TNF-alpha and IL-10 downregulation and marked oxidative stress in Neuromyelitis Optica

ABSTRACT: BACKGROUND: Neuromyelitis optica is a central nervous system demyelinating and inflammatory syndrome. The objective of this study is to identify cytokines related to the cellular immune response as well as blood brain barrier integrity and oxidative stress. METHODS: We performed a molecular characterization of cellular immune response and oxidative stress in serum from relapsing-NMO (R-NMO) patients and established the correlations between the clinical measurements and molecular parameters using the Bayesian approach.Serum samples from 11 patients with R-NMO diagnosed according to Wingerchuk criteria and matched in terms of age, gender and ethnicity with the healthy controls were analyzed. The levels of TNF-alpha, IFN-gamma, IL-10, MMP-9, TIMP-1 and oxidative stress markers: malondialdehyde, advanced oxidation protein products, peroxidation potential, superoxide dismutase, catalase, and total hydroperoxides were measured. RESULTS: We found almost undetectable levels of TNF-alpha, a decreased production of IL-10 and a significant up-regulation of every oxidative stress biomarker studied. The insufficient production of TNF-alpha and IL-10 in R-NMO patients, which are two important players of T cell mediated immunoregulation, suggest an effector - regulator imbalance. The overproduction of oxygen reactive species as a consequence of the chronic inflammatory milieu is reflected on the excess of oxidative damage mediators detected. Furthermore, Multidimensional Scaling and a Bayesian linear regression model revealed a significant linear dependence between Expanded Disability Status Scale Kurtzke and TIMP-1; pointing to a possible predictive or prognostic value of this clinical-molecular relationship. CONCLUSION: These results suggest that there is a breakdown in immunoregulatory mechanisms and noteworthy pro-oxidant environment contributing to NMO pathogenesis(AU)

Construction, purification, and characterization of a chimeric TH1 antagonist

TH1 immune response antagonism is a desirable approach to mitigate some autoimmune and inflammatory reactions during the course of several diseases where IL-2 and IFN-gamma are two central players. Therefore, the neutralization of both cytokines could provide beneficial effects in patients suffering from autoimmune or inflammatory illnesses. RESULTS: A chimeric antagonist that can antagonize the action of TH1 immunity mediators, IFN-gamma and IL-2, was designed, engineered, expressed in E. coli, purified and evaluated for its in vitro biological activities. The TH1 antagonist molecule consists of the extracellular region for the human IFNgamma receptor chain 1 fused by a four-aminoacid linker peptide to human 60 N-terminal aminoacid residues of IL-2. The corresponding gene fragments were isolated by RT-PCR and cloned in the pTPV-1 vector. E. coli (W3110 strain) was transformed with this vector. The chimeric protein was expressed at high level as inclusion bodies. The protein was partially purified by pelleting and washing. It was then solubilized with strong denaturant and finally refolded by gel filtration. In vitro biological activity of chimera was demonstrated by inhibition of IFN-gamma-dependent HLA-DR expression in Colo 205 cells, inhibition of IFN-gamma antiproliferative effect on HEp-2 cells, and by a bidirectional effect in assays for IL-2 T-cell dependent proliferation: agonism in the absence versus inhibition in the presence of IL-2. CONCLUSION: TH1 antagonist is a chimeric protein that inhibits the in vitro biological activities of human IFN-gamma, and is a partial agonist/antagonist of human IL-2. With these attributes, the chimera has the potential to offer a new opportunity for the treatment of autoimmune and inflammatory diseases(AU)

Respuesta inmune TH1 antagonismo es deseable un enfoque para mitigar algunas reacciones inflamatorias y autoinmunes durante el curso de varias enfermedades que la IL-2 e IFN-gamma son dos jugadores. Por lo tanto, la neutralización de citoquinas podrían proporcionar efectos beneficiosos en los pacientes que sufren de enfermedades autoinmunes o inflamatorias. RESULTADOS: Un antagonista quimérico que puede antagonizar la acción de los mediadores de inmunidad TH1, IFN-gamma e IL-2, se diseñan, planifican, expresado en E. coli, purificado y evaluados por su actividad biológica in vitro. El antagonista de TH1 molécula extracelular consiste en la región para el receptor de la cadena humana IFNgamma fundido 1 por un período de cuatro enlazador de aminoácidos del péptido a la N-terminal de 60 residuos de aminoácidos de la IL-2. El gen correspondiente fragmentos fueron aislados por RT-PCR y clonado en el vector pTPV-1. E. coli (cepa W3110) se transforma con este vector. El quimérico se expresó la proteína de alto nivel como cuerpos de inclusión. La proteína fue parcialmente purificada por granulación y lavado. Fue entonces disuelto con desnaturalizante fuerte y, por último, refolded por filtración en gel. Actividad biológica in vitro de la quimera fue demostrado por la inhibición de la IFN-gamma-dependiente de la expresión de HLA-DR en Colo 205 células, la inhibición de IFN-gamma efecto antiproliferativo sobre las células Hep-2, y por un efecto bidireccional en los ensayos para IL-2 T -dependiente de la proliferación celular: agonism en frente a la ausencia de inhibición en presencia de IL-2. CONCLUSIÓN: es un antagonista del TH1 quimérico proteína que inhibe la actividad biológica in vitro de la IFN-gamma, y es un agonista parcial / antagonista de la IL-2. Con estos atributos, la quimera tiene el potencial para ofrecer una nueva oportunidad para el tratamiento de enfermedades inflamatorias y autoinmunes