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1.
Microb Pathog ; 148: 104465, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32860878

RESUMO

In human ocular toxoplasmosis, serotype is related with greater severity. We analyzed Toxoplasma GRA6 serotype in 23 patients with ocular toxoplasmosis (13 confirmed, two co-infections- and eight unconfirmed cases) and 20 individuals chronically infected with Toxoplasma but without ocular involvement. In patients with ocular toxoplasmosis, we also studied host gene polymorphisms related to immune response (IL-1ß; IL-1α; IL-10; IFN-γ; TNF-α, IL-12), IL-17R, TLR-9, and P2RX7. Additionally, eight patients were studied for the production of TNFα, IL1-ß, IFN-γ and IL-10 by their peripheral leukocytes after ex vivo stimulation with soluble Toxoplasma antigens. There were no differences in the distribution of serotypes (GRA6-I versus GRA6 non-I) between infected individuals with- or without ocular involvement. Seropositivity for GRA6-I was associated with higher number of retinal lesions and higher levels of IL-1ß. Two polymorphisms were associated with specific clinical manifestations of ocular toxoplasmosis: IL-10 -819 C/T with bilateral lesions and IL-12 + 169,774 A/C with synechia. Higher levels of IL-10 were found in patients with the allele G/G at the polymorphic region IL-10 -1082. People with a GRA6 I serotype and possessing the allele G/G at the polymorphic region TNFα-857 suffered from an increased number of retinal lesions. We found a positive association between host cytokine genes polymorphisms and GRA6 serotypes correlated with specific clinical manifestations and immune response in ocular toxoplasmosis.


Assuntos
Toxoplasma , Toxoplasmose Ocular , Citocinas/genética , Humanos , Interleucina-12 , Polimorfismo Genético , Sorotipagem , Toxoplasma/genética , Toxoplasmose Ocular/genética
2.
Exp Parasitol ; 219: 108020, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33058858

RESUMO

Toxoplasma gondii is a parasite that can invade any cell in the human body. Here, we implemented and described an ex vivo model with human peripheral blood mononuclear cells (PBMCs) without using culture supplements/antibiotics and without cryopreserved cells (EXMOWS) to study the interactions between T. gondii and human cells. To establish the EXMOWS, three independent tests were carried out. Firstly, blood samples from 5 individuals were included to assess the viability and adherence of PBMCs in plate culture. In a second trial, blood samples from three seropositive and two seronegative individuals for T. gondii were used to evaluate human PBMCs cells: parasites, multiplicity of infection (MOI) 1:1, 1:3 and 1:5 at different times post infection (1 h, 6 h and 24 h). The possible immunomodulatory effect of the infection for this EXMOWS were evaluated in a third trial where HFF cells were infected with T. gondii and co-cultured with PBMCs obtained from anti-Toxoplasma IgG positive and IgG negative individuals. One hour was enough time for T. gondii infection of human PBMCs and 2 h was the minimum incubation time to guarantee adherence before carrying out any infection assay. A minimum of 1:3 MOI was necessary to guarantee efficient infection in human PBMCs with T. gondii RH-GFP. All protocols, including PBMCs isolation and stimulation, should be conducted the same day. This EXMOWS can be adapted to study the early stages of interaction with other microorganisms of human interest, without need of using cryopreservation and supplements/antibiotics.


Assuntos
Interações Hospedeiro-Parasita/fisiologia , Leucócitos Mononucleares/parasitologia , Toxoplasma/fisiologia , Adulto , Análise de Variância , Sobrevivência Celular , Células Cultivadas , Fibroblastos , Prepúcio do Pênis/citologia , Humanos , Imunoglobulina G/sangue , Masculino , RNA de Protozoário/química , RNA de Protozoário/isolamento & purificação , Adulto Jovem
3.
Ocul Immunol Inflamm ; : 1-10, 2023 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-37315178

RESUMO

This work analyzed exhaustion markers in CD8+ T-cell subpopulations in 21 samples of peripheral blood mononuclear cells (PBMCs) from individuals with ocular toxoplasmosis (n = 9), chronic asymptomatic toxoplasmosis (n = 7), and non-infected people (n = 5) by using RT-qPCR and flow cytometry techniques. The study found that gene expression of PD-1 and CD244, but not LAG-3, was higher in individuals with ocular toxoplasmosis versus individuals with asymptomatic infection or uninfected. Expression of PD1 in CD8+ central memory (CM) cells was higher in nine individuals with toxoplasmosis versus five uninfected individuals (p = .003). After ex vivo stimulation, an inverse correlation was found between the exhaustion markers and quantitative clinical characteristics (lesion size, recurrence index, and number of lesions). A total exhaustion phenotype was found in 55.5% (5/9) of individuals with ocular toxoplasmosis. Our results suggest that the CD8+ exhaustion phenotype is involved in the pathogenesis of ocular toxoplasmosis.

4.
Artigo em Inglês | MEDLINE | ID: mdl-30800644

RESUMO

In biology, models are experimental systems meant to recreate aspects of diseases or human tissue with the goal of generating inferences and approximations that can contribute to the resolution of specific biological problems. Although there are many models for studying intracellular parasites, their data have produced critical contradictions, especially in immunological assays. Peripheral blood mononuclear cells (PBMCs) represent an attractive tissue source in pharmacogenomics and in molecular and immunologic studies, as these cells are easily collected from patients and can serve as sentinel tissue for monitoring physiological perturbations due to disease. However, these cells are a very sensitive model due to variables such as temperature, type of stimulus and time of collection as part of posterior processes. PBMCs have been used to study Toxoplasma gondii and other apicomplexan parasites. For instance, this model is frequently used in new therapies or vaccines that use peptides or recombinant proteins derived from the parasite. The immune response to T. gondii is highly variable, so it may be necessary to refine this cellular model. This mini review highlights the major approaches in which PBMCs are used as a model of study for T. gondii and other apicomplexan parasites. The variables related to this model have significant implications for data interpretation and conclusions related to host-parasite interaction.


Assuntos
Apicomplexa/crescimento & desenvolvimento , Apicomplexa/imunologia , Interações Hospedeiro-Patógeno , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Modelos Teóricos , Infecções por Protozoários/fisiopatologia , Animais , Pesquisa Biomédica/tendências , Humanos , Infecções por Protozoários/imunologia , Infecções por Protozoários/parasitologia
5.
Artigo em Inglês | MEDLINE | ID: mdl-31867288

RESUMO

Toxoplasma gondii ROP16 and ROP18 proteins have been identified as important virulence factors for this parasite. Here, we describe the effect of ROP16 and ROP18 proteins on peripheral blood mononuclear cells (PBMCs) from individuals with different clinical status of infection. We evaluated IFN-γ, IL-10, and IL-1ß levels in supernatants from PBMCs cultures infected with tachyzoites of the T. gondii wild-type RH strain or with knock-out mutants of the rop16 and rop18 encoding genes (RHΔrop16 and RHΔrop18). Cytokine secretion was compared between PBMCs obtained from seronegative individuals (n = 10), with those with chronic asymptomatic (n = 8), or ocular infection (n = 12). We also evaluated if polymorphisms in the genes encoding for IFN-γ, IL-10, IL-1ß, Toll-like receptor 9 (TLR9), and purinoreceptor P2RX7 influenced the production of the encoded proteins after ex vivo stimulation. In individuals with chronic asymptomatic infection, only a moderate effect on IL-10 levels was observed when PBMCs were infected with RHΔrop16, whereas a significant difference in the levels of inflammatory cytokines IFN-γ and IL-1ß was observed in seronegative individuals, but this was also dependent on the host's cytokine gene polymorphisms. Infection with ROP16-deficient parasites had a significant effect on IFN-γ production in previously non-infected individuals, suggesting that ROP16 which is considered as a virulence factor plays a role during the primary infection in humans, but not in the secondary immune response.


Assuntos
Leucócitos Mononucleares/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Tirosina Quinases/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Toxoplasmose/parasitologia , Citocinas/metabolismo , Fibroblastos , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Leucócitos Mononucleares/metabolismo , Polimorfismo Genético , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT6/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/genética , Virulência , Fatores de Virulência/imunologia
6.
Eur J Med Chem ; 182: 111628, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31472473

RESUMO

Dengue fever is a mosquito-borne viral disease that has become a major public health concern worldwide. This disease presents with a wide range of clinical manifestations, from a mild cold-like illness to the more serious hemorrhagic dengue fever and dengue shock syndrome. Currently, neither an approved drug nor an effective vaccine for the treatment are available to fight the disease. The envelope protein (E) is a major component of the virion surface. This protein plays a key role during the viral entry process, constituting an attractive target for the development of antiviral drugs. The crystal structure of the E protein reveals the existence of a hydrophobic pocket occupied by the detergent n-octyl-ß-d-glucoside (ß-OG). This pocket lies at the hinge region between domains I and II and is important for the low pH-triggered conformational rearrangement required for the fusion of the virion with the host's cell. Aiming at the design of novel molecules which bind to E and act as virus entry inhibitors, we undertook a de novo design approach by "growing" molecules inside the hydrophobic site (ß-OG). From more than 240000 small-molecules generated, the 2,4 pyrimidine scaffold was selected as the best candidate, from which one synthesized compound displayed micromolar activity. Molecular dynamics-based optimization was performed on this hit, and thirty derivatives were designed in silico, synthesized and evaluated on their capacity to inhibit dengue virus entry into the host cell. Four compounds were found to be potent antiviral compounds in the low-micromolar range. The assessment of drug-like physicochemical and in vitro pharmacokinetic properties revealed that compounds 3e and 3h presented acceptable solubility values and were stable in mouse plasma, simulated gastric fluid, simulated intestinal fluid, and phosphate buffered saline solution.


Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Desenho de Fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas do Envelope Viral/antagonistas & inibidores , Células A549 , Animais , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Vírus da Dengue/metabolismo , Relação Dose-Resposta a Droga , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Solubilidade , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismo
8.
Rev. colomb. ortop. traumatol ; 34(1): 53-59, 2020. tab
Artigo em Espanhol | COLNAL, LILACS | ID: biblio-1117575

RESUMO

Introducción Las ulceras por presión (UPP) son una condición frecuente en los pacientes con movilidad restringida, su sobreinfección implica un esfuerzo adicional en el tratamiento, ya que se asocia a complicaciones como osteomielitis crónica y sepsis. El objetivo del estudio es describir las características clínicas y sociodemográficas de los pacientes con UPP enfatizando en el perfil microbiológico de aquellos con sobreinfección. Materiales y métodos Se realizó un estudio descriptivo retrospectivo que incluyó a 76 pacientes adultos con diagnóstico de UPP sobreinfectadas sometidos a cirugía entre 2013 y 2015. Se obtuvo información demográfica, clínica y microbiológica. Se describieron los resultados según la naturaleza de la variable. Resultados De los 76 pacientes, 56 (73.7%) fueron hombres. La mediana de edad fue de 45 años. La mediana de hospitalización fue de 22.5 días, el principal motivo de inmovilidad fue la lesión medular (69,7%). Los patógenos más comunes fueron Enterococcus faecalis (46%), Escherichia coli (43.4%), Streptococcus spp (35.5%) y Proteus spp (30.2%). En el 77,6% de los pacientes se aislaron dos o más microorganismos en los cultivos. Discusión La lesión medular en la condición más asociada a ulceras por presión en nuestro medio, principalmente en hombres jóvenes. La sobreinfección generalmente es polimicrobiana, lo que nos muestra la gran importancia de los cultivos para poder realizar un tratamiento específico y efectivo. Al conocer las características clínicas y demográficas se pueden direccionar estrategias de prevención y rehabilitación que impacten de manera positiva en la calidad de vida de los pacientes y la sostenibilidad del sistema de salud. Nivel de evidencia IV


Background Pressure ulcers (PU) are a frequent condition in patients with restricted mobility. Their infection requires an additional effort in the treatment, since it is associated with complications, such as chronic osteomyelitis and sepsis. The aim of this study is to describe the clinical and sociodemographic characteristics of patients with PU, emphasising the microbiological profile of those with infection. Materials and methods A retrospective descriptive study was carried out on 76 adult patients diagnosed with infected PU who underwent surgery between 2013 and 2015. Demographic, clinical, and microbiological information was obtained. The results were described according to the nature of the variable. Results Of the 76 patients, 56 (73.7%) were men. The median age was 45 years. The median number of days hospitalised was 22.5 days, and the main cause of immobility was spinal cord injury (69.7%). The most common pathogens were Enterococcus faecalis (46%), Escherichia coli (43.4%), Streptococcus spp (35.5%), and Proteus spp (30.2%). Two or more microorganisms were isolated in the cultures in 77.6% of the patients, Discussion Spinal cord injury is the condition most associated with pressure ulcers in this study, mainly in young men. Infection is usually polymicrobial, which shows the great importance of the cultures, in order to be able to carry out a specific and effective treatment. By knowing the clinical and demographic characteristics, prevention and rehabilitation strategies can be directed at having a positive impact on the quality of life of PU patients, and the sustainability of the health care system. Evidence Level IV


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Úlcera por Pressão/microbiologia , Fatores Socioeconômicos , Estudos Retrospectivos , Úlcera por Pressão/cirurgia , Úlcera por Pressão/tratamento farmacológico
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