Peripheral and central mechanisms in chronic human inflammatory and neuropathic pain and associated animal models

Imperial Users onl

Inflammatory Pathways in Parkinson's Disease; A BNE Microarray Study

The aetiology of Parkinson's disease (PD) is yet to be fully understood but it is becoming more and more evident that neuronal cell death may be multifactorial in essence. The main focus of PD research is to better understand substantia nigra homeostasis disruption, particularly in relation to the wide-spread deposition of the aberrant protein α-synuclein. Microarray technology contributed towards PD research with several studies to date and one gene, ALDH1A1 (Aldehyde dehydrogenase 1 family, member A1), consistently reappeared across studies including the present study, highlighting dopamine (DA) metabolism dysfunction resulting in oxidative stress and most probably leading to neuronal cell death. Neuronal cell death leads to increased inflammation through the activation of astrocytes and microglia. Using our dataset, we aimed to isolate some of these pathways so to offer potential novel neuroprotective therapeutic avenues. To that effect our study has focused on the upregulation of P2X7 (purinergic receptor P2X, ligand-gated ion channel, 7) receptor pathway (microglial activation) and on the NOS3 (nitric oxide synthase 3) pathway (angiogenesis). In summary, although the exact initiator of striatal DA neuronal cell death remains to be determined, based on our analysis, this event does not remain without consequence. Extracellular ATP and reactive astrocytes appear to be responsible for the activation of microglia which in turn release proinflammatory cytokines contributing further to the parkinsonian condition. In addition to tackling oxidative stress pathways we also suggest to reduce microglial and endothelial activation to support neuronal outgrowth

Innate Immunity in multiple sclerosis white matter lesions: expression of natural cytotoxicity triggering receptor 1 (NCR1)

<p>Abstract</p> <p>Background</p> <p>Pathogenic or regulatory effects of natural killer (NK) cells are implicated in many autoimmune diseases, but evidence in multiple sclerosis (MS) and its murine models remains equivocal. In an effort to illuminate this, we have here analysed expression of the prototypic NK cell marker, NCR1 (natural cytotoxicity triggering receptor; NKp46; CD335), an activating receptor expressed by virtually all NK cells and therefore considered a pan-marker for NK cells. The only definitive ligand of NCR1 is influenza haemagglutinin, though there are believed to be others. In this study, we investigated whether there were differences in NCR1⁺cells in the peripheral blood of MS patients and whether NCR1⁺cells are present in white matter lesions.</p> <p>Results</p> <p>We first investigated the expression of NCR1 on peripheral blood mononuclear cells and found no significant difference between healthy controls and MS patients. We then investigated mRNA levels in central nervous system (CNS) tissue from MS patients: NCR1 transcripts were increased more than 5 times in active disease lesions. However when we performed immunohistochemical staining of this tissue, few NCR1⁺NK cells were identified. Rather, the major part of NCR1 expression was localised to astrocytes, and was considerably more pronounced in MS patients than controls. In order to further validate <it>de novo </it>expression of NCR1 in astrocytes, we used an <it>in vitro </it>staining of the human astrocytoma U251 cell line grown to model whether cell stress could be associated with expression of NCR1. We found up-regulation of NCR1 expression in U251 cells at both the mRNA and protein levels.</p> <p>Conclusions</p> <p>The data presented here show very limited expression of NCR1⁺NK cells in MS lesions, the majority of NCR1 expression being accounted for by expression on astrocytes. This is compatible with a role of this cell-type and NCR1 ligand/receptor interactions in the innate immune response in the CNS in MS patients. This is the first report of NCR1 expression on astrocytes in MS tissue: it will now be important to unravel the nature of cellular interactions and signalling mediated through innate receptor expression on astrocytes.</p

COX-2, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord

BACKGROUND: While multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) are primarily inflammatory and degenerative disorders respectively, there is increasing evidence for shared cellular mechanisms that may affect disease progression, particularly glial responses. Cyclooxygenase 2 (COX-2) inhibition prolongs survival and cannabinoids ameliorate progression of clinical disease in animal models of ALS and MS respectively, but the mechanism is uncertain. Therefore, three key molecules known to be expressed in activated microglial cells/macrophages, COX-2, CB2 and P2X7, which plays a role in inflammatory cascades, were studied in MS and ALS post-mortem human spinal cord. METHODS: Frozen human post mortem spinal cord specimens, controls (n = 12), ALS (n = 9) and MS (n = 19), were available for study by immunocytochemistry and Western blotting, using specific antibodies to COX-2, CB2 and P2X7, and markers of microglial cells/macrophages (CD 68, ferritin). In addition, autoradiography for peripheral benzodiazepine binding sites was performed on some spinal cord sections using [3H] (R)-PK11195, a marker of activated microglial cells/macrophages. Results of immunostaining and Western blotting were quantified by computerized image and optical density analysis respectively. RESULTS: In control spinal cord, few small microglial cells/macrophages-like COX-2-immunoreactive cells, mostly bipolar with short processes, were scattered throughout the tissue, whilst MS and ALS specimens had significantly greater density of such cells with longer processes in affected regions, by image analysis. Inflammatory cell marker CD68-immunoreactivity, [3H] (R)-PK11195 autoradiography, and double-staining against ferritin confirmed increased production of COX-2 by activated microglial cells/macrophages. An expected 70-kDa band was seen by Western blotting which was significantly increased in MS spinal cord. There was good correlation between the COX-2 immunostaining and optical density of the COX-2 70-kDa band in the MS group (r = 0.89, P = 0.0011, n = 10). MS and ALS specimens also had significantly greater density of P2X7 and CB2-immunoreactive microglial cells/macrophages in affected regions. CONCLUSION: It is hypothesized that the known increase of lesion-associated extracellular ATP contributes via P2X7 activation to release IL-1 beta which in turn induces COX-2 and downstream pathogenic mediators. Selective CNS-penetrant COX-2 and P2X7 inhibitors and CB2 specific agonists deserve evaluation in the progression of MS and ALS

Prostanoid receptor EP1 and Cox-2 in injured human nerves and a rat model of nerve injury: a time-course study

BACKGROUND: Recent studies show that inflammatory processes may contribute to neuropathic pain. Cyclooxygenase-2 (Cox-2) is an inducible enzyme responsible for production of prostanoids, which may sensitise sensory neurones via the EP1 receptor. We have recently reported that while macrophages infiltrate injured nerves within days of injury, they express increased Cox-2-immunoreactivity (Cox-2-IR) from 2 to 3 weeks after injury. We have now investigated the time course of EP1 and Cox-2 changes in injured human nerves and dorsal root ganglia (DRG), and the chronic constriction nerve injury (CCI) model in the rat. METHODS: Tissue sections were immunostained with specific antibodies to EP1, Cox-2, CD68 (human macrophage marker) or OX42 (rat microglial marker), and neurofilaments (NF), prior to image analysis, from the following: human brachial plexus nerves (21 to 196 days post-injury), painful neuromas (9 days to 12 years post-injury), avulsion injured DRG, control nerves and DRG, and rat CCI model tissues. EP1 and NF-immunoreactive nerve fibres were quantified by image analysis. RESULTS: EP1:NF ratio was significantly increased in human brachial plexus nerve fibres, both proximal and distal to injury, in comparison with uninjured nerves. Sensory neurones in injured human DRG showed a significant acute increase of EP1-IR intensity. While there was a rapid increase in EP1-fibres and CD-68 positive macrophages, Cox-2 increase was apparent later, but was persistent in human painful neuromas for years. A similar time-course of changes was found in the rat CCI model with the above markers, both in the injured nerves and ipsilateral dorsal spinal cord. CONCLUSION: Different stages of infiltration and activation of macrophages may be observed in the peripheral and central nervous system following peripheral nerve injury. EP1 receptor level increase in sensory neurones, and macrophage infiltration, appears to precede increased Cox-2 expression by macrophages. However, other methods for detecting Cox-2 levels and activity are required. EP1 antagonists may show therapeutic effects in acute and chronic neuropathic pain, in addition to inflammatory pain

Deciphering the genomic, epigenomic, and transcriptomic landscapes of pre-invasive lung cancer lesions.

The molecular alterations that occur in cells before cancer is manifest are largely uncharted. Lung carcinoma in situ (CIS) lesions are the pre-invasive precursor to squamous cell carcinoma. Although microscopically identical, their future is in equipoise, with half progressing to invasive cancer and half regressing or remaining static. The cellular basis of this clinical observation is unknown. Here, we profile the genomic, transcriptomic, and epigenomic landscape of CIS in a unique patient cohort with longitudinally monitored pre-invasive disease. Predictive modeling identifies which lesions will progress with remarkable accuracy. We identify progression-specific methylation changes on a background of widespread heterogeneity, alongside a strong chromosomal instability signature. We observed mutations and copy number changes characteristic of cancer and chart their emergence, offering a window into early carcinogenesis. We anticipate that this new understanding of cancer precursor biology will improve early detection, reduce overtreatment, and foster preventative therapies targeting early clonal events in lung cancer

Cortical grey matter demyelination can be induced by elevated pro-inflammatory cytokines in the subarachnoid space of MOG-immunized rats

A substantial proportion of cases with secondary progressive multiple sclerosis have extensive inflammation in the leptomeninges that is associated with increased subpial demyelination, neuronal loss and an exacerbated disease course. However, the mechanisms underlying this extensive subpial pathology are poorly understood. We hypothesize that pro-inflammatory cytokine production within the meninges may be a key to this process. Post-mortem cerebrospinal fluid and dissected cerebral leptomeningeal tissue from patients with multiple sclerosis were used to study the presence of tumour necrosis factor and interferon gamma protein and messenger RNA levels. A novel model of subpial cortical grey matter demyelination was set up in Dark Agouti rats and analysed using quantitative immunohistochemistry. Increased expression of the pro-inflammatory cytokines tumour necrosis factor and interferon gamma was found in the meninges of cases with secondary progressive multiple sclerosis exhibiting tertiary lymphoid-like structures. Injection of tumour necrosis factor and interferon gamma into the subarachnoid space of female Dark Agouti rats pre-immunized with a subclinical dose of myelin oligodendrocyte glycoprotein mimicked the pathology seen in multiple sclerosis, including infiltration of lymphocytes (CD4+ and CD8+ T cells and CD79+ B cells) into the meninges and extensive subpial demyelination. Extensive microglial/macrophage activation was present in a gradient from the pial surface to deeper cortical layers. Demyelination did not occur in control animals immunized with incomplete Freund's adjuvant and injected with cytokines. These results support the hypothesis that pro-inflammatory molecules produced in the meninges play a major role in cortical demyelination in multiple sclerosis, but also emphasize the involvement of an anti-myelin immune response

The 18-kDa Mitochondrial Translocator Protein in Human Gliomas: An 11C-(R)PK11195 PET Imaging and Neuropathology Study.

The 18-kDa mitochondrial translocator protein (TSPO) is up-regulated in high grade astrocytomas and can be imaged by positron emission tomography (PET) using the selective radiotracer 11C-(R)PK11195. We investigated 11C-(R)PK11195 binding in human gliomas and its relationship with TSPO expression in tumor tissue and glioma associated microglia/macrophages within the tumors. METHODS: Twenty-two glioma patients underwent dynamic 11C-(R)PK11195 PET scans and perfusion MRI acquisition. Parametric maps of 11C-(R)PK11195 binding potential (BPND) were generated. Co-registered MR/PET images were used to guide tumor biopsy. The tumor tissue was quantitatively assessed for TSPO expression and infiltration of glioma associated microglia/macrophages (GAMs) using immunohistochemistry and double immunofluorescence. The imaging and histopathologic parameters were compared among different histotypes and grades, and correlated with each other. RESULTS: BPND of 11C-(R)PK11195 in high-grade gliomas were significantly higher than in low-grade astrocytomas and low-grade oligodendrogliomas. TSPO in gliomas was expressed predominantly by neoplastic cells, and its expression correlated positively with BPND in the tumors. Glioma associated microglia/macrophages only partially contributed to the overall TSPO expression within the tumors, and TSPO expression in GAMs did not correlate with tumor BPND. CONCLUSION: PET with 11C-(R)PK11195 in human gliomas predominantly reflects TSPO expression in tumor cells. It therefore has the potential to effectively stratify patients that are suitable for TSPO targeted treatment