Stress-induced brain activity, brain atrophy, and clinical disability in multiple sclerosis.

Prospective clinical studies support a link between psychological stress and multiple sclerosis (MS) disease severity, and peripheral stress systems are frequently dysregulated in MS patients. However, the exact link between neurobiological stress systems and MS symptoms is unknown. To evaluate the link between neural stress responses and disease parameters, we used an arterial-spin-labeling functional MRI stress paradigm in 36 MS patients and 21 healthy controls. Specifically, we measured brain activity during a mental arithmetic paradigm with performance-adaptive task frequency and performance feedback and related this activity to disease parameters. Across all participants, stress increased heart rate, perceived stress, and neural activity in the visual, cerebellar and insular cortex areas compared with a resting condition. None of these responses was related to cognitive load (task frequency). Consistently, although performance and cognitive load were lower in patients than in controls, stress responses did not differ between groups. Insula activity elevated during stress compared with rest was negatively linked to impairment of pyramidal and cerebral functions in patients. Cerebellar activation was related negatively to gray matter (GM) atrophy (i.e., positively to GM volume) in patients. Interestingly, this link was also observed in overlapping areas in controls. Cognitive load did not contribute to these associations. The results show that our task induced psychological stress independent of cognitive load. Moreover, stress-induced brain activity reflects clinical disability in MS. Finally, the link between stress-induced activity and GM volume in patients and controls in overlapping areas suggests that this link cannot be caused by the disease alone.

Brain activity, regional gray matter loss, and decision-making in multiple sclerosis.

BACKGROUND: Decision-making (DM) abilities deteriorate with multiple sclerosis (MS) disease progression which impairs everyday life and is thus clinically important. OBJECTIVE: To investigate the underlying neurocognitive processes and their relation to regional gray matter (GM) loss induced by MS. METHODS: We used a functional magnetic resonance imaging (fMRI) Iowa Gambling Task to measure DM-related brain activity in 36 MS patients and 21 healthy controls (HC). From this activity, we determined neural parameters of two cognitive stages, a deliberation ("choice") period preceding a choice and a post-choice ("feedback") stage reporting decision outcomes. These measures were related to DM separately in intact and damaged GM areas as determined by a voxel-based morphometry analysis. RESULTS: Severely affected patients (with high lesion burden) showed worse DM-learning than HC ( t = -1.75, p = 0.045), moderately affected (low lesion burden) did not. Activity in the choice stage in intact insular ( t = 4.60, pFamily-Wise Error [FWE] corrected = 0.034), anterior cingulate ( t = 4.50, pFWE = 0.044), and dorsolateral prefrontal areas ( t = 4.43, pFWE = 0.049) and in insular areas with GM loss ( t = 3.78, pFWE = 0.011) was positively linked to DM performance across patients with severe tissue damage and HC. Furthermore, activity in intact orbitofrontal areas was positively linked to DM-learning during the feedback stage across these participants ( t = 4.49, pFWE = 0.032). During none of the stages, moderately affected patients showed higher activity than HC, which might have indicated preserved DM due to compensatory activity. CONCLUSION: We identified dysregulated activity linked to impairment in specific cognitive stages of reward-related DM. The link of brain activity and impaired DM in areas with MS-induced GM loss suggests that this deficit might be tightly coupled to MS neuropathology.

Low 25-hydroxyvitamin D, but not the bioavailable fraction of 25-hydroxyvitamin D, is a risk factor for multiple sclerosis.

BACKGROUND AND PURPOSE: Low 25-hydroxyvitamin D [25(OH)D] levels correlate with higher disease activity in patients with multiple sclerosis (MS). However, it is not clear whether low 25(OH)D levels directly contribute to increased disease activity or merely represent a consequence of reduced endogenous vitamin D synthesis in more disabled MS patients. Furthermore, recent data suggest that bioavailable vitamin D, which also integrates the levels of vitamin D binding proteins and albumin, could be a biologically more relevant parameter than 25(OH)D. METHODS: Measured de-seasonalized 25(OH)D3 and vitamin D binding protein and calculated bioavailable and free vitamin D were compared in the baseline serum samples of 76 patients with clinically isolated syndrome enrolled in a longitudinal observational study and in 76 age- and sex-matched healthy controls (HC). RESULTS: 25(OH)D3 levels were lower in patients with clinically isolated syndrome (P = 0.002) than in HC, and more patients (8/76, 10.5%) than HC (1/76, 1.3%) had 25(OH)D3 levels <25 nmol/l (P = 0.03). In contrast, levels of 25(OH)D2, vitamin D binding protein and calculated levels of free and bioavailable vitamin D did not differ between the two groups. CONCLUSIONS: Lower 25(OH)D3 levels already in the earliest phase of disease and in clinically hardly affected patients suggest that low 25(OH)D3 levels are rather a risk factor for than a consequence of MS. Nevertheless, because bioavailable vitamin D levels did not differ between the two groups, the mechanism underlying the association of 25(OH)D3 and MS does not appear to be related to reduced bioavailability of vitamin D.

Next-generation sequencing identifies altered whole blood microRNAs in neuromyelitis optica spectrum disorder which may permit discrimination from multiple sclerosis.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) have a similar clinical phenotype but represent distinct diseases, requiring different therapies. MicroRNAs (miRNAs) are short non-coding RNAs whose expression profiles can serve as diagnostic biomarkers and which may be involved in the pathophysiology of neuroinflammatory diseases. Here, we analyzed miRNA profiles in serum and whole blood of patients with NMOSD and clinically isolated syndrome (CIS)/relapsing-remitting MS (RRMS) as well as healthy controls by next-generation sequencing (NGS). METHODS: MiRNA expression profiles were determined by NGS in sera of patients with aquaporin-4 antibody-positive NMOSD (n = 20), CIS/RRMS (n = 20), and healthy controls (n = 20) and in whole blood of patients with NMOSD (n = 11), CIS/RRMS (n = 60), and healthy controls (n = 43). Differentially expressed miRNAs were calculated by analysis of variance and t tests. All significance values were corrected for multiple testing. Selected miRNAs were validated in whole blood of patients with NMOSD (n = 18) and CIS/RRMS (n = 19) by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: None of 261 miRNAs detected in serum but 178 of 416 miRNAs detected in whole blood showed significantly different expression levels among the three groups. Pairwise comparisons revealed 115 (NMOSD vs. CIS/RRMS), 141 (NMOSD vs. healthy controls), and 44 (CIS/RRMS vs. healthy controls) miRNAs in whole blood with significantly different expression levels. qRT-PCR confirmed different expression levels in whole blood of patients with NMOSD and CIS/RRMS for 9 out of 10 exemplarily chosen miRNAs. In silico enrichment analysis demonstrated an accumulation of altered miRNAs in NMOSD in particular in CD15(+) cells (i.e., neutrophils and eosinophils). CONCLUSIONS: This study identifies a set of miRNAs in whole blood, which may have the potential to discriminate NMOSD from CIS/RRMS and healthy controls. In contrast, miRNA profiles in serum do not appear to be promising diagnostic biomarkers for NMOSD. Enrichment of altered miRNAs in CD15(+) neutrophils and eosinophils, which were previously implicated in the pathophysiology of NMOSD, suggests that miRNAs could be involved in the regulation of these cells in NMOSD.

The Diagnostic Performance of Multiparametric Ultrasound in the Qualitative Assessment of Inconclusive Cervical Lymph Nodes.

BACKGROUND: Enlarged cervical lymph nodes (CLNs) can result from infection or malignancies, and a definitive diagnosis requires histological examination. Ultrasound (US) remains the first-line imaging modality for detection, and new US techniques may improve characterization. The aim of our study was to investigate whether the qualitative assessment of multiparametric US (mpUS) can improve diagnostic performance in the differentiation of benign and malignant CLNs. METHODS: 107 CLNs in 105 patients were examined by preoperative mpUS consisting of B-mode US, color-coded duplex sonography (CCDS), shear wave elastography (SWE) and contrast-enhanced US (CEUS). US images were evaluated in consensus by two experienced US operators. Histopathological examination was used as reference standard. RESULTS: SWE and CEUS combined showed the highest overall diagnostic performance (91% sensitivity, 77% specificity, 87% positive predictive value (PPV), 83% negative predictive value (NPV), 90% accuracy, χ2 (1) = 51.485, p < 0.001) compared to B-mode US and CCDS (87% sensitivity, 44% specificity, 73% PPV, 65% NPV, 73% accuracy χ2 (1) = 12.415, p < 0.001). In terms of individual techniques, SWE had higher specificity than B-mode and CCDS (71% sensitivity, 90% specificity, 92% PPV, 64% NPV, 78% accuracy, χ2 (1) = 36.115, p < 0.001), while qualitative CEUS showed the best diagnostic performance of all investigated US techniques (93% sensitivity, 85% specificity, 91% PPV, 87% NPV, 90% accuracy, χ2 (1) = 13.219, p < 0.001). Perfusion patterns, homogeneity, presence of necrosis, and malignancy differed significantly between malignant and benign CLNs (p < 0.001). CONCLUSIONS: SWE and CEUS can facilitate the differentiation of inconclusive CLNs when performed to supplement B-mode US and CCDS. MpUS may thus aid the decision between surgery and a watch-and-scan strategy in enlarged CLNs.

Contrast-enhanced ultrasound (CEUS) in characterization of inconclusive cervical lymph nodes: a meta-analysis and systematic review.

Lymph node metastases are common in malignant neoplasms of head and neck. Since cervical lymph nodes (cLN) are localized superficially, ultrasound (US) represents the primary imaging modality. The aim of the study is to report the value of US and contrast-enhanced ultrasound (CEUS) and their diagnostic confidence in the characterization of inconclusive cLN. A systematic review was performed using the literature data base PubMed. Results were filtered (published in a peer-reviewed journal, full-text available, published within the last ten years, species human, English or German full-text) and inclusion criteria were clearly defined (cohort with lymphadenopathy or malignancy in head and neck ≥ 50 patients, histological confirmation of malignant imaging findings, performance of CEUS as outcome variable). The results were quantified in a meta-analysis using a random-effects model. Overall, five studies were included in qualitative and quantitative analysis. The combination of non-enhanced US and CEUS enlarges the diagnostic confidence in the characterization of lymph nodes of unclear dignity. The pooled values for sensitivity and specificity in the characterization of a malignant cervical lymph node using US are 76% (95%-CI 66-83%, I2 = 63%, p < 0.01) and 80% (95%-CI 45-95%, I2 = 92%, p < 0.01), compared to 92% (95%-CI 89-95%, I2 = 0%, p = 0.65) and 91% (95%-CI 87-94%, I2 = 0%, p = 0.40) for the combination of US and CEUS, respectively. Consistent results of the included studies show improved diagnostic performance by additional CEUS. Nevertheless, more prospective studies are needed to implement CEUS in the diagnostic pathway of cLN.

Assessment of Parotid Gland Tumors by Means of Quantitative Multiparametric Ultrasound (mpUS).

OBJECTIVE: The preoperative diagnostical differentiation of parotid gland tumor (PGT) is not always simple due to several different entities. B-mode-ultrasound (US) remains the imaging modality of choice, while histopathology serves as the gold standard for finalizing the diagnosis. We aimed to evaluate the use of multiparametric US (mpUS) in the assessment of PGT. METHODS: We included 97 PGTs from 96 patients. A standardized mpUS protocol using B-mode-US, shear-wave elastography (SWE), and standardized contrast-enhanced ultrasound (CEUS) was performed prior to surgical intervention. SWE was assessed by real-time measurement conducting a minimum of five measurements, while quantitative CEUS parameters were assessed with a post-processing perfusion software. RESULTS: SWE allowed differentiation between benign PGT (Warthin's Tumor (WT) paired with lymph nodes (LN) and pleomorphic adenoma (PA)), and WT and LN were softer compared to PA. WT showed lower velocities than squamous cell carcinoma (SCC): the most common malignant PGT. CEUS parameters showed significant group differences between WT and PA, WT and malignant lesions, WT and SCC, WT paired with LN versus PA, and WT paired with LN versus SCC. CONCLUSION: MpUS seems to be beneficial in the assessment of PGT characterization, with benign PGT appearing to be softer in SWE than tumors with malignant tendencies. The quantitative CEUS parameter shows higher perfusion in WT than in PA, and malignant PGTs are less vascularized than WTs.

Quantitative Multiparametric Ultrasound (mpUS) in the Assessment of Inconclusive Cervical Lymph Nodes.

Background: Enlarged cervical lymph nodes (CLN) are preferably examined by ultrasound (US) by using criteria such as size and echogenicity to assess benign and suspicious CLN, which should be histologically evaluated. This study aims to assess the differentiation of malign and benign CLN by using multiparametric US applications (mpUS). Methods: 101 patients received a standardized US protocol prior to surgical intervention using B-mode−US, shear-wave elastography (SWE) and contrast-enhanced ultrasound (CEUS). SWE was assessed by 2D real-time SWE conducting a minimum of five measurements, CEUS parameters were assessed with post-processing perfusion software. Histopathological confirmation served as the gold standard. Results: B-mode−US and SWE analysis of 104 CLN (36 benign, 68 malignant) showed a significant difference between benign and malignant lesions, presenting a larger long axis and higher tissue stiffness (both p < 0.001). Moreover, tissue stiffness assessed by SWE was significantly higher in CLN with regular B-mode−US criteria (Solbiati Index > 2 and short-axis < 1 cm, p < 0.001). No perfusion parameter on CEUS showed a significant differentiation between benign and malignant CLN. Discussion: As the only multiparametric parameter, SWE showed higher tissue stiffness in malignant CLN, also in subgroups with regular B-mode criteria. This fast and easy application may be a promising noninvasive tool to US examination to ameliorate the sonographic differentiation of inconclusive CLN.

Altered Coupling of Psychological Relaxation and Regional Volume of Brain Reward Areas in Multiple Sclerosis.

Background: Psychological stress can influence the severity of multiple sclerosis (MS), but little is known about neurobiological factors potentially counteracting these effects. Objective: To identify gray matter (GM) brain regions related to relaxation after stress exposure in persons with MS (PwMS). Methods: 36 PwMS and 21 healthy controls (HCs) reported their feeling of relaxation during a mild stress task. These markers were related to regional GM volumes, heart rate, and depressive symptoms. Results: Relaxation was differentially linked to heart rate in both groups (t = 2.20, p = 0.017), i.e., both markers were only related in HCs. Relaxation was positively linked to depressive symptoms across all participants (t = 1.99, p = 0.045) although this link differed weakly between groups (t = 1.62, p = 0.108). Primarily, the volume in medial temporal gyrus was negatively linked to relaxation in PwMS (t = -5.55, pfamily-wise-error(FWE)corrected = 0.018). A group-specific coupling of relaxation and GM volume was found in ventromedial prefrontal cortex (VMPFC) (t = -4.89, pFWE = 0.039). Conclusion: PwMS appear unable to integrate peripheral stress signals into their perception of relaxation. Together with the group-specific coupling of relaxation and VMPFC volume, a key area of the brain reward system for valuation of affectively relevant stimuli, this finding suggests a clinically relevant misinterpretation of stress-related affective stimuli in MS.

Neural mechanisms of perceptual decision-making and their link to neuropsychiatric symptoms in multiple sclerosis.

BACKGROUND: Decision-making (DM) capabilities are impaired in multiple sclerosis (MS). A variety of researchers hypothesized that this impairment is associated with reduced quality of life (QoL) and neuropsychiatric symptoms. Studies explicitly testing this hypothesis, however, are rare, provided inconclusive results, or evaluated only a limited selection of DM domains. Consequently, we conducted the first MS study on perceptual DM (e.g. deciding whether a car will fit into a parking lot based on a visual percept) to test this assumption. METHODS: Specifically, we used an fMRI task that measured brain activity in 30 MS patients and 19 healthy controls (HCs) while the participants repeatedly decided whether objects referenced indirectly via their written object names would fit into a shoebox to investigate neural mechanisms of perceptual DM. The objects varied in size and thus decision difficulty. From these data, we determined voxel-wise brain activity parameters reflecting (i) decision difficulty and (ii) decision speed and related them to behavioral DM performance, QoL, mild to moderate depressive symptoms, and fatigue. RESULTS: Patients showed reduced DM performance. Activity reflecting decision difficulty in the middle temporal gyrus was negatively related to DM performance across MS patients and HCs; activity reflecting decision speed in MS patients was associated with depressive symptoms and fatigue in areas of the dorsal visual stream. CONCLUSION: The study shows that the perceptual DM capacity is reduced in MS. Moreover, the link between neural mechanisms of perceptual DM and neuropsychiatric symptoms suggests that an impairment in this domain is clinically relevant.

Fine specificity of the antibody response to Epstein-Barr nuclear antigen-2 and other Epstein-Barr virus proteins in patients with clinically isolated syndrome: A peptide microarray-based case-control study.

We analyzed the fine specificity of antibodies to Epstein-Barr nuclear antigen-2 (EBNA-2) and other Epstein-Barr virus (EBV) proteins in 29 patients with clinically isolated syndrome (CIS, the first clinical manifestation of multiple sclerosis [MS]) and 29 controls with a peptide microarray containing 117 overlapping peptides representing the full-length EBNA-2 protein and 71 peptides from 8 further EBV proteins. While EBV peptide antibodies were elevated in CIS, suggesting that EBV contributes to MS early during disease development, they discriminated groups only slightly better than EBNA-1 antibodies. Thus, the additional value of EBV peptide antibodies as diagnostic biomarkers for CIS appears moderate.

Analysis of Lymphocytic DNA Damage in Early Multiple Sclerosis by Automated Gamma-H2AX and 53BP1 Foci Detection: A Case Control Study.

BACKGROUND: In response to DNA double-strand breaks, the histone protein H2AX becomes phosphorylated at its C-terminal serine 139 residue, referred to as γ-H2AX. Formation of γ-H2AX foci is associated with recruitment of p53-binding protein 1 (53BP1), a regulator of the cellular response to DNA double-strand breaks. γ-H2AX expression in peripheral blood mononuclear cells (PBMCs) was recently proposed as a diagnostic and disease activity marker for multiple sclerosis (MS). OBJECTIVE: To evaluate the significance of γ-H2AX and 53BP1 foci in PBMCs as diagnostic and disease activity markers in patients with clinically isolated syndrome (CIS) and early relapsing-remitting MS (RRMS) using automated γ-H2AX and 53BP1 foci detection. METHODS: Immunocytochemistry was performed on freshly isolated PBMCs of patients with CIS/early RRMS (n = 25) and healthy controls (n = 27) with γ-H2AX and 53BP1 specific antibodies. Nuclear γ-H2AX and 53BP1 foci were determined using a fully automated reading system, assessing the numbers of γ-H2AX and 53BP1 foci per total number of cells and the percentage of cells with foci. Patients underwent contrast enhanced 3 Tesla magnetic resonance imaging (MRI) and clinical examination including expanded disability status scale (EDSS) score. γ-H2AX and 53BP1 were also compared in previously frozen PBMCs of each 10 CIS/early RRMS patients with and without contrast enhancing lesions (CEL) and 10 healthy controls. RESULTS: The median (range) number of γ-H2AX (0.04 [0-0.5]) and 53BP1 (0.005 [0-0.2]) foci per cell in freshly isolated PBMCs across all study participants was low and similar to previously reported values of healthy individuals. For both, γ-H2AX and 53BP1, the cellular focus number as well as the percentage of positive cells did not differ between patients with CIS/RRMS and healthy controls. γ-H2AX and 53BP1 levels neither correlated with number nor volume of T2-weighted lesions on MRI, nor with the EDSS. Although γ-H2AX, but not 53BP1, levels were higher in previously frozen PBMCs of patients with than without CEL, γ-H2AX values of both groups overlapped and γ-H2AX did not correlate with the number or volume of CEL. CONCLUSION: γ-H2AX and 53BP1 foci do not seem to be promising diagnostic or disease activity biomarkers in patients with early MS. Lymphocytic DNA double-strand breaks are unlikely to play a major role in the pathophysiology of MS.

Association of serum Epstein-Barr nuclear antigen-1 antibodies and intrathecal immunoglobulin synthesis in early multiple sclerosis.

Multiple sclerosis (MS) is associated with Epstein-Barr virus (EBV) infection. A characteristic feature of MS is an intrathecal synthesis of immunoglobulin (Ig)G. In 90 patients with clinically isolated syndromes/early relapsing-remitting MS, serum antibodies to Epstein-Barr nuclear antigen-1, but not to EBV viral capsid antigen, rubella, or varicella zoster virus, were higher (p=0.03) in those with than those without a calculated intrathecal IgG synthesis >0% and correlated with the percentage (r=0.27, p=0.009) and concentration (r=0.27, p=0.012) of intrathecally produced IgG. These findings suggest a link between EBV infection and the events leading to intrathecal IgG synthesis in patients with MS.