RESUMO
BACKGROUND: Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis. Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms. OBJECTIVES: To synthesize and evaluate the anti-Acanthamoeba activity of a panel of S-MGBs, and therefore determine the potential of this class for further development. METHODS: A panel of 12 S-MGBs was synthesized and anti-Acanthamoeba activity was determined using an alamarBlue™-based trophocidal assay against Acanthamoeba castellanii. Cross-screening against Trypanosoma brucei brucei, Staphylococcus aureus and Escherichia coli was used to investigate selective potency. Cytotoxicity against HEK293 cells allowed for selective toxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays. RESULTS AND DISCUSSION: S-MGB-241 has an IC50 of 6.6 µM against A. castellanii, comparable to the clinically used miltefosine (5.6 µM) and negligible activity against the other organisms. It was also found to have an IC50â>â100 µM against HEK293 cells, demonstrating low cytotoxicity. S-MGB-241 binds to DNA as a dimer, albeit weakly compared to other S-MGBs previously studied. This was confirmed by DNA thermal shift assay with a ΔTmâ=â1â±â0.1°C. CONCLUSIONS: Together, these data provide confidence that S-MGBs can be further optimized to generate new, potent treatments for Acanthameoba spp. infections. In particular, S-MGB-241, has been identified as a 'hit' compound that is selectively active against A. castellanii, providing a starting point from which to begin optimization of DNA binding and potency.
Assuntos
Acanthamoeba castellanii , Acanthamoeba castellanii/efeitos dos fármacos , Humanos , Células HEK293 , Escherichia coli/efeitos dos fármacos , Antiprotozoários/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Testes de Sensibilidade Parasitária , Sobrevivência Celular/efeitos dos fármacosRESUMO
SARS-CoV-2 is the causative agent of COVID-19 in humans and has resulted in the death of millions of people worldwide. Similar numbers of infections have been documented in males and females; males, however, are more likely than females to be hospitalized, require intensive care unit, or die from COVID-19. The mechanisms that account for this are multi-factorial and are likely to include differential expression of ACE2 and TMPRSS2 molecules that are required for viral entry into hosts cells and sex differences in the immune response, which are due to modulation of cellular functions by sex hormones and differences in chromosomal gene expression. Furthermore, as comorbidities are also associated with poorer outcomes to SARS-CoV-2 infection and several comorbidities are overrepresented in males, these are also likely to contribute to the observed sex differences. Despite their relative better prognosis following infection with SARS-CoV-2, females do have poorer outcomes during pregnancy. This is likely to be due to pregnancy-induced changes in the immune system that adversely affect viral immunity and disruption of the renin-angiotensin system. Importantly, vaccination reduces the severity of disease in males and females, including pregnant females, and there is no evidence that vaccination has any adverse effects on the outcomes of pregnancy.
Assuntos
COVID-19 , Vacinas , Gravidez , Humanos , Feminino , Masculino , SARS-CoV-2/genética , COVID-19/prevenção & controle , Vacinação , Internalização do VírusRESUMO
AIM: Acanthamoeba infections are characterized by an intense localized innate immune response associated with an influx of macrophages. Acanthamoeba protease production is known to affect virulence. Herein, the ability of Acanthamoeba trophozoite proteases, of either the laboratory Neff strain or a recently isolated clinical strain, to stimulate IL-12 and IL-6 and to activate protease-activated receptors, PAR1 and PAR2 expressed on murine macrophages, was investigated. METHOD AND RESULTS: Using selected protease inhibitors, leupeptin and E64, we showed that Acanthamoeba proteases can stimulate IL-12 and IL-6 by murine macrophages. Subsequently, using specific antagonists to inhibit PAR1 , and bone marrow-derived macrophages from PAR2 gene-deficient mice, we demonstrate that PAR1 , but not PAR2 contributes to macrophage IL-12 production in response to Acanthamoeba. In contrast, Acanthamoeba-induced IL-6 production is PAR1 and PAR2 independent. CONCLUSION: This study shows for the first time the involvement of PARs, expressed on macrophages, in the response to Acanthamoeba trophozoites and might provide useful insight into Acanthamoeba infections and their future treatments.
Assuntos
Acanthamoeba/enzimologia , Acanthamoeba/imunologia , Amebíase/imunologia , Proteínas de Ciclo Celular/metabolismo , Ativação de Macrófagos , Macrófagos/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptor PAR-2/metabolismo , Animais , Imunidade Inata , Interleucina-12/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peptídeo Hidrolases/metabolismo , Transdução de SinaisRESUMO
Toxoplasma gondii is a zoonotic parasite which, depending on the geographical location, can infect between 10% and 90% of humans. Infection during pregnancy may result in congenital toxoplasmosis. The effects on the foetus vary depending on the stage of gestation in which primary maternal infection arises. A large body of research has focused on understanding immune response to toxoplasmosis, although few studies have addressed how it is affected by pregnancy or the pathological consequences of infection at the maternal-foetal interface. There is a lack of knowledge about how maternal immune cells, specifically macrophages, are modulated during infection and the resulting consequences for parasite control and pathology. Herein, we discuss the potential of T. gondii infection to affect the maternal-foetal interface and the potential of pregnancy to disrupt maternal immunity to T. gondii infection.
Assuntos
Relações Materno-Fetais/fisiologia , Complicações Parasitárias na Gravidez/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Feminino , Feto/imunologia , Humanos , Macrófagos/imunologia , Gravidez , Complicações Parasitárias na Gravidez/parasitologia , Toxoplasmose/parasitologiaRESUMO
Acanthamoeba castellanii is a ubiquitous free-living amoeba with a worldwide distribution that can occasionally infect humans, causing particularly severe infections in immunocompromised individuals. Dissecting the immunology of Acanthamoeba infections has been considered problematic due to the very low incidence of disease, despite the high exposure rates. While macrophages are acknowledged as playing a significant role in Acanthamoeba infections, little is known about how this facultative parasite influences macrophage activity. Therefore, in this study we investigated the effects of Acanthamoeba on the activation of resting macrophages. Consequently, murine bone marrow-derived macrophages were cocultured with trophozoites of either the laboratory Neff strain or a clinical isolate of A. castellaniiIn vitro real-time imaging demonstrated that trophozoites of both strains often established evanescent contact with macrophages. Both Acanthamoeba strains induced a proinflammatory macrophage phenotype characterized by the significant production of interleukin-12 (IL-12) and IL-6. However, macrophages cocultured with the clinical isolate of Acanthamoeba produced significantly less IL-12 and IL-6 than the Neff strain. The utilization of macrophages derived from MyD88-, TRIF-, Toll-like receptor 2 (TLR2)-, TLR4-, and TLR2/4-deficient mice indicated that Acanthamoeba-induced proinflammatory cytokine production was through MyD88-dependent, TRIF-independent, TLR4-induced events. This study shows for the first time the involvement of TLRs expressed on macrophages in the recognition of and response to Acanthamoeba trophozoites.
Assuntos
Acanthamoeba castellanii/imunologia , Interleucina-12/imunologia , Interleucina-6/imunologia , Macrófagos/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 4 Toll-Like/metabolismo , Amebíase/imunologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/genéticaRESUMO
Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, such as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages during the early phase of infection. Here, THP-1 cells, primary human monocytes isolated from peripheral blood, and human monocyte-derived macrophages were either coincubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell-free conditioned medium. Production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-12), anti-inflammatory cytokine (IL-10), and chemokine (IL-8) was evaluated at specific hours poststimulation (ranging from 1.5 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both proinflammatory cytokines and chemokine production, suggesting that this protozoan infection results from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of proinflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response.
Assuntos
Acanthamoeba castellanii/imunologia , Amebíase/imunologia , Citocinas/metabolismo , Interleucina-10/metabolismo , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , Análise de Variância , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Genótipo , HumanosRESUMO
The dual specific phosphatase, MAP kinase phosphatase-2 (MKP-2) has recently been demonstrated to negatively regulate macrophage arginase-1 expression, while at the same time to positively regulate iNOS expression. Consequently, MKP-2 is likely to play a significant role in the host interplay with intracellular pathogens. Here we demonstrate that MKP-2(-/-) mice on the C57BL/6 background have enhanced susceptibility compared with wild-type counterparts following infection with type-2 strains of Toxoplasma gondii as measured by increased parasite multiplication during acute infection, increased mortality from day 12 post-infection onwards and increased parasite burdens in the brain, day 30 post-infection. MKP-2(-/-) mice did not, however, demonstrate defective type-1 responses compared with MKP-2(+/+) mice following infection although they did display significantly reduced serum nitrite levels and enhanced tissue arginase-1 expression. Early resistance to T. gondii in MKP-2(+/+), but not MKP-2(-/-), mice was nitric oxide (NO) dependent as infected MKP-2(+/+), but not MKP-2(-/-) mice succumbed within 10 days post-infection with increased parasite burdens following treatment with the iNOS inhibitor L-NAME. Conversely, treatment of infected MKP-2(-/-) but not MKP-2(+/+) mice with nor-NOHA increased parasite burdens indicating a protective role for arginase-1 in MKP-2(-/-) mice. In vitro studies using tachyzoite-infected bone marrow derived macrophages and selective inhibition of arginase-1 and iNOS activities confirmed that both iNOS and arginase-1 contributed to inhibiting parasite replication. However, the effects of arginase-1 were transient and ultimately the role of iNOS was paramount in facilitating long-term inhibition of parasite multiplication within macrophages.
Assuntos
Arginase/metabolismo , Macrófagos/parasitologia , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Toxoplasma/fisiologia , Toxoplasmose/parasitologia , Animais , Arginase/antagonistas & inibidores , Arginase/genética , Western Blotting , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Toxoplasmose/metabolismoRESUMO
Macrophage migration inhibitory factor (MIF) is a proinflammatory molecule in mammals that, unusually for a cytokine, exhibits tautomerase and oxidoreductase enzymatic activities. Homologues of this well conserved protein are found within diverse phyla including a number of parasitic organisms. Herein, we produced recombinant histidine-tagged Toxoplasma gondii MIF (TgMIF), a 12-kDa protein that lacks oxidoreductase activity but exhibits tautomerase activity with a specific activity of 19.3 µmol/min/mg that cannot be inhibited by the human MIF inhibitor ISO-1. The crystal structure of the TgMIF homotrimer has been determined to 1.82 Å, and although it has close structural homology with mammalian MIFs, it has critical differences in the tautomerase active site that account for the different inhibitor sensitivity. We also demonstrate that TgMIF can elicit IL-8 production from human peripheral blood mononuclear cells while also activating ERK MAPK pathways in murine bone marrow-derived macrophages. TgMIF may therefore play an immunomodulatory role during T. gondii infection in mammals.
Assuntos
Fatores Inibidores da Migração de Macrófagos , Macrófagos , Proteínas de Protozoários , Toxoplasma , Toxoplasmose , Animais , Cristalografia por Raios X , Humanos , Interleucina-8/química , Interleucina-8/genética , Interleucina-8/imunologia , Interleucina-8/metabolismo , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Toxoplasma/química , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasma/metabolismo , Toxoplasmose/genética , Toxoplasmose/imunologia , Toxoplasmose/metabolismoRESUMO
The enoyl acyl-carrier protein reductase (ENR) enzyme of the apicomplexan parasite family has been intensely studied for antiparasitic drug design for over a decade, with the most potent inhibitors targeting the NAD(+) bound form of the enzyme. However, the higher affinity for the NADH co-factor over NAD(+) and its availability in the natural environment makes the NADH complex form of ENR an attractive target. Herein, we have examined a benzimidazole family of inhibitors which target the NADH form of Francisella ENR, but despite good efficacy against Toxoplasma gondii, the IC50 for T. gondii ENR is poor, with no inhibitory activity at 1 µM. Moreover similar benzimidazole scaffolds are potent against fungi which lack the ENR enzyme and as such we believe that there may be significant off target effects for this family of inhibitors.
Assuntos
Benzimidazóis/química , Benzimidazóis/farmacologia , Sistemas de Liberação de Medicamentos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Toxoplasma/efeitos dos fármacos , Antiparasitários/química , Antiparasitários/farmacologia , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Estrutura MolecularRESUMO
Acanthamoeba keratitis (AK) is a severe infection of the cornea. Prevention and treatment are difficult due to the inefficacy of currently available compounds. The impact of many commonly used compounds for routine examinations of Acanthamoeba is unexplored but might offer insight useful in combatting AK. In this study, we demonstrate that sodium metabisulfite, a common preservation constituent of eye care solutions, was found to be active against Acanthamoeba trophozoites at concentrations lower than that commonly found in eye drops (IC50 0.03 mg/mL). We demonstrate that sodium metabisulfite depletes thiamine from growth medium and that Acanthamoeba is a thiamine auxotroph, requiring thiamine salvage for growth. The inhibitory effects of sodium metabisulfite can be overcome by thiamine supplementation. These results are consistent with the lack of key enzymes for thiamine biosynthesis in the genome of Acanthamoeba, an area which might prove exploitable using new or existing compounds. Indeed, this study highlights sodium metabisulfite as a useful inhibitor of Acanthamoeba castellanii trophozoites in vitro and that it acts, at least in part, by limiting available thiamine.
RESUMO
Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.
RESUMO
Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.
Assuntos
Desenho de Fármacos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Toxoplasma/enzimologia , Triclosan/farmacologia , Relação Dose-Resposta a Droga , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Triclosan/síntese química , Triclosan/químicaRESUMO
Toxoplasma gondii is a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery of N-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range against T. gondii in vitro and in vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be useful as a new scaffold for development of novel and improved inhibitors of T. gondii. Our genome-wide investigations reveal a specific mechanism of resistance to N-benzoyl-2-hydroxybenzamides mediated by adaptin-3ß, a large protein from the secretory protein complex. N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs). N-Benzoyl-2-hydroxybenzamide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs. Furthermore, QQ-437 is active against chloroquine-resistant Plasmodium falciparum. Our studies reveal a novel class of compounds that disrupts a unique secretory pathway of T. gondii, with the potential to be used as scaffolds in the search for improved compounds to treat the devastating diseases caused by apicomplexan parasites.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Antiprotozoários/farmacologia , Benzamidas/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Toxoplasma/efeitos dos fármacos , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Antimaláricos/síntese química , Antimaláricos/farmacologia , Antiprotozoários/síntese química , Benzamidas/síntese química , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/parasitologia , Humanos , Concentração Inibidora 50 , Organelas/efeitos dos fármacos , Organelas/genética , Organelas/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Relação Quantitativa Estrutura-Atividade , Via Secretória/efeitos dos fármacos , Via Secretória/fisiologia , Toxoplasma/genética , Toxoplasma/metabolismoRESUMO
The role of progesterone in modulating dendritic cell (DC) function following stimulation of different TLRs is relatively unknown. We compared the ability of progesterone to modulate murine bone marrow-derived DC cytokine production (IL-6 and IL-12) and costimulatory molecule expression (CD40, CD80, and CD86) induced by either TLR3 or TLR4 ligation and determined whether activity was via the progesterone receptor (PR) or glucocorticoid receptor (GR) by comparative studies with the PR-specific agonist norgestrel and the GR agonist dexamethasone. Progesterone was found to downregulate, albeit with different sensitivities, both TLR3- and TLR4-induced IL-6 production entirely via the GR, but IL-12p40 production via either the GR or PR. Of particular significance was that progesterone was able to significantly inhibit TLR3- but not TLR4-induced CD40 expression in bone marrow-derived DCs. Stimulation of the PR (with progesterone and norgestrel) by pretreatment of DCs was found to sustain IFN regulatory factor-3 phosphorylation following TLR3 ligation, but not TLR4 ligation. Overall, these studies demonstrate that progesterone can differentially regulate the signaling pathways employed by TLR3 and TLR4 agonists to affect costimulatory molecule expression and cytokine production.
Assuntos
Células Dendríticas/metabolismo , Progesterona/metabolismo , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Progesterona/farmacologia , Receptores de Glucocorticoides/imunologia , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/imunologia , Receptores de Progesterona/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 3 Toll-Like/efeitos dos fármacos , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/imunologiaRESUMO
The efficacy of RNA-based vaccines has been recently demonstrated, leading to the use of mRNA-based COVID-19 vaccines. The application of self-amplifying mRNA within these formulations may offer further enhancement to these vaccines, as self-amplifying mRNA replicons enable longer expression kinetics and more potent immune responses compared to non-amplifying mRNAs. To investigate the impact of administration route on RNA-vaccine potency, we investigated the immunogenicity of a self-amplifying mRNA encoding the rabies virus glycoprotein encapsulated in different nanoparticle platforms (solid lipid nanoparticles (SLNs), polymeric nanoparticles (PNPs) and lipid nanoparticles (LNPs)). These were administered via three different routes: intramuscular, intradermal and intranasal. Our studies in a mouse model show that the immunogenicity of our 4 different saRNA vaccine formulations after intramuscular or intradermal administration was initially comparable; however, ionizable LNPs gave higher long-term IgG responses. The clearance of all 4 of the nanoparticle formulations from the intramuscular or intradermal administration site was similar. In contrast, immune responses generated after intranasal was low and coupled with rapid clearance for the administration site, irrespective of the formulation. These results demonstrate that both the administration route and delivery system format dictate self-amplifying RNA vaccine efficacy.
Assuntos
COVID-19 , Nanopartículas , Animais , Vacinas contra COVID-19 , Humanos , Lipossomos , Camundongos , RNA Mensageiro , SARS-CoV-2 , Potência de Vacina , Vacinas Sintéticas , Vacinas de mRNARESUMO
The formation of a protein layer "corona" on the nanoparticle surface upon entry into a biological environment was shown to strongly influence the interactions with cells, especially affecting the uptake of nanomedicines. In this work, we present the impact of the protein corona on the uptake of PEGylated zein micelles by cancer cells, macrophages, and dendritic cells. Zein was successfully conjugated with poly(ethylene glycol) (PEG) of varying chain lengths (5K and 10K) and assembled into micelles. Our results demonstrate that PEGylation conferred stealth effects to the zein micelles. The presence of human plasma did not impact the uptake levels of the micelles by melanoma cancer cells, regardless of the PEG chain length used. In contrast, it decreased the uptake by macrophages and dendritic cells. These results therefore make PEGylated zein micelles promising as potential drug delivery systems for cancer therapy.
RESUMO
Progesterone is the female sex hormone necessary for the maintenance of pregnancy, and is known to modulate macrophage activation. However, studies have concentrated exclusively on the ability of progesterone to negatively regulate the innate and classical pathways of activation, associated with nitric oxide (NO) and interleukin (IL)-12 production. Our aim was to examine the ability of progesterone to modulate alternative macrophage activation. Bone marrow cells were isolated and differentiated from male BALB/c mice, exposed to varying concentrations of progesterone and stimulated with lipopolysaccharide (LPS) (innate activation), IL-4 (alternative activation) or LPS in combination with IL-4. Our present study demonstrates that progesterone not only down-regulates inducible nitric oxide synthase 2 (iNOS) activity in macrophages but also arginase activity, in a dose-dependent manner, independent of the stimuli, whether it is induced by LPS (innate activation), IL-4 (alternative activation) or LPS in combination with IL-4. The ability of progesterone to down-modulate IL-4-induced cell surface expression of the mannose receptor further suggested a negative regulation of alternative macrophage activation by this hormone. Analysis of mRNA expression, by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), of genes associated with innate and alternative macrophage activation revealed that progesterone down-regulated LPS-induced macrophage nos2, argI and p40 (IL-12/IL-23) expression and IL-4-induced argI, mrc-1 and fizz1 expression. However, progesterone up-regulated IL-4-induced macrophage expression of ym1, while dectin-1 expression remained unaltered. Following treatment of macrophages with LPS and IL-4 in combination a similar pattern was observed, with the exception that progesterone up-regulated macrophage expression of fizz1 as well as ym1 and did not modify mrc-1 expression. Our data demonstrate for the first time that a hormone has the ability to regulate selectively the expression of different genes associated with alternative macrophage activation.
Assuntos
Expressão Gênica/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Progesterona/farmacologia , Animais , Arginase/genética , Arginase/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Expressão Gênica/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-4/farmacologia , Lectinas/genética , Lectinas/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Progestinas/farmacologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
T1/ST2 is an immunoregulatory protein of the IL-1 receptor family that has recently been reported as being a component of the IL-33 receptor. IL-33 is a newly described cytokine known to amplify the Th2 response and reduce production of Th1 cytokines. The function of T1/ST2 during Toxoplasma gondii infection is as yet undescribed. Given the requirement of a balanced type 1/type 2 response for effective control of parasite number and immunopathology, it is likely that T1/ST2 may play a part in aiding this process. Accordingly, we have shown that T1/ST2 mRNA transcripts are upregulated in the brains of mice infected with T. gondii and that mice deficient in T1/ST2 demonstrated increased susceptibility to infection with T. gondii that correlated with increased pathology and greater parasite burden in the brains. Real-time PCR analysis of cerebral cytokine levels revealed increased mRNA levels of iNOS, IFN-gamma and TNF-alpha in infected T1/ST2(-/-) mice. These effects were independent of changes in IL-10 production. This study provides the first evidence of a specific role for IL-33 receptor signalling in the brain as well as highlighting the requirement of this mechanism in limiting infection with an intracellular parasite.
Assuntos
Encefalite/imunologia , Receptores de Interleucina/imunologia , Transdução de Sinais/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Animais , Peso Corporal , Encéfalo/metabolismo , Encéfalo/parasitologia , Encéfalo/patologia , Encefalite/parasitologia , Ensaio de Imunoadsorção Enzimática , Feminino , Interferon gama/genética , Interferon gama/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose Animal/parasitologia , Ativação Transcricional , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Studies indicate that female mice are more susceptible to T. gondii infection, as defined by higher mortality rates in comparison to male mice. However, whether this is due to an inability to control initial parasite multiplication or due to detrimental effects of the immune system has not been determined. Therefore, the following studies were undertaken to determine the influence of sex on early parasite multiplication and the immune response during T. gondii infection and to correlate this with disease outcome. Early parasite replication was studied through applying an in vivo imaging system (IVIS) with luciferase expressing T. gondii. In parallel immunological events were studied by cytometric bead array to quantify key immunological mediators. The results confirmed the previous findings that female mice are more susceptible to acute infection, as determined by higher mortality rates and weight loss compared with males. However, conflicting with expectations, female mice had lower parasite burdens during the acute infection than male mice. Female mice also exhibited significantly increased production of Monocyte Chemoattractant Protein-1 (MCP-1), Interferon (IFN)-γ, and Tumour Necrosis Factor (TNF)-α than male mice. MCP-1 was found to be induced by T. gondii in a dose dependent manner suggesting that the observed increased levels detected in female mice was due to a host-mediated sex difference rather than due to parasite load. However, MCP-1 was not affected by physiological concentration of estrogen or testosterone, indicating that MCP-1 differences observed between the sexes in vivo are due to an as yet unidentified intermediary factor that in turn influences MCP-1 levels. These results suggest that a stronger immune response in female mice compared with male mice enhances their ability to control parasite replication but increases their morbidity and mortality.
RESUMO
A range of cationic delivery systems have been investigated as vaccine adjuvants, though few direct comparisons exist. To investigate the impact of the delivery platform, we prepared four cationic systems (emulsions, liposomes, polymeric nanoparticles and solid lipid nanoparticles) all containing equal concentrations of the cationic lipid dimethyldioctadecylammonium bromide in combination with the Neisseria adhesin A variant 3 subunit antigen. The formulations were physicochemically characterized and their ability to associate with cells and promote antigen processing (based on degradation of DQ-OVA, a substrate for proteases which upon hydrolysis is fluorescent) was compared in vitro and their vaccine efficacy (antigen-specific antibody responses and IFN-γ production) and biodistribution (antigen and adjuvant) were evaluated in vivo. Due to their cationic nature, all delivery systems gave high antigen loading (> 85%) with liposomes, lipid nanoparticles and emulsions being <200 nm, whilst polymeric nanoparticles were larger (~350 nm). In vitro, the particulate systems tended to promote cell uptake and antigen processing, whilst emulsions were less effective. Similarly, whilst the particulate delivery systems induced a depot (of both delivery system and antigen) at the injection site, the cationic emulsions did not. However, out of the systems tested the cationic emulsions induced the highest antibody responses. These results demonstrate that while cationic lipids can have strong adjuvant activity, their formulation platform influences their immunogenicity.