An ANXA11 P93S variant dysregulates TDP-43 and causes corticobasal syndrome.

INTRODUCTION: Variants of uncertain significance (VUS) surged with affordable genetic testing, posing challenges for determining pathogenicity. We examine the pathogenicity of a novel VUS P93S in Annexin A11 (ANXA11) - an amyotrophic lateral sclerosis/frontotemporal dementia-associated gene - in a corticobasal syndrome kindred. Established ANXA11 mutations cause ANXA11 aggregation, altered lysosomal-RNA granule co-trafficking, and transactive response DNA binding protein of 43 kDa (TDP-43) mis-localization. METHODS: We described the clinical presentation and explored the phenotypic diversity of ANXA11 variants. P93S's effect on ANXA11 function and TDP-43 biology was characterized in induced pluripotent stem cell-derived neurons alongside multiomic neuronal and microglial profiling. RESULTS: ANXA11 mutations were linked to corticobasal syndrome cases. P93S led to decreased lysosome colocalization, neuritic RNA, and nuclear TDP-43 with cryptic exon expression. Multiomic microglial signatures implicated immune dysregulation and interferon signaling pathways. DISCUSSION: This study establishes ANXA11 P93S pathogenicity, broadens the phenotypic spectrum of ANXA11 mutations, underscores neuronal and microglial dysfunction in ANXA11 pathophysiology, and demonstrates the potential of cellular models to determine variant pathogenicity. HIGHLIGHTS: ANXA11 P93S is a pathogenic variant. Corticobasal syndrome is part of the ANXA11 phenotypic spectrum. Hybridization chain reaction fluorescence in situ hybridization (HCR FISH) is a new tool for the detection of cryptic exons due to TDP-43-related loss of splicing regulation. Microglial ANXA11 and related immune pathways are important drivers of disease. Cellular models are powerful tools for adjudicating variants of uncertain significance.

Neurofilament light chain in cerebrospinal fluid as a novel biomarker in evaluating both clinical severity and therapeutic response in Niemann-Pick disease type C1.

PURPOSE: Niemann-Pick disease type C1 (NPC1) is a neurodegenerative lysosomal disorder caused by pathogenic variants in NPC1. Disease progression is monitored using the NPC Neurological Severity Scale, but there are currently no established validated or qualified biomarkers. Neurofilament light chain (NfL) is being investigated as a biomarker in multiple neurodegenerative diseases. METHODS: Cross-sectional and longitudinal cerebrospinal fluid (CSF) samples were obtained from 116 individuals with NPC1. NfL levels were measured using a solid-phase sandwich enzyme-linked immunosorbent assay and compared with age-appropriate non-NPC1 comparison samples. RESULTS: Median levels of NfL were elevated at baseline (1152 [680-1840] pg/mL) in NPC1 compared with controls (167 [82-372] pg/mL; P < .001). Elevated NfL levels were associated with more severe disease as assessed by both the 17-domain and 5-domain NPC Neurological Severity Score. Associations were also observed with ambulation, fine motor, speech, and swallowing scores. Although treatment with the investigational drug 2-hydroxypropyl-ß-cyclodextrin (adrabetadex) did not decrease CSF NfL levels, miglustat therapy over time was associated with a decrease (odds ratio = 0.77, 95% CI = 0.62-0.96). CONCLUSION: CSF NfL levels are increased in individuals with NPC1, associated with clinical disease severity, and decreased with miglustat therapy. These data suggest that NfL is a biomarker that may have utility in future therapeutic trials.

Outcomes in Previously Healthy Cryptococcal Meningoencephalitis Patients Treated With Pulse Taper Corticosteroids for Post-infectious Inflammatory Syndrome.

BACKGROUND: Cryptococcal meningoencephalitis (CM) is a major cause of mortality in immunosuppressed patients and previously healthy individuals. In the latter, a post-infectious inflammatory response syndrome (PIIRS) is associated with poor clinical response despite antifungal therapy and negative cerebrospinal fluid (CSF) cultures. Data on effective treatment are limited. METHODS: Between March 2015 and March 2020, 15 consecutive previously healthy patients with CM and PIIRS were treated with adjunctive pulse corticosteroid taper therapy (PCT) consisting of intravenous methylprednisolone 1 gm daily for 1 week followed by oral prednisone 1 mg/kg/day, tapered based on clinical and radiological response plus oral fluconazole. Montreal cognitive assessments (MOCA), Karnofsky performance scores, magnetic resonance imaging (MRI) brain scanning, ophthalmic and audiologic exams, and CSF parameters including cellular and soluble immune responses were compared at PIIRS diagnosis and after methylprednisolone completion. RESULTS: The median time from antifungal treatment to steroid initiation was 6 weeks. The most common symptoms at PIIRS diagnosis were altered mental status and vision changes. All patients demonstrated significant improvements in MOCA and Karnofsky scores at 1 month (P < .0003), which was accompanied by improvements in CSF glucose, white blood cell (WBC) count, protein, cellular and soluble inflammatory markers 1 week after receiving corticosteroids (CS) (P < .003). All patients with papilledema and visual field deficits also exhibited improvement (P < .0005). Five out of 7 patients who underwent audiological testing demonstrated hearing improvement. Brain MRI showed significant improvement of radiological findings (P = .001). CSF cultures remained negative. CONCLUSIONS: PCT in this small cohort of PIIRS was associated with improvements in CM-related complications with minimal toxicity in the acute setting.

Neurofilament light chain levels correlate with clinical measures in CLN3 disease.

PURPOSE: CLN3 disease is a neurodegenerative disorder with onset in childhood. It affects multiple functions at different developmental stages. Incomplete understanding of the pathophysiology hampers identification of cell and tissue biochemical compounds reflective of the disease process. As treatment approaches are being explored, more sensitive, objective, quantifiable, and clinically relevant biomarkers are needed. METHODS: We collected prospective biosamples from 21 phenotyped individuals with CLN3. We measured neurofilament light chain (NEFL) levels, a marker of neuronal damage, in cross-sectional CSF and serum samples from individuals with CLN3 and in pediatric non-CLN3 controls using two different assays. RESULTS: Cerebrospinal fluid (CSF) and serum NEFL levels are significantly higher in CLN3 (CSF: 2096 ± 1202; serum: 29.0 ± 18.0 pg/mL) versus similarly aged non-CLN3 (CSF: 345 ± 610; serum: 6.7 ± 3.2 pg/mL) samples. NEFL levels correlate with Unified Batten Disease Rating Scale and adaptive behavior composite scores, and magnetic resonance (MR) spectroscopy markers. NEFL levels from CSF and serum are strongly correlated (rp = 0.83; p < 0.0001). CONCLUSION: CSF and serum NEFL levels increase in multiple neurologic conditions. Here, we show that CSF and serum NEFL levels also increase in CLN3 (versus non-CLN3) and correlate with other disease-relevant measures. These findings suggest NEFL as a relevant and feasible biomarker for applications in CLN3 clinical trials and management.

Seizure phenotype in CLN3 disease and its relation to other neurologic outcome measures.

CLN3 disease is a pediatric neurodegenerative condition wherein seizures are common. The most common disease-causing variant is an ~1-kb deletion in CLN3. We investigated seizure phenotype in relation to genotype and to adaptive behavior, MR spectroscopy and CSF biochemical markers in a CLN3 cohort. We performed seizure phenotyping using clinical history, EEG, and the Unified Batten Disease Rating Scale (UBDRS) seizure score. We assessed correlations of seizure severity with disease severity (UBDRS capability), adaptive behavior composite score (ABC; Vineland-3), glutamate+glutamine+GABA and N-acetylaspartate+N-acetylaspartyl glutamate (MR spectroscopy), and CSF neurofilament light chain (NEFL) levels. In 20 participants, median age was 10.7 years (IQR = 7.8). Eighteen completed baseline EEG; 12 had a 1-year follow-up. Seizures were reported in 14 (8 1-kb deletion homozygotes), with median age at onset of 10.0 (IQR = 6.8). Epileptiform discharges were noted in 15 (9 homozygotes). Bilateral tonic clonic (n = 11) and nonmotor seizures (n = 7) were most common. UBDRS seizure score correlated with age (rp = 0.50; [0.08,0.77]; P = .02), UBDRS capability (rp = -0.57; [-0.81,-0.17]; P = .009) and ABC (rp = -0.66; [-0.85,-0.31]; P = .001) scores, glutamate+glutamine+GABA (rp = -0.54; [-0.80,-0.11]; P = .02) and N-acetylaspartate+N-acetylaspartyl glutamate (rp = -0.54; [-0.80,-0.11]; P = .02), and CSF NEFL (rp = 0.65; [0.29,0.85]; P = .002) levels. After controlling for age, correlations with ABC and CSF NEFL remained significant. In our CLN3 cohort, seizures and epileptiform discharges were frequent and often started by age 10 years without significant difference between genotypes. ABC and CSF NEFL correlate with UBDRS seizure score, reflecting the role of seizures in the neurodegenerative process. Longitudinal evaluations in a larger cohort are needed to confirm these findings.

Genetic model of MS severity predicts future accumulation of disability.

No genetic modifiers of multiple sclerosis (MS) severity have been independently validated, leading to a lack of insight into genetic determinants of the rate of disability progression. We investigated genetic modifiers of MS severity in prospectively acquired training (N = 205) and validation (N = 94) cohorts, using the following advances: (1) We focused on 113 genetic variants previously identified as related to MS severity; (2) We used a novel, sensitive outcome: MS Disease Severity Scale (MS-DSS); (3) Instead of validating individual alleles, we used a machine learning technique (random forest) that captures linear and complex nonlinear effects between alleles to derive a single Genetic Model of MS Severity (GeM-MSS). The GeM-MSS consists of 19 variants located in vicinity of 12 genes implicated in regulating cytotoxicity of immune cells, complement activation, neuronal functions, and fibrosis. GeM-MSS correlates with MS-DSS (r = 0.214; p = 0.043) in a validation cohort that was not used in the modeling steps. The recognized biology identifies novel therapeutic targets for inhibiting MS disability progression.

Molecular-based diagnosis of multiple sclerosis and its progressive stage.

OBJECTIVE: Biomarkers aid diagnosis, allow inexpensive screening of therapies, and guide selection of patient-specific therapeutic regimens in most internal medicine disciplines. In contrast, neurology lacks validated measurements of the physiological status, or dysfunction(s) of cells of the central nervous system (CNS). Accordingly, patients with chronic neurological diseases are often treated with a single disease-modifying therapy without understanding patient-specific drivers of disability. Therefore, using multiple sclerosis (MS) as an example of a complex polygenic neurological disease, we sought to determine whether cerebrospinal fluid (CSF) biomarkers are intraindividually stable, cell type-, disease- and/or process-specific, and responsive to therapeutic intervention. METHODS: We used statistical learning in a modeling cohort (n = 225) to develop diagnostic classifiers from DNA-aptamer-based measurements of 1,128 CSF proteins. An independent validation cohort (n = 85) assessed the reliability of derived classifiers. The biological interpretation resulted from in vitro modeling of primary or stem cell-derived human CNS cells and cell lines. RESULTS: The classifier that differentiates MS from CNS diseases that mimic MS clinically, pathophysiologically, and on imaging achieved a validated area under the receiver operating characteristic curve (AUROC) of 0.98, whereas the classifier that differentiates relapsing-remitting from progressive MS achieved a validated AUROC of 0.91. No classifiers could differentiate primary progressive from secondary progressive MS better than random guessing. Treatment-induced changes in biomarkers greatly exceeded intraindividual and technical variabilities of the assay. INTERPRETATION: CNS biological processes reflected by CSF biomarkers are robust, stable, disease specific, or even disease stage specific. This opens opportunities for broad utilization of CSF biomarkers in drug development and precision medicine for CNS disorders. Ann Neurol 2017;82:795-812.

Spinal Arachnoiditis as a Complication of Cryptococcal Meningoencephalitis in Non-HIV Previously Healthy Adults.

BACKGROUND: Cryptococcus can cause meningoencephalitis (CM) among previously healthy non-HIV adults. Spinal arachnoiditis is under-recognized, since diagnosis is difficult with concomitant central nervous system (CNS) pathology. METHODS: We describe 6 cases of spinal arachnoiditis among 26 consecutively recruited CM patients with normal CD4 counts who achieved microbiologic control. We performed detailed neurological exams, cerebrospinal fluid (CSF) immunophenotyping and biomarker analysis before and after adjunctive immunomodulatory intervention with high dose pulse corticosteroids, affording causal inference into pathophysiology. RESULTS: All 6 exhibited severe lower motor neuron involvement in addition to cognitive changes and gait disturbances from meningoencephalitis. Spinal involvement was associated with asymmetric weakness and urinary retention. Diagnostic specificity was improved by MRI imaging which demonstrated lumbar spinal nerve root enhancement and clumping or lesions. Despite negative fungal cultures, CSF inflammatory biomarkers, sCD27 and sCD21, as well as the neuronal damage biomarker, neurofilament light chain (NFL), were elevated compared to healthy donor (HD) controls. Elevations in these biomarkers were associated with clinical symptoms and showed improvement with adjunctive high dose pulse corticosteroids. CONCLUSIONS: These data suggest that a post-infectious spinal arachnoiditis is an important complication of CM in previously healthy individuals, requiring heightened clinician awareness. Despite microbiological control, this syndrome causes significant pathology likely due to increased inflammation and may be amenable to suppressive therapeutics.

Cerebrospinal fluid markers reveal intrathecal inflammation in progressive multiple sclerosis.

OBJECTIVE: The management of complex patients with neuroimmunological diseases is hindered by an inability to reliably measure intrathecal inflammation. Currently implemented laboratory tests developed >40 years ago either are not dynamic or fail to capture low levels of central nervous system (CNS) inflammation. Therefore, we aimed to identify and validate biomarkers of CNS inflammation in 2 blinded, prospectively acquired cohorts of untreated patients with neuroimmunological diseases and embedded controls, with the ultimate goal of developing clinically useful tools. METHODS: Because biomarkers with maximum utility reflect immune phenotypes, we included an assessment of cell specificity in purified primary immune cells. Biomarkers were quantified by optimized electrochemiluminescent immunoassays. RESULTS: Among markers with cell-specific secretion, soluble CD27 is a validated biomarker of intrathecal T-cell activation, with an area under the receiver operating characteristic curve of 0.97. Comparing the quantities of cerebrospinal fluid (CSF) immune cells and their respective cell-specific soluble biomarkers (released by CSF cells as well as their counterparts in CNS tissue) provided invaluable information about stationary CNS immune responses, previously attainable via brain biopsy only. Unexpectedly, progressive and relapsing-remitting multiple sclerosis (MS) patients have comparable numbers of activated intrathecal T and B cells, which are preferentially embedded in CNS tissue in the former group. INTERPRETATION: The cell-specific biomarkers of intrathecal inflammation may improve diagnosis and management of neuroimmunological diseases and provide pharmacodynamic markers for future therapeutic developments in patients with intrathecal inflammation that is not captured by imaging, such as in progressive MS.

Comprehensive immunophenotyping of cerebrospinal fluid cells in patients with neuroimmunological diseases.

We performed unbiased, comprehensive immunophenotyping of cerebrospinal fluid (CSF) and blood leukocytes in 221 subjects referred for the diagnostic work-up of neuroimmunological disorders to obtain insight about disease-specific phenotypes of intrathecal immune responses. Quantification of 14 different immune cell subsets, coupled with the assessment of their activation status, revealed physiological differences between intrathecal and systemic immunity, irrespective of final diagnosis. Our data are consistent with a model where the CNS shapes intrathecal immune responses to provide effective protection against persistent viral infections, especially by memory T cells, plasmacytoid dendritic cells, and CD56(bright) NK cells. Our data also argue that CSF immune cells do not simply reflect cells recruited from the periphery. Instead, they represent a mixture of cells that are recruited from the blood, have been activated intrathecally and leave the CNS after performing effector functions. Diagnosis-specific differences provide mechanistic insight into the disease process in the defined subtypes of multiple sclerosis (MS), neonatal onset multisystem inflammatory disease, and Aicardi-Goutières syndrome. This analysis also determined that secondary-progressive MS patients are immunologically closer to relapsing-remitting patients as compared with patients with primary-progressive MS. Because CSF immunophenotyping captures the biology of the intrathecal inflammatory processes, it has the potential to guide optimal selection of immunomodulatory therapies in individual patients and monitor their efficacy. Our study adds to the increasing number of publications that demonstrate poor correlation between systemic and intrathecal inflammatory biomarkers in patients with neuroimmunological diseases and stresses the importance of studying immune responses directly in the intrathecal compartment.

Retinoid X receptor activation reverses age-related deficiencies in myelin debris phagocytosis and remyelination.

The efficiency of central nervous system remyelination declines with age. This is in part due to an age-associated decline in the phagocytic removal of myelin debris, which contains inhibitors of oligodendrocyte progenitor cell differentiation. In this study, we show that expression of genes involved in the retinoid X receptor pathway are decreased with ageing in both myelin-phagocytosing human monocytes and mouse macrophages using a combination of in vivo and in vitro approaches. Disruption of retinoid X receptor function in young macrophages, using the antagonist HX531, mimics ageing by reducing myelin debris uptake. Macrophage-specific RXRα (Rxra) knockout mice revealed that loss of function in young mice caused delayed myelin debris uptake and slowed remyelination after experimentally-induced demyelination. Alternatively, retinoid X receptor agonists partially restored myelin debris phagocytosis in aged macrophages. The agonist bexarotene, when used in concentrations achievable in human subjects, caused a reversion of the gene expression profile in multiple sclerosis patient monocytes to a more youthful profile and enhanced myelin debris phagocytosis by patient cells. These results reveal the retinoid X receptor pathway as a positive regulator of myelin debris clearance and a key player in the age-related decline in remyelination that may be targeted by available or newly-developed therapeutics.

Evolution of tumefactive lesions in multiple sclerosis: a 12-year study with serial imaging in a single patient.

We describe the acute presentation and the long-term evolution of recurrent tumefactive lesions (TLs) in a patient with relapsing-remitting multiple sclerosis. Five TLs occurred on three different occasions over a period of 12 years and these were followed by 73 serial magnetic resonance images (MRI). TL evolution was described by means of magnetization transfer imaging (MTI) and cerebrospinal fluid tissue specific imaging (TSI) over the follow-up period. During the study period, the patient had three clinical relapses with only minimal disability progression. MTI demonstrated that only the peripheral portion of each TL reverted to pre-lesional MT ratios within six months' post-enhancement. Recurring TLs may present a similar pattern of evolution that may be associated with a long-term favourable clinical outcome.

Unexpected role for granzyme K in CD56bright NK cell-mediated immunoregulation of multiple sclerosis.

Functional NK cell deficiencies are associated with autoimmune diseases, including multiple sclerosis. NK cells can promote or inhibit adaptive immunity via either cytokine production or cytotoxicity toward immature dendritic cells and activated T cells. In humans, this immunoregulatory role resides in the CD56(bright) NK cell subset, which is selectively expanded by daclizumab, a CD25-blocking Ab that suppresses multiple sclerosis-associated inflammation. The objective of this study was to investigate the molecular mechanisms underlying the cytotoxicity of NK cells toward activated T cells. We demonstrated that NK cells induce caspase-independent apoptosis that requires NK cell degranulation and causes mitochondrial dysfunction in activated T cells. Although both granzyme A and granzyme K (GrK) can mediate this form of apoptosis, quantitatively we observed preferential transfer of GrK to target cells. Consequently, gene silencing of GrK in the NK-92 cell line, which retains functional characteristics of CD56(bright) NK cells, profoundly inhibited the ability of NK-92 cells to kill activated syngeneic T cells. Finally, we demonstrated that daclizumab treatment significantly enhanced this newly defined mechanism of cytotoxicity by CD56(bright) NK cells. Our study describes the important physiological role that GrK plays in immunoregulation of adaptive immunity in humans and indicates that therapeutic exploitation of this pathway is beneficial in controlling autoimmunity.

An ANXA11 P93S variant dysregulates TDP-43 and causes corticobasal syndrome.

As genetic testing has become more accessible and affordable, variants of uncertain significance (VUS) are increasingly identified, and determining whether these variants play causal roles in disease is a major challenge. The known disease-associated Annexin A11 (ANXA11) mutations result in ANXA11 aggregation, alterations in lysosomal-RNA granule co-trafficking, and TDP-43 mis-localization and present as amyotrophic lateral sclerosis or frontotemporal dementia. We identified a novel VUS in ANXA11 (P93S) in a kindred with corticobasal syndrome and unique radiographic features that segregated with disease. We then queried neurodegenerative disorder clinic databases to identify the phenotypic spread of ANXA11 mutations. Multi-modal computational analysis of this variant was performed and the effect of this VUS on ANXA11 function and TDP-43 biology was characterized in iPSC-derived neurons. Single-cell sequencing and proteomic analysis of iPSC-derived neurons and microglia were used to determine the multiomic signature of this VUS. Mutations in ANXA11 were found in association with clinically diagnosed corticobasal syndrome, thereby establishing corticobasal syndrome as part of ANXA11 clinical spectrum. In iPSC-derived neurons expressing mutant ANXA11, we found decreased colocalization of lysosomes and decreased neuritic RNA as well as decreased nuclear TDP-43 and increased formation of cryptic exons compared to controls. Multiomic assessment of the P93S variant in iPSC-derived neurons and microglia indicates that the pathogenic omic signature in neurons is modest compared to microglia. Additionally, omic studies reveal that immune dysregulation and interferon signaling pathways in microglia are central to disease. Collectively, these findings identify a new pathogenic variant in ANXA11, expand the range of clinical syndromes caused by ANXA11 mutations, and implicate both neuronal and microglia dysfunction in ANXA11 pathophysiology. This work illustrates the potential for iPSC-derived cellular models to revolutionize the variant annotation process and provides a generalizable approach to determining causality of novel variants across genes.

Prognostic performance of blood neurofilament light chain protein in hospitalized COVID-19 patients without major central nervous system manifestations: an individual participant data meta-analysis.

BACKGROUND AND AIMS: To investigate the prognostic value of blood neurofilament light chain protein (NfL) levels in the acute phase of coronavirus disease 2019 (COVID-19). METHODS: We conducted an individual participant data (IPD) meta-analysis after screening on MEDLINE and Scopus to May 23rd 2022. We included studies with hospitalized adult COVID-19 patients without major COVID-19-associated central nervous system (CNS) manifestations and with a measurement of blood NfL in the acute phase as well as data regarding at least one clinical outcome including intensive care unit (ICU) admission, need of mechanical ventilation (MV) and death. We derived the age-adjusted measures NfL Z scores and conducted mixed-effects modelling to test associations between NfL Z scores and other variables, encompassing clinical outcomes. Summary receiver operating characteristic curves (SROCs) were used to calculate the area under the curve (AUC) for blood NfL. RESULTS: We identified 382 records, of which 7 studies were included with a total of 669 hospitalized COVID-19 cases (mean age 66.2 ± 15.0 years, 68.1% males). Median NfL Z score at admission was elevated compared to the age-corrected reference population (2.37, IQR: 1.13-3.06, referring to 99th percentile in healthy controls). NfL Z scores were significantly associated with disease duration and severity. Higher NfL Z scores were associated with a higher likelihood of ICU admission, need of MV, and death. SROCs revealed AUCs of 0.74, 0.80 and 0.71 for mortality, need of MV and ICU admission, respectively. CONCLUSIONS: Blood NfL levels were elevated in the acute phase of COVID-19 patients without major CNS manifestations and associated with clinical severity and poor outcome. The marker might ameliorate the performance of prognostic multivariable algorithms in COVID-19.

Chromosomal microarray analysis supplements exome sequencing to diagnose children with suspected inborn errors of immunity.

Purpose: Though copy number variants (CNVs) have been suggested to play a significant role in inborn errors of immunity (IEI), the precise nature of this role remains largely unexplored. We sought to determine the diagnostic contribution of CNVs using genome-wide chromosomal microarray analysis (CMA) in children with IEI. Methods: We performed exome sequencing (ES) and CMA for 332 unrelated pediatric probands referred for evaluation of IEI. The analysis included primary, secondary, and incidental findings. Results: Of the 332 probands, 134 (40.4%) received molecular diagnoses. Of these, 116/134 (86.6%) were diagnosed by ES alone. An additional 15/134 (11.2%) were diagnosed by CMA alone, including two likely de novo changes. Three (2.2%) participants had diagnostic molecular findings from both ES and CMA, including two compound heterozygotes and one participant with two distinct diagnoses. Half of the participants with CMA contribution to diagnosis had CNVs in at least one non-immune gene, highlighting the clinical complexity of these cases. Overall, CMA contributed to 18/134 diagnoses (13.4%), increasing the overall diagnostic yield by 15.5% beyond ES alone. Conclusion: Pairing ES and CMA can provide a comprehensive evaluation to clarify the complex factors that contribute to both immune and non-immune phenotypes. Such a combined approach to genetic testing helps untangle complex phenotypes, not only by clarifying the differential diagnosis, but in some cases by identifying multiple diagnoses contributing to the overall clinical presentation.

Evolution of the blood-brain barrier in newly forming multiple sclerosis lesions.

OBJECTIVE: Multiple sclerosis (MS) lesions develop around small, inflamed veins. New lesions enhance with gadolinium on magnetic resonance imaging (MRI), reflecting disruption of the blood-brain barrier (BBB). Single time point results from pathology and standard MRI cannot capture the spatiotemporal expansion of lesions. We investigated the development and expansion of new MS lesions, focusing on the dynamics of BBB permeability. METHODS: We performed dynamic contrast-enhanced (DCE) MRI in relapsing-remitting MS. We obtained data over 65 minutes, during and after gadolinium injection. We labeled spatiotemporal enhancement dynamics as centrifugal when initially central enhancement expanded outward and centripetal when initially peripheral enhancement gradually filled the center. RESULTS: We detected 34 enhancing lesions in 200 DCE-MRI scans. In 65%, enhancement first appeared as a closed ring; in 18%, as a nodule; and in 18%, as an open ring. Lesions with initially nodular enhancement were smaller than those initially enhancing as rings (p < 0.0001). All initially nodular lesions enhanced centrifugally, whereas initially ringlike lesions enhanced centripetally, becoming nodular if small (82%) or nearly nodular if larger (18%). Open-ring lesions were periventricular or juxtacortical and enhanced centripetally. Centrifugally enhancing lesions evolved into centripetally enhancing lesions over several days. INTERPRETATION: The rapid change of enhancement dynamics from centrifugal to centripetal reflects the outward growth of MS lesions around their central vein and suggests that factors mediating lesion growth and tissue repair derive from different locations at different times. We propose a model of new lesion growth that unites our imaging observations with existing pathology data.

An IL-2 paradox: blocking CD25 on T cells induces IL-2-driven activation of CD56(bright) NK cells.

Daclizumab (Dac), an Ab against the IL-2R alpha-chain, inhibits brain inflammation in patients with multiple sclerosis, while expanding CD56(bright) immunoregulatory NK cells in vivo. We hypothesized that this unexpected expansion is paradoxically IL-2 driven; caused by the increased availability of T cell-derived IL-2 for NK cell signaling. To this end, we performed ex vivo functional analyses of CD56(bright) NK cells and T cells from patients in clinical trials with Dac. We developed in vitro models to investigate mechanisms for ex vivo observations. We observed that Dac treatment caused decreased numbers and proliferation of FoxP3(+) T regulatory cells (Tregs), a model T cell population known to be dependent on IL-2 for proliferation and survival. As anticipated, Dac therapy inhibited IL-2 signaling in all T cells; however, we also observed functional adaptation of T cells to low IL-2 signal in vivo, characterized by the concomitant enhancement of IL-7 signaling on all T cells and parallel increase of CD127 expression by Tregs. In contrast, IL-2 signaling on CD56(bright) NK cells was not inhibited by Dac and their in vivo proliferation and cytotoxicity actually increased. Mechanistic studies indicated that the activation of CD56(bright) NK cells was likely IL-2 driven, as low doses of IL-2, but not IL-15, mimicked this activation in vitro. Our study provides insight into the role that IL-2 and CD25 play in functional regulation of two important immunoregulatory cell populations in humans: FoxP3(+) Tregs and CD56(bright) NK cells.

Current Status and Future Opportunities in Modeling Clinical Characteristics of Multiple Sclerosis.

Development of effective treatments requires understanding of disease mechanisms. For diseases of the central nervous system (CNS), such as multiple sclerosis (MS), human pathology studies and animal models tend to identify candidate disease mechanisms. However, these studies cannot easily link the identified processes to clinical outcomes, such as MS severity, required for causality assessment of candidate mechanisms. Technological advances now allow the generation of thousands of biomarkers in living human subjects, derived from genes, transcripts, medical images, and proteins or metabolites in biological fluids. These biomarkers can be assembled into computational models of clinical value, provided such models are generalizable. Reproducibility of models increases with the technical rigor of the study design, such as blinding, control implementation, the use of large cohorts that encompass the entire spectrum of disease phenotypes and, most importantly, model validation in independent cohort(s). To facilitate the growth of this important research area, we performed a meta-analysis of publications (n = 302) that model MS clinical outcomes extracting effect sizes, while also scoring the technical quality of the study design using predefined criteria. Finally, we generated a Shiny-App-based website that allows dynamic exploration of the data by selective filtering. On average, the published studies fulfilled only one of the seven criteria of study design rigor. Only 15.2% of the studies used any validation strategy, and only 8% used the gold standard of independent cohort validation. Many studies also used small cohorts, e.g., for magnetic resonance imaging (MRI) and blood biomarker predictors, the median sample size was <100 subjects. We observed inverse relationships between reported effect sizes and the number of study design criteria fulfilled, expanding analogous reports from non-MS fields, that studies that fail to limit bias overestimate effect sizes. In conclusion, the presented meta-analysis represents a useful tool for researchers, reviewers, and funders to improve the design of future modeling studies in MS and to easily compare new studies with the published literature. We expect that this will accelerate research in this important area, leading to the development of robust models with proven clinical value.

Confounder-adjusted MRI-based predictors of multiple sclerosis disability.

Introduction: Both aging and multiple sclerosis (MS) cause central nervous system (CNS) atrophy. Excess brain atrophy in MS has been interpreted as "accelerated aging." Current paper tests an alternative hypothesis: MS causes CNS atrophy by mechanism(s) different from physiological aging. Thus, subtracting effects of physiological confounders on CNS structures would isolate MS-specific effects. Methods: Standardized brain MRI and neurological examination were acquired prospectively in 646 participants enrolled in ClinicalTrials.gov Identifier: NCT00794352 protocol. CNS volumes were measured retrospectively, by automated Lesion-TOADS algorithm and by Spinal Cord Toolbox, in a blinded fashion. Physiological confounders identified in 80 healthy volunteers were regressed out by stepwise multiple linear regression. MS specificity of confounder-adjusted MRI features was assessed in non-MS cohort (n = 158). MS patients were randomly split into training (n = 277) and validation (n = 131) cohorts. Gradient boosting machine (GBM) models were generated in MS training cohort from unadjusted and confounder-adjusted CNS volumes against four disability scales. Results: Confounder adjustment highlighted MS-specific progressive loss of CNS white matter. GBM model performance decreased substantially from training to cross-validation, to independent validation cohorts, but all models predicted cognitive and physical disability with low p-values and effect sizes that outperform published literature based on recent meta-analysis. Models built from confounder-adjusted MRI predictors outperformed models from unadjusted predictors in the validation cohort. Conclusion: GBM models from confounder-adjusted volumetric MRI features reflect MS-specific CNS injury, and due to stronger correlation with clinical outcomes compared to brain atrophy these models should be explored in future MS clinical trials.