RESUMO
Toxoplasma gondii (T. gondii) is a protozoan parasite that infects approximately one-third of the global human population, often leading to chronic infection. While acute T. gondii infection can cause neural damage in the central nervous system and result in toxoplasmic encephalitis, the consequences of T. gondii chronic infection (TCI) are generally asymptomatic. However, emerging evidence suggests that TCI may be linked to behavioral changes or mental disorders in hosts. Astrocyte polarization, particularly the A1 subtype associated with neuronal apoptosis, has been identified in various neurodegenerative diseases. Nevertheless, the role of astrocyte polarization in TCI still needs to be better understood. This study aimed to establish a mouse model of chronic TCI and examine the transcription and expression levels of glial fibrillary acidic protein (GFAP), C3, C1q, IL-1α, and TNF-α in the brain tissues of the mice. Quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay, and Western blotting were employed to assess these levels. Additionally, the expression level of the A1 astrocyte-specific marker C3 was evaluated using indirect fluorescent assay (IFA). In mice with TCI, the transcriptional and expression levels of the inflammatory factors C1q, IL-1α, and TNF-α followed an up-down-up pattern, although they remained elevated compared to the control group. These findings suggest a potential association between astrocyte polarization towards the A1 subtype and synchronized changes in these three inflammatory mediators. Furthermore, immunofluorescence assay (IFA) revealed a significant increase in the A1 astrocytes (GFAP+C3+) proportion in TCI mice. This study provides evidence that TCI can induce astrocyte polarization, a biological process that may be influenced by changes in the levels of three inflammatory factors: C1q, IL-1α, and TNF-α. Additionally, the release of neurotoxic substances by A1 astrocytes may be associated with the development of TCI.
Assuntos
Astrócitos , Encéfalo , Toxoplasma , Animais , Astrócitos/metabolismo , Astrócitos/parasitologia , Astrócitos/patologia , Camundongos , Toxoplasma/patogenicidade , Toxoplasma/fisiologia , Encéfalo/parasitologia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Doença Crônica , Polaridade Celular , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/genética , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia , Toxoplasmose/patologia , Fator de Necrose Tumoral alfa/metabolismo , Toxoplasmose Cerebral/parasitologia , Toxoplasmose Cerebral/patologia , Toxoplasmose Cerebral/metabolismoRESUMO
Various opportunistic infections develop during immunodeficiency due to human immunodeficiency virus (HIV) infection. The treatment options for malignant lymphoma (ML) and toxoplasmic encephalitis (TE) are completely different; therefore, their discrimination is critical. A 25-year-old female of foreign nationality had been experiencing headaches for several weeks and suddenly developed convulsions. Brain computed tomography revealed multiple intracranial lesions; therefore, the patient was referred to the neurosurgery department. Brain magnetic resonance imaging (MRI) revealed multiple masses with surrounding edema, accompanied by enhanced contrast. The largest mass (2 cm) in the left occipital lobe exhibited ringed contrast enhancement. Her blood test results showed a CD4 count of 40/µL, positive HIV Ag/Ab, HIV-RNA level of 56 × 104 copies/mL, positive anti-Toxoplasma IgG (63 IU/mL), and negative anti-Toxoplasma IgM. 201Tl- single photon emission computed tomography (201Tl-SPECT) revealed abnormal accumulation only in the tumor in the left occipital lobe (early T/N ratio, 3.034; delayed T/N ratio, 2.738; retention index, 0.9), which was suspected to be a ML. Both tumors, with or without high accumulation of 201Tl, were subjected to craniotomy biopsy. Pathological examination revealed infiltration of small lymphocytes with a necrotic background. The patient was diagnosed with TE based on a positive result of a tissue polymerase chain reaction test for Toxoplasma gondii. Two weeks after sulfamethoxazole and trimethoprim combination therapy, MRI imaging showed dramatic improvement in multiple brain tumors. This case is atypical because ML was ruled out despite high 201Tl-SPECT uptake and retention. Careful diagnosis through pathological examination and DNA testing is important.
Assuntos
Infecções por HIV , Linfoma , Toxoplasmose Cerebral , Humanos , Feminino , Adulto , Toxoplasmose Cerebral/diagnóstico , Toxoplasmose Cerebral/tratamento farmacológico , Toxoplasmose Cerebral/parasitologia , Toxoplasmose Cerebral/diagnóstico por imagem , Linfoma/diagnóstico , Infecções por HIV/complicações , Imageamento por Ressonância Magnética , Diagnóstico Diferencial , Tomografia Computadorizada de Emissão de Fóton Único , Toxoplasma/isolamento & purificaçãoRESUMO
Introducción. No existen reportes sobre las variaciones en la secuencia de los genes blanco de los medicamentos anti- Toxoplasma en aislamientos provenientes de Suramérica. Objetivo. Clonar y secuenciar los genes de la dihidrofolato-reductasa ( dhfr ) y la dihidropteroato-sintetasa ( dhps ) de la cepa de referencia RH y de dos aislamientos colombianos de Toxoplasma gondii. Materiales y métodos. Se obtuvieron dos aislamientos de T. gondii en líquido céfalorraquídeo de pacientes colombianos positivos para HIV con toxoplasmosis cerebral. Se extrajo el ADN de los genes dhfr y dhps y se amplificaron mediante reacción en cadena de la polimerasa (PCR). Los productos fueron clonados en el vector pGEM-T y secuenciados. Resultados. Se encontró un cambio de adenina por guanina (A « G) en la posición 235 del exón 2 del gen dhps , dos cambios de guanina por citocina (G « C) en las posiciones 259 y 260 y un cambio de timina por guanina (T « G) en la posición 371 del exón 4 del gen dhps. Por análisis bioinformático, en este último exón se identificó un polimorfismo no sinónimo en la región codificante, que podría llevar al cambio de una Glu (CAA o CAG) por una His (codificada por los codones AAU o AAC). Se calculó el modelo estructural de la enzima dihidropteroato-sintetasa (DHPS) de T. gondii y se identificaron las modificaciones en la estructura secundaria ocasionadas por las mutaciones. Conclusiones. La metodología estandarizada puede servir como base para la búsqueda de polimorfismos en muestras de pacientes con diferentes manifestaciones clínicas de toxoplasmosis y para establecer su posible relación con los cambios en la sensibilidad a los antifolatos y la reacción al tratamiento.
Introduction: There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates. Objective: This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii . Materials and methods: Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced. Results: One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations. Conclusions: The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.
Assuntos
Animais , Humanos , Masculino , Camundongos , Di-Hidropteroato Sintase/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , Toxoplasma/enzimologia , Infecções Oportunistas Relacionadas com a AIDS/líquido cefalorraquidiano , Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Substituição de Aminoácidos , Sequência de Bases , Clonagem Molecular , Colômbia , Líquido Cefalorraquidiano/parasitologia , DNA de Protozoário/genética , DNA Recombinante/genética , Di-Hidropteroato Sintase/química , Éxons/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas de Protozoários/química , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Toxoplasma/genética , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/parasitologia , Toxoplasmose Cerebral/líquido cefalorraquidiano , Toxoplasmose Cerebral/parasitologiaRESUMO
As cerebral toxoplasmosis is the most common cerebral focal lesion in AIDS patients, this study evaluated three PCR markers for diagnosis, since some limitations remain present, such as low parasite levels in some clinical samples. The molecular markers were B22-B23 and Tg1-Tg2 (based on the B1 gene) and Tox4-Tox5 (non-coding fragment, repeated 200-300-fold). DNA samples from 102 AIDS patients with previously known diagnosis were analyzed. The cerebral toxoplasmosis group was constituted of DNA extracted from the blood of 66 AIDS patients, which was collected before or until the third day of the therapy for toxoplasmosis. DNA from the blood of 36 AIDS patients with other neurologic opportunistic infections was used as control group. Sensitivities of B22-B23, Tg1-Tg2, and Tox4-Tox5 markers were of 95.5 percent, 93.9 percent, and 89.3 percent, respectively. In the control group, the specificities were of 97.2 percent (B22-B23), 88.9 percent (Tg1-Tg2), and 91.7 percent (Tox4-Tox5). The association of at least two markers increased the PCR sensitivity and specificity. The concordance index between two markers varied from 83.3 percent to 93.1 percent. These data demonstrated that all markers evaluated here were highly sensitive for T. gondii determination, although B22-B23 has been shown to be the best. The association of two markers increases PCR sensitivity, but the procedure was more expensive and time-consuming.
Assuntos
Humanos , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Genes de Protozoários/genética , Marcadores Genéticos/genética , Toxoplasma/genética , Toxoplasmose Cerebral/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Estudos de Casos e Controles , Primers do DNA/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Toxoplasmose Cerebral/parasitologiaRESUMO
Cerebral toxoplasmosis is a major cause of morbidity and mortality among HIV-infected patients, particularly from developing countries. This article summarizes current literature on cerebral toxoplasmosis. It focuses on: Toxoplasma gondii genetic diversity and its possible relationship with disease presentation; host responses to the parasite antigens; host immunosupression in HIV and cerebral toxoplasmosis as well as different diagnostic methods; clinical and radiological features; treatment; and the direction that studies on cerebral toxoplasmosis will likely take in the future.
Assuntos
Toxoplasma/genética , Toxoplasma/patogenicidade , Variação Genética , Infecções por HIV/complicações , Infecções por HIV/imunologia , Toxoplasmose Cerebral/diagnóstico , Toxoplasmose Cerebral/parasitologia , Toxoplasmose Cerebral/patologia , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Pesquisa Biomédica/tendências , AnimaisRESUMO
Toxoplasma gondii strains displaying the Type I/III genotype are associated with acquired ocular toxoplasmosis in humans. Here, we used a mice model to characterize some immunological mechanisms involved in host resistance to infection with such strains. We have chosen the Type I/III strains D8, G2 and P-Br, which cause a chronic infection in mice that resembles human toxoplamosis. Mice deficient of molecules MyD88, IFN-gamma, and IL-12 were susceptible to all three parasite strains. This finding indicates the importance of innate mechanisms in controlling infection. On the other hand, MHC haplotype did not influenced resistance/susceptibility; since mice lineages displaying a same genetic background but different MHC haplotypes (H2b or H2d) developed similar mortality and cyst numbers after infection with those strains. In contrast, the C57BL/6 genetic background, and not MHC haplotype, was critical for development of intestinal inflammation caused by any of the studied strains. Finally, regarding effector mechanisms, weobserved that B and CD8+ T lymphocytes controlled survival,whereas the inducible nitric oxide synthase influenced cyst numbers in brains of mice infected with Type I/III strains. These findings are relevant to further understanding of the immunologic mechanisms involved in host protection and pathogenesis during infection with T. gondii.
Cepas de Toxoplasma gondii que apresentam o genótipo I/III são associadas a toxoplasmose ocular adquirida em humanos. No presente trabalho, nós utilizamos um modelo da doença em camundongos para caracterizar mecanismos imunológicos envolvidos na resistência do hospedeiro à infecção por aquelas cepas. Escolhemos as cepas D8, G2 e P-Br, que causam infecção crônica em camundongos, semelhante à toxoplasmose humana. Camundongos deficientes em MyD88, IFN-G e IL-12 foram susceptíveis a infecções com todas as três linhagens do parasita. Esses dados indicam a importância de mecanismos inatos no controle da infecção. Por outro lado, o haplótipo do MHC não influenciou na resistência/susceptibilidade, na medida em que linhagens de camundongos com um mesmo "background'' genético, mas diferentes haplótipos de MHC (H2b e H2d) apresentam o índice de mortalidade e número de cistos semelhantes após a infecção com aquelas cepas do parasita. Em contraste, o "background'' genético de C57BL/6, mas não o haplótipo de MHC, foi crítico para o desenvolvimento de inflamação intestinal causada pelas cepas estudadas. Finalmente, com relação aos mecanismos efetores, observamos que linfócitos B e T CD8+ controlam a sobrevivência após infecção. Por outro lado, a ativação da enzima óxido nítrico sintase induzida foi um fator importante para controle do número de cistos cerebrais em camundongos infectados com cepas do Tipo I/III. Esses achados são relevantes para o melhor entendimento dos mecanismos imunológicos envolvidos na proteção e patogênese durante infecção com T. gondii.
Assuntos
Animais , Camundongos , Haplótipos/genética , Complexo Principal de Histocompatibilidade/genética , Camundongos Endogâmicos/imunologia , Toxoplasma/genética , Toxoplasmose Animal/imunologia , Toxoplasmose Cerebral/imunologia , Modelos Animais de Doenças , Genótipo , Interferon gama/deficiência , Interferon gama/imunologia , /deficiência , /imunologia , Complexo Principal de Histocompatibilidade/imunologia , Camundongos Endogâmicos/genética , /deficiência , /imunologia , Fatores de Tempo , Receptores Toll-Like/imunologia , Toxoplasma/patogenicidade , Toxoplasmose Animal/parasitologia , Toxoplasmose Animal/patologia , Toxoplasmose Cerebral/parasitologia , Toxoplasmose Cerebral/patologia , Virulência/genéticaRESUMO
El presente trabajo abarcó el período comprendido entre el año 1987 y agosto de 1994. Se diagnosticaron 25 pacientes con toxoplasmosis cerebrales (23 hombres y 2 mujeres), con una media de 35 años. Doce fueron heterosexuales y 11 homosexuales, todos en etapa Sida con repercusión general. Las manifestaciones clínicas más frecuentes fueron síndrome neurológico en 19 pacientes (síndrome motor deficitario 12, síndrome arquineocerebeloso 6, síndrome frontal 3, estado confusional l); síndrome febril prolongado 11; síndrome de hipertensión endocraneana 14; síndrome poliadenomegálico 7. La TAC mostró en 24 pacientes imágenes compatibles con toxoplasmosis. Se observaron frecuentemente infecciones asociadas de las cuales 14 fueron marcadoras de etapa evolutiva SIDA; (Candidiasis orofaríngea P pacientes, neumocistosis 6 pacientes), otras infecciones asociadas (lúes, Bk, CMV, herpes). Siete pacientes cursaron con clínica de toxoplasmosis sin otras infecciones intercurrentes. El diagnóstico fue tomográfico, clínico y serológico. No hubo confirmación parasitológica, mediante biopsia cerebral. La determinación de las poblaciones linfocitarias mostró cifras de T4 menores a 200 en 15 pacientes. La serología para toxoplasmosis mostró títulos altos de anticuerpos en 13 pacientes, con IgM positiva en 2 de ellos. La mayor eficacia terapéutica se obtuvo con la combinación de pirimetamina, sulfadiazina y ácido folínico, obteniéndose en 10 pacientes buena respuesta clínica y paraclínica