RESUMO
Systemic candidiasis remains a significant public health concern worldwide, with high mortality rates despite available antifungal drugs. Drug-resistant strains add to the urgency for alternative therapies. In this context, vaccination has reemerged as a prominent immune-based strategy. Extracellular vesicles (EVs), nanosized lipid bilayer particles, carry a diverse array of native fungal antigens, including proteins, nucleic acids, lipids, and glycans. Previous studies from our laboratory demonstrated that Candida albicans EVs triggered the innate immune response, activating bone marrow-derived dendritic cells (BMDCs) and potentially acting as a bridge between innate and adaptive immunity. Vaccination with C. albicans EVs induced the production of specific antibodies, modulated cytokine production, and provided protection in immunosuppressed mice infected with lethal C. albicans inoculum. To elucidate the mechanisms underlying EV-induced immune activation, our study investigated pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) involved in EVs-phagocyte engagement. EVs from wild-type and mutant C. albicans strains with truncated mannoproteins were compared for their ability to stimulate BMDCs. Our findings revealed that EV decoration with O- and N-linked mannans and the presence of ß-1,3-glucans and chitin oligomers may modulate the activation of specific PRRs, in particular Toll-like receptor 4 (TLR4) and dectin-1. The protective effect of vaccination with wild-type EVs was found to be dependent on TLR4. These results suggest that fungal EVs can be harnessed in vaccine formulations to selectively activate PRRs in phagocytes, offering potential avenues for combating or preventing candidiasis.IMPORTANCESystemic candidiasis is a serious global health concern with high mortality rates and growing drug resistance. Vaccination offers a promising solution. A unique approach involves using tiny lipid-coated particles called extracellular vesicles (EVs), which carry various fungal components. Previous studies found that Candida albicans EVs activate the immune response and may bridge the gap between innate and adaptive immunity. To understand this better, we investigated how these EVs activate immune cells. We demonstrated that specific components on EV surfaces, such as mannans and glucans, interact with receptors on immune cells, including Toll-like receptor 4 (TLR4) and dectin-1. Moreover, vaccinating with these EVs led to strong immune responses and full protection in mice infected with Candida. This work shows how harnessing fungal EVs might lead to effective vaccines against candidiasis.
Assuntos
Candida albicans , Candidíase , Células Dendríticas , Vesículas Extracelulares , Vacinas Fúngicas , Receptores de Reconhecimento de Padrão , Receptor 4 Toll-Like , Animais , Candida albicans/imunologia , Vesículas Extracelulares/imunologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Camundongos , Candidíase/imunologia , Candidíase/prevenção & controle , Candidíase/microbiologia , Vacinas Fúngicas/imunologia , Vacinas Fúngicas/administração & dosagem , Células Dendríticas/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Camundongos Endogâmicos C57BL , Feminino , Imunidade Inata , Modelos Animais de DoençasRESUMO
We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of A. castellanii EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for Acanthamoeba-related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of A. castellanii EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of Acanthamoeba-related diseases.
Assuntos
Acanthamoeba castellanii , Vesículas Extracelulares , Proteômica , Acanthamoeba castellanii/metabolismo , Acanthamoeba castellanii/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Humanos , Metabolismo dos Lipídeos/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteoma/metabolismo , Proteoma/genéticaRESUMO
The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.
Assuntos
Anticorpos Antifúngicos , Anticorpos Monoclonais , Cryptococcus neoformans , Hibridomas , Cryptococcus neoformans/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Animais , Humanos , Hibridomas/imunologia , Anticorpos Antifúngicos/imunologia , Anticorpos Antifúngicos/isolamento & purificação , Camundongos , Linfócitos B/imunologia , Criptococose/imunologia , Criptococose/diagnóstico , Antígenos de Fungos/imunologia , ImunizaçãoRESUMO
We tested the hypothesis that increasing the exercise dose or changing the exercise mode would augment hypotensive effects when traditional aerobic exercise training failed to produce it in postmenopausal women. Sixty-five postmenopausal women with essential hypertension were randomly allocated into the continuous aerobic training (CAT) and non-exercising control (CON) groups. CAT group cycled at moderate intensity 3 times a week for 12 weeks. Individuals who failed to decrease systolic blood pressure (BP) were classified as non-responders (n = 34) and performed an additional 12 weeks of exercise training with either increasing the exercise dose or changing the exercise mode. The 3 follow-up groups were continuous aerobic training 3 times a week, continuous aerobic training 4 times a week, and high-intensity interval training. After the first 12 weeks of exercise training, systolic BP decreased by 1.5 mmHg (NS) with a wide range of inter-individual responses (-23 to 23 mmHg). Sixty-seven percent of women who were initially classified as non-responders participated in the second training period. Sixty percent of women who participated in continuous exercise training 3 or 4 times a week at greater exercise intensities reduced systolic BP. All (100%) of the women who performed high-intensity interval training experienced significant reductions in systolic BP. Traditional aerobic exercise was not sufficient to decrease BP significantly in the majority of postmenopausal women. However, those women who were not sensitive to recommended exercise may reduce BP if they were exposed to continuous aerobic exercise at higher intensities and/or volumes or a different mode of exercise.
Assuntos
Pressão Sanguínea , Exercício Físico , Hipertensão , Pós-Menopausa , Humanos , Feminino , Pós-Menopausa/fisiologia , Hipertensão/terapia , Hipertensão/fisiopatologia , Pessoa de Meia-Idade , Pressão Sanguínea/fisiologia , Exercício Físico/fisiologia , Idoso , Terapia por Exercício/métodos , Resultado do TratamentoRESUMO
OBJECTIVE: The aim of the present study was to assess the impact of orthodontic retainers on oral health-related quality of life (OHRQoL) in the short and long terms after orthodontic treatment. METHODS: Data from 45 patients up to three years after orthodontic treatment (T0) were analyzed. Patients were reassessed four years (T1) after T0. OHRQoL was measured using the OHIP-14 (Oral Health Impact Profile-14) questionnaire. The presence of a fixed retainer in the upper and/or lower arches, sex, and age were the predictive variables evaluated at T0 and T1. The occurrence of retainer fracture at T0 was clinically evaluated. Due to the COVID-19 pandemic, clinical examination on T1 was not possible, so the OHIP-14 and the self-perception of changes in teeth position and fracture of retainers were examined using an on-line questionnaire. RESULTS: At the initial examination, the presence of upper retainers had a negative impact on quality of life (p=0.018). The OHIP-14 value increased significantly from T0 to T1 (p=0.014), regardless of the presence of retainers. The fracture or debonding of the retainer reported by the patient was the only variable that had a negative impact on OHRQoL (p=0.05). CONCLUSION: The use of fixed upper retainers suggests a negative impact on the quality of life of the orthodontic patient after the end of orthodontic treatment. This impact, however, is negligible in the long term, except when associated with fracture or debonding. This study emphasizes the need for continuous follow-up of orthodontic patients during the retention period.
Assuntos
COVID-19 , Qualidade de Vida , Humanos , Contenções Ortodônticas , Pandemias , Estudos ProspectivosRESUMO
In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal ß-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface ß-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a ß-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble ß-1,3-glucan substantially inhibited this adhesion, indicating the involvement of ß-1,3-glucan recognition. Biotinylated ß-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the ß-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding ß-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to ß-1,3-glucans. These findings underscore the complexity of binding via ß-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of ß-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for ß-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.
Assuntos
Acanthamoeba castellanii , Amoeba , beta-Glucanas , Amoeba/metabolismo , Manose/metabolismo , Proteômica , beta-Glucanas/metabolismo , Glucanos/metabolismo , Histoplasma/metabolismoRESUMO
ABSTRACT Objective: The aim of the present study was to assess the impact of orthodontic retainers on oral health-related quality of life (OHRQoL) in the short and long terms after orthodontic treatment. Methods: Data from 45 patients up to three years after orthodontic treatment (T0) were analyzed. Patients were reassessed four years (T1) after T0. OHRQoL was measured using the OHIP-14 (Oral Health Impact Profile-14) questionnaire. The presence of a fixed retainer in the upper and/or lower arches, sex, and age were the predictive variables evaluated at T0 and T1. The occurrence of retainer fracture at T0 was clinically evaluated. Due to the COVID-19 pandemic, clinical examination on T1 was not possible, so the OHIP-14 and the self-perception of changes in teeth position and fracture of retainers were examined using an on-line questionnaire. Results: At the initial examination, the presence of upper retainers had a negative impact on quality of life (p=0.018). The OHIP-14 value increased significantly from T0 to T1 (p=0.014), regardless of the presence of retainers. The fracture or debonding of the retainer reported by the patient was the only variable that had a negative impact on OHRQoL (p=0.05). Conclusion: The use of fixed upper retainers suggests a negative impact on the quality of life of the orthodontic patient after the end of orthodontic treatment. This impact, however, is negligible in the long term, except when associated with fracture or debonding. This study emphasizes the need for continuous follow-up of orthodontic patients during the retention period.
RESUMO Objetivo: O objetivo deste estudo foi avaliar o impacto das contenções ortodônticas na qualidade de vida relacionada à saúde bucal (QVRSB) em curto e longo prazos após o tratamento ortodôntico. Métodos: Foram analisados dados de 45 pacientes até três anos após o tratamento ortodôntico (T0). Os pacientes foram reavaliados quatro anos (T1) após T0. A QVRSB foi mensurada usando o questionário OHIP-14 (Oral Health Impact Profile-14). A presença da contenção fixa nas arcadas superior e/ou inferior, o sexo e a idade foram as variáveis preditoras em T0 e T1. A ocorrência de quebra da contenção em T0 foi avaliada clinicamente. Em razão da pandemia da COVID-19, não foi possível o exame clínico em T1; assim, a autopercepção das alterações na posição dos dentes e a quebra ou descolagem das contenções foram registradas por meio de um questionário online. Resultados: No exame inicial, a presença da contenção superior apresentou um impacto negativo na qualidade de vida (p=0,018). Em T1, o valor de OHIP-14 aumentou significativamente em comparação a T0 (p=0,014), independentemente da presença das contenções. A quebra ou descolagem da contenção relatada pelo paciente foi a única variável que apresentou um impacto negativo na QVRSB (p=0,05). Conclusão: O uso de contenção fixa superior sugere um impacto inicial negativo na qualidade de vida do paciente após o fim do tratamento ortodôntico. Esse impacto, entretanto, é insignificante em longo prazo, exceto quando associado à descolagem ou quebra das contenções ortodônticas. Esse estudo enfatiza a necessidade de acompanhamento contínuo das contenções fixas após a finalização do tratamento ortodôntico.
RESUMO
Blood pressure (BP) responses to recommended aerobic training can vary widely between individuals. Although studies demonstrate the role of exercise training in regulating BP responsiveness, predictive models are still unknown. This study aimed to identify hemodynamic predictive markers for the diagnosis of BP responsiveness based on baseline characteristics and postexercise ambulatory blood pressure (ABP) before an aerobic training program in postmenopausal women. Sixty-five postmenopausal women with essential hypertension were randomly allocated into the continuous aerobic training (CAT, n = 51) and nonexercising control (CON, n = 14) groups. CAT group cycled at moderate intensity three times a week for 12 weeks. Individuals who failed to decrease systolic blood pressure (SBP) were classified as nonresponders (NRs; n = 34) based on typical error of measurement. Baseline anthropometric, metabolic, cardiovascular, hemodynamic variables, and postexercise ABP was measured to predict BP responsiveness. A logistic regression model based on Baseline SBP [odds ratio (OR) = 1.202; 95% confidence interval (CI) = 1.080-1.338], SBP Nighttime (OR = 0.889; 95% CI = 0.811-0.975), and heart rate (HR) Nighttime (OR = 1.127; 95% CI = 1.014-1.254) were able to diagnose responders and NR individuals to BP reduction in response to CAT with 92.6% accuracy (P < 0.001; Sensitivity = 94.1%; Specificity = 79.4%). The findings highlight the potential value of baseline clinical characteristics as Baseline SBP, SBP, and HR Nighttime as markers for diagnosing BP responsiveness to recommended CAT in hypertension postmenopausal women. Clinical Trial Registration number: RBR-3xnqxs8.
Assuntos
Sistema Cardiovascular , Hipertensão , Feminino , Humanos , Pressão Sanguínea/fisiologia , Monitorização Ambulatorial da Pressão Arterial , Pós-Menopausa , Hipertensão/diagnóstico , Hipertensão/terapiaRESUMO
The objective of this study was to identify the meanings that Brazilians attribute to the vaccines against COVID-19. A non-probabilistic sample of 1,817 Brazilians was used, each of whom answered a sociodemographic questionnaire and then the free word association technique was applied, with the inducing stimulus "vaccine for COVID-19". This data was analyzed using descriptive statistics in the SPSS software and the textual analysis was completed using IRaMuTeQ. The results were organized into classes: (1) "Vaccine - a glimmer of hope", portraying the expectation of healing and freedom; (2) "Vaccine - a scientific response to prevention", showing the role of research; and (3) "Vaccine - fears and political aspects", highlighting obstacles to vaccination. The results also demonstrated differences in responses depending on the region; adhesion to isolation; history of infection and hospitalization for COVID-19; being a frontline health professional; and being from a risk group. It is concluded that understanding the meanings of vaccination is essential for planning more effective interventions in immunization. (AU)
O objetivo deste estudo foi identificar os significados que brasileiros atribuem às vacinas contra a COVID-19. Contou-se com amostra não probabilística de 1.817 brasileiros, que responderam a um questionário sociodemográfico e à técnica de associação livre de palavras, com estímulo indutor "vacina para COVID-19", analisados por meio de estatística descritiva no software SPSS e análise textual no IRaMuTeQ. Os resultados organizaram-se em classes: (1) "Vacina - um sopro de esperança", retratando a expectativa de cura e liberdade; (2) "Vacina - uma resposta da ciência para a prevenção", lembrando o papel das pesquisas; e (3) "Vacina - temores e aspectos políticos", ressaltando entraves à vacinação. Também trouxe diferenças nas evocações em função da região; adesão ao isolamento; histórico de infecção e internação por COVID-19; ser profissional de saúde da linha de frente e ser do grupo de risco. Conclui-se que compreender os significados da vacinação é essencial ao planejamento de intervenções mais efetivas na imunização. (AU)
El objetivo de este estudio fue identificar los significados que los brasileños atribuyen a las vacunas contra la COVID-19. Contamos con una muestra de 1.817 brasileños, quienes respondieron un cuestionario sociodemográfico y la técnica de asociación libre de palabras, con el estímulo inductor "vacuna para COVID-19", analizado mediante estadística descriptiva en el SPSS y análisis textual en el IRaMuTeQ. Los resultados se organizaron en clases: (1) "Vacuna - un soplo de esperanza", retratando la expectativa de curación y libertad; (2) "Vacuna - una respuesta científica a la prevención", recordando el papel de la investigación; y (3) "Vacuna: miedos y aspectos políticos", destacando los obstáculos para la vacunación. También se identificaron diferencias en las respuestas según la región; la adherencia al aislamiento; los antecedentes de infección y hospitalización por COVID-19; ser un profesional de la salud de primera línea; y pertenecer a un grupo de riesgo. Se concluye que comprender los significados de la vacunación es fundamental para planificar intervenciones más efectivas de inmunización. (AU)
Assuntos
Humanos , Masculino , Feminino , Adulto , Adulto Jovem , Pandemias , Fatores Socioeconômicos , Grupos de Risco , Estudos Transversais , Inquéritos e Questionários , Pessoal de Saúde , Programas de Imunização , Associação LivreRESUMO
We aimed to evaluate the effect of supplemental CP on the nutritional characteristics and performance of Holstein × Gyr crossbreed heifers grazing intensively-managed Brachiaria decumbens throughout the year. Thirty-eight heifers with average initial body weight of 172.5 ± 11.15 kg (mean ± SE) and 8.2 ± 0.54 mo of age were randomly assigned to four treatments: three protein supplements (SUP) composed of soybean meal and ground corn fed at 5g/kg of BW, plus a control group (CON). The supplements had 12, 24 and 36% of CP for treatments S12 (n = 9), S24 (n = 10), and S36 (n = 9), respectively. The experiment latest one year, subdivided into four seasons: rainy, dry, rainy-dry transition (RDT), and dry-rainy transition (DRT). Feces and pasture samples were collected for 4 days in each season, using chromium oxide, titanium dioxide, and indigestible neutral detergent fiber (NDF) to estimate fecal excretion, supplement, and pasture intake, respectively. The data were analyzed using PROC GLIMMIXED of the SAS with repeated measures. No effects of supplementation were detected on pasture and NDF intake. However, SUP animals had a greater intake of DM, metabolizable energy, and metabolizable protein. A positive linear response on metabolizable protein intake was observed among SUP animals. We observed an interaction between treatment and season for all digestibility variables, with a positive linear response in CP digestibility among SUP animals during all seasons. For neutral detergent fiber (NDF) digestibility, we observed a positive linear response in RDT and rainy seasons and a quadratic response during the dry season. Furthermore, SUP animals had greater average daily gain (ADG) than non-supplemented animals, and among SUP animals, there was a quadratic response to ADG, with the greatest gain observed in S24. We observed greater nitrogen retention coefficient in SUP animals than in non-supplemented animals and a positive linear effect among SUP animals. Supplemental CP did not affect microbial protein production and efficiency. We observed an interaction between treatment and season for blood glucose, with SUP animals having greater glucose concentration in all seasons than non-supplemented animals. Additionally, we observed a quadratic response among SUP animals only during RDT and dry season, with the greatest glucose concentration in S24. SUP animals had greater blood concentrations of urea and IGF-1. In conclusion, SUP animals had greater intake, digestibility, and performance than non-supplemented animals, with the 24% CP supplement demonstrating the best metabolic responses and performance.