A 31-year-old woman was diagnosed with relapsing-remitting multiple sclerosis (RRMS) in December 2006 (first lumbar puncture, LP), according to revised McDonald criteria [5] after an onset with severe tactil hypoaesthesia in the left limbs. No oligoclonal bands (OCB) or increased intrathecal Ig synthesis were detected in the CSF; other possible causes were excluded. Her past medical history was unremarkable except for autoimmune thyroiditis in replacement therapy since 2004. At the time of diagnosis, brain MRI showed several T2-weighted hyperintense lesions in supratentorial and infratentorial white matter, some of them characterized by gadolinium (Gd)-enhancement on T1-weighted images The patient started glatiramer acetate treatment. In June and December 2008 she had two relapses (Fig. 1a); brain and spinal MRI confirmed sustained disease activity, characterized by new T2 weighted lesions and Gd + T1-weighted enhancing lesions. In 2009 her neurological examination showed only mild hyposthenia in right limb. The Expanded Disability Status Scale (EDSS) score was 2.0 [6]. Although switched to IFNB1-a therapy (22 mcg 3/week and then 44 mcg 3/week), she showed sustained disease activity with two annual relapses and new typical brain lesions.
From April to August 2013 (time of second LP) she was treated with Natalizumab, stopped due to a relapse with atypical brain MRI lesions. CSF examination, repeated for suspected brain infection, excluded viral or opportunistic infections; neither OCB or increased intrathecal Ig synthesis were detected. Non conventional 3 T MRI analysis, with DIR sequences, showed significant cortical/iuxtacortical lesion load (Fig. 1b): 23 GM lesions (GM lesion volume: 2438.0 cm3), of which 6 probably intracortical, were detected. After a new optical relapse with multiple active brain lesions, plasma exchange therapy allowed recovery of visual impairment.
In May 2014, after a sensitive relapse, the patient started cyclophosphamide treatment (800 mg/m2 i.v. monthly) which she carried on until March 2015. Thereafter for 3 months she was given oral dimethyl-fumarate, without effects in reducing disease activity (Fig. 1b). At that time the patient showed severe ataxic gait with reduced vibratory and tactile sensation, EDSS was 4.5. A specific battery of cognitive tests [7, 8] revealed cognitive impairment, in particular with regards to memory and attention domains. In June 2016 she started Alemtuzumab therapy. At the last follow-up in December 2017, no adverse effects nor disease activity were noticed; neurological examination showed moderate ataxic gait and patient had resumed most of her usual activities.
Advanced protein analysis technology (Bio-Plex System, BioRad) was used to assess the presence and levels of 70 inflammatory/cytotoxic proteins in paired serum and CSF samples obtained from the examined MS case at the time of diagnosis (2006, t0) and in 2013 (t1), following the procedures previously optimized [4]. By using t test and the non-parametric Mann-Whitney test to detect potential significant differences (p-value < 0.05) in protein levels in the two CSF samples collected at t0 and t1, we found that at time of diagnosis 42 inflammatory molecules overexpressed (at least 2 fold change, p < 0.05) in CSF of our patient (Fig. 2) respect to a control group of 26 patients including 12 with non-inflammatory neurological diseases and 14 with other inflammatory neurological diseases previous examined in detail [4]. Interestingly, with the exception of TNF, BAFF and IL8 that remained unchanged, 11 out of these 42 inflammatory molecules, including CXCL13, CXCL12, IFNγ, TNF, sTNFR1, IL8, sCD163, APRIL, BAFF, pentraxin III and MMP2, were found significantly increased (at least 2 fold change, p < 0.05) after 7 years (Fig. 2, Additional file 1: Table S1). On the other hand we found at the second time point (t1) a decrease in the levels of GM-CSF, sTNFR2, TWEAK, LIGTH, sCD30, IFNλ1, sIL6-Rβ, IL6, IL19, IL22 and IL34 (Fig. 2).
On the contrary, no substantial changes in levels of the same inflammatory proteins have been observed in the two consecutive paired serum samples and when comparing these with a pool of control sera (data not shown).
In addition, by using ELISA procedure previously optimized for the analysis of CSF protein expression of neurofilament light chains (Nf-L) [4], we found significant increase of CSF levels of Nf-L from t0 (4400 pg/ml) to t1 (6300 pg/ml).