Clinical neurobiology

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Group leader:  Antonio Bertolotto

Clinical neurobiology

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) and comprises a heterogeneous spectrum of disease subtypes.

The distinctive variability of clinical presentations, histopathologic and immunologic patterns, as well as neuroradiologic phenotypes in MS poses a diagnostic challenge to the attending physician and claims a more differentiated typing of MS patients by diagnostic biomarkers in order to anticipate the expected disease course and to stratify patients for specifically tailored therapies.

With the perspective of predictive monitoring our research group aims at providing new tools for management of various aspects of MS, and hence the possibility of individualized (ideally preventive) therapeutic procedures. Our work should also contribute to a better understanding of altered physiological pathways in MS pathogenesis, and define targets for novel treatment approaches.

Experimental approaches

Our research focuses on the molecular and immunological analysis of MS in close collaboration with a clinical research group. Taking advantage of its active and often leading role in international multi-centre therapeutic and diagnostic MS studies, the Clinical Research Group (based on a large multidisciplinary MS Clinic) provides access to biological samples from well characterized and systematically followed patients with different clinical courses and treatments.  

Cerebrospinal fluid diagnostic activity

The Clinical Neurobiology Laboratory deals with routine CSF analysis from AOU San Luigi Gonzaga patients and from all over Piedmont centers. Even if CSF analysis is no more  required for MS diagnosis, it is still important to offer diagnostic and prognostic information ( to confirm a diagnosis of MS early in the disease course and be used to identify patients with a high probability of developing MS after a first clinical event) and to rule out differential diagnoses; furthermore it’s important to provide an important research tool. In particular, analysis performed in or laboratory are cytological and biochemical CSF analysis, and oligoclonal IgG bands detection for evaluation of intrathecal IgG synthesis.

Research activity

A wide range of topics are covered by the group:

Biomarkers in MS

The study of multiple sclerosis can benefit from the use of biomarkers because of the disease’s inherent heterogeneity. Biomarkers in multiple sclerosis might assist with diagnosis, prediction of disease course, or identification of response outcome to treatments. One of the main features of the Clinical Neurobiology Lab is to search and validate biomarkers that could be useful for clinicians in the daily management of MS patients.

Diagnostic and prognostic biomarkers
The Clinical neurobiology Lab is involved in setting up and validation of assays for the detection of autoantibodies related to demyelinating diseases of the CNS in differential diagnosis with MS, such as Neuromyelitis Optica (NMO) and related diseases (NMO spectrum disorders). In our laboratory we have evaluated a multiparametric immunofluorescence assay for NMO serology ("Neurology Mosaic 17", Euroimmun), as a powerful tool with high specificity, sensibility and reproducibility, that allows the identification of different AQP4 specific and non-specific patterns. Our research is ongoing to facilitate early diagnosis and appropriate treatment for these patients; in particular, we set up a FACS assay to evaluate serum anti-myelin olygodendrocytic glycoprotein (MOG) in patients with NMOsd, negative for anti-AQP4 antibodies. 

The hunt for biomarkers to predict MS disease evolution and identify patient subsets that may benefit from specific therapeutic regimens is a continuous effort in MS research.  A test measuring biochemical biomarkers could be a useful tool in the diagnostic process, but, at present no validated biomarkers are available to diagnose disease, to monitor or predict disease progression, or to aid in assessment of early treatment effects. Recently, anti-potassium channel KIR4.1 antibodies have been reported in a substantial proportion of MS patients: this finding could have a tremendous impact in diagnosis and management of MS patients. In our laboratory we are working to set up the assay for quantification of anti-KIR4.1 antibodies.

One of the aims of our research is the identification and clinical validation of new molecular CSF biomarkers for predicting MS disease progression. Among environmental factors involved in MS pathogenesis, Epstein Barr virus (EBV) as gained credibility as the most plausible candidate agent. CNS could be the main site of a dysregulated EBV infection: one of the aims of our research is to understand how EBV disregulation may contribute to MS, analysing at molecular level EBV regulation in CNS and PBMCs of MS patients, and trying to relate it to immune response.

Treatment-response biomarkers
Treatment-response biomarkers are measured in patients receiving MS treatments to monitor the single patient biological response to therapy and identify risk factors for treatment failure.

Recombinant homologues of human proteins have the potential to induce an immunogenic response when used therapeutically. Some drugs actually used in MS, as Beta-IFN, Natalizumab and Rituximab, have the potential to induce the development of anti-drug antibodies, which are able to abrogate the biological and clinical action of the drug. Treatment-response biomarkers are useful to identify individuals who are at risk for treatment failure or serious adverse drug reactions and, therefore, are eligible for a treatment change. Furthermore, identification of patient non-responders to these treatments can avoid the cost associated with failed therapy.

One of the aim of our research is the evaluation of biological activity of treatment used in MS, and identification of new specific markers to monitor clinical efficacy and response to each treatment.
Actually, in our laboratory, biomarkers are analyzed for the following therapies:

  • Beta-IFN: evaluation of biological activity by MxA mRNA analysis, and detection of binding and neutralizing anti-drug antibodies.
  • Glatiramer Acetate (GA): evaluation of biological activity, measuring T-reg cells induction.
  • Natalizumab: neutralizing antibodies detection; evaluation of risk to develop PML by detection of anti-JCV antibodies and L-selectin analysis on T CD4+ lymphocytes.
  • Rituximab: neutralizing antibodies detection, evaluation of biological activity by the quantification of circulating drug, and of CD19 expression on B lymphocytes.
Pregnancy

Pregnancy represents a physiological transitory state of immune tolerance to avoid the rejection of the fetus, and is frequently associated with reduced activity of autoimmune diseases, including MS. Nevertheless, the biological mechanisms underlying the pregnancy-related decrease in disease activity are poorly understood. It is of interest to evaluate these changes through different strategies both cellular and molecular. By using a wide range of molecular technologies and by flow cytometric analysis and in vitro essays, we seek to determine the cellular interaction and molecular factors, which are modulated during pregnancy in MS.

Immunopathogenesis

A genome wide transcriptional analysis enabled the identification of a panel of anti-inflammatory genes differentially expressed in blood from not treated MS patients compared to healthy controls.  We aim to investigate the role of these genes in the disease, the causes underlying their deregulation (genetic, epigenetic and hormonal) and the cell population mainly affected. To address these issues we take advantage of human hematic and brain post mortem tissues and a MS mouse model (Experimental autoimmune encephalomyelitis, EAE).

Bio-Bank

Despite considerable investment in biological clinical research, very few laboratory results are transformed in drugs. Biological research suffers from poor reproducibility of published data, even in prestigious journals, because of the lack of rigor in the collection of biological samples, the insufficient validation of the methods according to the instructions of FDA (Food and Drug Administration) and limited sharing of data.

In a chronic disease such as MS is difficult to collect biological samples because the patients need careful follow-ups through the collection of clinical, biological, neuroimaging (MRI), neuro-cognitive and neurophysiological data. Our laboratory created a bio-bank that aims to collect biological materials from more than 1800 MS patients followed at CRESM, patients with other neurological disease and healthy controls. The collected biological material includes serum, plasma, cerebrospinal fluid (CSF), cells from blood and CSF, DNA and RNA. Patients included in the bio-bank sign the Informed Consent approved by the AOU San Luigi Ethics Committee. Biological samples are collected according to strict criteria and recorded in a database. iMED database is established for each patient with clinical, biological, neurophysiological, neuroimaging, cognitive testing data. 

The researcher who receives samples from the bio-bank is committed to: i) providing the bio-bank with detailed protocols of the methods used in the research, ii ) sharing data of his experiments with the bio-bank so that could be available for others studies.

The Clinical neurobiology Laboratory will assure: i) co-validation of methods; ii) replication of data obtained by researchers who have used biological samples of the bio-bank , iii) implementation of educational courses for technicians/biologists on biological methods.

Finally, the CRESM bio-bank has been included in an European network of bio-banks dedicated to MS research.

CRESM Bio-Bank has been funded by Fondazione Italiana Sclerosi Multipla (FISM) Cod. 2014/PMS/1.  

Methodological  Approaches

Molecular Biology

  • Nucleic acid extraction from cells, tissues, extracellular fluids, whole blood
  • Real Time PCR: gene expression analysis, microRNA detection, SNP genotyping, high-throughput projects

Cell Biology

  • Cell culture and stimulation
  • Magnetic beads cell separation
  • Flow cytometry analysis
  • Expression and purification of recombinant proteins.

Biochemical analysis

  • Western Blot and Immunoprecipitation
  • Capture ELISA, bridging ELISA
  • CSF cytochemical examination
  • Cytopatic Effect Assay (for anti-IFN NABs evaluation)
  • Immunoisoelettrofocusing
  • Immunofluorescence
  • Immunohistochemistry
  • Human post mortem MS brain characterization and analysis

Mouse models

  • EAE induction (MOG 35-55) using C57 BL6 and transgenic mice
  • Clinical and neuropathological evaluation
  • Gene expression analysis and protein analysis from tissues
  • Tissues storage