Faiez Al Nimer

Sustained survival of xenografted human neural stem/progenitor cells in experimental brain trauma despite discontinuation of immunosuppression

Neural stem cells have emerged as a promising therapeutic tool in CNS disease and injuries. In the clinical setting, cultured human neural stem/progenitor cells (hNSC) are an attractive possibility for transplantation to the damaged brain. However, transplantation of hNSC requires toxic immunosuppressive treatment to avoid rejection. The aim of the current study was to evaluate if shortening the duration of immunosuppression by cyclosporin A would affect hNSC survival and differentiation after transplantation to the site of a focal brain injury in the rat. hNSC were xenografted to the hippocampus and the medial limit of an experimentally induced cortical contusion. The animals received immunosuppression for either 6 or 3 weeks or no immunosuppression. The status of the grafted human cells was analysed by immunohistochemistry. No statistically significant differences were observed between the two immunosuppressed groups regarding graft survival, migration or proliferation at 6 weeks post-transplantation. In contrast, the graft survival was extremely poor in the non-immunosuppressed group. Furthermore, the expression of the differentiation markers nestin, neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) in the transplanted cells did not differ significantly between the two immunosuppressed groups. Moreover, a fourth group of eight animals that were immunosuppressed for 3 weeks were allowed to survive for 6 months. Five of these rats demonstrated robust graft survival in the hippocampus and scattered cells in the cortex. This study demonstrates the importance of immunosuppression but also the possibility of shortening immunosuppression without impacting on the phenotype of the grafted hNSC.

Intense Inflammation and Nerve Damage in Early Multiple Sclerosis Subsides at Older Age: A Reflection by Cerebrospinal Fluid Biomarkers

Inflammatory mediators have crucial roles in leukocyte recruitment and subsequent central nervous system (CNS) neuroinflammation. The extent of neuronal injury and axonal loss are associated with the degree of CNS inflammation and determine physical disability in multiple sclerosis (MS). The aim of this study was to explore possible associations between a panel of selected cerebrospinal fluid biomarkers and robust clinical and demographic parameters in a large cohort of patients with MS and controls (nu200a=u200a1066) using data-driven multivariate analysis. Levels of matrix metalloproteinase 9 (MMP9), chemokine (C–X–C motif) ligand 13 (CXCL13), osteopontin (OPN) and neurofilament-light chain (NFL) were measured by ELISA in 548 subjects comprising different MS subtypes (relapsing-remitting, secondary progressive and primary progressive), clinically isolated syndrome and persons with other neurological diseases with or without signs of inflammation/infection. Principal component analyses and orthogonal partial least squares methods were used for unsupervised and supervised interrogation of the data. Models were validated using data from a further 518 subjects in which one or more of the four selected markers were measured. There was a significant association between increased patient age and lower levels of CXCL13, MMP9 and NFL. CXCL13 levels correlated well with MMP9 in the younger age groups, but less so in older patients, and after approximately 54 years of age the levels of CXCL13 and MMP9 were consistently low. CXCL13 and MMP9 levels also correlated well with both NFL and OPN in younger patients. We demonstrate a strong effect of age on both inflammatory and neurodegenerative biomarkers in a large cohort of MS patients. The findings support an early use of adequate immunomodulatory disease modifying drugs, especially in younger patients, and may provide a biological explanation for the relative inefficacy of such treatments in older patients at later disease stages.

Comparative Assessment of the Prognostic Value of Biomarkers in Traumatic Brain Injury Reveals an Independent Role for Serum Levels of Neurofilament Light

Traumatic brain injury (TBI) is a common cause of death and disability, worldwide. Early determination of injury severity is essential to improve care. Neurofilament light (NF-L) has been introduced as a marker of neuroaxonal injury in neuroinflammatory/-degenerative diseases. In this study we determined the predictive power of serum (s-) and cerebrospinal fluid (CSF-) NF-L levels towards outcome, and explored their potential correlation to diffuse axonal injury (DAI). A total of 182 patients suffering from TBI admitted to the neurointensive care unit at a level 1 trauma center were included. S-NF-L levels were acquired, together with S100B and neuron-specific enolase (NSE). CSF-NF-L was measured in a subcohort (n = 84) with ventriculostomies. Clinical and neuro-radiological parameters, including computerized tomography (CT) and magnetic resonance imaging, were included in the analyses. Outcome was assessed 6 to 12 months after injury using the Glasgow Outcome Score (1-5). In univariate proportional odds analyses mean s-NF-L, -S100B and -NSE levels presented a pseudo-R2 Nagelkerke of 0.062, 0.214 and 0.074 in correlation to outcome, respectively. In a multivariate analysis, in addition to a model including core parameters (pseudo-R2 0.33 towards outcome; Age, Glasgow Coma Scale, pupil response, Stockholm CT score, abbreviated injury severity score, S100B), S-NF-L yielded an extra 0.023 pseudo-R2 and a significantly better model (p = 0.006) No correlation between DAI or CT assessed-intracranial damage and NF-L was found. Our study thus demonstrates that S-NF-L correlates to TBI outcome, even if used in models with S100B, indicating an independent contribution to the prediction, perhaps by reflecting different pathophysiological processes, not possible to monitor using conventional neuroradiology. Although we did not find a predictive value of NF-L for DAI, this cannot be completely excluded. We suggest further studies, with volume quantification of axonal injury, and a prolonged sampling time, in order to better determine the connection between NF-L and DAI.

Complement Component C3 and Butyrylcholinesterase Activity Are Associated with Neurodegeneration and Clinical Disability in Multiple Sclerosis

Dysregulation of the complement system is evident in many CNS diseases but mechanisms regulating complement activation in the CNS remain unclear. In a recent large rat genome-wide expression profiling and linkage analysis we found co-regulation of complement C3 immediately downstream of butyrylcholinesterase (BuChE), an enzyme hydrolyzing acetylcholine (ACh), a classical neurotransmitter with immunoregulatory effects. We here determined levels of neurofilament-light (NFL), a marker for ongoing nerve injury, C3 and activity of the two main ACh hydrolyzing enzymes, acetylcholinesterase (AChE) and BuChE, in cerebrospinal fluid (CSF) from patients with MS (n = 48) and non-inflammatory controls (n = 18). C3 levels were elevated in MS patients compared to controls and correlated both to disability and NFL. C3 levels were not induced by relapses, but were increased in patients with ≥9 cerebral lesions on magnetic resonance imaging and in patients with progressive disease. BuChE activity did not differ at the group level, but was correlated to both C3 and NFL levels in individual samples. In conclusion, we show that CSF C3 correlates both to a marker for ongoing nerve injury and degree of disease disability. Moreover, our results also suggest a potential link between intrathecal cholinergic activity and complement activation. These results motivate further efforts directed at elucidating the regulation and effector functions of the complement system in MS, and its relation to cholinergic tone.

iNOS-mediated secondary inflammatory response differs between rat strains following experimental brain contusion

BackgroundNitric oxide is a key mediator of post-traumatic inflammation in the brain. We examined the expressions of iNOS, nNOS, and eNOS in inbred DA and PVGa rat strains where DA is susceptible to autoimmune neuroinflammation and PVGa-resistant.MethodsParietal contusions using a weight drop model were produced in five rats per genotype. After 24 h, the brains were removed and analyzed using a range of immunohistochemical methods.ResultsPVGa presented significantly increased iNOS expression in infiltrating inflammatory cells in the perilesional area compared to DA (pu2009<u20090.05). The amount of w3/13-positive infiltrating inflammatory cells did not differ between strains. eNOS and nNOS expression did not differ between strains. iNOS-positive cells coexpressed neuronal (NeuN), macrophage (ED-1), and leucocyte (w3/13) markers. MnSOD was significantly increased in PVGa (pu2009<u20090.05). 3-Nitrotyrosine, a measure of peroxynitrite levels, and fluoro-jade stained neuronal degeneration, did not differ between strains.ConclusionsTwo inbred rat strains with genetically determined differences in susceptibility to develop autoimmune disease displayed different levels of the inflammatory and anti-inflammatory mediators iNOS and MnSOD, indicating genetic regulation. Interestingly, the increased levels of iNOS did not lead to elevated expression of the neuronal cell-death marker fluoro-jade. The increased iNOS expression was correlated with increased expression of superoxide scavenger MnSOD. Excessive peroxynitrite formation was probably prevented by limitation of available superoxide. Subsequently, the higher expression of potentially deleterious iNOS in PVGa did not result in increased neuronal death.

Genetic variability in the rat Aplec C-type lectin gene cluster regulates lymphocyte trafficking and motor neuron survival after traumatic nerve root injury.

BackgroundC-type lectin (CLEC) receptors are important for initiating and shaping immune responses; however, their role in inflammatory reactions in the central nervous system after traumatic injuries is not known. The antigen-presenting lectin-like receptor gene complex (Aplec) contains a few CLEC genes, which differ genetically among inbred rat strains. It was originally thought to be a region that regulates susceptibility to autoimmune arthritis, autoimmune neuroinflammation and infection.MethodsThe inbred rat strains DA and PVG differ substantially in degree of spinal cord motor neuron death following ventral root avulsion (VRA), which is a reproducible model of localized nerve root injury. A large F2 (DAxPVG) intercross was bred and genotyped after which global expressional profiling was performed on spinal cords from F2 rats subjected to VRA. A congenic strain, Aplec, created by transferring a small PVG segment containing only seven genes, all C-type lectins, ontoDA background, was used for further experiments together with the parental strains.ResultsGlobal expressional profiling of F2 (DAxPVG) spinal cords after VRA and genome-wide eQTL mapping identified a strong cis-regulated difference in the expression of Clec4a3 (Dcir3), a C-type lectin gene that is a part of the Aplec cluster. Second, we demonstrate significantly improved motor neuron survival and also increased T-cell infiltration into the spinal cord of congenic rats carrying Aplec from PVG on DA background compared to the parental DA strain. In vitro studies demonstrate that the Aplec genes are expressed on microglia and upregulated upon inflammatory stimuli. However, there were no differences in expression of general microglial activation markers between Aplec and parental DA rats, suggesting that the Aplec genes are involved in the signaling events rather than the primary activation of microglia occurring upon nerve root injury.ConclusionsIn summary, we demonstrate that a genetic variation in Aplec occurring among inbred strains regulates both survival of axotomized motor neurons and the degree of lymphocyte infiltration. These results demonstrate a hitherto unknown role for CLECs for intercellular communication that occurs after damage to the nervous system, which is relevant for neuronal survival.

Complement Receptor 2 is increased in cerebrospinal fluid of multiple sclerosis patients and regulates C3 function

Besides its vital role in immunity, the complement system also contributes to the shaping of the synaptic circuitry of the brain. We recently described that soluble Complement Receptor 2 (sCR2) is part of the nerve injury response in rodents. We here study CR2 in context of multiple sclerosis (MS) and explore the molecular effects of CR2 on C3 activation. Significant increases in sCR2 levels were evident in cerebrospinal fluid (CSF) from both patients with relapsing-remitting MS (n=33; 6.2ng/mL) and secondary-progressive MS (n=9; 7.0ng/mL) as compared to controls (n=18; 4.1ng/mL). Furthermore, CSF sCR2 levels correlated significantly both with CSF C3 and C1q as well as to a disease severity measure. In vitro, sCR2 inhibited the cleavage and down regulation of C3b to iC3b, suggesting that it exerts a modulatory role in complement activation downstream of C3. These results propose a novel function for CR2/sCR2 in human neuroinflammatory conditions.

Complement receptor 2 is up regulated in the spinal cord following nerve root injury and modulates the spinal cord response

BackgroundActivation of the complement system has been implicated in both acute and chronic states of neurodegeneration. However, a detailed understanding of this complex network of interacting components is still lacking.MethodsLarge-scale global expression profiling in a rat F2(DAxPVG) intercross identified a strong cis-regulatory influence on the local expression of complement receptor 2 (Cr2) in the spinal cord after ventral root avulsion (VRA). Expression of Cr2 in the spinal cord was studied in a separate cohort of DA and PVG rats at different time-points after VRA, and also following sciatic nerve transection (SNT) in the same strains. Consequently, Cr2−/− mice and Wt controls were used to further explore the role of Cr2 in the spinal cord following SNT. The in vivo experiments were complemented by astrocyte and microglia cell cultures.ResultsExpression of Cr2 in naïve spinal cord was low but strongly up regulated at 5–7xa0days after both VRA and SNT. Levels of Cr2 expression, as well as astrocyte activation, was higher in PVG rats than DA rats following both VRA and SNT. Subsequent in vitro studies proposed astrocytes as the main source of Cr2 expression. A functional role for Cr2 is suggested by the finding that transgenic mice lacking Cr2 displayed increased loss of synaptic nerve terminals following nerve injury. We also detected increased levels of soluble CR2 (sCR2) in the cerebrospinal fluid of rats following VRA.ConclusionsThese results demonstrate that local expression of Cr2 in the central nervous system is part of the axotomy reaction and is suggested to modulate subsequent complement mediated effects.

Depression and fatigue in multiple sclerosis: Relation to exposure to violence and cerebrospinal fluid immunomarkers

Multiple sclerosis (MS) is a neuroinflammatory condition characterized by chronic dysregulation of immune responses leading to repeated episodes of inflammation in the central nervous system. Depression and fatigue are common among MS patients, even in early disease phases, and the disease course can be negatively affected by stressful events. IL-6 and IL-8 have been associated with depression and stressful life events in non-MS patients. The aim of this study was to examine the relationships between depression, fatigue, and exposure to violence, with IL-6 and IL-8 levels in the cerebrospinal fluid (CSF) of MS patients. Levels of IL-6 and -8 were analyzed in the CSF of 47 patients with relapsing-remitting MS. Correlations between IL-6 and IL-8 levels and self-rated depression and fatigue symptoms, as well as clinician-rated history of being exposed to interpersonal violence, were analyzed with correction for age, sex and MS disability status. IL-6 correlated significantly (pu202f<u202f0.05) with depressive symptoms (adjusted Spearmans ρu202f=u202f0.39), fatigue (ρu202f=u202f0.39), and exposure to violence in adult life (ρu202f=u202f0.35). Depression correlated with both fatigue and being exposed to violence. Associations were not present among patients exposed to disease modifying drugs. In exploratory analyses, the relationship between exposure to violence and IL-6 was non-significant when controlled for depression. Further research should focus on replication of these results, as well as exploring the impact of stressful life events on immune regulation and the clinical characteristics and prognosis of MS patients.

Memory B Cells Activate Brain-Homing, Autoreactive CD4+ T Cells in Multiple Sclerosis

Summary Multiple sclerosis is an autoimmune disease that is caused by the interplay of genetic, particularly the HLA-DR15 haplotype, and environmental risk factors. How these etiologic factors contribute to generating an autoreactive CD4+ T cell repertoire is not clear. Here, we demonstrate that self-reactivity, defined as “autoproliferation” of peripheral Th1 cells, is elevated in patients carrying the HLA-DR15 haplotype. Autoproliferation is mediated by memory B cells in a HLA-DR-dependent manner. Depletion of B cells in vitro and therapeutically in vivo by anti-CD20 effectively reduces T cell autoproliferation. T cell receptor deep sequencing showed that in vitro autoproliferating T cells are enriched for brain-homing T cells. Using an unbiased epitope discovery approach, we identified RASGRP2 as target autoantigen that is expressed in the brain and B cells. These findings will be instrumental to address important questions regarding pathogenic B-T cell interactions in multiple sclerosis and possibly also to develop novel therapies.