Construction, purification, and characterization of a chimeric TH1 antagonist

BACKGROUND: 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-γ 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-γ 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 IFNγ 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-γ-dependent HLA-DR expression in Colo 205 cells, inhibition of IFN-γ 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-γ, 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

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>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-<it>α</it>, IFN-<it>γ</it>, 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.</p> <p>Results</p> <p>We found almost undetectable levels of TNF-<it>α</it>, a decreased production of IL-10 and a significant up-regulation of every oxidative stress biomarker studied. The insufficient production of TNF-<it>α </it>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.</p> <p>Conclusion</p> <p>These results suggest that there is a breakdown in immunoregulatory mechanisms and noteworthy pro-oxidant environment contributing to NMO pathogenesis.</p

Cytoprotection in Multiple Sclerosis and Ischemic Stroke with C-Phycocyanin and Phycocyanobilin

Cytoprotection in human diseases can be achieved by avoiding and ameliorating tissue damage or by restoring the homeostatic balance either as a local or a systemic defense response. Multiple Sclerosis (MS) and Ischemic Stroke (IS) although being different central nervous system diseases, have common pathogenic aspects such as a deregulated inflammatory response, a toxic redox imbalance and a prominent neuronal dysfunction. C-Phycocyanin (C-PC), the main biliprotein of the Spirulina platensis cyanobacteria, and its associated chromophore named Phycocyanobilin (PCB), has shown strong antioxidant, anti-inflammatory and immunomodulatory properties. In this review, we describe the main experimental findings of our group supporting the medical application of C-PC/PCB as effective diseasemodifying therapies for MS and IS. We demonstrated that C-PC induced regulatory T cells and protected both mice and rats against the progression of experimental autoimmune encephalomyelitis. Both compounds exerted beneficial actions in several models of IS, either in vitro or in vivo. We also addressed the hypothesis of possible combinations of C-PC/PCB with already approved treatments for MS, such as beta IFN, to improve the effectiveness, lower the cost and achieve patient relief or recovery. The safety and tolerability of these compounds are also stressed. The gathered evidence supports the implementation of clinical trials to demonstrate the potential therapeutic effect of C-PC/PCB against these diseases.</p

Report on the Symposium “Molecular Mechanisms Involved in Neurodegeneration”

The prevalence of neurodegenerative diseases is currently a major concern in public health because of the lack of neuroprotective and neuroregenerative drugs. The symposium on Molecular Mechanisms Involved in Neurodegeneration held in Varadero, Cuba, updated the participants on the basic mechanisms of neurodegeneration, on the different approaches for drug discovery, and on early research results on therapeutic approaches for the treatment of neurodegenerative diseases. Alzheimer’s disease and in silico research were covered by many of the presentations in the symposium, under the umbrella of the “State of the Art of Non-clinical Models for Neurodegenerative Diseases” International Congress, held from 20 to 24 June 2017. This paper summarizes the highlights of the symposium

C-Phycocyanin and Phycocyanobilin as Remyelination Therapies for Enhancing Recovery in Multiple Sclerosis and Ischemic Stroke: A Preclinical Perspective

Myelin loss has a crucial impact on behavior disabilities associated to Multiple Sclerosis (MS) and Ischemic Stroke (IS). Although several MS therapies are approved, none of them promote remyelination in patients, limiting their ability for chronic recovery. With no available therapeutic options, enhanced demyelination in stroke survivors is correlated with a poorer behavioral recovery. Here, we show the experimental findings of our group and others supporting the remyelinating effects of C-Phycocyanin (C-PC), the main biliprotein of Spirulina platensis and its linked tetrapyrrole Phycocyanobilin (PCB), in models of these illnesses. C-PC promoted white matter regeneration in rats and mice affected by experimental autoimmune encephalomyelitis. Electron microscopy analysis in cerebral cortex from ischemic rats revealed a potent remyelinating action of PCB treatment after stroke. Among others biological processes, we discussed the role of regulatory T cell induction, the control of oxidative stress and pro-inflammatory mediators, gene expression modulation and COX-2 inhibition as potential mechanisms involved in the C-PC and PCB effects on the recruitment, differentiation and maturation of oligodendrocyte precursor cells in demyelinated lesions. The assembled evidence supports the implementation of clinical trials to demonstrate the recovery effects of C-PC and PCB in these diseases

Evaluación de la ficocianobilina y sus combinaciones en un modelo animal de esclerosis múltiple

INTRODUCTION: Multiple sclerosis is a chronic inflammatory disease of the central nervous system characterized by generalized areas of focal demyelination. The mainly used animal model is experimental autoimmune encephalomyelitis.OBJECTIVE: To demonstrate the effects of treatment with phycocyanobilin and their combinations. The PCB is a biliproteina derived from the cyanobacteria Spirulina platensis.METHODS: EAE was induced in mice C57BL / 6 with MOG35-55 in adjuvant full of Freund/emulsion of Mycobacterium tuberculosis and pertussis toxin. In this model was evaluated the effect of PCB/IFNbeta compared with the active ingredients in an independent way. The daily clinical progression was evaluated, and the concentrations of CCL5, CXCL10, IFN-γ, CXCL2, CCL2, IL-17A, IL-6 and IL-10 in homogenized tissue were determined by ELISA.RESULTS: EAE mice treated with the PCB/IFN-beta combination showed a significant decrease of the clinical score. On the other hand, PCB/IFN-beta combination treatment induced a significant decrease in the levels of IL-10 and IL-17 in homogenized's brain.CONCLUSION: The PCB/IFN-beta combination reduces the clinical progression of disease EAE, an effect possibly mediated in the brain by a decrease in the levels of IL-17A and IL-10.INTRODUCCIÓN: La esclerosis múltiple es una enfermedad inflamatoria crónica del sistema nervioso central caracterizada por áreas generalizadas de desmielinización focal. El modelo animal principalmente utilizado es la encefalomielitis autoinmune experimental.OBJETIVO: Demostrar los efectos del tratamiento con ficocianobilina y sus combinaciones. La PCB es una biliproteína derivada de la cianobacteria Spirulina platensis.MÉTODOS: Se indujo EAE en ratones C57BL / 6 con MOG35–55 en adyuvante completo de Freund/emulsión de Mycobacterium tuberculosis y la toxina pertussis. En este modelo fue evaluado el efecto de la PCB/IFNbeta en comparación con los ingredientes activos de forma independiente. La progresión clínica diaria fue evaluada, y se determinaron, mediante ELISA, las concentraciones de CCL5, CXCL10, IFN–γ, CXCL2, CCL2, IL–17A, IL–6 e IL–10 en homogeneizados de tejido.RESULTADOS: Los ratones con EAE tratados con la combinación PCB/IFN–beta mostraron una disminución significativa de la puntuación clínica. Por otro lado, el tratamiento de combinación PCB/IFN–beta indujo una disminución significativa de los niveles de IL–10 y IL–17 en homogeneizados de cerebro.CONCLUSIÓN: La combinación PCB/IFN–beta aminora la progresión clínica de la enfermedad EAE, un efecto posiblemente mediado en el cerebro por una disminución de los niveles de IL–17A e IL–10