Kappa free light chains is a valid tool in the diagnostics of MS: A large multicenter study.

OBJECTIVE: To validate kappa free light chain (KFLC) and lambda free light chain (LFLC) indices as a diagnostic biomarker in multiple sclerosis (MS). METHODS: We performed a multicenter study including 745 patients from 18 centers (219 controls and 526 clinically isolated syndrome (CIS)/MS patients) with a known oligoclonal IgG band (OCB) status. KFLC and LFLC were measured in paired cerebrospinal fluid (CSF) and serum samples. Gaussian mixture modeling was used to define a cut-off for KFLC and LFLC indexes. RESULTS: The cut-off for the KFLC index was 6.6 (95% confidence interval (CI) = 5.2-138.1). The cut-off for the LFLC index was 6.9 (95% CI = 4.5-22.2). For CIS/MS patients, sensitivity of the KFLC index (0.88; 95% CI = 0.85-0.90) was higher than OCB (0.82; 95%CI = 0.79-0.85; p < 0.001), but specificity (0.83; 95% CI = 0.78-0.88) was lower (OCB = 0.92; 95% CI = 0.89-0.96; p < 0.001). Both sensitivity and specificity for the LFLC index were lower than OCB. CONCLUSION: Compared with OCB, the KFLC index is more sensitive but less specific for diagnosing CIS/MS. Lacking an elevated KFLC index is more powerful for excluding MS compared with OCB but the latter is more important for ruling in a diagnosis of CIS/MS.

Anti-inflammatory disease-modifying treatment and disability progression in primary progressive multiple sclerosis: a cohort study.

BACKGROUND AND PURPOSE: Treatment options in primary progressive multiple sclerosis (PPMS) are scarce and, with the exception of ocrelizumab, anti-inflammatory agents have failed to show efficacy in ameliorating disability progression. The aim of this study was to investigate a potential effect of anti-inflammatory disease-modifying treatment on disability outcomes in PPMS. METHODS: Using MSBase, a large, international, observational database, we identified patients with PPMS who were either never treated or treated with a disease-modifying agent. Propensity score matching was used to select subpopulations with similar baseline characteristics. Expanded Disability Status Scale (EDSS) outcomes were compared with an intention-to-treat and an as-treated approach in paired, pairwise-censored analyses. RESULTS: Of the 1284 included patients, 533 were matched (treated, n = 195; untreated n = 338). Median on-study pairwise-censored follow-up was 3.4 years (quartiles 1.2-5.5). No difference in the hazard of experiencing 3-month confirmed EDSS progression events was observed between the groups [hazard ratio (HR), 1.0; 95% confidence interval (CI), 0.6-1.7, P = 0.87]. We did not find significant differences in the hazards of confirmed EDSS improvement (HR, 1.0; 95% CI, 0.6-1.6, P = 0.91) or reaching a confirmed EDSS step ≥7 (HR, 1.1; 95% CI, 0.7-1.6, P = 0.69). CONCLUSION: Our pooled analysis of disease-modifying agents suggests that these therapies have no substantial effect on short- to medium-term disability outcomes in PPMS.

Free Kappa light chains in neuroinflammatory disorders: Complement rather than substitute?

OBJECTIVES: The detection of cerebrospinal fluid (CSF)-specific IgG oligoclonal bands (OCB) by isoelectric focusing (IEF) is widely used to help diagnose inflammatory neurological disorders (IND), including multiple sclerosis. However, the quantification of free light chains (FLC) is increasingly evaluated as a surrogate method to determine the presence of an intrathecal inflammatory process. The objective of this study was to evaluate the diagnostic performance of kappa (κ) FLC measurement in comparison with OCB detection by IEF. MATERIAL AND METHODS: We measured serum and CSF κFLCs by turbidimetry using the SPAplus automated analyser and calculated the κ index in 142 samples from OCB-positive and negative MS, as well as from patients with inflammatory and non-inflammatory neurological disorders (IND and NIND). RESULTS: The κFLC index was significantly increased in OCB-positive MS and IND patients versus OCB-negative patients. Its performance was relatively comparable to that of IEF for MS diagnosis. When using a κFLC index cutoff value of 6.29, sensitivity increased from 61.2% to 75.7% in comparison with IEF for diagnosing IND (P = .0051), with a slightly lower non-statistically significant specificity (82.1% vs 100%). When considering both OCB status positivity or a κFLC index superior to 6.29 to diagnose IND status, sensitivity raised to 80.6% (P < .05) with an equal specificity. CONCLUSION: Our results demonstrate that the κFLC index does not discriminate MS from other IND patients, but is a reliable technique to detect intrathecal inflammation. However, κFLC quantification should probably be considered as a complementary method, rather than a substitute, to OCB detection.

Decrease of blood anti-α1,3 Galactose Abs levels in multiple sclerosis (MS) and clinically isolated syndrome (CIS) patients.

The etiology of multiple sclerosis (MS) remains elusive. Among the possible causes, the increase of anti-Neu5Gc antibodies during EBV primo-infection of Infectious mononucleosis (IMN) may damage the integrity of the blood-brain barrier facilitating the transfer of EBV-infected B cells and anti-EBV T cell clones in the brain. We investigated the change in titers of anti-Neu5Gc and anti-α1,3 Galactose antibodies in 49 IMN, in 76 MS, and 73 clinically isolated syndrome (CIS) patients, as well as age/gender-matched healthy individuals. Anti-Gal and anti-Neu5Gc are significantly increased during IMN (p=0.02 and p<1.10-4 respectively), but not in acute CMV primo-infection. We show that, whereas there was no change in anti-Neu5Gc in MS/CIS, the two populations exhibit a significant decrease in anti-Gal (combined p=2.7.10-3), in contrast with patients with non-MS/CIS central nervous system pathologies. Since anti-Gal result from an immunization against α1,3 Gal, lacking in humans but produced in the gut, our data suggest that CIS and MS patients have an altered microbiota or an altered response to this microbiotic epitope.

Anti-SPAG16 antibodies in primary progressive multiple sclerosis are associated with an elevated progression index.

BACKGROUND AND PURPOSE: Sperm-associated antigen 16 (SPAG16), a sperm protein which is upregulated in reactive astrocytes in multiple sclerosis (MS) lesions, has recently been identified as a novel autoantibody target in MS. The aim of this study was to investigate whether anti-SPAG16 antibody levels differ between MS subtypes (relapsing-remitting, RR; primary or secondary progressive, PP, SP) and whether antibody positivity is associated with clinical characteristics. METHODS: Plasma anti-SPAG16 antibody levels were determined by recombinant protein enzyme-linked immunosorbent assay (ELISA) in 374 MS patients (274 RRMS, 39 SPMS and 61 PPMS) and 106 healthy controls. RESULTS: Significantly elevated anti-SPAG16 antibodies were found in 22% of MS patients with 93% specificity. Anti-SPAG16 seropositivity was associated with an increased Expanded Disability Status Scale (EDSS) in overall MS. A higher proportion of PPMS patients showed anti-SPAG16 antibody reactivity (34%) compared to RRMS (19%) and SPMS (26%), and presented with higher anti-SPAG16 antibody levels. Seropositive PPMS patients had a significantly increased progression index compared to seronegative patients. CONCLUSIONS: Anti-SPAG16 antibodies are associated with an increased EDSS in overall MS, indicating that they are linked to a worse MS disease outcome. Moreover, the presence of anti-SPAG16 antibodies may be a biomarker for a more severe disease in PPMS patients, as indicated by an increased progression index.

A basic overview of multiple sclerosis immunopathology.

Multiple sclerosis (MS) is a multi-component disease characterized by inflammation, neurodegeneration and failure of central nervous system (CNS) repair mechanisms. Immune dysregulation appears to originate with dendritic cells (antigen-presenting cells) which have an activated phenotype in individuals with MS. Dendritic cells migrate across the blood-brain barrier and induce differentiation of memory T cells into pro-inflammatory T helper 1 (Th1) and Th17 lymphocytes. In turn, induction of macrophage and microglial activation produces other pro-inflammatory cytokines and oxygen and nitric oxide radicals responsible for the demyelination and axonal loss. Other known mediators of MS pathology include CD8+ T cells and memory B cells within the CNS. Some pathological hallmarks of MS are early axonal degeneration and progressive decline of brain volume in patients with clinically isolated syndromes who progress to clinically definite MS. Many new options to interfere with the course of MS have become available in recent years. To limit inflammatory demyelinating processes and delay disease progression, intervention to control inflammation must begin as early as possible. Each distinct type of immunotherapy (immunomodulation, immunosuppression and immune-selective intervention - blockade type, sequestering type or depleting type) corresponds to a specific underlying immunopathology of MS.

[Physiotherapy and neurogenic lower urinary tract dysfunction in multiple sclerosis patients: a randomized controlled trial]. / Kinésithérapie et symptômes du bas appareil urinaire chez des patients atteints de la sclérose en plaques : étude contrôlée randomisée.

AIM: This randomized controlled trial compare the efficacy of pelvic floor muscle training vs. transcutaneous posterior tibial nerve stimulation. PATIENTS AND METHODS: Inclusion criteria were EDSS score<7 and presence of lower urinary tract symptoms. Exclusion criteria were multiple sclerosis relapse during the study, active urinary tract infection and pregnancy. The primary outcome was quality of life (SF-Qualiveen questionnaire). Secondary outcomes included overactive bladder (USP questionnaire) score and frequency of urgency episodes (3-day bladder diary). Sample size was calculated after 18 patients were included. Data analysis was blinded. Each patient received 9 sessions of 30 minutes weekly. Patients were randomized in pelvic floor muscles exercises with biofeedback group (muscle endurance and relaxation) or transcutaneous posterior tibial nerve stimulation group (rectangular alternative biphasic current with low frequency). RESULTS: A total of 31 patients were included. No difference appeared between groups for quality of life, overactive bladder and frequency of urgency episodes (respectively P=0.197, P=0.532 et P=0.788). These parameters were significantly improved in pelvic floor muscle training group (n=16) (respectively P=0.004, P=0.002 et P=0.006) and in transcutaneous posterior tibial nerve stimulation group (n=15) (respectively P=0.001, P=0.001 et P=0.031). CONCLUSIONS: Pelvic floor muscle training and transcutaneous posterior tibial nerve stimulation improved in the same way symptoms related to urgency in MS patients with mild disability. LEVEL OF EVIDENCE: 2.

The combined administration of LNC-encapsulated retinoic acid and calcitriol stimulates oligodendrocyte progenitor cell differentiation in vitro and in vivo after intranasal administration.

Intranasal administration is an efficient strategy for bypassing the BBB, favoring drug accumulation in the brain, and improving its efficiency. Lipid nanocapsules (LNC) are suitable nanocarriers for the delivery of lipophilic drugs via this route and can be used to encapsulate lipophilic molecules such as retinoic acid (RA) and calcitriol (Cal). As the hallmarks of multiple sclerosis (MS) are neuroinflammation and oligodendrocyte loss, our hypothesis was that by combining two molecules known for their pro-differentiating properties, encapsulated in LNC, and delivered by intranasal administration, we would stimulate oligodendrocyte progenitor cells (OPC) differentiation into oligodendrocytes and provide a new pro-remyelinating therapy. LNC loaded with RA (LNC-RA) and Cal (LNC-Cal) were stable for at least 8 weeks. The combination of RA and Cal was more efficient than the molecules alone, encapsulated or not, on OPC differentiation in vitro and decreased microglia cell activation in a dose-dependent manner. After the combined intranasal administration of LNC-RA and LNC-Cal in a mouse cuprizone model of demyelination, increased MBP staining was observed in the corpus callosum. In conclusion, intranasal delivery of lipophilic drugs encapsulated in LNC is a promising strategy for myelinating therapies.

Comparative effectiveness and cost-effectiveness of natalizumab and fingolimod in rapidly evolving severe relapsing-remitting multiple sclerosis in the United Kingdom.

AIM: To evaluate the real-world comparative effectiveness and the cost-effectiveness, from a UK National Health Service perspective, of natalizumab versus fingolimod in patients with rapidly evolving severe relapsing-remitting multiple sclerosis (RES-RRMS). METHODS: Real-world data from the MSBase Registry were obtained for patients with RES-RRMS who were previously either naive to disease-modifying therapies or had been treated with interferon-based therapies, glatiramer acetate, dimethyl fumarate, or teriflunomide (collectively known as BRACETD). Matched cohorts were selected by 3-way multinomial propensity score matching, and the annualized relapse rate (ARR) and 6-month-confirmed disability worsening (CDW6M) and improvement (CDI6M) were compared between treatment groups. Comparative effectiveness results were used in a cost-effectiveness model comparing natalizumab and fingolimod, using an established Markov structure over a lifetime horizon with health states based on the Expanded Disability Status Scale. Additional model data sources included the UK MS Survey 2015, published literature, and publicly available sources. RESULTS: In the comparative effectiveness analysis, we found a significantly lower ARR for patients starting natalizumab compared with fingolimod (rate ratio [RR] = 0.65; 95% confidence interval [CI], 0.57-0.73) or BRACETD (RR = 0.46; 95% CI, 0.42-0.53). Similarly, CDI6M was higher for patients starting natalizumab compared with fingolimod (hazard ratio [HR] = 1.25; 95% CI, 1.01-1.55) and BRACETD (HR = 1.46; 95% CI, 1.16-1.85). In patients starting fingolimod, we found a lower ARR (RR = 0.72; 95% CI, 0.65-0.80) compared with starting BRACETD, but no difference in CDI6M (HR = 1.17; 95% CI, 0.91-1.50). Differences in CDW6M were not found between the treatment groups. In the base-case cost-effectiveness analysis, natalizumab dominated fingolimod (0.302 higher quality-adjusted life-years [QALYs] and £17,141 lower predicted lifetime costs). Similar cost-effectiveness results were observed across sensitivity analyses. CONCLUSIONS: This MSBase Registry analysis suggests that natalizumab improves clinical outcomes when compared with fingolimod, which translates to higher QALYs and lower costs in UK patients with RES-RRMS.

There are several medications used to treat people with relapsing remitting multiple sclerosis, such as interferon-based therapies (Betaferon/Betaseron (US), Rebif, Avonex, Extavia), glatiramer acetate (Copaxone), teriflunomide (Aubagio), and dimethyl fumarate (Tecfidera), collectively named BRACETD. Other treatments for multiple sclerosis (MS) have a narrower use, such as natalizumab (Tysabri) or fingolimod (Gilenya), among others.This study objective was to assess how well natalizumab and fingolimod helped treating MS (clinical effectiveness) and subsequently estimate what the cost of these treatments is in comparison to the benefit they bring to people with rapidly evolving severe MS that use them in the United Kingdom (UK) (cost-effectiveness).We used an international disease registry (MSBase), which collects clinical data from people with MS in various centers around the world to compare the effectiveness of natalizumab, fingolimod and BRACETD treatments. We used a technique called propensity score matching to obtain results from comparable patient groups. People treated with natalizumab had better disease control, namely with fewer relapses and higher improvement on their disability level, than patients on fingolimod or BRACETD. Conversely, there were no differences between each group of people on a measure called disability worsening.Based on these clinical results, we built an economic model that simulates the lifetime costs and consequences of treating people with MS with natalizumab in comparison with fingolimod. We found that using natalizumab was less costly and was more effective compared to using fingolimod in UK patients.

Fluctuations of MS births and UV-light exposure.

BACKGROUND: Patients with multiple sclerosis (MS) are more frequently born in spring when compared to autumn. Fluctuation of UV-light has been hypothesized to drive this phenomenon. AIM: To assess the correlation between fluctuation of sunlight and birth season in persons with MS. METHODS: For this record-linkage study, we collected from the international MSBase and the Italian MS iMed-web databases the dates of birth of 11,415 patients with MS from 36 centres from 15 countries worldwide and compared these to dates of live-births from national registries. From all participating sites, we collected data on UV-light fluctuation and assessed its correlation with seasonal fluctuation in MS births. RESULTS: Compared with the reference cohort, an increased proportion of persons with MS were born in spring and a decreased proportion in autumn (odds ratio (OR) to be born in spring versus autumn = 1.158, χ² = 36.347, P < 0.001). There was no significantly increased fluctuation of MS births with increased quartile of ambient UV-light fluctuation (Ptrend = 0.086). CONCLUSION: Seasonal fluctuation of MS births as found in this worldwide cohort of patients with MS did not correlate with variation in seasonal fluctuation of UV-light. Most likely, it results from a complex interplay between fluctuation of sunlight, behavioural factors, other environmental factors and (epi)genetic factors.

Transient perioperative visual loss after an elective neurosurgical procedure.

OBJECTIVE: To describe a case of reversible visual loss after a neurosurgical intervention and to discuss the role of the prone position as a potential risk factor. OBSERVATION: A 63-year-old woman without significant medical previous history underwent elective resection of a left parieto-occipital meningioma. Preoperatively, the patient presented a right homonymous lower quadranopsia. The surgical procedure was not complicated. The patient was positioned in prone with a mild inclination of the table in reverse Trendelenburg position. The head was maintained in a Mayfield skull clamp, and ocular compression was excluded. There was no significant hypotension, hemodilution or vasopressors infusion during the procedure. Immediately after recovery from anesthesia, the patient experienced total blindness and flash visual evoked potentials confirmed the absence of retinal, primary or late occipital activities. A progressive, but finally complete recovery started after 24 hours. CONCLUSION: This case illustrates the individual risk for visual injury after the prone position during some neurosurgical interventions.

The frequency of CSF oligoclonal banding in multiple sclerosis increases with latitude.

BACKGROUND: With the advent of MRI scanning, the value of lumbar puncture to assess oligoclonal band (OCB) status-for the diagnosis of multiple sclerosis (MS) is increasingly uncertain. One major issue is that the reported frequency of cerebrospinal fluid (CSF)-restricted oligoclonal banding for the diagnosis of MS varies considerably in different studies. In addition, the relationship between OCB positivity and disease outcome remains uncertain, as reported studies are generally too small to assess comparative disability outcomes with sufficient power. METHODS: In order to further investigate variation of OCB positivity in patients with MS, we utilized MSBase, a longitudinal, Web-based collaborative MS outcomes registry following clinical cohorts in several continents and latitudes. We also assessed whether OCB positivity affects long-term disability outcome. RESULTS: A total of 13,242 patient records were obtained from 37 MS specialist centres in 19 different countries. OCB status was documented in 4481 (34%) patients and 80% of these were OCB positive. The presence of OCB was associated with degree of latitude (p = 0.02). Furthermore, the outcome of patients negative for CSF-specific OCB was significantly better in comparison to the OCB positive patients, as assessed by Expanded Disability Status Scale change (p < 0.001). CONCLUSIONS: The results of this study indicate that latitude could explain some of the inconsistencies in OCB status reported in different populations. The study confirms that OCB positivity in MS is associated with a worse long-term prognosis.

Recurrent Miller Fisher syndrome with vestibular involvement.

We describe a patient who had four relapses of Miller Fisher syndrome over a period of 20 years. The classical triad - ophthalmoparesis, ataxia and areflexia - was present during the first two attacks; ataxia was not observed during the third episode. The final recurrence was characterized by signs suggestive of a central involvement of the oculomotor pathways, subclinical slowing of the visual-evoked potentials, and peripheral vestibular hyporeactivity. Brain imaging was normal, but high levels of anti-GQ1b IgG antibodies were detectable during the second relapse and persisted after the fourth recurrence despite complete clinical recovery.

Mitoxantrone-related acute leukemia in two MS patients.

We report two new cases of mitoxantrone-related leukemia occurring in two patients with multiple sclerosis (MS), 14 and 18 months after the last infusion of the drug. One patient was successfully treated. We were able to collect 29 other cases in the literature. Most of them were single reports but some were described within cohorts of mitoxantrone-treated MS patients. The incidence rate was 0.65% from all cohorts totalizing 2299 patients. Acute promyelocytic leukemia with the translocation t(15;17) was over-represented in the MS population in comparison with cancer patients also treated with mitoxanrone. The occurrence of leukemia was dose-independent and appeared with a mean delay of 20 months after the end of the treatment.

Corticosteroids in the management of acute multiple sclerosis exacerbations.

Multiple sclerosis (MS) is an autoimmune, inflammatory demyelinating disease of the central nervous system characterized in the majority of the patients by a relapsing-remitting disease course. For decades high-dosage corticosteroids (CS) are considered the cornerstone in the management of acute MS relapses. However, many unanswered questions remain when it comes to the exact modalities of CS administration. In this review on behalf of the Belgian Study Group for MS we define the efficacy of CS in reducing MS-related morbidity and examine whether the effect is different according to type of CS, route of administration, cumulative dosage, timing of initiation and disease course. We also review the use of CS in combination with other MS treatments and during pregnancy and lactation. Furthermore, we delineate the relevant adverse events due to a pulse CS regimen and present a decision tree that can be used when treating MS relapses in clinical practice.

Polymerase chain reaction analysis and oligoclonal antibody in the cerebrospinal fluid from 34 patients with varicella-zoster virus infection of the nervous system.

OBJECTIVE: To study cerebrospinal fluid (CSF) and serum samples from 34 consecutive patients suspected of having varicella-zoster virus (VZV) infection of the central nervous system (CNS). POPULATION AND METHODS: The patients were divided into three groups. The first group consisted of 27 patients with a rash in one to three dermatomes and clinical suspicion of meningitis and radiculitis; among them, three subgroups were distinguished according to the affected dermatome: ophthalmicus (n = 9), oticus (n = 11) and cervico-thoraco-lumbar zoster (n = 7). Four cases of zoster sine herpete (ZSH) were included in the second group: these patients presented with either radiculitis (n = 2) or meningoencephalitis (n = 2), without cutaneous eruption. The third group consisted of three patients with a generalised rash and encephalitis. A polymerase chain reaction (PCR) for VZV DNA and antigen-driven immunoblots for oligoclonal anti-VZV antibodies were carried out on all CSF samples. RESULTS: PCR of the CSF was positive in 44% of the patients from the first group, mainly within the first 7 days after eruption. In addition, intrathecal synthesis of anti-VZV antibodies was detected in 37% of patients, always after an interval of 7 days (p<0.0001). Among the four patients with ZSH, a positive VZV PCR was detected in three patients and CSF-specific oligoclonal anti-VZV antibodies in two. PCR was also positive in the CSF of two of the three patients with generalised rash and encephalitis; local production of anti-VZV antibodies was seen in a second CSF sample in one patient, and was also present in the third patient. CONCLUSION: Amplification of VZV DNA by PCR in the CSF and antigen-driven immunoblots have important diagnostic value in suspected VZV infection, although their presence depends on the timing of the CSF sampling. VZV is thought to be a causative agent in unexplained cases of meningitis associated with radiculitis or focal CNS symptoms, even in the absence of skin manifestations. In such patients, rapid diagnosis by this combined approach permits early antiviral treatment.

Clinical, electrophysiological and brain imaging features during recurrent ictal cortical blindness associated with chronic liver failure.

Transient neuroimaging features indicating primary cortical and secondary subcortical white matter cytotoxic oedema have been described in association with prolonged or intense seizures. We describe the unusual condition of recurrent ictal cortical blindness due to focal occipital status epilepticus, in the context of chronic hepatic failure. There was a close association between the onset and disappearance of clinical, electrophysiological and magnetic resonance imaging abnormalities.

[Type I interferons]. / Les interférons de type I.

Type I interferons (IFNa/b) form a family of related cytokines that include INFa, b, e/s, j, x (human) and limitin (mouse). These cytokines exert a potent antiviral activity, control cell proliferation and modulate the immune response. They are used in the fight against viral infections, tumors, and multiple sclerosis. Expression of IFNs is typically induced by viral infections. Cells express cytoplasmic helicases as well as endosomial toll-like receptors acting as sensors to detect endogenous and exogenous viral infections, respectively. Signal transduction from these sensors induces the transcription of IFN genes. IFNs are secreted and bind to a cell surface receptor expressed by most cells of the organism. Upon receptor binding, IFNs induce the transcription of hundreds of genes whose products exert antiviral, antiproliferative and immunomodulatory functions. Antiviral activity of IFNs is so potent that most (if not all) viruses developed strategies to antagonize the IFN response.

Susac-like syndrome in a chronic cocaine abuser: could levamisole play a role?

INTRODUCTION: Toxic leukoencephalopathy is a possible but rare complication of chronic cocaine abuse. The role of adulterants, mainly levamisole, is still debated. CASE REPORT: We describe an atypical case of fatal leukoencephalopathy mimicking Susac syndrome in a 22-year-old man who was chronically abusing cannabis and cocaine. Exposure to levamisole as adulterant to cocaine was proven by hair analysis. Despite cessation of exposure to cocaine and aggressive immunosuppressive therapy, the patient remained in a minimally conscious state until death. DISCUSSION: Susac syndrome is a rare entity, and its etiology is not yet fully elucidated. The toxic etiologies have been poorly investigated to date. Further observations are required to determine if cocaine and/or adulterants might play a significant role.