Diego Franciotta

A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking

There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in CSF are being used in clinical practice. One of the most critical factors in CSF biomarker research is the inadequate powering of studies because of the lack of sufficient samples that can be obtained in single-center studies. Therefore, collaboration between investigators is needed to establish large biobanks of well-defined samples. Standardized protocols for biobanking are a prerequisite to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by preanalytical factors. Here, a consensus report on recommendations for CSF collection and biobanking is presented, formed by the BioMS-eu network for CSF biomarker research in multiple sclerosis. We focus on CSF collection procedures, preanalytical factors, and high-quality clinical and paraclinical information. The biobanking protocols are applicable for CSF biobanks for research targeting any neurologic disease.

Mechanisms of Disease: aquaporin-4 antibodies in neuromyelitis optica

Neuromyelitis optica (NMO) is a rare CNS inflammatory disorder that predominantly affects the optic nerves and spinal cord. Recent serological findings strongly suggest that NMO is a distinct disease rather than a subtype of multiple sclerosis. In NMO, serum antibodies, collectively known as NMO-IgG, characteristically bind to cerebral microvessels, pia mater and Virchow–Robin spaces. The main target antigen for this immunoreactivity has been identified as aquaporin-4 (AQP4). The antibodies are highly specific for NMO, and they are also found in patients with longitudinally extensive transverse myelitis without optic neuritis, which is thought to be a precursor to NMO in some cases. An antibody-mediated pathogenesis for NMO is supported by several observations, including the characteristics of the AQP4 antibodies, the distinct NMO pathology—which includes IgG and complement deposition and loss of AQP4 from spinal cord lesions—and emerging evidence of the beneficial effects of B-cell depletion and plasma exchange. Many aspects of the pathogenesis, however, remain unclear.

B cells and multiple sclerosis.

Clonal expansion of B cells and the production of oligoclonal IgG in the brain and cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) have long been interpreted as circumstantial evidence of the immune-mediated pathogenesis of the disease and suggest a possible infectious cause. Extensive work on intrathecally produced antibodies has not yet clarified whether they are pathogenetically relevant. Irrespective of antibody specificity, however, the processes of antibody synthesis in the CNS of patients with MS are becoming increasingly clear. Likewise, targeting B cells might be therapeutically relevant in MS and other autoimmune diseases that are deemed to be driven predominantly by T cells. Accumulating evidence indicates that in MS, similar to rheumatoid arthritis, B cells aggregate into lymphoid-like structures in the target organ. The process of aggregation is mediated through the expression of lymphoid-homing chemokines. In the brain of a patient with MS, ectopic B-cell follicles preferentially adjoin the pial membrane within the subarachnoid space. Recent findings indicate that substantial numbers of B cells that are infected with Epstein-Barr virus (EBV) accumulate in these intrameningeal follicles and in white matter lesions and are probably the target of a cytotoxic immune response. These findings, which await confirmation, could be an explanation for the continuous B-cell and T-cell activation in MS, but leave open concerns about the possible pathogenicity of autoantibodies. Going beyond the antimyelin-antibody dogma, the above data warrant further work on various B-cell-related mechanisms, including investigation of B-cell effector and regulatory functions, definition of the consistency of CNS colonisation by Epstein-Barr virus-infected B cells, and understanding of the mechanisms that underlie the formation and persistence of tertiary lymphoid tissues in patients with MS and other chronic autoimmune diseases (ectopic follicle syndromes). This work will stimulate new and unconventional ways of reasoning about MS pathogenesis.

Antibody to aquaporin 4 in the diagnosis of neuromyelitis optica.

Background Neuromyelitis optica (NMO) is a demyelinating disease of the central nervous system (CNS) of putative autoimmune aetiology. Early discrimination between multiple sclerosis (MS) and NMO is important, as optimum treatment for both diseases may differ considerably. Recently, using indirect immunofluorescence analysis, a new serum autoantibody (NMO-IgG) has been detected in NMO patients. The binding sites of this autoantibody were reported to colocalize with aquaporin 4 (AQP4) water channels. Thus we hypothesized that AQP4 antibodies in fact characterize NMO patients. Methods and Findings Based on these observations we cloned human water channel AQP4, expressed the protein in a eukaryotic transcription/translation system, and employed the recombinant AQP4 to establish a new radioimmunoprecipitation assay (RIPA). Indeed, application of this RIPA showed that antibodies against AQP4 exist in the majority of patients with NMO (n = 37; 21 positive) as well as in patients with isolated longitudinally extensive transverse myelitis (n = 6; six positive), corresponding to a sensitivity of 62.8% and a specificity of 98.3%. By contrast, AQP4 antibodies were virtually absent in 291 other participants, which included patients with MS (n = 144; four positive), patients with other inflammatory and noninflammatory neurological diseases (n = 73; one positive), patients with systemic autoimmune diseases (n = 45; 0 positive), and healthy participants (n = 29; 0 positive). Conclusions In the largest series reported so far to our knowledge, we quantified AQP4 antibodies in patients with NMO versus various other diseases, and showed that the aquaporin 4 water channel is a target antigen in a majority of patients with NMO. The newly developed assay represents a highly specific, observer-independent, and easily reproducible detection method facilitating clinically relevant discrimination between NMO, MS, and other inflammatory diseases.

Intrathecal Delivery of IFN-γ Protects C57BL/6 Mice from Chronic-Progressive Experimental Autoimmune Encephalomyelitis by Increasing Apoptosis of Central Nervous System-Infiltrating Lymphocytes

The exclusive detrimental role of proinflammatory cytokines in demyelinating diseases of the CNS, such as multiple sclerosis, is controversial. Here we show that the intrathecal delivery of an HSV-1-derived vector engineered with the mouse IFN-γ gene leads to persistent (up to 4 wk) CNS production of IFN-γ and inhibits the course of a chronic-progressive form of experimental autoimmune encephalomyelitis (EAE) induced in C57BL/6 mice by myelin oligodendrocyte glycoprotein (MOG)35–55. Mice treated with the IFN-γ-containing vector before EAE onset showed an earlier onset but a milder course of the disease compared with control mice treated with the empty vector. In addition, 83% of IFN-γ-treated mice completely recovered within 25 days post immunization, whereas control mice did not recover up to 60 days post immunization. Mice treated with the IFN-γ-containing vector within 1 wk after EAE onset partially recovered from the disease within 25 days after vector injection, whereas control mice worsened. Recovery from EAE in mice treated with IFN-γ was associated with a significant increase of CNS-infiltrating lymphocytes undergoing apoptosis. During the recovery phase, the mRNA level of TNFR1 was also significantly increased in CNS-infiltrating cells from IFN-γ-treated mice compared with controls. Our results further challenge the exclusive detrimental role of IFN-γ in the CNS during EAE/multiple sclerosis, and indicate that CNS-confined inflammation may induce protective immunological countermechanisms leading to a faster clearance of encephalitogenic T cells by apoptosis, thus restoring the immune privilege of the CNS.

NMO-IgG in the diagnosis of neuromyelitis optica

Recently, a newly detected serum autoantibody (called NMO-IgG) has been reported to distinguish between neuromyelitis optica (NMO) and multiple sclerosis (MS),1 and revised diagnostic criteria for NMO giving a crucial role to this antibody have been suggested.2 This proposal has important clinical implications and therefore demands independent confirmation. We studied 36 unselected patients with NMO (33 relapsing, three monophasic). All patients met the 1999 criteria of Wingerchuk et al.3 Spinal cord lesions in three or more segments were present in 35 of 36. Median follow-up from onset was 38 months. In addition, we tested 80 patients with MS according to the revised McDonald criteria4 (71% relapsing-remitting, 20% secondary progressive, 9% primary progressive), five patients with longitudinally extensive transverse myelitis (LETM),1,5 11 patients with non-LETM myelitis, 21 with miscellaneous neurologic disorders, and 25 healthy controls. NMO-IgG was detected by indirect immunofluorescence.1 Briefly, adult mouse cerebellum cryosections (10 μm) were incubated with 10% phosphate-buffered formalin for 4 minutes. After three washes in phosphate-buffered saline (PBS), detergent (1% CHAPS in PBS) was applied for 4 minutes. After …

Serum and CSF levels of MCP-1 and IP-10 in multiple sclerosis patients with acute and stable disease and undergoing immunomodulatory therapies.

The two chemokines, monocyte chemoattractant protein (MCP)-1 and gamma-interferon inducible protein (IP)-10, are thought to be involved in the pathogenesis of multiple sclerosis (MS). We measured MCP-1 and IP-10 levels in serum and CSF samples from 38 acute and 25 stable MS patients and from 40 controls. The latter consisted in patients with other inflammatory neurological diseases (OIND) or with non-inflammatory neurological diseases, and healthy controls. CSF MCP-1 levels exceeded those found in serum in all the patients studied as well as in healthy controls. CSF MCP-1 levels were significantly lower in acute MS [468+/-(S.E.M.) 18 pg/ml] than in stable MS (857+/-104 pg/ml). When detectable, serum and CSF IP-10 levels were significantly higher in acute MS (serum 331+/-66 pg/ml; CSF 118+/-16 pg/ml) than in stable MS (serum 69+/-7 pg/ml; CSF 25+/-2 pg/ml). Among OIND patients, those with HIV-1-associated dementia showed high serum and CSF levels of both MCP-1 and IP-10. Those with encephalitis showed high serum and CSF levels of IP-10 and CSF mononuclear pleiocytosis. We also evaluated the effects of 6-methylprednisolone or IFN-beta1a therapy on circulating MCP-1 and IP-10 levels. Neither MCP-1 nor IP-10 post-therapy levels varied significantly from baseline values. Our findings suggest that (a) MCP-1 could be constitutively produced within the brain; (b) MCP-1 and IP-10 CSF levels in acute MS vary significantly from those in stable MS, and these variations are inverse; and (c) current MS therapies do not modify circulating levels of MCP-1 and IP-10.

Cerebrospinal fluid findings in aquaporin-4 antibody positive neuromyelitis optica: results from 211 lumbar punctures.

BACKGROUND Neuromyelitis optica (NMO, Devic disease) is a severely disabling autoimmune disorder of the CNS, which was considered a subtype of multiple sclerosis (MS) for many decades. Recently, however, highly specific serum autoantibodies (termed NMO-IgG or AQP4-Ab) have been discovered in a subset (60-80%) of patients with NMO. These antibodies were subsequently shown to be directly involved in the pathogenesis of the condition. AQP4-Ab positive NMO is now considered an immunopathogenetically distinct disease in its own right. However, to date little is known about the cerebrospinal fluid (CSF) in AQP4-Ab positive NMO. OBJECTIVE To describe systematically the CSF profile of AQP4-Ab positive patients with NMO or its formes frustes, longitudinally extensive myelitis and optic neuritis. MATERIAL AND METHODS Cytological and protein biochemical results from 211 lumbar punctures in 89 AQP4-Ab positive patients of mostly Caucasian origin with neuromyelitis optica spectrum disorders (NMOSD) were analysed retrospectively. RESULTS CSF-restricted oligoclonal IgG bands, a hallmark of MS, were absent in most patients. If present, intrathecal IgG (and, more rarely, IgM) synthesis was low, transient, and, importantly, restricted to acute relapses. CSF pleocytosis was present in around 50% of samples, was mainly mild (median, 19 cells/μl; range 6-380), and frequently included neutrophils, eosinophils, activated lymphocytes, and/or plasma cells. Albumin CSF/serum ratios, total protein and CSF L-lactate levels correlated significantly with disease activity as well as with the length of the spinal cord lesions in patients with acute myelitis. CSF findings differed significantly between patients with acute myelitis and patients with acute optic neuritis at the time of LP. Pleocytosis and blood CSF barrier dysfunction were also present during remission in some patients, possibly indicating sustained subclinical disease activity. CONCLUSION AQP4-Ab positive NMOSD is characterized by CSF features that are distinct from those in MS. Our findings are important for the differential diagnosis of MS and NMOSD and add to our understanding of the immunopathogenesis of this devastating condition.

Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen

BACKGROUND Recently, a highly specific serum autoantibody was discovered in patients with neuromyelitis optica, called NMO-IgG, and aquaporin-4, the most abundant water channel in the CNS, was identified as the target antigen. Several assays for the detection of NMO-IgG/AQP4-Ab have been described. Tests based on recombinant human AQP4 have been repeatedly demonstrated to be more sensitive than the previous gold standard assay, i.e. immunohistochemistry (IHC) on mouse brain tissue. However, the sophisticated techniques applied restrict their availability to few laboratories worldwide. OBJECTIVE To develop an easy-to-use, recombinant immunofluorescence assay (rIFA) suitable for standardized and high-throughput detection of NMO-IgG/AQP4-Ab. METHODS HEK293 cells seeded on cover glasses were transfected with full-length recombinant human AQP4 at large scale. Cover glasses with the immobilized cells were cut into millimetre-sized fragments and transferred to microscopy slides. 151 serum samples from patients with NMO spectrum disorders (NMOSD) and controls were analysed both in the standard IHC assay and in the newly developed rIFA. RESULTS 25/32 (78.1%) patients with clinically definite NMO and 36/51 (70.6%) of total patients with NMOSD were positive for NMO-IgG/AQP4-Ab in the rIFA compared to 65.6% and 58.8%, respectively, in the IHC assay. CONCLUSION The recombinant IFA presented here provides laboratories familiar with indirect immunofluorescence microscopy with a highly sensitive and reproducible diagnostic tool for standardized detection of antibodies to AQP4. This new approach could make AQP4-Ab testing, which is of high clinical relevance, more widely available.

Cerebrospinal fluid antibodies to aquaporin-4 in neuromyelitis optica and related disorders: frequency, origin, and diagnostic relevance

BackgroundIn 70-80% of cases, neuromyelitis optica (NMO) is associated with highly specific serum auto-antibodies to aquaporin-4 (termed AQP4-Ab or NMO-IgG). Recent evidence strongly suggests that AQP4-Ab are directly involved in the immunopathogenesis of NMO.ObjectiveTo assess the frequency, syndrome specificity, diagnostic relevance, and origin of cerebrospinal fluid (CSF) AQP4-Ab in patients with NMO spectrum disorders (NMOSD).Methods87 CSF samples from 37 patients with NMOSD and 42 controls with other neurological diseases were tested for AQP4-Ab in a cell based assay using recombinant human AQP4. Twenty-three paired CSF and serum samples from AQP4-Ab seropositive NMOSD patients were further analysed for intrathecal IgG synthesis to AQP4.ResultsAQP4-Ab were detectable in 68% of CSF samples from AQP4-Ab seropositive patients with NMOSD, but in none of the CSF samples from AQP4-Ab seronegative patients with NMOSD and in none of the control samples. Acute disease relapse within 30 days prior to lumbar puncture, AQP4-Ab serum titres >1:250, and blood-CSF barrier dysfunction, but not treatment status, predicted CSF AQP4-Ab positivity. A positive AQP4-specific antibody index was present in 1/23 samples analysed.ConclusionsAQP4-Ab are detectable in the CSF of most patients with NMOSD, mainly during relapse, and are highly specific for this condition. In the cohort analysed in this study, testing for CSF AQP4-Ab did not improve the sensitivity and specificity of the current diagnostic criteria for NMO. The substantial lack of intrathecal AQP4-Ab synthesis in patients with NMOSD may reflect the unique localisation of the target antigen at the blood brain barrier, and is important for our understanding of the immunopathogenesis of the disease.