Astrocytes play a vital role in neuronal protection, homeostasis, vascular interchange and the local immune response. Some viruses and parasites can cross the blood-brain barrier and infect glia. Trypanosoma cruzi, the aetiological agent of Chagas disease, can seriously compromise the central nervous system, mainly in immune-suppressed individuals, but also during the acute phase of the infection. In this report, the infective capacity of T. cruzi in a human astrocyte tumour-derived cell line was studied. Astrocytes exposed to trypomastigotes (1:10 ratio) produced intracellular amastigotes and new trypomastigotes emerged by day 4 post-infection (p.i.). At day 6 p.i., 93% of the cells were infected. Using flow cytometry, changes were observed in both the expression of major histocompatibility complex class I and II molecules and the chemokine secretion pattern of astrocytes exposed to the parasite. Blocking the low-density lipoprotein receptor on astrocytes did not reduce parasite intracellular infection. Thus, T. cruzi can infect astrocytes and modulate the immune response during central nervous system infection.

Introducción. El interferón beta (IFN-β) se usa para tratar la forma recaída-remisión de la esclerosis múltiple. Sin embargo, el uso de proteínas recombinantes como medicamentos puede generar la producción de anticuerpos, disminuyendo así la efectividad del tratamiento. Objetivo. Estandarizar una técnica de detección de anticuerpos neutralizantes contra IFN-β, mediante citometría de flujo. Materiales y métodos. Se cultivaron dos líneas tumorales humanas (U937 y K562) con IFN-β1a humano recombinante y mediante citometría de flujo se determinó la expresión de la proteína ISG15 intracelular. Los sueros se obtuvieron de un conejo Nueva Zelanda antes y después de la inmunización con 100.000 UI de IFN-β1a en adyuvante de Freund. Para la detección de anticuerpos neutralizantes, se estimularon células K562 con IFN-β1a preincubado con sueros de conejos a una dilución 1:20. Después de 24 horas de incubación se determinó la expresión de la proteína ISG15. Resultados. La expresión de ISG15 fue mayor en células K562 estimuladas. La intensidad media de fluorescencia para la ISG-15 entre células en ausencia IFN-β1a, mostró una mediana de 198 unidades arbitrarias (UA) (p25-75= 173-231 UA) y, en presencia de IFN-β1a, 430 UA (p25-75 =316-611,5), con una diferencia estadísticamente significativa (p=0,008). La presencia de anticuerpos anti-IFN-β1a en el suero del conejo inmunizado, hizo disminuir de forma acentuada la expresión de la ISG15 en células K562 cultivadas con IFN-β1a, en comparación con el control (mediana=3,040 UA Vs. 43,644 UA, respectivamente). Conclusiones. Este trabajo muestra la detección de anticuerpos neutralizantes contra IFN-β en conejos, utilizando la expresión de la proteína ISG15 por citometría de flujo.

Introduction. Interferon beta (IFN-β) is a treatment for relapsing remitting multiple sclerosis. However, the therapeutic use of recombinant proteins induces a humoral immunologic response resulting in the induction of binding (BAb) or neutralizing (NAb) antibodies against the biological product. The presence of neutralizing antibodies has been associated with decreased IFN-β treatment efficacy. Materials and methods. Two tumor cell lines (K562 and U937) were cultivated with human recombinant IFN-β1a at different concentrations and lengths of time in order to measure the expression of intracellular ISG15, an inducible molecule in the IFN-β1a signaling cascade. Blood was obtained from non-immunized and IFN-β1a immunized (100,000 IU) New Zealand rabbits. The presence of BAb was evaluated by ELISA. For NAb detection, sera 1:20 dilution were added to the IFN-β1a-stimulated cell lines, and ISG15 expression was evaluated by flow cytometry. Results. K562 cells provided the better cell line for the assay, stimulated with a dose of 1,000 IU of IFN-β1a, and a 1:100 dilution for the primary antibody and a 1:200 dilution for the secondary antibody. ISG15 expression was compared between cells alone or cultivated with IFN-β1a. Mean fluorescence intensity (MFI) for ISG-15 expression median was 198 arbitrary units (AU) with interquartile ranges of 173-231 AU for non-stimulated cells and 430 AU with interquartile ranges of 316-611.5 AU for IFN-β1a stimulated cells (p<0.01). Immunized rabbit sera decreased the expression of ISG-15 in K562 cells stimulated with IFN-β1a, whereas non-immunized rabbit sera did not. Conclusions. This rabbit model demonstrates that ISG15 expression evaluated with flow cytometry can be used as a detection assay for NAb.