Chemokine-mediated cell migration into the central nervous system in progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) has been associated with different forms of immune compromise. This study analyzes the chemokine signals and attracted immune cells in cerebrospinal fluid (CSF) during PML to define immune cell subpopulations relevant for the PML immune response. In addition to chemokines that indicate a general state of inflammation, like CCL5 and CXCL10, the CSF of PML patients specifically contains CCL2 and CCL4. Single-cell transcriptomics of CSF cells suggests an enrichment of distinct CD4+ and CD8+ T cells expressing chemokine receptors CCR2, CCR5, and CXCR3, in addition to ITGA4 and the genetic PML risk genes STXBP2 and LY9. This suggests that specific immune cell subpopulations migrate into the central nervous system to mitigate PML, and their absence might coincide with PML development. Monitoring them might hold clues for PML risk, and boosting their recruitment or function before therapeutic immune reconstitution might improve its risk-benefit ratio.

Absence of JC polyomavirus in stool samples of patients with multiple sclerosis despite high anti-JCV antibodies in serum.

BACKGROUND: Natalizumab is an effective treatment for relapsing multiple sclerosis (MS). During therapy, individuals are at increased risk of developing progressive multifocal leukoencephalopathy (PML). So far, the relevant reservoir for PML-type JC polyomavirus (JCV) remains elusive. We here tested if the detection of JCV-DNA in stool of persons with MS treated with natalizumab could be a future tool for PML risk assessment. METHODS: The presence of JCV-DNA in stool, urine, and whole blood of MS patients treated with natalizumab and known serum anti-JCV antibodies index values (IV) was studied. Different DNA extraction methods, real-time (RT) and droplet digital (dd) PCR techniques were compared. JCV isolates were screened for PML-associated variants by sequencing. RESULTS: Thirty MS patients treated with natalizumab were screened. For 21 patients, blood, stool, and urine samples were available. These patients were stratified according to their serum anti-JCV antibody IV (high (>1.5, n = 12); medium (1.5-0.9, n = 2); low (<0.9, n = 1); negative (n = 6)). JCV-DNA could not be detected in the whole blood or stool samples. Four urine samples had measurable JCV-DNA, ranging from 1.71×104-1.07×108 international units (IU)/mL detected by RT-PCR, corresponding to 4.62×104-9.85×106 copies/mL measured by ddPCR. All JCV variants were wild-type and derived from patients with high antibody IV. CONCLUSION: Stool-specific DNA extraction methods provided the highest quality of DNA, while the sensitivity of ddPCR and RT- PCR was comparable. Our findings do not support assessing stool samples for PML risk stratification in persons with MS. Further studies are needed to explore where PML-associated viral variants arise.

Comprehensive proteomic analysis of JC polyomavirus-infected human astrocytes and their extracellular vesicles.

IMPORTANCE: Progressive multifocal leukoencephalopathy is a crimpling demyelinating disease of the central nervous system caused by JC polyomavirus (JCPyV). Much about JCPyV propagation in the brain remains obscure because of a lack of proper animal models to study the virus in the context of the disease, thus hampering efforts toward the development of new antiviral strategies. Here, having established a robust and representative model of JCPyV infection in human-induced pluripotent stem cell-derived astrocytes, we are able to fully characterize the effect of JCPyV on the biology of the cells and show that the proteomic signature observed for JCPyV-infected astrocytes is extended to extracellular vesicles (EVs). These data suggest that astrocyte-derived EVs found in body fluids might serve as a rich source of information relevant to JCPyV infection in the brain, opening avenues toward better understanding the pathogenesis of the virus and, ultimately, the identification of new antiviral targets.

High levels of endothelial ICAM-1 prohibit natalizumab mediated abrogation of CD4+ T cell arrest on the inflamed BBB under flow in vitro.

INTRODUCTION: The humanized anti-α4 integrin blocking antibody natalizumab (NTZ) is an effective treatment for relapsing-remitting multiple sclerosis (RRMS) that is associated with the risk of progressive multifocal leukoencephalopathy (PML). While extended interval dosing (EID) of NTZ reduces the risk for PML, the minimal dose of NTZ required to maintain its therapeutic efficacy remains unknown. OBJECTIVE: Here we aimed to identify the minimal NTZ concentration required to inhibit the arrest of human effector/memory CD4+ T cell subsets or of PBMCs to the blood-brain barrier (BBB) under physiological flow in vitro. RESULTS: Making use of three different human in vitro BBB models and in vitro live-cell imaging we observed that NTZ mediated inhibition of α4-integrins failed to abrogate T cell arrest to the inflamed BBB under physiological flow. Complete inhibition of shear resistant T cell arrest required additional inhibition of ß2-integrins, which correlated with a strong upregulation of endothelial intercellular adhesion molecule (ICAM)-1 on the respective BBB models investigated. Indeed, NTZ mediated inhibition of shear resistant T cell arrest to combinations of immobilized recombinant vascular cell adhesion molecule (VCAM)-1 and ICAM-1 was abrogated in the presence of tenfold higher molar concentrations of ICAM-1 over VCAM-1. Also, monovalent NTZ was less potent than bivalent NTZ in inhibiting T cell arrest to VCAM-1 under physiological flow. In accordance with our previous observations ICAM-1 but not VCAM-1 mediated T cell crawling against the direction of flow. CONCLUSION: Taken together, our in vitro observations show that high levels of endothelial ICAM-1 abrogate NTZ mediated inhibition of T cell interaction with the BBB. EID of NTZ in MS patients may thus require consideration of the inflammatory status of the BBB as high levels of ICAM-1 may provide an alternative molecular cue allowing for pathogenic T cell entry into the CNS in the presence of NTZ.

A single-cell analysis framework allows for characterization of CSF leukocytes and their tissue of origin in multiple sclerosis.

Peripheral central nervous system (CNS)-infiltrating lymphocytes are a hallmark of relapsing-remitting multiple sclerosis. Tissue-resident memory T cells (TRM) not only populate the healthy CNS parenchyma but also are suspected to contribute to multiple sclerosis pathology. Because cerebrospinal fluid (CSF), unlike CNS parenchyma, is accessible for diagnostics, we evaluated whether human CSF, apart from infiltrating cells, also contains TRM cells and CNS-resident myeloid cells draining from the parenchyma or border tissues. Using deep generative models, we integrated 41 CSF and 14 CNS parenchyma single-cell RNA sequencing (scRNAseq) samples from eight independent studies, encompassing 120,629 cells. By comparing CSF immune cells collected during multiple sclerosis relapse with cells collected during therapeutic very late antigen-4 blockade, we could identify immune subsets with tissue provenance across multiple lineages, including CNS border-associated macrophages, CD8 and CD4 TRM cells, and tissue-resident natural killer cells. All lymphocytic CNS-resident cells shared expression of CXCR6 but showed differential ITGAE expression (encoding CD103). A common signature defined CD4 and CD8 TRM cells by expression of ZFP36L2, DUSP1, and ID2. We further developed a user interface-driven application based on this analysis framework for atlas-level cell identity transfer onto new CSF scRNAseq data. Together, these results define CNS-resident immune cells involved in multiple sclerosis pathology that can be detected and monitored in CSF. Targeting these cell populations might be promising to modulate immunopathology in progressive multiple sclerosis and other neuroinflammatory diseases.

Remyelination trial failures: Repercussions of ignoring neurorehabilitation and exercise in repair.

Several putative neurorestorative therapies in multiple sclerosis (MS) have recently failed; this includes high-dose biotin, bexarotene, a retinoic acid receptor gamma agonist, and opicinumab (anti-LINGO-1). Are these failures biological or due to poor trial design? We argue that the failure to include exercise in these trials and selecting participants without the capacity for repair may explain these disappointing results. We propose the need for mapping the biological mechanisms of recovery within trials, understanding the critical window when remyelination/repair occurs in terms of targeting interventions at the right time and selecting subjects who are capable of repair. We also make the case for testing combinations that include other pro-repair interventions such as exercise, Nrf2 inducers and possibly neurostimulation. The MS community can't afford for any more treatments to fail because of poor trial design and ignoring biology.

Assessing Blood-Based Biomarkers to Define a Therapeutic Window for Natalizumab.

Natalizumab is a monoclonal antibody that binds CD49d. Although it is one of the most effective treatments for Relapsing-Remitting Multiple Sclerosis (RRMS), a dosing regimen has not been optimized for safety and efficacy in individual patients. We aimed to identify biomarkers to monitor Natalizumab treatment and to establish a personalized dose utilizing an ongoing longitudinal study in 29 RRMS patients under Natalizumab with standard interval dose (SD) of 300 mg/4wks or extended interval dose (EID) of 300 mg/6wks. Blood samples were analyzed by flow cytometry to determine CD49d saturation and expression in several T and B lymphocytes subpopulations. Each patient was analyzed at two different timepoints separated by 3 Natalizumab administrations. Natalizumab and sVCAM-1 levels in serum were also analyzed using ELISA. To determine the reproducibility of various markers, two different timepoints were compared and no significant differences were observed for CD49d expression nor for saturation; SD patients had higher saturation levels (~80%) than EID patients (~60%). A positive correlation exists between CD49d saturation and Natalizumab serum levels. CD49d expression and saturation are stable parameters that could be used as biomarkers in the immunomonitoring of Natalizumab treatment. Moreover, Natalizumab and sVCAM-1 serum levels could be used to optimize an individual's dosing schedule.

Evaluation of natalizumab pharmacokinetics and pharmacodynamics with standard and extended interval dosing.

BACKGOUND: Natalizumab (NTZ) is a highly efficacious therapeutic for multiple sclerosis (MS), but treatment is associated with an increased risk of progressive multifocal leukoencephalopathy (PML). Extended interval dosing (EID) of NTZ has been proposed as an alternative dosing strategy to reduce PML risk. Pharmacokinetic (PK) and pharmacodynamic (PD) profiles of standard interval dosing (SID) and EID under real-world circumstances remain poorly characterized. OBJECTIVE: To evaluate PK and PD parameters of NTZ for SID and EID in the context of patient and treatment characteristics. METHODS: An observational cohort study was conducted to measure NTZ serum concentrations in MS patients at SID and EID nadir timepoints. NTZ occupancy of α4-integrin receptor sites, and cell surface expression of α4-integrin, was also measured. Patient body weight, age, and treatment exposure metrics were collected. RESULTS: NTZ serum concentrations were lower for EID than SID (mean = 18.2 versus 35.7 µg/ml, respectively; p < 0.001). Patient body weight, age, and treatment duration impacted concentrations with SID, though their influences were reduced or absent with EID. α4-integrin receptor occupancy by NTZ was lower for EID than SID (mean = 78.2 versus 87.4%, respectively; p < 0.001). Body weight impacted α4-integrin receptor occupancy differentially for EID versus SID. α4-integrin cell surface expression was modestly higher for EID than SID (267.2 versus 238.1 MFI, respectively; p < 0.001). CONCLUSIONS: EID of NTZ reduces nadir serum drug levels and α4-integrin receptor occupancy, as well as increases α4-integrin cell surface expression. The resulting increase in the number of open α4-intregrin receptors may enhance immune surveillance of JCV and prevention of PML. Body weight plays a significant role in the pharmacokinetic and pharmacodynamic responses to NTZ treatment. Further research is warranted to help establish pharmacological thresholds of NTZ efficacy and safety, which could help guide decision-making for dosing of NTZ.

A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity.

In obesity, inflammation of white adipose tissue (AT) is associated with diminished generation of beige adipocytes ('beige adipogenesis'), a thermogenic and energy-dissipating function mediated by beige adipocytes that express the uncoupling protein UCP1. Here we delineated an inflammation-driven inhibitory mechanism of beige adipogenesis in obesity that required direct adhesive interactions between macrophages and adipocytes mediated by the integrin α4 and its counter-receptor VCAM-1, respectively; expression of the latter was upregulated in obesity. This adhesive interaction reciprocally and concomitantly modulated inflammatory activation of macrophages and downregulation of UCP1 expression dependent on the kinase Erk in adipocytes. Genetic or pharmacological inactivation of the integrin α4 in mice resulted in elevated expression of UCP1 and beige adipogenesis of subcutaneous AT in obesity. Our findings, established in both mouse systems and human systems, reveal a self-sustained cycle of inflammation-driven impairment of beige adipogenesis in obesity.

Pilomotor function is impaired in patients with Parkinson's disease: A study of the adrenergic axon-reflex response and autonomic functions.

INTRODUCTION: Autonomic nervous system disturbances including sweating abnormalities and cardiovascular symptoms are frequent in Parkinson's disease (PD) and often precede motor involvement. Cholinergic vasomotor and sudomotor skin nerves are impaired in patients with PD even at early disease stages. We hypothesized that adrenergic pilomotor nerve function is similarly impaired in early PD and might constitute a novel diagnostic target. METHODS: We conducted a study in 12 PD patients (Hoehn&Yahr 1-2) and 12 healthy control subjects. Pilomotor function was evaluated after iontophoresis of phenylephrine on the dorsal forearm to elicit axon-reflex mediated pilomotor erection (goose bumps). Silicone impressions were obtained, scanned and quantified for pilomotor muscle impressions by number, area and axon-reflex spread. Vasomotor function was evaluated using laser Doppler flowmetry and sudomotor function via sympathetic skin response. Cardiac autonomic function was assessed via heart rate variability. Severity of autonomic symptoms was evaluated using the Scales for Outcomes in Parkinson's disease-Autonomic questionnaire. RESULTS: Pilomotor response was reduced in PD patients compared to control subjects (impression number: 12.2 ± 8.2 vs. 16.5 ± 5.9, p < 0.05; impression area: 10.8 ± 2.2 mm2 vs. 24.8 ± 3.1 mm2, p < 0.01; axon-reflex spread: 89.0 ± 10.6 mm2 vs. 185.9 ± 10.8 mm2, p < 0.01) and correlated negatively with severity of autonomic symptoms (p < 0.01). Similarly, sudomotor (p < 0.01) and vasomotor (p < 0.05) but not cardiac autonomic (p = n.s.) function were reduced in PD patients versus control subjects. CONCLUSION: Pilomotor function is impaired in early stages of PD. Pilomotor axon-reflex assessment might be useful in the investigation of disease related pathology and supplement other clinical markers of autonomic neuropathy in PD.

JC Polyomavirus Abundance and Distribution in Progressive Multifocal Leukoencephalopathy (PML) Brain Tissue Implicates Myelin Sheath in Intracerebral Dissemination of Infection.

Over half of adults are seropositive for JC polyomavirus (JCV), but rare individuals develop progressive multifocal leukoencephalopathy (PML), a demyelinating JCV infection of the central nervous system. Previously, PML was primarily seen in immunosuppressed patients with AIDS or certain cancers, but it has recently emerged as a drug safety issue through its association with diverse immunomodulatory therapies. To better understand the relationship between the JCV life cycle and PML pathology, we studied autopsy brain tissue from a 70-year-old psoriasis patient on the integrin alpha-L inhibitor efalizumab following a ~2 month clinical course of PML. Sequence analysis of lesional brain tissue identified PML-associated viral mutations in regulatory (non-coding control region) DNA, capsid protein VP1, and the regulatory agnoprotein, as well as 9 novel mutations in capsid protein VP2, indicating rampant viral evolution. Nine samples, including three gross PML lesions and normal-appearing adjacent tissues, were characterized by histopathology and subject to quantitative genomic, proteomic, and molecular localization analyses. We observed a striking correlation between the spatial extent of demyelination, axonal destruction, and dispersion of JCV along white matter myelin sheath. Our observations in this case, as well as in a case of PML-like disease in an immunocompromised rhesus macaque, suggest that long-range spread of polyomavirus and axonal destruction in PML might involve extracellular association between virus and the white matter myelin sheath.

Brain Endothelial- and Epithelial-Specific Interferon Receptor Chain 1 Drives Virus-Induced Sickness Behavior and Cognitive Impairment.

Sickness behavior and cognitive dysfunction occur frequently by unknown mechanisms in virus-infected individuals with malignancies treated with type I interferons (IFNs) and in patients with autoimmune disorders. We found that during sickness behavior, single-stranded RNA viruses, double-stranded RNA ligands, and IFNs shared pathways involving engagement of melanoma differentiation-associated protein 5 (MDA5), retinoic acid-inducible gene 1 (RIG-I), and mitochondrial antiviral signaling protein (MAVS), and subsequently induced IFN responses specifically in brain endothelia and epithelia of mice. Behavioral alterations were specifically dependent on brain endothelial and epithelial IFN receptor chain 1 (IFNAR). Using gene profiling, we identified that the endothelia-derived chemokine ligand CXCL10 mediated behavioral changes through impairment of synaptic plasticity. These results identified brain endothelial and epithelial cells as natural gatekeepers for virus-induced sickness behavior, demonstrated tissue specific IFNAR engagement, and established the CXCL10-CXCR3 axis as target for the treatment of behavioral changes during virus infection and type I IFN therapy.

Feasibility of the use of combinatorial chemokine arrays to study blood and CSF in multiple sclerosis.

Meningeal inflammation, including the presence of semi-organized tertiary lymphoid tissue, has been associated with cortical pathology at autopsy in secondary progressive multiple sclerosis (SPMS). Accessible and robust biochemical markers of cortical inflammation for use in SPMS clinical trials are needed. Increased levels of chemokines in the cerebrospinal fluid (CSF) can report on inflammatory processes occurring in the cerebral cortex of MS patients. A multiplexed chemokine array that included BAFF, a high sensitivity CXCL13 assay and composite chemokine scores were developed to explore differences in lymphoid (CXCL12, CXCL13, CCL19 and CCL21) and inflammatory (CCL2, CXCL9, CXCL10 and CXCL11) chemokines in a small pilot study. Paired CSF and serum samples were obtained from healthy controls (n=12), relapsing-remitting MS (RRMS) (n=21) and SPMS (N=12). A subset of the RRMS patients (n = 9) was assessed upon disease exacerbation and 1 month later following iv methylprednisone. SPMS patients were sampled twice to ascertain stability. Both lymphoid and inflammatory chemokines were elevated in RRMS and SPMS with the highest levels found in the active RRMS group. Inflammatory and lymphoid chemokine signatures were defined and generally correlated with each other. This small exploratory clinical study shows the feasibility of measuring complex and potentially more robust chemokine signatures in the CSF of MS patients during clinical trials. No differences were found between stable RRMS and SPMS. Future trials with larger patient cohorts with this chemokine array are needed to further characterize the differences, or the lack thereof, between stable RRMS and SPMS.

L-selectin is a possible biomarker for individual PML risk in natalizumab-treated MS patients.

OBJECTIVE: To find biomarkers identifying patients at risk for the development of progressive multifocal leukoencephalopathy (PML) during natalizumab treatment. METHODS: Patients were recruited from 10 European and US cohorts. Of 289 patients with multiple sclerosis (MS), 224 had been treated with natalizumab (18-80 months), 21 received other immune-modulatory treatments, and 28 were untreated. We had access to samples from 16 natalizumab PML patients. Eight of these patients had given blood before the diagnosis of PML. We also analyzed non-natalizumab-treated patients who developed PML (n = 10) and age- and sex-matched healthy donors (n = 31). All flow cytometric assessments were done on previously cryopreserved, viable peripheral blood mononuclear cells. RESULTS: The percentage of l-selectin-expressing CD4+ T cells was significantly lower in patients treated long-term with natalizumab (40.2%) when compared with patients not receiving natalizumab treatment (47.2%; p = 0.016) or healthy controls (61.0%; p < 0.0001). An unusually low percentage (9-fold lower; 4.6%) was highly correlated with the risk of developing PML in the patient group with available pre-PML samples when compared with non-PML natalizumab-treated patients (p ≤ 0.0001). Samples were gathered between 4 and 26 months before PML diagnosis. CONCLUSIONS: The cell-based assessment of the percentage of l-selectin-expressing CD4 T cells could provide an urgently needed biomarker for individual PML risk assessment.

Use of a standardized MxA protein measurement-based assay for validation of assays for the assessment of neutralizing antibodies against interferon-ß.

Effective monitoring of the development of neutralizing antibodies (NAbs) against IFN-ß in multiple sclerosis (MS) patients on IFN-ß therapy is important for clinical decision making and disease management. To date, antiviral assays have been the favored approach for NAb determination, but variations in assay conditions between laboratories and the increasing use of novel assays have contributed to the reporting of inconsistent antibody data between laboratories and between products. This study, undertaken at the request of the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA), is a joint effort by manufacturers of IFN-ß products (approved in Europe) towards harmonization of a NAb assay that facilitates generation of comparable NAb data, which, in conjunction with clinical outcomes, should prove useful for clinicians treating MS patients with IFN-ß products. This article describes the standardized cellular myxovirus resistance protein A (MxA) protein measurement-based assay for detection of IFN-ß NAbs and its use for the validation of assays used for the quantitative determination of such antibodies. Although titers varied between laboratories and the products used, utilization of IFN-ß1a rather than IFN-ß1b as the challenge antigen produced more consistent results in the NAb assay. Adoption of the standardized assay improves comparability between laboratories circumventing problems that arise when different, nonstandardized assays are employed for immunogenicity assessment. Based on the data, the EMA recommended for standardization purposes, the use of IFN-ß1a in NAb assays, independent of the therapeutic product used for therapy and validation of new NAb procedures against the standardized assay described.

Changes to anti-JCV antibody levels in a Swedish national MS cohort.

BACKGROUND: The anti-JC virus (JCV) antibody status has been introduced to stratify patients with multiple sclerosis (MS) for higher or lower risk of progressive multifocal leukoencephalopathy (PML). OBJECTIVE: To assess the potential utility of anti-JCV antibody levels for earlier diagnosis or prediction of PML. METHODS: An analytically validated antibody assay was used to determine serological status, normalised optical density values, and dilution titres for anti-JCV antibodies. The method was applied to stored sera of 1157 patients with MS including five cases of PML, all enrolled in the Swedish pharmacovigilance study for natalizumab (NAT). Anticytomegalovirus (CMV) and antivaricella-zoster (VZV) antibody levels served as controls. RESULTS: Prior to treatment with NAT, anti-JCV antibody levels were stable in the anti-JCV positive patients. During therapy, a slight decrease in anti-JCV and anti-VZV antibody levels, but not anti-CMV antibody levels, was observed. All five patients who developed PML showed a mild to moderate increase in anti-JCV antibody levels at time of PML diagnosis; pre-PML samples suggested that this increase might start already prior to diagnosis of PML. CONCLUSIONS: Treatment initiation with NAT may lead to a slight decrease in anti-JCV and anti-VZV antibody levels, suggestive of a mild suppressive effect of NAT on antibody levels. Our findings in five cases of PML demonstrate that the onset of PML can be accompanied by increasing anti-JCV antibodies in serum. Monitoring of anti-JCV antibody levels could potentially be used as a tool for prediction or earlier diagnosis of PML during NAT treatment for MS. Further studies are warranted.

The effect of plasma exchange on serum anti-JC virus antibodies.

OBJECTIVE: Natalizumab, a highly effective treatment for multiple sclerosis (MS) and Crohn's disease, is associated with progressive multifocal leukoencephalopathy (PML). Upon suspicion or diagnosis of PML, plasma exchange (PLEX) is performed to remove natalizumab from the circulation, allowing immune reconstitution of the central nervous system. Since PLEX may also remove other circulating antibodies, we examined the effects of PLEX on serum immunoglobulin (IgG) and anti-JC virus (JCV) antibody levels in MS patients with and without PML. METHODS: Serum samples from 12 natalizumab-treated patients without PML collected before, during and after PLEX were tested for IgG isotypes using a commercial assay, and for anti-JCV antibodies using a two-step enzyme-linked immunosorbent assay. Five natalizumab-treated PML patients who underwent PLEX were also tested for anti-JCV antibodies. RESULTS: PLEX produced a two- to three-fold reduction in all IgG isotypes. Among patients without PML, 42% (five of 12 patients) had detectable anti-JCV antibodies before PLEX; in these patients, anti-JCV antibodies were reduced approximately two- to five-fold, with levels returning to 50-100 percent of baseline two weeks after the final PLEX. The five PML patients, all of whom had detectable anti-JCV antibodies before PLEX, experienced similar reductions in anti-JCV antibody levels following PLEX. CONCLUSIONS: Our results indicate that PLEX effectively removes circulating antibodies; however, levels of endogenous anti-JCV antibody, unlike exogenously administered natalizumab, were replenished relatively quickly following PLEX. While interpretation of anti-JCV antibody levels during or within two weeks after PLEX may be problematic, humoral JCV immunity is not abolished by PLEX and antibody levels are rapidly restored.

Risk of natalizumab-associated progressive multifocal leukoencephalopathy.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is associated with natalizumab treatment. We quantified the risk of PML in patients with multiple sclerosis, according to the presence or absence of three risk factors: positive status with respect to anti-JC virus antibodies, prior use of immunosuppressants, and increasing duration of natalizumab treatment. METHODS: We used data from postmarketing sources, clinical studies, and an independent Swedish registry to estimate the incidence of PML among natalizumab-treated patients with multiple sclerosis, according to positive or negative status with respect to anti-JC virus antibodies, prior or no prior use of immunosuppressants, and duration of treatment (1 to 24 months vs. 25 to 48 months). Blood samples were available for anti-JC virus antibody testing from 5896 patients with multiple sclerosis and from 54 patients with multiple sclerosis who were treated with natalizumab and in whom PML later developed. RESULTS: As of February 29, 2012, there were 212 confirmed cases of PML among 99,571 patients treated with natalizumab (2.1 cases per 1000 patients). All 54 patients with PML for whom samples were available before the diagnosis were positive for anti-JC virus antibodies. When the risk of PML was stratified according to three risk factors, the risk of PML was lowest among the patients who were negative for anti-JC virus antibodies, with the incidence estimated to be 0.09 cases or less per 1000 patients (95% confidence interval [CI], 0 to 0.48). Patients who were positive for anti-JC virus antibodies, had taken immunosuppressants before the initiation of natalizumab therapy, and had received 25 to 48 months of natalizumab treatment had the highest estimated risk (incidence, 11.1 cases per 1000 patients [95% CI, 8.3 to 14.5]). CONCLUSIONS: Positive status with respect to anti-JC virus antibodies, prior use of immunosuppressants, and increased duration of natalizumab treatment, alone or in combination, were associated with distinct levels of PML risk in natalizumab-treated patients with multiple sclerosis. (Funded by Biogen Idec and Elan Pharmaceuticals.).

Anatomic closure of the premature patent ductus arteriosus: The role of CD14+/CD163+ mononuclear cells and VEGF in neointimal mound formation.

Permanent closure of the newborn ductus arteriosus requires the development of neointimal mounds to completely occlude its lumen. VEGF is required for neointimal mound formation. The size of the neointimal mounds (composed of proliferating endothelial and migrating smooth muscle cells) is directly related to the number of VLA4 mononuclear cells that adhere to the ductus lumen after birth. We hypothesized that VEGF plays a crucial role in attracting CD14/CD163 mononuclear cells (expressing VLA4) to the ductus lumen and that CD14/CD163 cell adhesion to the ductus lumen is important for neointimal growth. We used neutralizing antibodies against VEGF and VLA-4 to determine their respective roles in remodeling the ductus of premature newborn baboons. Anti-VEGF treatment blocked CD14/CD163 cell adhesion to the ductus lumen and prevented neointimal growth. Anti-VLA-4 treatment blocked CD14/CD163 cell adhesion to the ductus lumen, decreased the expression of PDGF-B (which promotes smooth muscle migration), and blocked smooth muscle influx into the neointimal subendothelial space (despite the presence of increased VEGF in the ductus wall). We conclude that VEGF is necessary for CD14/CD163 cell adhesion to the ductus lumen and that CD14/CD163 cell adhesion is essential for VEGF-induced expansion of the neointimal subendothelial zone.

Sequencing and analysis of JC virus DNA from natalizumab-treated PML patients.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) in natalizumab-treated MS patients is linked to JC virus (JCV) infection. JCV sequence variation and rearrangements influence viral pathogenicity and tropism. To better understand PML development, we analyzed viral DNA sequences in blood, CSF and/or urine of natalizumab-treated PML patients. METHODS: Using biofluid samples from 17 natalizumab-treated PML patients, we sequenced multiple isolates of the JCV noncoding control region (NCCR), VP1 capsid coding region, and the entire 5 kb viral genome. RESULTS: Analysis of JCV from multiple biofluids revealed that individuals were infected with a single genotype. Across our patient cohort, multiple PML-associated NCCR rearrangements and VP1 mutations were present in CSF and blood, but absent from urine-derived virus. NCCR rearrangements occurred in CSF of 100% of our cohort. VP1 mutations were observed in blood or CSF in 81% of patients. Sequencing of complete JCV genomes demonstrated that NCCR rearrangements could occur without VP1 mutations, but VP1 mutations were not observed without NCCR rearrangement. CONCLUSIONS: These data confirm that JCV in natalizumab-PML patients is similar to that observed in other PML patient groups, multiple genotypes are associated with PML, individual patients appear to be infected with a single genotype, and PML-associated mutations arise in patients during PML development.