Serum and cerebrospinal fluid neurofilament light chains measured by SIMOA™, Ella™, and Lumipulse™ in multiple sclerosis naïve patients.

BACKGROUND: Neurofilament light chains (NfL) are cytoskeletal biomarkers of axonal damage, about 40-fold higher in cerebrospinal fluid (CSF) compared to serum, and requiring ultrasensitive techniques to be measured in this latter fluid. OBJECTIVES: To compare CSF and serum NfL levels in multiple sclerosis (MS) patients using different platforms. METHODS: 60 newly diagnosed relapsing-remitting MS patients (38 females; median age: 36.5 years, range: 15-60) were enrolled before steroid or disease-modifying treatments. CSF and serum NfL were measured with: the commercial Ella™ microfluidic platform (Bio-Techne), the Lumipulse™ Chemiluminescent Enzyme ImmunoAssay (Fujirebio), and the SIMOA™ on the SR-X instrument using NF-light assays (Quanterix). RESULTS: CSF and serum NfL absolute levels strongly correlated between assays, although being more elevated with Ella™. Passing-Bablok regression showed high agreement in measuring CSF NfL between assays (with greater proportional difference using Ella™), and very high agreement for serum comparing SIMOA™ and Lumipulse™. Similarly, the Bland-Altman comparison evidenced lower biases for Lumipulse™ for both fluids. CONCLUSIONS: CSF and serum NfL in naïve MS patients are reliably measured with all assays. Although not interchangeable, SIMOA™ and Lumipulse™ showed high agreement for serum and CSF values.

Serum Neurofilaments are a reliable biomarker to early detect PML in Multiple Sclerosis patients.

BACKGROUND: The earliest detection of progressive multifocal leukoencephalopathy (PML) is crucial in Natalizumab (NTZ)-treated Multiple Sclerosis (MS) patients. This study aims to assess serum Neurofilaments (sNFL) ability to early detect PML in longitudinal patients' follow-up. METHODS: NFL were retrospectively measured in four PML cases occurred at the Regional Referring Center for MS (CRESM, Italy), in samples collected since one year before PML diagnosis, at PML diagnosis, during PML and in post-PML follow-up. sNFL levels were interpreted according to previously defined reference values. Clinical examination and EDSS were performed at each NTZ infusion. Routinary MRI was undertaken every six months; after PML diagnosis, MRI was performed according to clinical evaluation. sNFL were also measured in 45 NTZ-treated patients experiencing NEDA-3 status for at least 12 months. RESULTS: Patients showed different PML onsets and manifestations: in 3 patients routinary brain MRI revealed radiological signs of PML preceding different clinical manifestations, while in one patient brain MRI was performed after the clinical onset. PML diagnosis was defined at the time of the first detection of JCV DNA in cerebrospinal fluid. The following different PML phases were considered: 1. Basal (up to 4 months before PML diagnosis): sNFL values were in the normal range in all patients' samples, except for one (median 9.1 pg/ml, range 6.2-15.1 pg/ml) 2. Pre-PML (within 3 months before PML diagnosis): sNFL were elevated in all available samples (median 19.50 pg/ml, range 15.50-33.80 pg/ml). 3. PML diagnosis: sNFL were elevated in all patients (median 59.20 pg/ml, range 11.1-101.50 pg/ml). 4. PML/IRIS: during this phase, sNFL levels reached their peak (median 96.35 pg/ml, range 20.5-272.9) in all patients. 5. Post-PML (recovery phase, starting from the first MRI without enhancement, up to the end of follow-up): sNFL levels showed a decrease (median 12.80 pg/ml, range 9.30-30.60); however, based on reference values, sNFL were still elevated in 2 out of 4 patients at the end of their follow-up (622 and 887 days after PML diagnosis). sNFL were always elevated when MRI scan suggested a suspicious of PML. In NEDA-3 patients, sNFL levels were in the normal range in all patients' samples (median 4.7 pg/ml, range 1.4-8.6 pg/ml). CONCLUSION: Elevated sNFL were observed not only at PML diagnosis, but also in pre-PML phase. At PML recovery, sNFL weren't normalized in all patients' samples, suggesting ongoing neuronal degeneration. sNFL represent a reliable biomarker and should be introduced in clinical practice as an additional/alternative parameter to MRI to early detect and monitor PML.

Study of the NR4A family gene expression in patients with multiple sclerosis treated with Fingolimod.

BACKGROUND AND PURPOSE: Fingolimod is a drug approved for treatment of relapsing-remitting multiple sclerosis (RRMS) that exerts its effects via sequestering lymphocytes within the lymph nodes. The drug, acting on the sphingosine-1-phosphate pathway, is involved in a plethora of processes and, to date, its mechanism of action is not completely understood. Recently, it has been demonstrated that Fingolimod increases the expression of transcription factor NR4A2 in murine brain. NR4A2 belongs to nuclear receptor family 4, group A (NR4A) along with NR4A1 and NR4A3. The role of NR4A2 in the pathogenesis of multiple sclerosis is already known and supported by its down-regulation observed in blood obtained from patients with RRMS compared with healthy controls (HCs). It is notable that NR4A2 impairment is reversed in patients with RRMS during pregnancy, which represents a transitory state of immune tolerance, associated with reduced disease activity. An inverse correlation between NR4A2 gene expression levels and clinical parameters indicates that more aggressive forms of the disease are characterized by lower levels of NR4A2. METHODS: Gene expression levels of NR4A in blood obtained from HCs, treatment-naive (T0) and Fingolimod-treated patients with RRMS were evaluated to determine their contribution to drug response. RESULTS: Gene expression levels of NR4A were down-regulated in T0 patients compared with HCs. Patients treated with Fingolimod for >2 years were characterized by higher levels of NR4A2 compared with the T0 group, approaching those of HCs. NR4A1 and NR4A3 levels were not altered. CONCLUSIONS: Involvement of the NR4A family in the pathogenesis of multiple sclerosis and a role of Fingolimod in the recovery from NR4A2 deficit can be hypothesized based on our data.

Anti-inflammatory genes associated with multiple sclerosis: a gene expression study.

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system caused by a complex interaction between multiple genes and environmental factors. HLA region is the strongest susceptibility locus, but recent huge genome-wide association studies identified new susceptibility genes. Among these, BACH2, PTGER4, RGS1 and ZFP36L1 were highlighted. Here, a gene expression analysis revealed that three of them, namely BACH2, PTGER4 and ZFP36L1, are down-regulated in MS patients' blood cells compared to healthy subjects. Interestingly, all these genes are involved in the immune system regulation with predominant anti-inflammatory role and their reduction could predispose to MS development.

Exogenous Sonic hedgehog modulates the pool of GABAergic interneurons during cerebellar development.

All cerebellar GABAergic interneurons were derived from a common pool of precursor cells residing in the embryonic ventricular zone (VZ) and migrating in the prospective white matter (PWM) after birth, where both intrinsic and extrinsic factors contribute to regulate their amplification. Among the environmental factors, we focused on Sonic hedgehog (Shh), a morphogen well known to regulate neural progenitor cell proliferation. We asked if and how exogenous Shh treatment affects the lineage of cerebellar GABAergic interneurons. To address these issues, exogenous Shh was administered to embryonic and postnatal organotypic slices. We found that Shh is able to expand the pool of interneuron progenitors residing in the embryonic epithelium and in the postnatal PWM. In particular, Shh signalling pathway was highly mitogenic at early developmental stages of interneuron production, whereas its effect decreased after the first postnatal week. Gene expression analysis of sorted cells and in situ hybridization further showed that immature interneurons express both the Shh receptor patched and the Shh target gene Gli1. Thus, within the interneuron lineage, Shh might exert regulatory functions also in postmitotic cells. On the whole, our data enlighten the role of Shh during cerebellar maturation and further broaden our knowledge on the amplification mechanisms of the interneuron progenitor pool.

Monocytes and CD4+ T cells contribution to the under-expression of NR4A2 and TNFAIP3 genes in patients with multiple sclerosis.

We recently found a gene signature for multiple sclerosis (MS) that reverted to normal during pregnancy in MS patients and included NR4A2 and TNFAIP3, key molecules in anti-inflammatory processes. Here we focus on the expression levels of these two genes in monocytes and CD4+ T cells from healthy controls and treatment-naïve RRMS patients. Our findings show that monocytes play a key role in the dysregulated anti-inflammatory response, being the expression of both genes down-regulated in these cells in RRMS patients with respect to healthy individuals. CD4+ T cells seem to have only a marginal part, because we can observe only a slight down-regulation in NR4A2.

Labile and stable glycosylated hemoglobin during OGTT in healthy subjects and maturity-onset diabetics.

HbA1 levels were determined by a rapid chromatographic column test in 15 healthy subjects (HS) and in 15 maturity-onset diabetics (MOD), fasting and 2 h after glucose ingestion (100 g for HS, 50 g for MOD). Chromatography was carried out both before and after 6 h of red cell incubation in saline at 37 degrees C. HbA1 in HS at 0 and 120 min of OGTT was not significantly different, either before (6.24 +/- 0.61% and 6.22 +/- 0.62%) or after red cell incubation in saline (5.85 +/- 0.61% and 5.87 +/- 0.55%). Red cell incubation in saline significantly reduced HbA1 levels both at 0 and 120 min (2p less than 0.001). HbA1 in MOD before red cell incubation in saline, was slightly but significantly higher at 120 min (8.61 +/- 1.03%) than at 0 min (8.39 +/- 1.01%): 2p less than 0.001. After incubation in saline, this difference was cancelled (7.86 +/- 0.85% at 0 min and 7.97 +/- 0.83% at 120 min: n.s.). Post-incubation levels were lower than pre-incubation ones both at 0 and 120 min (2p less than 0.001). The HbA1 increment observed in MOD is significantly correlated (p less than 0.01; r=0.64) to the blood glucose increment observed at the glycemic peak. We conclude that hemoglobin glycosylation may show rapid changes also in MOD, reflecting blood glucose changes, whereas in HS the physiological glycemic excursions are not wide enough to produce rapid HbA1 changes. Since labile and stable HbA1 co-elute in the rapid chromatographic methods, red cell incubation in saline for 6 h at 37 degrees C is recommended as a simple procedure which allows the measurement of the stable fraction alone, i.e. the real index of long-term glycemic control, independent of rapid glycemic fluctuations.