial cells and neurons in damaged myenteric ganglia is a frequent finding. Glial cells have crucial roles in many neuropathological situations and are potential sources of NO. Here, we investigate peripheral glial cell response to Trypanosoma cruzi infection to clarify the role of these cells in the neuronal lesion pathogenesis of Chagas disease. We used primary glial cell cultures from superior cervical ganglion to investigate cell activation and NO production after T. cruzi infection or lipopolysaccharide (LPS) exposure in comparison to peritoneal macrophages. T. cruzi infection was greater in glial cells, despite similar levels of NO production in both cell types. Glial cells responded similarly to T. cruzi and LPS, but were less responsive to LPS than macrophages were. Our observations contribute to the understanding of Chagas disease pathogenesis, as based on the high susceptibility of autonomic glial cells to T. cruzi infection with subsequent NO production. Moreover, our findings will facilitate future research into the immune responses and activation mechanisms of peripheral glial cells, which are important for understanding the paradoxical responses of this cell type in neuronal lesions and neuroprotection.


Subject(s)
Animals , Chagas Disease/immunology , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/parasitology , Neuroglia/parasitology , Nitric Oxide/biosynthesis , Trypanosoma cruzi/immunology , Chagas Disease/etiology , Fluorescent Antibody Technique , Mice, Inbred BALB C , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/immunology , Neuroglia/drug effects , Neuroglia/immunology
14.
Mem. Inst. Oswaldo Cruz ; 109(1): 61-69, 02/2014. graf
Article in English | LILACS | ID: lil-703640

ABSTRACT

Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L.) amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internalise Leishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis.


Subject(s)
Animals , Fibroblasts/parasitology , Leishmania/physiology , Leishmaniasis/physiopathology , Leukocytes, Mononuclear/parasitology , Analysis of Variance , Flow Cytometry , Fibroblasts/ultrastructure , Host-Parasite Interactions/physiology , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Mesoderm/cytology , Mice, Inbred BALB C/parasitology , Nitric Oxide/analysis , Nitric Oxide/biosynthesis , Primary Cell Culture , Statistics, Nonparametric , Time Factors
15.
Biol. Res ; 47: 1-6, 2014. graf, tab
Article in English | LILACS | ID: biblio-950765

ABSTRACT

BACKGROUND: Ligularia fischeri (common name Gomchwi) is known for its pharmaceutical properties and used in the treatment of jaundice, scarlet-fever, rheumatoidal arthritis, and hepatic diseases; however, little is known about its anti-inflammatory effect. In this study the influence of blanching and pan-frying on the anti-inflammatory activity of Ligularia fischeri (LF) was evaluated. RESULTS: Fresh LF and cooked LF showed no significant effect on the viability of macrophages after 24 h incubation. Fresh LF was found to be the most potent inhibitor of nitric oxide (NO) production at 100 µg/ml, while pan-fried LF showed little inhibitory effect on lipoloysaccharide (LPS) stimulated murine machrophage RAW264.7 cells. In contrast with its effect on NO production, pan-fried LF showed significant attenuation of the expression of inducible nitiric oxide synthase (iNOS) compared with fresh LF. In the cooking method of LF, PGE2 production was not affected in the LPS-induced RAW 264.7 cells. In LPS-induced RAW 264.7 cells, pretreatment by fresh and cooked LF increased COX2 mRNA expression. The 3-O-caffeoylquinic acid content of blanching and pan-frying LF increased by 4.92 and 9.7 fold with blanching and pan-frying respectively in comparison with uncooked LF. CONCLUSIONS: Regardless of the cooking method, Ligularia fischeri exhibited potent inhibition of NO production through expression of iNOS in LPS-induced RAW264.7 cells.


Subject(s)
Animals , Mice , Cooking/methods , Asteraceae/chemistry , Plant Preparations/pharmacology , Nitric Oxide Synthase Type II/metabolism , Macrophages/drug effects , Nitric Oxide/biosynthesis , Quinic Acid/analysis , Quinic Acid/analogs & derivatives , Quinic Acid/classification , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Dinoprostone/analysis , Dinoprostone/biosynthesis , Cell Survival , Lipopolysaccharides , Chromatography, High Pressure Liquid , Asteraceae/classification , Cyclooxygenase 2/analysis , Cyclooxygenase 2/metabolism , RAW 264.7 Cells , Hot Temperature , Macrophages/physiology , Anti-Inflammatory Agents/pharmacology
16.
Rev. méd. Chile ; 141(9): 1150-1157, set. 2013. ilus, graf, tab
Article in Spanish | LILACS | ID: lil-699682

ABSTRACT

Background: The rainforest is an important source of natural compounds with therapeutic properties. Although there are many anti-inflammatory and antineoplastic drugs available to the clinician, there is an ongoing need for new therapeutic drugs with fewer serious adverse effects. Aim: To evaluate the in vitro cytotoxic effects of lupeol and casearin G on tumor cells, on phagocytic activity and nitric oxide (NO) production by blood mononuclear cells. Material and Methods: The cytotoxic effect of these compounds on cell lines MCF-7 (human breast adenocarcinoma) and PC-3 (human prostate cancer) was measured by a colorimetric assay (MTS/PMS) and the sulphorhodamine B assay. Peripheral blood mononuclear cells were obtained from eight healthy volunteers. The effect of these compounds on nitric oxide (NO) production was measured using the Griess reaction. Their effect on phagocytic activity of PBMC was also evaluated. Results: Lupeol (≥ 2 mM) resulted in a reduction of both the phagocytic index and the percentage of phagocytic monocytes and macrophages. Treatment of monocytes/macrophages with lupeol (72 µM) and casearin G (4 µM) reduced the production of NO. Neither lupeol (< 969 µM) nor casearin G (< 55 µM) had cytotoxic effects on PBMC. Casearin G showed both cytotoxic (IC50, LC50) and cytostatic (GI50) effects against tumor cells, PC-3 (IC50 = 12.5 µM; GI50 = 13.3 µM; LC50 = 51.9 µM) and MCF-7 (IC50 = 112.8 µM; GI50 = 11.8 µM; LC50 = 49.4 µM), as well as a hemolytic effect (≥ 182 µM). Conclusions: These observations indicate that lupeol and casearin G might be useful compounds in the preparation of anti-inflammatory drugs, whereas casearin G might be useful in the elaboration of antitumor drugs.


Subject(s)
Humans , Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Diterpenes/pharmacology , Leukocytes, Mononuclear/drug effects , Nitric Oxide/biosynthesis , Pentacyclic Triterpenes/pharmacology , Phagocytosis/drug effects , Antineoplastic Agents, Phytogenic/isolation & purification , Casearia/chemistry , Cell Line, Tumor/drug effects , Drug Screening Assays, Antitumor , Diterpenes/isolation & purification , Pentacyclic Triterpenes/isolation & purification , Zanthoxylum/chemistry
17.
Mem. Inst. Oswaldo Cruz ; 108(2): 172-177, abr. 2013. tab, graf
Article in English | LILACS | ID: lil-670406

ABSTRACT

Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10 2 and 2.5 x 10 6 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.


Subject(s)
Animals , Leishmania mexicana/immunology , Leishmaniasis, Cutaneous/immunology , Macrophages, Peritoneal/parasitology , Nitric Oxide/biosynthesis , Peromyscus/metabolism , Disease Models, Animal , Macrophages, Peritoneal/immunology , Peromyscus/parasitology
18.
Article in English | WPRIM | ID: wpr-148472

ABSTRACT

The endothelial nitric oxide synthase (eNOS) gene plays an important role in several biological functions. Polymorphisms of the eNOS gene have been associated with cancer. It has been suggested that the VNTR 4 a/b polymorphism may affect the expression of eNOS and contributes to tumor promotion in the mammary gland. We examined the role of the eNOS4 a/b polymorphism by comparing the genotypes of 281 healthy Mexican women with the genotypes of 429 Mexican women with breast cancer (BC). The observed genotype frequencies for control and BC patients were 0.6% and 0.7% for a/a (polymorphic); 87% and 77% for a/a (wild type); and 12% and 22% for a/b respectively. We found that the odds ratio (OR) was 1.9, with a 95% confidence interval (95%CI) of 1.29-2.95, P = 0.001 for genotypes a/a-a/b, b/c. The association was also evident when comparing the distribution of the a/a-a/b genotypes in patients with high levels of glutamate-oxaloacetate transaminase (SGOT) (OR, 1.93; 95% CI, 1.14-3.28; P = 0.015); undergoing menopause with high levels of SGOT (OR, 2.0; 95% CI, 1.1-3.84); and with high levels of glutamic-pyruvic transaminase (SGPT) (OR, 3.5; 95% CI, 1.56-8.22). The genotypes a/a-a/b are associated with BC susceptibility in the analyzed samples from the Mexican population.


Subject(s)
Adult , Alanine Transaminase/blood , Aspartate Aminotransferases/blood , Breast Neoplasms/blood , Female , Gene Frequency , Genetic Predisposition to Disease , Genotype , Humans , Mexico , Middle Aged , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type III/genetics , Polymorphism, Single Nucleotide
19.
Arq. neuropsiquiatr ; 70(11): 874-879, Nov. 2012. graf
Article in English | LILACS | ID: lil-655926

ABSTRACT

The effects of a high estradiol dose on memory and on nitric oxide metabolites in hippocampal tissues were investigated. Sham-Est and OVX-Est Groups were treated with 4 mg/kg of estradiol valerate for 12 weeks. Time latency and path length were significantly higher in the Sham-Est and OVX-Est Groups than in the Sham and OVX Groups, respectively (p<0.001). The animals in the Sham-Est and OVX-Est Groups spent lower time in the target quadrant (Q1) than those of the Sham and OVX Groups during the probe trial test (p<0.05 and <0.001, respectively). Significantly lower nitric oxide metabolite levels in the hippocampi of the Sham-Est and OVX-Est Groups were observed than in the Sham and OVX ones (p<0.001). These results suggest that decreased nitric oxide levels in the hippocampus may play a role in the learning and memory deficits observed after treatment with a high dose of estradiol, although the precise underlying mechanisms remain to be elucidated.


Os efeitos de uma alta dose de estradiol na memória e nos metabólitos do óxido nítrico de tecidos hipocampais foram estudados. Os Grupos Sham-Est e OVX-Est foram tratados com 4 mg/kg de valerato de estradiol por 12 semanas. O tempo de latência e o comprimento do caminho foram significativamente maiores nos Grupos Sham-Est e OVX-Est em relação aos Grupos Sham e OVX, respectivamente (p<0,001). Os animais dos Grupos Sham-Est e OVX-Est passaram menos tempo na meta do quadrante (Q1) do que aqueles dos Grupos Sham e OVX durante o teste inicial (p<0,05 e <0,001, respectivamente). Níveis significativamente menores de metabólitos do óxido nítrico foram observados nos hipocampos dos Grupos Sham-Est e OVX-Est em relação aos Grupos Sham e OVX (p<0,001). Esses resultados sugerem que os níveis diminuídos de óxido nítrico no hipocampo podem ter um papel nos déficits de aprendizado e de memória, que são observados após tratamento com alta dose de estradiol, embora os mecanismos específicos envolvidos nestes achados ainda precisam ser elucidados.


Subject(s)
Animals , Female , Humans , Rats , Contraceptive Agents/administration & dosage , Estradiol/analogs & derivatives , Hippocampus/metabolism , Learning Disabilities/etiology , Memory Disorders/etiology , Nitric Oxide/metabolism , Analysis of Variance , Dose-Response Relationship, Drug , Estradiol/administration & dosage , Hippocampus/drug effects , Learning Disabilities/metabolism , Maze Learning/drug effects , Memory Disorders/metabolism , Memory/drug effects , Nitric Oxide/biosynthesis , Ovariectomy , Random Allocation , Rats, Wistar
20.
Braz. j. med. biol. res ; 45(11): 1017-1024, Nov. 2012. ilus, tab
Article in English | LILACS | ID: lil-650575

ABSTRACT

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.


Subject(s)
Adult , Female , Humans , Male , Middle Aged , Antimicrobial Cationic Peptides/biosynthesis , Neutrophils/metabolism , Nitric Oxide/biosynthesis , Periodontitis/immunology , alpha-Defensins/biosynthesis , Case-Control Studies , Chronic Disease , Dental Plaque Index , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Lipopolysaccharides , Neutrophils/immunology , Periodontal Index , Periodontitis/metabolism , Reverse Transcriptase Polymerase Chain Reaction
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