CX(3)CL1 (fractalkine) and CX(3)CR1 expression in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis: kinetics and cellular origin

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). It is associated with local activation of microglia and astroglia, infiltration of activated macrophages and T cells, active degradation of myelin and damage to axons and neurons. The proposed role for CX(3)CL1 (fractalkine) in the control of microglia activation and leukocyte infiltration places this chemokine and its receptor CX(3)CR1 in a potentially strategic position to control key aspects in the pathological events that are associated with development of brain lesions in MS. In this study, we examine this hypothesis by analyzing the distribution, kinetics, regulation and cellular origin of CX(3)CL1 and CX(3)CR1 mRNA expression in the CNS of rats with an experimentally induced MS-like disease, myelin oligodendrocyte glycoprotein (MOG)-induced autoimmune encephalomyelitis (EAE). METHODS: The expression of CX(3)CL1 and its receptor CX(3)CR1 was studied with in situ hybridization histochemical detection of their mRNA with radio labeled cRNA probes in combination with immunohistochemical staining of phenotypic cell markers. Both healthy rat brains and brains from rats with MOG EAE were analyzed. In defined lesional stages of MOG EAE, the number of CX(3)CR1 mRNA-expressing cells and the intensity of the in situ hybridization signal were determined by image analysis. Data were statistically evaluated by ANOVA, followed by Tukey\primes multiple comparison test. RESULTS: Expression of CX(3)CL1 mRNA was present within neuronal-like cells located throughout the neuraxis of the healthy rat. Expression of CX(3)CL1 remained unaltered in the CNS of rats with MOG-induced EAE, with the exception of an induced expression in astrocytes within inflammatory lesions. Notably, the brain vasculature of healthy and encephalitic animals did not exhibit signs of CX(3)CL1 mRNA expression. The receptor, CX(3)CR1, was expressed by microglial cells in all regions of the healthy brain. Induction of MOG-induced EAE was associated with a distinct accumulation of CX(3)CR1 mRNA expressing cells within the inflammatory brain lesions, the great majority of which stained positive for markers of the microglia-macrophage lineage. Analysis in time-staged brain lesions revealed elevated levels of CX(3)CR1 mRNA in microglia in the periplaque zone, as well as a dramatically enhanced accumulation of CX(3)CR1 expressing cells within the early-active, late-active and inactive, demyelinated lesions. CONCLUSION: Our data demonstrate constitutive and regulated expression of the chemokine CX(3)CL1 and its receptor CX(3)CR1 by neurons/astrocytes and microglia, respectively, within the normal and inflamed rat brain. Our findings propose a mechanism by which neurons and reactive astrocytes may control migration and function of the surrounding microglia. In addition, the accumulation of CX(3)CR1 expressing cells other than microglia within the inflammatory brain lesions indicate a possible role for CX(3)CL1 in controlling invasion of peripheral leucocytes to the brain

Temporal expression and cellular origin of CC chemokine receptors CCR1, CCR2 and CCR5 in the central nervous system: insight into mechanisms of MOG-induced EAE

<p>Abstract</p> <p>Background</p> <p>The CC chemokine receptors CCR1, CCR2 and CCR5 are critical for the recruitment of mononuclear phagocytes to the central nervous system (CNS) in multiple sclerosis (MS) and other neuroinflammatory diseases. Mononuclear phagocytes are effector cells capable of phagocytosing myelin and damaging axons. In this study, we characterize the regional, temporal and cellular expression of CCR1, CCR2 and CCR5 mRNA in the spinal cord of rats with myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE). While resembling human MS, this animal model allows unique access to CNS-tissue from various time-points of relapsing neuroinflammation and from various lesional stages: early active, late active, and inactive completely demyelinated lesions.</p> <p>Methods</p> <p>The expression of CCR1, CCR2 and CCR5 mRNA was studied with <it>in situ </it>hybridization using radio labelled cRNA probes in combination with immunohistochemical staining for phenotypic cell markers. Spinal cord sections from healthy rats and rats with MOG-EAE (acute phase, remission phase, relapse phase) were analysed. In defined lesion stages, the number of cells expressing CCR1, CCR2 and CCR5 mRNA was determined. Data were statistically analysed by the nonparametric Mann-Whitney U test.</p> <p>Results</p> <p>In MOG-EAE rats, extensive up-regulation of CCR1 and CCR5 mRNA, and moderate up-regulation of CCR2 mRNA, was found in the spinal cord during episodes of active inflammation and demyelination. Double staining with phenotypic cell markers identified the chemokine receptor mRNA-expressing cells as macrophages/microglia. Expression of all three receptors was substantially reduced during clinical remission, coinciding with diminished inflammation and demyelination in the spinal cord. Healthy control rats did not show any detectable expression of CCR1, CCR2 or CCR5 mRNA in the spinal cord.</p> <p>Conclusion</p> <p>Our results demonstrate that the acute and chronic-relapsing phases of MOG-EAE are associated with distinct expression of CCR1, CCR2, and CCR5 mRNA by cells of the macrophage/microglia lineage within the CNS lesions. These data support the notion that CCR1, CCR2 and CCR5 mediate recruitment of both infiltrating macrophages and resident microglia to sites of CNS inflammation. Detailed knowledge of expression patterns is crucial for the understanding of therapeutic modulation and the validation of CCR1, CCR2 and CCR5 as feasible targets for therapeutic intervention in MS.</p

Neurotripanosomiasis americana: aspectos clínicos de un problema básico.

Trypanosoma cruzi, causative agent of Chagas disease, affects not only cardiac and intestinal structures but also neurological structures. A high prevalence of T. cruzi infection occurs in Colombia, prompting the present study. First, a qualitative metaanalysis was undertaken using the PubMed database, the electronic internet engine Altavista, Colombian journals indexed by Colciencias, and three relevant textbooks. The following key words were used: Trypanosoma, Chagas disease, nervous system, spinal cord, central nervous system, peripheral nervous system, neuromuscular junction, autonomic nervous system, muscle, muscle disorders, neuromuscular disease, neuromuscular disorders, synapticopathies and dysautonomia. The documents analyzed numbered 116 and included original papers, reviews, case reports, editorials, brief communications, conferences and book chapters. At minimum, each document included data involving ELISA testing, indirect immunofluorescense, or parasitemia levels in the clinical, serological or histopathological studies. Polymerase chain reaction (PCR) studies were not included because of the recent introduction of PCR as a confirmatory technique for Chagas disease in Colombia. Chagas disease affects the central, the peripheral and the autonomic nervous system in humans, although its effects on the antonomic system is most commonly investigated in Colombia. Neurological lesions must be evaluated carefully, because patients may be misdiagnosed and treated as carriers of 'idiopathic' diseases. Neurological pathologies poses a serious threat in Colombia due to the prevalence of Chagas disease.Trypanosoma cruzi es el agente causal de la enfermedad de Chagas, patología que afecta principalmente estructuras cardiacas e intestinales. Sin embargo, las complicaciones neurológicas no han sido adecuadamente identificadas y estudiadas en Colombia, a pesar de existir allí áreas geográficas que presentan prevalencias de infección iguales o mayores de las informadas en otras latitudes, en donde se le ha dado una mayor atención a este tipo de complicaciones, desde hace ya varios años. Realizamos un metanálisis cualitativo sobre el tema, en la base de datos PubMed, en el motor de búsqueda Altavista y en las revistas colombianas indexadas por Colciencias, así como en tres libros que trataban el tópico de manera específica. Usamos las palabras claves: Trypanosoma, Chagas? disease, nervous system, spinal cord, central nervous system, peripheral nervous system, neuromuscular junction, autonomic nervous system, muscle, muscle disorders, neuromuscular disease, neuromuscular disorders, synapticopathies y dysautonomia. Como criterio de inclusión se debía haber realizado e informado la prueba de ELISA, inmunofluorescencia indirecta, presencia de parasitemia o presencia de parásitos en los tejidos, dependiendo de si se trataba de un estudio clínicoserológico o histopatólogico. No tuvimos en cuenta como criterio de inclusión la realización de la prueba de reacción en cadena de la polimerasa, dado que sólo hasta épocas recientes se introdujo esta técnica en el estudio de esta patología en Colombia. Encontramos 116 manuscritos con los términos antes descritos; éstos incluían artículos originales, revisiones, informe de casos, editoriales y comunicaciones breves, así como conferencias y capítulos de libros que cumplieron con los requisitos planteados. En ellos se apreció claramente cómo la enfermedad de Chagas afecta todos los niveles del sistema nervioso central, periférico y autonómico, siendo este último sistema el que se ha estudiado con mayor profundidad en nuestro país. Consideramos que el compromiso neurosistémico producido por T. cruzi debe ser evaluado de una manera más profunda a partir de la fecha, dado que muchos de los pacientes pueden estar siendo diagnosticados, tratados y seguidos como portadores de enfermedades ?idiopáticas?. Dichas patologías pueden llegar a convertirse en una seria amenaza para la salud de muchos colombianos si no se toman las medidas de prevención y control adecuadas. Por tanto, es necesario que actuemos en consecuencia, de acuerdo con el espectro de anormalidades neurológicas que se presentan en estos pacientes, como lo demostramos en el presente trabajo

Immunopathogenesis of experimental Chagas' disease

Chagas' disease is a serious, life-threatening disease caused by the protozoan parasite Trypanosoma cruzi in South and Central America. Neither vaccine candidate(s) nor efficient, nontoxic chemotherapeutical agents exist at the present time. Thus 20-30% of infected individuals eventually succumb to chronic Chagas' cardiomyopathy (50 000 deaths annually). Understanding the immunopathogenesis initiating and driving the disease is therefore of primary importance, and this thesis has sought to address these issues by making use of experimental animal models. The expression of cytokines and their receptors during the initial acute phase of the disease, at which time parasites are present in blood and organs, was investigated both at mRNA processing and post-transcriptional levels. Expression of these molecules, e.g. IL-2, IL-2R and IFN-y was demonstrated to be organ-specific, indicating that T. cruzi parasites themselves can modulate the host response during infection. In order to extrapolate experimental findings to patient scenarios, good experimental models which mimic the human disease course are required. With this in mind, a new, chronic model was developed in CBA mice. A series of studies aimed at characterizing pathology in this model revealed that it is a most appropriate experimental model for Chagas' disease. Histological analysis demonstrated extensive calcified infarctions and an extensive infiltration of immunocompetent blood cells into the myocardium of chronically-infected mice. These infiltrates comprised mainly macrophages and T cells, and an associated production of type 2 cytokines was recorded by in situ immunocytochemistry. Subsequent investigation of the expression of T cell receptors by the infiltrating T cells revealed a preferential usage of TCRBV8S2 and TCRBV8S3 elements, implicating a specific character of T cells in the genesis and/or perpetuation of the observed pathology during late-stage disease. The extent of inflammation in the target organ the heart, already during the acute phase of the disease, prompted further study of the aorta. Prominent vasculitis was recorded in aortas of infected mice, with associated endothelial changes and an infiltration of macrophages and T cells into the adventitial layer. This observation led to investigation of whether this observed inflammation, in combination with an excess of lipids, could lead to development of atherosclerosis, a debilitating condition in which arteries become occluded due to inflammatory injury of the intima. Thus a combined regime of fat-enriched diet and T. cruzi infection was applied in CBA mice, and already after 3 months a significant development of aortic atherosclerotic lesions was observed. No such pathology was seen in mice fed only the fatty diet. Immunohistochemical analysis revealed extensive infiltration in both adventitial and intimal layers underlying the developing lesions, with associated production of IL-6 and TNF a. These novel observations indicate that a combination of hyperlipidemia and infection-induced cardiovascular inflammation in CBA mice could provide new means for study of experimental atherosclerosis, and potentially serve to explain the changes in macro- and microvasculatures associated with chronic Chagas' disease pathology

Identification and in vitro characterization of C05-01, a PPB3 derivative with improved affinity for alpha-synuclein

The neuropathological hallmark of Parkinsońs disease, multiple system atrophy and dementia with Lewy bodies is the accumulation of α-synuclein. The development of an imaging biomarker for α-synuclein is an unmet need. To date, no selective α-synuclein imaging agent has been identified, though initial studies suggest that the tau tracer [11C]PBB3 displays some degree of binding to α-synuclein. In this study, a series of compounds derived from the PBB3 scaffold were examined using fluorescence imaging and tissue microarrays (TMAs) derived from brain samples with different proteinopathies. One compound, C05-01, was selected based on its higher fluorescence signal associated with Lewy body aggregates compared with other PBB3 analogues. In vitro binding assays using human brain homogenates and recombinant fibrils indicated that C05-01 had higher affinity for α-synuclein (KD/Ki 25 nM for fibrils, Ki 3.5 nM for brain homogenates) as compared with PBB3 (KD 58 nM). In autoradiography (ARG) studies using fresh frozen human tissue and TMAs, [3H]C05-01 displayed specific binding in cases with α-synuclein pathology.C05-01 is the first PBB3 analogue developed as a potential compound targeting α-synuclein. Despite improved affinity for α-synuclein, C05-01 showed specific binding in AD tissue with Amyloid β and tau pathology, as well as relatively high non-specific and off-target binding. Additional efforts are needed to optimize the pharmacological and physicochemical properties of this series of compounds as ligands for α-synuclein. This study also showed that the construction of TMAs from different proteinopathies provides a tool for evaluation of fluorescent or radiolabelled compounds binding to misfolded proteins

[C-11]PBB3 binding in A beta(-) or A beta(+) corticobasal syndrome

Corticobasal syndrome (CBS) is associated with 4-repeat tauopathy and/or Alzheimers disease pathologies. To examine tau and amyloid-beta (A beta) deposits in CBS patients using positron emission tomography (PET). Eight CBS patients and three healthy individuals lacking amyloid pathology underwent PET with [C-11]PBB3 for tau imaging, and [C-11]AZD2184 for A beta. Subcortical and cortical binding of [C-11]PBB3 was compared between A beta(-) and A beta(+) CBS patients and reference group. Postmortem analysis was done in one CBS patient. Three CBS patients were considered A beta(+). Total binding was higher in all patients compared to the reference group. Similar regional binding profiles of [C-11]PBB3 in A beta(+) and A beta(-) CBS patients were found. Elevated [C-11]PBB3 binding in pallidum was observed in all CBS patients. Cortical [C-11]PBB3 binding was higher in A beta(+) compared to A beta(-) patients. Postmortem analysis of a CBS patient revealed corticobasal degeneration neuropathology and [C-11]PBB3 autofluorescence in some tau-positive structures. [C-11]PBB3 is elevated in CBS patients with binding in relevant areas capturing some, but not all, 4-repeat tauopathy in CBS