Altered CSF Albumin Quotient Links Peripheral Inflammation and Brain Damage in MS

OBJECTIVE CNS damage can increase the susceptibility of the blood-brain barrier (BBB) to changes induced by systemic inflammation. The aim of this study is to better understand BBB permeability in patients with MS and to examine whether compromised BBB integrity in some of these patients is associated with CNS damage and systemic inflammation. METHODS Routine CSF measurements of 121 patients with MS were analyzed including number and type of infiltrating cells, total protein, lactate, and oligoclonal bands, as well as intrathecal production of immunoglobulins and CSF/serum quotients for albumin, immunoglobulins, and glucose. In addition, in a subcohort of these patients, we performed ex vivo immunophenotyping of CSF-infiltrating and paired circulating lymphocytes using a panel of 13 monoclonal antibodies, we quantified intrathecal neurofilament light chain (NF-L) and chitinase 3-like 1 (CHI3L1), and we performed intrathecal lipidomic analysis. RESULTS Patients with MS with abnormal high levels of albumin in the CSF showed a distinct CSF cell infiltrate and markers of CNS damage such as increased intrathecal levels of NF-L and CHI3L1 as well as a distinct CSF lipidomic profile. In addition, these patients showed higher numbers of circulating proinflammatory Th1 and Th1* cells compatible with systemic inflammation. Of interest, the abnormally high levels of albumin in the CSF of those patients were preserved over time. CONCLUSIONS Our results support the hypothesis that CNS damage may increase BBB vulnerability to systemic inflammation in a subset of patients and thus contribute to disease heterogeneity

Broadly neutralizing human monoclonal JC polyomavirus VP1-specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy

In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show "recognition holes"; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML

Brain Citrullination Patterns and T Cell Reactivity of Cerebrospinal Fluid-Derived CD4+ T Cells in Multiple Sclerosis

Immune responses to citrullinated peptides have been described in autoimmune diseases like rheumatoid arthritis (RA) and multiple sclerosis (MS). We investigated the post-translational modification (PTM), arginine to citrulline, in brain tissue of MS patients and controls (C) by proteomics and subsequently the cellular immune response of cerebrospinal fluid (CSF)-infiltrating T cells to citrullinated and unmodified peptides of myelin basic protein (MBP). Using specifically adapted tissue extraction- and combined data interpretation protocols we could establish a map of citrullinated proteins by identifying more than 80 proteins with two or more citrullinated peptides in human brain tissue. We report many of them for the first time. For the already described citrullinated proteins MBP, GFAP, and vimentin, we could identify additional citrullinated sites. The number of modified proteins in MS white matter was higher than control tissue. Citrullinated peptides are considered neoepitopes that may trigger autoreactivity. We used newly identified epitopes and previously reported immunodominant myelin peptides in their citrullinated and non-citrullinated form to address the recognition of CSF-infiltrating CD4+ T cells from 22 MS patients by measuring proliferation and IFN-γ secretion. We did not detect marked responses to citrullinated peptides, but slightly more strongly to the non-modified version. Based on these data, we conclude that citrullination does not appear to be an important activating factor of a T cell response, but could be the consequence of an immune- or inflammatory response. Our approach allowed us to perform a deep proteome analysis and opens new technical possibilities to analyze complex PTM patterns on minute quantities of rare tissue samples

MAPK pathway and B cells overactivation in multiple sclerosis revealed by phosphoproteomics and genomic analysis

Dysregulation of signaling pathways in multiple sclerosis (MS) can be analyzed by phosphoproteomics in peripheral blood mononuclear cells (PBMCs). We performed in vitro kinetic assays on PBMCs in 195 MS patients and 60 matched controls and quantified the phosphorylation of 17 kinases using xMAP assays. Phosphoprotein levels were tested for association with genetic susceptibility by typing 112 single-nucleotide polymorphisms (SNPs) associated with MS susceptibility. We found increased phosphorylation of MP2K1 in MS patients relative to the controls. Moreover, we identified one SNP located in the PHDGH gene and another on IRF8 gene that were associated with MP2K1 phosphorylation levels, providing a first clue on how this MS risk gene may act. The analyses in patients treated with disease-modifying drugs identified the phosphorylation of each receptor’s downstream kinases. Finally, using flow cytometry, we detected in MS patients increased STAT1, STAT3, TF65, and HSPB1 phosphorylation in CD19+ cells. These findings indicate the activation of cell survival and proliferation (MAPK), and proinflammatory (STAT) pathways in the immune cells of MS patients, primarily in B cells. The changes in the activation of these kinases suggest that these pathways may represent therapeutic targets for modulation by kinase inhibitors

Signaling mechanisms underlying neural stem cell differentiation. Implications of BMPs, retinoids, and MAP kinases

Introduction: Neural stem cells are proliferating cells, derived from the nervous system with the ability to give rise to neurons, astrocytes, and oligodendrocytes. The molecular events and signaling pathways regulating neural stem cell differentiation are largely unknown. However, understanding the mechanisms underlying lineage commitment of neural stem cells is a crucial prerequisite for the potential future use of these cells in cell-based therapeutic strategies to neurodegenerative diseases.Aims: In the present thesis, the implications of bone morphogenetic protein (BMP)-, retinoid- and mitogen-activated protein (MAP) kinase-signaling on neural stem cell development were investigated in embryonic neural primary culture and in adult hippocampal progenitors (AHPs).Results: Paper I: BMPs but not GDFs were shown to significantly increase the number of tyrosine hydroxylase (TH) positive cells in embryonic mesencephalic primary culture. BMPs directly targeted and differentiated the neuronal cell population without affecting cell survival. The inability of GDFs to increase the number of dopaminergic neurons was reflected by a lack of activation of intracellular smad proteins in neurons of GDF-treated cultures.Paper II: The significance of BMP-signaling on survival and differentiation of AHPs was investigated by adenoviral expression of dominant-negative BMP type I receptors with non-functional intracellular kinase domains. Signaling via the BMP receptor type Ib (ALK6) was required for the generation and survival of astrocytes and inhibition of oligodendrocyte differentiation. Furthermore, blockage of BMP type Ia receptor (ALK3)-signaling enhanced ALK6-mediated smad-phosphorylation due to increased expression of ALK6.Paper III: Overexpression of a constitutively active form of ASK1 (ASK1-Delta) significantly increased the number of neurons in AHP cultures, while decreasing the number of astroglial cells. We also show that ASK1 inhibits astrocyte differentiation by STAT3-independent inhibition of the GFAP-promoter. Finally, ASK1-induced neuronal differentiation and inhibition of astroglial differentiation were found to be critically dependent on p38 MAP kinase activation.Paper IV: The roles of retinoic acid (RA) and related retinoids on CNTF-induced instructive gliogenesis were investigated in progenitor cell cultures derived from the developing cortex. Pre- but not co-incubation of RA significantly reduced the number of CNTF-induced astrocytes in E13 cortical progenitors. Furthermore, RAR-alpha activation was sufficient to mimic the inhibitory effect of RA on gliogenesis in E13 cortical progenitors. In contrast, RA-signaling enhanced CNTF-induced astrocyte differentiation in progenitor cell cultures derived from the E17 cortex. Conclusions: The findings presented in this thesis suggest a novel role for ASK1 in the regulation of neuronal and astroglial differentiation in progenitor cells derived from the adult hippocampus. Our data also indicate a pivotal role for ALK6- as well as retinoid-signaling in the differentiation of endogenously generated and cytokine-induced astrocytes. Taken together, the data presented here provide novel insight into the regulation of neural stem and progenitor cell differentiation and may be of potential benefit for future therapeutic approaches to the diseased CNS

Data from: Lower carotid revascularization rates after stroke in ethnic minority-serving US hospitals

Objective: We sought to determine whether the use of carotid revascularization procedures after stroke due to carotid stenosis differs between minority-serving hospitals and hospitals serving predominantly white patients. Methods: We identified ischemic stroke cases due to carotid disease, identified by ICD9-CM codes, from 2007-2011 in the Nationwide Inpatient Sample. The use of carotid endarterectomy (CEA) and carotid artery stenting (CAS) was recorded. Hospitals with ≥40% ethnic minority patients (minority-serving hospitals) were compared to hospitals with <40% minority patients (white hospitals). Logistic regression was used to evaluate the use of CEA/CAS among minority-serving and white hospitals. Results: Of the 26,189 ischemic stroke cases meeting inclusion criteria, 20,870 (79.7%) were treated at 1,113 white hospitals, and 5,319 (20.3%) received care at 325 hospitals minority-serving hospitals. Compared to patients in white hospitals, patients in minority hospitals were less likely to undergo CEA/CAS (17.6%, 95% CI 16.6%-18.6%, in minority vs. 21.2%, 95% CI 20.7%-21.8%, in white hospitals, p<0.001). In fully adjusted logistic regression models, the odds of CEA/CAS were lower in minority compared to white hospitals (OR 0.81, 95% CI 0.70-0.93), independent of individual patient race and other measured hospital characteristics. Whites and Hispanics had significantly lower odds of CEA/CAS in minority compared to white hospitals. Patient-level racial differences in the use of carotid revascularization procedures remained within each hospital stratum. Conclusions: The odds of carotid revascularization after stroke is lower in minority-serving compared to white hospitals, suggesting system-level factors as a major contributor to explain race disparities in the use of carotid revascularization

Immunology of progressive multifocal leukoencephalopathy

The high prevalence of asymptomatic JC polyomavirus (JCV) infection in the general population indicates coexistence with the human host and efficient immune control in healthy individuals. For unknown reasons, kidney-resident archetypic JCV strains can turn into neurotropic JCV strains which in hereditary or acquired states of immunodeficiency cause opportunistic infection and cytolytic destruction of glial cells or granule cell neurons resulting in progressive multifocal demyelination in the central nervous system (CNS) or cerebellar atrophy, respectively. Immunomodulatory or immunosuppressive therapies with specific monoclonal antibodies including natalizumab, efalizumab, and rituximab have increased the risk of progressive multifocal leukoencephalopathy (PML) among treated patients, highlighting that symptomatic JCV infection of the CNS is associated with disturbances of adaptive immunity affecting B cells, antibodies, and CD4(+) and/or CD8(+) T cells. To date, no specific therapy to overcome PML is available and the only way to eliminate the virus from the CNS is to reconstitute global immune function. However, since the identification of JCV as the causative agent of PML 40 years ago, it is still not fully understood which components of the immune system prevent the development of PML and which immune mechanisms are involved in eliminating the virus from the CNS. This review gives an update about adaptive JCV-specific immune responses

Immunology of progressive multifocal leukoencephalopathy

The high prevalence of asymptomatic JC polyomavirus (JCV) infection in the general population indicates coexistence with the human host and efficient immune control in healthy individuals. For unknown reasons, kidney-resident archetypic JCV strains can turn into neurotropic JCV strains which in hereditary or acquired states of immunodeficiency cause opportunistic infection and cytolytic destruction of glial cells or granule cell neurons resulting in progressive multifocal demyelination in the central nervous system (CNS) or cerebellar atrophy, respectively. Immunomodulatory or immunosuppressive therapies with specific monoclonal antibodies including natalizumab, efalizumab, and rituximab have increased the risk of progressive multifocal leukoencephalopathy (PML) among treated patients, highlighting that symptomatic JCV infection of the CNS is associated with disturbances of adaptive immunity affecting B cells, antibodies, and CD4(+) and/or CD8(+) T cells. To date, no specific therapy to overcome PML is available and the only way to eliminate the virus from the CNS is to reconstitute global immune function. However, since the identification of JCV as the causative agent of PML 40years ago, it is still not fully understood which components of the immune system prevent the development of PML and which immune mechanisms are involved in eliminating the virus from the CNS. This review gives an update about adaptive JCV-specific immune responses