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Pegylated interferon beta in the treatment of the Theiler's murine encephalomyelitis virus mouse model of multiple sclerosis
Abstract
We evaluated the effects of pegylated-interferonβ-1a (pegIFNβ) therapy on intrathecal antibody responses, disability progression, and viral load in the CNS in mice infected with the Theiler's virus (TMEV), an animal model of progressive disability in Multiple Sclerosis (MS). The lack of a direct antiviral activity in the CNS, the absence of any effect upon the intrathecal immune response, and the failure to treat disease progression, indicate that the immunomodulatory effects of pegIFNβ-1a likely occur in the systemic circulation rather than within the CNS. These results may be relevant to the relative lack of effect of IFNβ in progressive MS relative to relapsing MS.
... TMEV-IDD was induced by intracerebral injection of 4 × 10 6 plaque-forming units of TMEV strain BeAn into 6-to 8-week-old SJL/J female mice, as previously described [7][8][9][10]. All experiments were approved by the Dartmouth Institutional Animal Care and Use Committee. ...
... None of these immunological measures correlated with the development of enlarged ventricular volumes. This is consistent with our previous results in TMEV-IDD, where multiple immunosuppressive or immunomodulatory medications have downregulated neuroinflammation without ameliorating disability progression [7][8][9][10]. It is also consistent with results in human MS, where most disability accrues in the secondary progressive phase of the disease. ...
Background
There is a lack of understanding of the mechanisms by which the CNS is injured in multiple sclerosis (MS). Since Theiler’s murine encephalomyelitis virus (TMEV) infection in SJL/J mice is an established model of progressive disability in MS, and CNS atrophy correlates with progressive disability in MS, we used in vivo MRI to quantify total ventricular volume in TMEV infection. We then sought to identify immunological and virological biomarkers that correlated with increased ventricular size.
Methods
Mice, both infected and control, were followed for 6 months. Cerebral ventricular volumes were determined by MRI, and disability was assessed by Rotarod. A range of immunological and virological measures was obtained using standard techniques.
Results
Disability was present in infected mice with enlarged ventricles, while infected mice without enlarged ventricles had Rotarod performance similar to sham mice. Ventricular enlargement was detected as soon as 1 month after infection. None of the immunological and virological measures correlated with the development of ventricular enlargement.
Conclusions
These results support TMEV infection with brain MRI monitoring as a useful model for exploring the biology of disability progression in MS, but they did not identify an immunological or virological correlate with ventricular enlargement.
... Therefore, in our analyses, we only included mice effectively infected with TMEV. Effective infection was proven by demonstrating viral infection of the spinal cord at necropsy by real-time reverse transcription quantitative PCR (RT-qPCR) for TMEV mRNA [30,31] and anti-TMEV antibody in serum by ELISA [31,32]. Levels of serum anti-TMEV antibody were tested at 30, 90, and 120 days p.i to further confirm viral persistence. ...
... Therefore, in our analyses, we only included mice effectively infected with TMEV. Effective infection was proven by demonstrating viral infection of the spinal cord at necropsy by real-time reverse transcription quantitative PCR (RT-qPCR) for TMEV mRNA [30,31] and anti-TMEV antibody in serum by ELISA [31,32]. Levels of serum anti-TMEV antibody were tested at 30, 90, and 120 days p.i to further confirm viral persistence. ...
Background
The mechanisms driving multiple sclerosis (MS), the most common cause of non-traumatic disability in young adults, remain unknown despite extensive research. Especially puzzling are the underlying molecular processes behind the two major disease patterns of MS: relapsing-remitting and progressive. The relapsing-remitting course is exemplified by acute inflammatory attacks, whereas progressive MS is characterized by neurodegeneration on a background of mild-moderate inflammation. The molecular and cellular features differentiating the two patterns are still unclear, and the role of inflammation during progressive disease is a subject of active debate.
Methods
We performed a comprehensive analysis of the intrathecal inflammation in two clinically distinct mouse models of MS: the PLP139-151-induced relapsing experimental autoimmune encephalomyelitis (R-EAE) and the chronic progressive, Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Microarray technology was first used to examine global gene expression changes in the spinal cord.
Inflammation in the spinal cord was further assessed by immunohistochemical image analysis and flow cytometry. Levels of serum and cerebrospinal fluid (CSF) immunoglobulin (Ig) isotypes and chemokines were quantitated using Luminex Multiplex technology, whereas a capture ELISA was used to measure serum and CSF albumin levels. Finally, an intrathecal Ig synthesis index was established with the ratio of CSF and serum test results corrected as a ratio of their albumin concentrations.
Results
Microarray analysis identified an enrichment of B cell- and Ig-related genes upregulated in TMEV-IDD mice. We also demonstrated an increased level of intrathecal Ig synthesis as well as a marked infiltration of late differentiated B cells, including antibody secreting cells (ASC), in the spinal cord of TMEV-IDD, but not R-EAE mice. An intact blood-brain barrier in TMEV-IDD mice along with higher CSF levels of CXCL13, CXCL12, and CCL19 provides evidence for an intrathecal synthesis of chemokines mediating B cell localization to the central nervous system (CNS).
Conclusions
Overall, these findings, showing increased concentrations of intrathecally produced Igs, substantial infiltration of ASC, and the presence of B cell supporting chemokines in the CNS of TMEV-IDD mice, but not R-EAE mice, suggest a potentially important role for Igs and ASC in the chronic progressive phase of demyelinating diseases.
Electronic supplementary material
The online version of this article (10.1186/s12974-019-1501-9) contains supplementary material, which is available to authorized users.
... 18 Because of striking similarities with MS disease course and its pathology, the TMEV has been increasingly used to test efficacy of DMTs. [19][20][21][22][23][24] Interestingly, the BBB breakdown is rarely observed in the TMEV lesions using Gad, 25 whereas USPIO has been more successful to image inflammation and migration of specific cell types, including CD8 + T-cells and CD4 + T-cells, into the CNS. 26 However, no previous studies investigated the temporal patterns of USPIO enhancement during inflammatory and neurodegenerative phases of the disease, and explored its association with clinical and MRI measures of disease severity. ...
BACKGROUND AND PURPOSE
Blood‐derived monocytes/macrophages can be labeled with ultrasmall superparamagnetic iron oxides (USPIO) at periphery and subsequently migrate into areas of inflammation in the brain. We investigated temporal pattern of migration of peripheral immune cells in Theiler's murine encephalomyelitis virus (TMEV) model of chronic demyelination by USPIO‐enhanced imaging.
METHODS
Fifteen SJL mice (Envigo, Indianapolis, IN) were injected with TMEV (n = 12) or saline (n = 3) at 7 weeks of age. Brain MRI of 9.4 T was performed at 3 months postinfection (mpi) (the peak of inflammatory phase), at 4, 5, and 7 mpi (throughout neurodegenerative phase) using T2*‐weighted gradient echo MRI, and performed 24 hours after USPIO injection. Contrast enhancing lesion (CEL) number and volume were measured and development of brain atrophy was assessed across serial time points. Clinical disability scale and rotarod score assessed disease progression.
RESULTS
CEL was detected in a total of eight (66.7%) TMEV‐infected animals and none of the Controls. The CEL was present in four (33.3%) TMEV‐infected animals at 3 mpi, two (16.7%) at 4 mpi, six (54.5%) at 5 mpi, and four (44.4%) at 7 mpi, respectively. In TMEV‐infected animals, the CEL number and volume increased significantly from 3 to 7 mpi (P < .01 for both). The correlation between total CEL number and volume with clinical and MRI outcomes was trending (P < .05). On histopathology analysis, CEL showed increased density of Iba1 staining for microglia activity.
CONCLUSIONS
Serial USPIO imaging is a promising biomarker for investigating the effect of therapeutic interventions on monocytes/macrophages and microglia activation and neurodegeneration in TMEV‐infected animals.
Disease in the spinal cord is a major component of disability in multiple sclerosis, yet current techniques of imaging spinal cord injury are insensitive and nonspecific. This study seeks to remove this major impediment to research in multiple sclerosis and other spinal cord diseases by identifying reliable biomarkers of disability progression using diffusion tensor imaging (DTI), a magnetic resonance imaging technique, to evaluate the spinal cord in a model of multiple sclerosis, i.e. the Theiler's Murine Encephalitis Virus-Induced Demyelinating Disease (TMEV-IDD). Mice with TMEV-IDD with varying levels of clinical disease were imaged using a 9.4T small animal MRI scanner. Axial diffusivity, radial diffusivity, and fractional anisotropy were calculated. Disability was assessed periodically using Rotarod assay and data were expressed as a neurological function index. Correlation was performed between DTI measurements and disability scores. TMEV-IDD mice displayed significant increased neurological deficits over time when compared with controls (p<0.0001). Concurrently, the values of fractional anisotropy and axial diffusivity were both decreased compared to controls (both p<0.0001), while radial diffusivity was increased (p<0.0001). Overall, fractional anisotropy changes were larger in white matter than in grey matter and differences were more pronounced in the ventral region. Lower disability scores were associated with decreased fractional anisotropy values measured in the ventral (r = 0.68; p<0.0001) and ventral-lateral (r = 0.70; p<0.0001) regions of the white matter. These data demonstrate that DTI measures of the spinal cord contribute to strengthening the association between neuroradiological markers and clinical disability, and support the use of DTI measures in spinal cord imaging in MS patients.
The analysis of intrathecal IgG, IgA and IgM synthesis in cerebrospinal fluid (CSF) and evaluation in combined quotient diagrams provides disease-related patterns. The compilation with complementary parameters (barrier function, i.e., CSF flow rate, cytology, lactate, antibodies) in a cumulative CSF data report allows a knowledge-based interpretation and provides analytical and medical plausibility for the quality assessment in CSF laboratories. The diagnostic relevance is described for neurological and psychiatric diseases, for which CSF analysis can't be replaced by other diagnostic methods without loss of information. Dominance of intrathecal IgM, IgA or three class immune responses give a systematic approach for Facial nerve palsy, Neurotrypanosomiasis, Opportunistic diseases, lymphoma, Neurotuberculosis, Adrenoleucodystrophy or tumor metastases. Particular applications consider the diagnostic power of the polyspecific antibody response (MRZ-antibodies) in multiple sclerosis, a CSF-related systematic view on differential diagnostic of psychiatric diseases and the dynamics of brain- derived compared to blood-derived molecules in CSF for localization of paracytes.
Background:
Efficacy of interferon beta in multiple sclerosis (MS) can be dampened in patients who develop neutralizing antidrug antibodies (NAbs). Peginterferon beta1a is an interferon conjugated with a polyethylene glycol (PEG) moiety. Pegylation increases a drug's half life and exposure, and may also reduce immunogenicity.
Objective:
The objective of this study was to characterize the incidence and impact of immunogenicity to peginterferon beta1a over 2 years in patients with MS.
Methods:
Patients with relapsing-remitting MS (N = 1512) were randomized to subcutaneous peginterferon beta1a 125 μg every 2 or 4 weeks, or placebo, for 1 year; patients in the placebo group were rerandomized to active treatment in year 2. The incidence and titers of binding antibodies (BAbs) and NAbs to interferon and antibodies to PEG (anti-PEG) were assessed in analytically validated assays. The clinical impact of immunogenicity on relapse and magnetic resonance imaging endpoints was evaluated.
Results:
Over 2 years, 6%, less than 1%, and 7% of patients developed anti-interferon BAbs, NAbs, and anti-PEG antibodies, respectively. There was no discernible clinically meaningful effect of antibody status on the pharmacodynamic, efficacy, or safety parameters evaluated, although these analyses were limited by the low incidence of treatment-emergent antibodies.
Conclusion:
The treatment effect of peginterferon beta1a in patients with relapsing-remitting MS is not expected to be attenuated by immunogenicity.
Importance:
Herpes simplex virus type 1 (HSV-1) is a ubiquitous virus and a significant cause of morbidity and some mortality. It is the causative agent of benign cold sores, but can also cause blindness and life-threatening encephalitis. The success of HSV-1 is largely due to its ability to establish lifelong latent infections in neurons and to occasionally reactivate. The exact mechanisms by which neurons defend against virus infection is poorly understood, but is at least partially mediated by autophagy, an intracellular pathway by which pathogens and other unwanted cargoes are degraded. The study demonstrates and investigates a new autophagic structure that appears specific to the interaction between neurotropic herpesviruses and murine primary sensory neurons. This work may therefore have important implications for our understanding of latency and reactivation.
Since the late 18th century, the murine model has been widely used in biomedical research (about 59% of total animals used) as it is compact, cost-effective, and easily available, conserving almost 99% of human genes and physiologically resembling humans. Despite the similarities, mice have a diminutive lifespan compared to humans. In this study, we found that one human year is equivalent to nine mice days, although this is not the case when comparing the lifespan of mice versus humans taking the entire life at the same time without considering each phase separately. Therefore, the precise correlation of age at every point in their lifespan must be determined. Determining the age relation between mice and humans is necessary for setting up experimental murine models more analogous in age to humans. Thus, more accuracy can be obtained in the research outcome for humans of a specific age group, although current outcomes are based on mice of an approximate age. To fill this gap between approximation and accuracy, this review article is the first to establish a precise relation between mice age and human age, following our previous article, which explained the relation in ages of laboratory rats with humans in detail.
Objectives:
To explore long-term effects of treatment and prognostic relevance of variables assessed at baseline and during the European secondary progressive multiple sclerosis (SPMS) trial of interferon beta 1b (IFNB-1b).
Methods:
We assessed 362 patients (60% female; median age 41 years; Expanded Disability Status Scale (EDSS): 5.5; 51% randomized to IFNB-1b) for their EDSS and treatment history after 10 years. Non-parametric analysis of covariance (ANCOVA) and multivariate linear regression models were applied.
Results:
Median EDSS was 6.0 at the end of the randomized controlled trial (RCT), in the IFNB-1b and placebo groups, and 7.0 in long-term follow-up patients (those receiving IFNB-1b in the RCT were 6.5 and those receiving placebo in the RCT were 7.0; p = 0.086). 24 patients (6.6%) were deceased. The EDSS at baseline and the EDSS change during the RCT were the most important predictors of the EDSS 10 years later (partial R(2): 0.47). The ability to predict changes in EDSS 10 years after the RCT was limited (R(2): 0.12). Magnetic resonance imaging (MRI) measures remained in the predictive models, but explained < 5% of the variability.
Conclusions:
The results from this analysis did not provide convincing evidence to support a favorable long-term outcome in those patients allocated IFNB-1b during the RCT, in our SPMS cohort. The progressive stage of the disease remains largely unpredictable by clinical and conventional MRI measures, so better prognostic markers are needed.
Unlabelled:
Previously we found that following intranasal (i.n.) infection with neurotropic vesicular stomatitis virus (VSV) type I interferon receptor (IFNAR) triggering of neuroectodermal cells was critically required to constrain intracerebral virus spread. To address whether locally active IFN-β was induced proximally, we studied spatiotemporal conditions of VSV-mediated IFN-β induction. To this end, we performed infection studies with IFN-β reporter mice. One day after intravenous (i.v.) VSV infection, luciferase induction was detected in lymph nodes. Upon i.n. infection, luciferase induction was discovered at similar sites with delayed kinetics, whereas on days 3 and 4 postinfection enhanced luciferase expression additionally was detected in the foreheads of reporter mice. A detailed analysis of cell type-specific IFN-β reporter mice revealed that within the olfactory bulb IFN-β was expressed by neuroectodermal cells, primarily by astrocytes and to a lesser extent by neurons. Importantly, locally induced type I IFN triggered distal parts of the brain as indicated by the analysis of ISRE-eGFP mice which after i.n. VSV infection showed enhanced green fluorescent protein (eGFP) expression throughout the brain. Compared to wild-type mice, IFN-β(-/-) mice showed increased mortality to i.n. VSV infection, whereas upon i.v. infection no such differences were detected highlighting the biological significance of intracerebrally expressed IFN-β. In conclusion, upon i.n. VSV instillation, IFN-β responses mounted by astrocytes within the olfactory bulb critically contribute to the antiviral defense by stimulating distal IFN-β-negative brain areas and thus arresting virus spread.
Importance:
The central nervous system has long been considered an immune privileged site. More recently, it became evident that specialized immune mechanisms are active within the brain to control pathogens. Previously, we showed that virus, which entered the brain via the olfactory route, was arrested within the olfactory bulb by a type I IFN-dependent mechanism. Since peripheral type I IFN would not readily cross the blood-brain barrier and within the brain thus far no abundant type I IFN responses have been detected, here we addressed from where locally active IFN originated from. We found that upon intranasal VSV instillation, primarily astrocytes, and to a lesser extent neurons, were stimulated within the olfactory bulb to mount IFN-β responses that also activated and protected distal brain areas. Our results are surprising because in other infection models astrocytes have not yet been identified as major type I IFN producers.
AimsTo evaluate the pharmacokinetics and pharmacodynamics of subcutaneous peginterferon beta-1a in patients with relapsing-remitting multiple sclerosis (RRMS) in the Phase 3 ADVANCE study (n=1512).Methods
During Year 1, patients were randomised (1:1:1) to placebo or peginterferon beta-1a 125 μg every 2 or 4 weeks. After Year 1, patients randomised to placebo were re-randomised to 125 μg peginterferon beta-1a administered every 2 weeks or every 4 weeks for Year 2. Patients randomised to peginterferon beta-1a in Year 1 remained on the same dosing regimen in Year 2. Intensive blood samples for pharmacokinetic and pharmacodynamic (neopterin elevation; a biomarker of pharmacological activity induced by interferon beta-1a) measurements were collected from 44 patients pre-dosing and at intervals over 240 hours post-dosing at Weeks 4 and 24. Sparse samples were collected from all patients after each dosing at Weeks 4, 12, 24, 56, and 84.ResultsThe pharmacokinetic profile of peginterferon beta-1a did not change over time or between dosing regimens. No accumulation was observed. Peak serum concentrations were reached 1–1.5 days post-dosing, with a mono-phasic decline, and a median half-life of approximately 2–3 days. Dosing every 2 weeks provided approximately 2-fold greater monthly cumulative area-under-the-curve than every 4 weeks. Neopterin elevation was sustained for 10−14 days following each dose, indicating doubled cumulative duration of pharmacological activity for dosing every 2 weeks versus every 4 weeks.Conclusions
These pharmacokinetic/pharmacodynamic profiles potentially explain the enhanced efficacy of dosing every 2 weeks in patients with RRMS.
The pathogenesis of multiple sclerosis (MS) is as yet unknown. Commonly, MS is assumed to be due to an autoimmune inflammation of the central nervous system (CNS). Neurodegeneration is regarded to be a secondary reaction. This concept is increasingly being challenged. Human endogenous retroviruses (HERV) that could be locally activated in the CNS have been proposed as an alternative concept. HERV-encoded envelope proteins (env) can act as strong immune stimulators (superantigens). Thus, slow disease progression following neurodegeneration might be induced by re-activation of HERV expression directly, while relapses in parallel to inflammation might be secondary to the expression of HERV-encoded superantigens. It has been shown previously that T-cell superantigens are capable to induce a cellular inflammatory reaction in the CNS of experimental animals similar to that in MS. Furthermore, B-cell superantigens have been shown to activate blood leucocytes in vitro to produce immunoglobulin in an oligoclonal manner. It remains to be established, whether the outlined hypothesis accords with all known features of MS. Furthermore, anti-HERV agents may be taken into consideration to enrich and improve MS therapy.
Subcutaneous pegylated interferon (peginterferon) beta-1a is being developed for treatment of relapsing multiple sclerosis, with less frequent dosing than currently available first-line injectable treatments. We assessed the safety and efficacy of peginterferon beta-1a after 48 weeks of treatment in the placebo-controlled phase of the ADVANCE trial, a study of patients with relapsing-remitting multiple sclerosis.
We did this 2-year, double-blind, parallel group, phase 3 study, with a placebo-controlled design for the first 48 weeks, at 183 sites in 26 countries. Patients with relapsing-remitting multiple sclerosis (age 18-65 years, with Expanded Disability Status Scale score ≤5) were randomly assigned (1:1:1) via an interactive voice response or web system, and stratified by site, to placebo or subcutaneous peginterferon beta-1a 125 μg once every 2 weeks or every 4 weeks. The primary endpoint was annualised relapse rate at 48 weeks. This trial is registered with ClinicalTrials.gov, number NCT00906399.
We screened 1936 patients and enrolled 1516, of whom 1512 were randomly assigned (500 to placebo, 512 to peginterferon every 2 weeks, 500 to peginterferon every 4 weeks); 1332 (88%) patients completed 48 weeks of treatment. Adjusted annualised relapse rates were 0·397 (95% CI 0·328-0·481) in the placebo group versus 0·256 (0·206-0·318) in the every 2 weeks group and 0·288 (0·234-0·355) in the every 4 weeks group (rate ratio for every 2 weeks group 0·644, 95% CI 0·500-0·831, p=0·0007; rate ratio for the every 4 weeks group 0·725, 95% CI 0·565-0·930, p=0·0114). 417 (83%) patients taking placebo, 481 (94%) patients taking peginterferon every 2 weeks, and 472 (94%) patients taking peginterferon every 4 weeks reported adverse events including relapses. The most common adverse events associated with peginterferon beta-1a were injection site reactions, influenza-like symptoms, pyrexia, and headache. 76 (15%) patients taking placebo, 55 (11%) patients taking study drug every 2 weeks, and 71 (14%) patients taking study drug every 4 weeks reported serious adverse events; relapse, pneumonia, and urinary tract infection were the most common.
After 48 weeks, peginterferon beta-1a significantly reduced relapse rate compared with placebo. The drug might be an effective treatment for relapsing-remitting multiple sclerosis with less frequent administration than available treatments.
Biogen Idec.
Interferon-beta (IFNβ) regulates the expression of a complex set of pro- as well as anti-inflammatory genes. In cohorts of MS patients unstratified for therapeutic response to IFNβ, normal vaccine-specific immune responses have been observed. Data capturing antigen-specific immune responses in cohorts of subjects defined by response to IFNβ-therapy are not available.
To assess antigen-specific immune responses in a cohort of MS patients responding clinically and radiologically to IFNβ.
In 26 MS patients, clinical and MRI disease activity were assessed before and under treatment with IFNβ. Humoral and cellular immune response to influenza vaccine was prospectively characterized in these individuals, and 33 healthy controls by influenza-specific Enzyme-Linked Immunosorbent Assay (ELISA) and Enzyme Linked Immuno Spot Technique (ELISPOT).
Related to pre-treatment disease activity, IFNβ reduced clinical and radiological MS disease-activity. Following influenza vaccination, frequencies of influenza-specific T cells and concentrations of anti-influenza A and B IgM and IgG increased comparably in MS-patients and in healthy controls.
By showing in a cohort of MS-patients responding to IFNβ vaccine-specific immune responses comparable to controls, this study indicates that antigen-specific immune responses can be preserved under successful IFNβ-therapy.
Multiple sclerosis is the most common autoimmune inflammatory demyelinating disease of the central nervous system, and its etiology is believed to have both genetic and environmental components. Several viruses have already been implicated as triggers and there are several studies that implicate members of the Herpesviridae family in the pathogenesis of MS. The most important characteristic of these viruses is that they have periods of latency and exacerbations within their biological sanctuary, the central nervous system. The Epstein-Barr, cytomegalovirus, human herpesvirus 6 and human herpesvirus 7 viruses are the members that are most studied as being possible triggers of multiple sclerosis. According to evidence in the literature, the herpesvirus family is strongly involved in the pathogenesis of this disease, but it is unlikely that they are the only component responsible for its development. There are probably multiple triggers and more studies are necessary to investigate and define these interactions.
Background
Clinical trials offer a unique opportunity to study human disease and response to therapy in a highly controlled setting. The application of high-throughput expression profiling to peripheral blood from clinical trial subjects could facilitate the identification of transcripts that function as prognostic or diagnostic markers of disease or treatment. The paramount issue for these methods is the ability to produce robust, reproducible, and timely mRNA expression profiles from peripheral blood. Single-stranded complementary DNA (sscDNA) targets derived from whole blood exhibit improved detection of transcripts and reduced variance as compared to their complementary RNA counterparts and therefore provide a better option for interrogation of peripheral blood on oligonucleotide arrays. High-throughput microarray technologies such as the high-throughput plate array platform offer several advantages compared with slide- or cartridge-based arrays; however, manufacturer’s protocols do not support the use of sscDNA targets.
Results
We have developed a highly reproducible, high-through put, whole blood expression profiling methodology based on sscDNA and used it to analyze human brain reference RNA and universal human reference RNA samples to identify experimental conditions that most highly correlated with a gold standard quantitative polymerase chain reaction reference dataset. We then utilized the optimized method to analyze whole blood samples from healthy clinical trial subjects treated with different versions of interferon (IFN) beta-1a. Analysis of whole blood samples before and after treatment with intramuscular [IM] IFN beta-1a or polyethylene glycol-conjugated IFN (PEG-IFN) beta-1a under optimized experimental conditions demonstrated that PEG-IFN beta-1a induced a more sustained and prolonged pharmacodynamic response than unmodified IM IFN beta-1a. These results provide validation of the utility of this new methodology and suggest the potential therapeutic benefit of a sustained pharmacodynamic response to PEG-IFN beta-1a.
Conclusions
This novel microarray methodology is ideally suited for utilization in large clinical studies to identify expressed transcripts for the elucidation of disease mechanisms of action and as prognostic, diagnostic, or toxicity markers.
Rabies virus (RABV) is a neurotropic virus transmitted by the bite of an infected animal that triggers a fatal encephalomyelitis. During its migration in the nervous system (NS), RABV triggers an innate immune response, including a type I IFN response well known to limit viral infections. We showed that although the neuroinvasive RABV strain CVS-NIV dampens type I IFN signaling by inhibiting IRF3 phosphorylation and STAT2 translocation, an early and transient type I IFN response is still triggered in the infected neuronal cells and NS. This urged us to investigate the role of type I IFN on RABV infection. We showed that primary mouse neurons (DRGs) of type I IFN(α/β) receptor deficient mice (IFNAR(-/-) mice) were more susceptible to RABV than DRGs of WT mice. In addition, exogenous type I IFN is partially efficient in preventing and slowing down infection in human neuroblastoma cells. Intra-muscular inoculation of type I IFNAR deficient mice [IFNAR(-/-) mice and NesCre ((+/-)) IFNAR ((flox/flox)) mice lacking IFNAR in neural cells of neuroectodermal origin only] with RABV reveals that the type I IFN response limits RABV dissemination in the inoculated muscle, slows down invasion of the spinal cord, and delays mortality. Thus, the type I IFN which is still produced in the NS during RABV infection is efficient enough to reduce neuroinvasiveness and pathogenicity and partially protect the host from fatal infection.
Multiple sclerosis (MS) is an inflammatory, autoimmune, demyelinating disease of the central nervous system (CNS) that usually starts as a relapsing-remitting disease. In most patients the disease evolves into a chronic progressive phase characterized by continuous accumulation of neurological deficits. While treatment of relapsing-remitting MS (RRMS) has improved dramatically over the last decade, the therapeutic options for chronic progressive MS, both primary and secondary, are still limited. In order to find new pharmacological targets for the treatment of chronic progressive MS, the mechanisms of the underlying neurodegenerative process that becomes apparent as the disease progresses need to be elucidated. New animal models with prominent and widespread progressive degenerative components of MS have to be established to study both inflammatory and non-inflammatory mechanisms of neurodegeneration. Here, we discuss disease mechanisms and treatment strategies for chronic progressive MS.
Some recent studies suggest that in progressive multiple sclerosis, neurodegeneration may occur independently from inflammation. The aim of our study was to analyse the interdependence of inflammation, neurodegeneration and disease progression in various multiple sclerosis stages in relation to lesional activity and clinical course, with a particular focus on progressive multiple sclerosis. The study is based on detailed quantification of different inflammatory cells in relation to axonal injury in 67 multiple sclerosis autopsies from different disease stages and 28 controls without neurological disease or brain lesions. We found that pronounced inflammation in the brain is not only present in acute and relapsing multiple sclerosis but also in the secondary and primary progressive disease. T- and B-cell infiltrates correlated with the activity of demyelinating lesions, while plasma cell infiltrates were most pronounced in patients with secondary progressive multiple sclerosis (SPMS) and primary progressive multiple sclerosis (PPMS) and even persisted, when T- and B-cell infiltrates declined to levels seen in age matched controls. A highly significant association between inflammation and axonal injury was seen in the global multiple sclerosis population as well as in progressive multiple sclerosis alone. In older patients (median 76 years) with long-disease duration (median 372 months), inflammatory infiltrates declined to levels similar to those found in age-matched controls and the extent of axonal injury, too, was comparable with that in age-matched controls. Ongoing neurodegeneration in these patients, which exceeded the extent found in normal controls, could be attributed to confounding pathologies such as Alzheimer's or vascular disease. Our study suggests a close association between inflammation and neurodegeneration in all lesions and disease stages of multiple sclerosis. It further indicates that the disease processes of multiple sclerosis may die out in aged patients with long-standing disease.
The IgG pattern in CSF was studied in 11 patients with multiple sclerosis who exhibited an oligoclonal banding upon thin-layer polyacrylamide gel isoelectric focusing followed by silver stain of unconcentrated CSF. Each patient received beta-interferon intrathecally during a 2 month period. No modification was observed over a 6 month period. In addition, the oligoclonal pattern was remarkably unique for each individual representing a typical "fingerprint" which allowed the identification of any single CSF.
Interferon beta (IFNB) is available in parenteral formulations for treatment of multiple sclerosis (MS). The purpose of this study was to evaluate safety, tolerability and effects on MRI lesions of three different doses of oral IFNB-1a compared with placebo over six months in relapsing-remitting (RR) MS patients.
In this multicenter; double-blind randomized trial, RR-MS patients received 0.06, 0.6 or 6 million international units (MIU) IFNB-1a or placebo every other day for up to six months. Gadolinium DTPA-enhanced brain MRI scans were performed at screening and monthly during treatment. The primary variable was the cumulative number of newly active lesions. Secondary variables included volume of enhancing lesions on T1-weighted images each month and lesion volume on T2-weighted images at months three and six. Safety measures included adverse events, laboratory variables, vital signs, ECG, physical examination, EDSS and number of relapses. Neopterin was measured in 21 patients and neutralizing antibodies in 24 patients.
Of 194 screened patients, 173 were randomized (42-44 patients per group) in 15 centers. Median cumulative numbers of newly active lesions over six months were 4.0 in the placebo and 0.6 MIU groups, compared with 7.5 and 9.0 in the 0.06 and 6 MIU groups (no significant differences). Secondary efficacy endpoints showed small and inconsistent differences between groups. Adverse events showed no notable group differences. Approximately two-thirds of patients in each group remained relapse free. No patients showed neutralizing antibodies. Neopterin levels were comparable between groups.
Oral IFNB-1a showed neither beneficial effects in RRMS nor any systemic biological effects. Treatment was safe and well tolerated.
After intracerebral infection with the Theiler’s Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, interlaboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.
Multiple sclerosis is caused by a complex interaction between genetic predisposition and environmental factors. Epstein–Barr virus (EBV) is an environmental risk factor that is strongly related to multiple sclerosis (MS), since EBV seropositivity is linked to a significant risk of developing MS. EBV may be involved in the pathogenesis of the disease and it is possibly a prerequisite for the development of MS. EBV infection persists in B-cells during the lifetime of the host and can modulate their function. In addition, MS patients might have a deficient capacity to eliminate latent EBV infection in the central nervous system and this would promote the accumulation of infected B cells. Several mechanisms of pathogenesis, including a direct and indirect function of infected B cells, have been postulated in inflammation and neurodegeneration. A relationship between EBV and human endogenous retroviruses in the pathogenesis of MS has also been reported. If EBV is important in the pathogenesis of MS, different therapeutic strategies seem possible for MS treatment.
Introduction:
Multiple sclerosis (MS) is a chronic immune-mediated disease of the CNS characterized in most cases by a relapsing and remitting disease course, often followed by a progressive course. There are many available injectable therapies for the relapsing-remitting form of MS (RRMS); however, efficacy can be affected by poor adherence and compliance, and an increased frequency of side effects as a result of frequent injections is seen.
Areas covered:
This review focuses on the use of subcutaneous PEGylated IFNβ-1a (PEGIFN β-1a) in RRMS. The pharmacological data in addition to clinical safety and tolerability are analyzed. The clinical efficacy is assessed by evaluating results of various end points used in the ADVANCE Phase III study and the differences between treatment groups over 2 years.
Expert opinion:
We discuss the significance of the ADVANCE trial results and how the results compare to other products on the market. Due to a lack of head-to-head comparison of PEGIFN β-1a with other types of drugs for RRMS, it is difficult to draw conclusion about the superiority of the drug. We also discuss whether the results can be applied to patients with more severe forms of the disease. Overall, PEGIFN β-1a is a promising addition to the repertoire of emerging drugs for the treatment of RRMS.
Objective The management of complex patients with neuroimmunological diseases is hindered by an inability to reliably measure intrathecal inflammation. Currently implemented laboratory tests developed more than 40 years ago are either 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 two 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 assessment of cell-specificity in purified primary immune cells. Biomarkers were quantified by optimized electrochemiluminescent immuno-assays. 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. This article is protected by copyright. All rights reserved.
© 2015 American Neurological Association.
Following reports of elevated antiviral antibodies in MS patient sera and viral DNA detection in MS plaques nearly two decades ago, the neurovirology community has actively explored how herpesviruses such as HHV-6 might be involved in MS disease pathogenesis. Though findings across the field are non-uniform, an emerging consensus of viral correlates with disease course and evidence of HHV-6-specific immune responses in the CNS provide compelling evidence for a role, direct or indirect, of this virus in MS. Ultimately, the only way to demonstrate the involvement, or lack thereof, of HHV-6 or other herpesviruses in this disease is through a controlled clinical trial of an efficacious antiviral drug.
Published by Elsevier B.V.
Although genetic susceptibility explains the clustering of multiple sclerosis (MS) cases within families, the changes in MS risk that occur with migration can be explained only by changes in the environment. The strongest known risk factor for MS is infection with Epstein-Barr virus (EBV). Compared with uninfected individuals, the hazard of developing MS is approximately 15-fold higher among individuals infected with EBV in childhood and about 30-fold higher among those infected with EBV in adolescence or later in life. Although the mechanisms underlying this association remain unclear, the data provide strong evidence of a causal relation between EBV infection and MS risk. Relevant aspects of MS epidemiology beyond genetics are not explained by EBV involvement, however, implying the involvement of other factors. Modifiable factors for MS risk include smoking and childhood obesity. Increased risk of MS in individuals with vitamin D insufficiency has been proposed to explain the strong latitude gradient in MS prevalence. Results of case-control studies that relied on prevalent MS cases have been mixed, however, and potentially influenced by selection and recall biases. In a recent case-control study of individuals presenting with a first demyelinating episode, higher levels of vitamin D, sun exposure or actinic damage were found to be associated with reduced MS risk. Two longitudinal studies have thus far been completed. In the first, based on assessment of vitamin D intake from diet and supplements, the risk of MS was found to be 30% lower among women in the highest quintile compared with those in the lowest quintile. In the second study, conducted among young adults in the US military, vitamin D status was assessed by averaging multiple season-adjusted measures of 25-hydroxyvitamin D (25[OH]D). During an average of 5 years' follow-up, MS risk among healthy young adults with serum levels of 25(OH) vitamin D >100 nmol/l was about 60% lower than in individuals of the same age and sex with serum 25(OH) vitamin D levels <100 nmol/l. If confirmed, these findings suggest that a high proportion of MS cases could be effectively prevented by vitamin D supplementation. Furthermore, there is growing evidence that vitamin D insufficiency is a risk factor for conversion from clinically isolated syndrome to MS and for MS progression. Both prevention and treatment trials with vitamin D are needed to confirm these findings and to determine optimal levels of vitamin D.
Background and objective: This report provides results of CSF analyses done in a subset of relapsing remitting MS patients participating in a placebo-controlled, double-blind, phase III clinical trial of IFNβ-Studies supported by the National Multiple Sclerosis Society (grants RG2019, RG2827),a (Avonex®, Biogen). The clinical trial demonstrated that IFNβ-1a treatment resulted in significantly reduced disability progression, annual relapse rate, and new brain lesions visualized by cranial magnetic resonance imaging. The objectives of the current study were to determine: (a) whether CSF abnormalities in MS patients correlated with disease or MRI characteristics, and (b) effects of IFNβ-1a therapy on these CSF abnormalities. Methods: CSF was analyzed from 262 (87%) of the 301 study subjects at entry into the clinical trial, and a second CSF sample was analyzed from 137 of these 262 subjects after 2 years of therapy. CSF cell counts, oligoclonal bands (OCB), IgG index, and free kappa light chains were measured using standard assays. Baseline CSF results were compared with demographic, disease, and MRI parameters. Differences in on-study relapse rate, gadolinium enhancement, and EDSS change according to baseline CSF status was used to determine the predictive value of CSF for subsequent clinical and MRI disease activity. Change in CSF parameters after 104 weeks were used to determine the effects of treatment. Results: (1) At study baseline, 37% of the subjects had abnormal CSF WBC counts, 61% had abnormal levels of CSF free kappa light chains, 84% had abnormal IgG index values, and 90% were positive for OCB. (2) Baseline IgG index, kappa light chains, and OCB showed weakly positive, statistically significant correlations with Gd-enhanced lesion volume and T2 lesion volume. WBC showed a statistically significant correlation with Gd-enhancing lesion volume but was uncorrelated with T2 lesion volume. (3) There was an associated between baseline CSF WBC counts and on-study clinical and MRI disease activity in placebo recipients. (4) IFNβ-1a treatment resulted in significantly reduced CSF WBC counts, but there was no treatment-related change in CSF IgG index, kappa light chains, or OCB, which remained relatively stable over time in both patient groups. Conclusions: The current study documents significant reductions in CSF WBC counts in patients treated with IFNβ-1a for 104 weeks. This finding is considered relevant to the therapeutic response, since CSF WBC counts were found to be positively correlated with subsequent clinical and MRI disease activity in placebo-treated relapsing MS patients.
Abstract Theiler's murine encephalomyelitis virus (TMEV) induces a demyelinating disease in susceptible SJL mice that has similarities to multiple sclerosis in humans. TMEV infection of susceptible mice leads to a persistent virus infection of the central nervous system (CNS), which promotes the development of demyelinating disease associated with an inflammatory immune response in the CNS. TMEV infection of resistant C57BL6 mice results in viral clearance without development of demyelinating disease. Interestingly, TMEV infection of resistant mice deficient in IFNγ leads to a persistent virus infection in the CNS and development of demyelinating disease. We have previously shown that the innate immune response affects development of TMEV- induced demyelinating disease, thus we wanted to determine the role of IFNγ during the innate immune response. TMEV-infected IFNγ-deficient mice had an altered innate immune response, including reduced expression of innate immune cytokines, especially type I interferons. Administration of type I interferons, IFNα and IFNß, to TMEV- infected IFNγ-deficient mice during the innate immune response restored the expression of innate immune cytokines. Most importantly, administration of type I interferons to IFNγ-deficient mice during the innate immune response decreased the virus load in the CNS and decreased development of demyelinating disease. Microglia are the CNS resident immune cells that express innate immune receptors. In TMEV- infected IFNγ-deficient mice, microglia had reduced expression of innate immune cytokines, and administration of type I interferons to these mice restored the innate immune response by microglia. In the absence of IFNγ, microglia from TMEV-infected mice had reduced expression of some innate immune receptors and signaling molecules, especially IRF1. These results suggest that IFNγ plays an important role in the innate immune response to TMEV by enhancing the expression of innate immune cytokines, especially type I interferons, which directly affects the development of demyelinating disease.
Multiple sclerosis (MS) is a chronic disease of unknown etiology that involves the central nervous system and affects an estimated 2.0 million to 2.5 million people worldwide.1 The majority of—but not all—patients with MS develop severe disability 10 to 20 years after diagnosis.2,3 Several controlled therapeutic trials have consistently shown that immunomodulatory treatments, interferon beta and glatiramer acetate, can reduce relapse frequency, relapse-related progression of impairment and disability, and inflammatory activity as depicted by magnetic resonance imaging.4 The common but disputed assumption has been that these clinical and radiographic findings in studies limited to 2 to 3 years' duration translate into long-term benefits, with delay or prevention of long-term disability in patients typically seen in a neurological practice. This is particularly important for a disease with a mean duration of 30 or more years.5,6
We performed yearly MRI analyses on 327 of the total 372 patients in a multicenter, randomized, double-blind, placebo-controlled trial of interferon beta-lb (IFNB). Clinical results are presented in the preceding companion paper. Baseline MRI characteristics were the same in all treatment groups. Fifty-two patients at one center formed a cohort for frequent MRIs (one every 6 weeks) for analysis of disease activity. The MRI results support the clinical results in showing a significant reduction in disease activity as measured by numbers of active scans (median 80% reduction, p = 0.0082) and appearance of new lesions. In addition, there was an equally significant reduction in MRI-detected burden of disease in the treatment as compared with placebo groups (mean group difference of 23%, p = 0.001). These results demonstrate that IFNB has made a significant impact on the natural history of MS in these patients.
ABSTRACT Multiple sclerosis (MS) is a chronic autoimmune disease of the CNS characterized by inflammation, demyelination, and axonal injury. These pathologic effects are manifested in clinical symptoms of relapse and disability. Various disease-modifying therapies have been developed in recent years to modulate the body's immune response. Among the most widely used are the beta interferons (IFNbeta). All produce comparable biological effects and are approved for the treatment of relapsing-remitting MS (RRMS). Although the precise mechanisms through which IFNbeta achieves its antiinflammatory and immunomodulatory effects remain uncertain, several modes of action have been proposed, including inhibition of T-cell activation and proliferation; apoptosis of autoreactive T cells; induction of regulatory T cells; inhibition of leukocyte migration across the blood-brain barrier; cytokine modulation; and potential antiviral activity. Endogenously produced IFNbeta in the injured brain is also now believed to contribute to mediation of antiinflammatory and regenerative effects. All these mechanisms are believed to underlie the therapeutic effect of IFNbeta in the treatment of RRMS.
Ten patients with multiple sclerosis who were treated with human fibroblast interferon (IFN-B) for 6 months showed a significant reduction in their exacerbation rates compared with their rates before treatment (P < .01). The IFN-B was administered intrathecally by serial lumbar punctures. There was no significant change in the exacerbation rates of ten multiple sclerosis control patients before and during the period of observation. The IFN-B recipients have now been on the study a mean of 1.5 years, the controls, 1.2 years. The clinical condition of five of the IFN-B recipients and one of the control patients has improved, whereas the condition of five of the controls and one of the IFN-B recipients has deteriorated (P < .036). These findings warrant cautious optimism about the efficacy of intrathecal IFN-B in altering the course of multiple sclerosis and support concepts of a viral or dysimmune etiology of the disease.
Immunoglobulin production within the central nervous system (CNS) is a prominent feature of multiple sclerosis and its animal model induced by infection with Theiler's meningoencephalitis virus, as well as of other inflammatory and infectious neurological diseases. However, relatively little is known about the plasma cells (PCs) responsible for producing Ig within the CNS.
We induced Theiler's-induced demyelinating disease, characterized by disability, inflammation, and demyelination. We used immunofluorescence to localize and characterize IgG-producing cells, and correlated the morphology with results from CSF and tissue analysis.
Confidence that IgG production occurred within the CNS was gained by demonstrating high levels of IgG in the cerebrospinal fluid in the absence of blood-CSF barrier or blood-brain barrier breakdown. CNS IgG-producing cells were CD138+, like IgG-producing cells elsewhere in the body. Less than 5% of IgG-positive cells were Ki67-positive, indicating that most were nonproliferative PCs. The PCs were present primarily in perivascular infiltrates and in the meninges. Isolated PCs could be found in the CNS parenchyma, and, when present, were largely in demyelinated regions of the cord.
These results demonstrate that PCs are a significant part of this chronic progressive disabling demyelinating disease, and suggest the possibility that these cells play a role in CNS injury by their secretion of immunoglobulin.
Vaccines are still one of the best approaches to manage infectious diseases. Despite the advances in drug therapies, prophylactic medicine is still more cost efficient and minimizes the burden in the heath system. Despite all the research in vaccine development, many infectious diseases are still without an effective vaccine. The use of adjuvants in vaccines has been one successful strategy to increase efficacy. IFNs are widely expressed cytokines that have potent antiviral effects. These cytokines are the first line of defense against viral infections and have important roles in immuno surveillance for malignant cells. One of the most promising uses of IFNs is as adjuvants that are co-applied with antigen in vaccines.
In this review, a cumulative analysis of many of the studies that have used IFN-α, -β, -γ and -λ as adjuvants between 1987 and the present suggests that many do possess the capacity to serve as potent immunoadjuvants for vaccination.
This review provides a very large collection of studies involving all types of IFNs used as adjuvants in vaccines using different vaccination strategies and various animal models.
It is clear that the use of IFNs not only improved the efficacy and safety of most vaccines, but also had important immunomodulatory effect directing T(H)1 immune responses.
White matter (WM) lesions are the classic pathological hallmarks of multiple sclerosis (MS). However, MRI-based WM lesion load shows relatively poor correlation with functional outcome, resulting in the "clinico-radiological paradox" of MS. Unlike lesion based measures, volumetric MRI assessment of brain atrophy shows a strong correlation with functional outcome, and the presence of early atrophy predicts a worse disease course. While extensive literature exists describing MRI characteristics of atrophy in MS, the exact pathogenesis and the substrate of atrophy-gray vs. WM loss, axonal/neuronal damage vs. demyelination, or a combination of the above-remain unclear. Animal models of atrophy would allow for detailed investigations of the pathomechanism, and would contribute to an enhanced understanding of structural-functional connections in this complex disease. We now report that in the Theiler's Murine Encephalitis Virus (TMEV) model of MS in SJL/J mice, significant brain atrophy accompanies the development of the progressive MS-like disease. We conducted volumetric MRI studies in 8 cases and 4 age, gender- and strain-matched control mice. While in controls we did not detect any brain atrophy, significant atrophy developed as early as 3 months into the disease course, and reached its peak by 6 months, resulting in ventricular enlargement by 118% (p=0.00003). A strong correlation (r=-0.88) between atrophy and disability, as assessed by rotarod assay, was also demonstrated. We earlier reported another neurodegenerative feature in this model, the presence of deep gray matter T2 hypointensity in thalamic nuclei. Future studies utilizing this model will allow us to investigate key components of MRI detectable neurodegenerative feature development, their tissue correlations and associations with functional outcome measures. These studies are expected to pave the way to a better understanding of the substrate of disability in MS models.
Multiple Sclerosis (MS) is a complex disease with an unknown etiology and no effective cure, despite decades of extensive research that led to the development of several partially effective treatments. Researchers have only limited access to early and immunologically active MS tissue samples, and the modification of experimental circumstances is much more restricted in human studies compared to studies in animal models. For these reasons, animal models are needed to clarify the underlying immune-pathological mechanisms and test novel therapeutic and reparative approaches. It is not possible for a single mouse model to capture and adequately incorporate all clinical, radiological, pathological and genetic features of MS. The three most commonly studied major categories of animal models of MS include: (1) the purely autoimmune experimental autoimmune/allergic encephalomyelitis (EAE); (2) the virally induced chronic demyelinating disease models, with the main model of Theiler's Murine Encephalomyelitis Virus (TMEV) infection and (3) toxin-induced models of demyelination, including the cuprizone model and focal demyelination induced by lyso-phosphatidyl choline (lyso-lecithine). EAE has been enormously helpful over the past several decades in our overall understanding of CNS inflammation, immune surveillance and immune-mediated tissue injury. Furthermore, EAE has directly led to the development of three approved medications for treatment in multiple sclerosis, glatiramer acetate, mitoxantrone and natalizumab. On the other hand, numerous therapeutical approaches that showed promising results in EAE turned out to be either ineffective or in some cases harmful in MS. The TMEV model features a chronic-progressive disease course that lasts for the entire lifespan in susceptible mice. Several features of MS, including the role and significance of axonal injury and repair, the partial independence of disability from demyelination, epitope spread from viral to myelin epitopes, the significance of remyelination has all been demonstrated in this model. TMEV based MS models also feature several MRI findings of the human disease. Toxin-induced demyelination models has been mainly used to study focal demyelination and remyelination. None of the three main animal models described in this review can be considered superior; rather, they are best viewed as complementary to one another. Despite their limitations, the rational utilization and application of these models to address specific research questions will remain one of the most useful tools in studies of human demyelinating diseases.
Interferon beta (IFNbeta) administered subcutaneously is immunogenic in some patients with multiple sclerosis (MS) and leads to the development of neutralizing antibodies (NAbs). Considerable evidence has accumulated that NAbs diminish or abolish IFNbeta bioactivity, but there is less evidence that NAbs impact clinical efficacy of the drug.
Because a robust effect of IFNbeta is a decrease in enhancing lesions on brain MRI scans, the Betaseron Copaxone in Multiple Sclerosis With Triple-Dose Gadolinium and 3-Tesla MRI Endpoints (BECOME) study, a head-to-head study of IFNbeta-1b vs glatiramer acetate with a primary endpoint of enhancing lesions on MRI, provided an excellent opportunity to determine the effect of NAbs on MRI activity. We measured NAbs and IFNbeta bioactivity by myxovirus resistance protein A gene expression and identified 2 groups of patients: one labeled "bioactivity preserved," with absent NAbs and robust IFNbeta bioactivity (n = 8), and the other labeled "bioactivity lost," with high levels of NAbs and diminished bioactivity (n = 7). The development of enhancing lesions in the groups was then compared.
The incidence of NAbs and effect of NAbs on bioactivity were consistent with previous studies. We analyzed MRI outcomes in patients with NAbs at levels high enough to abolish bioactivity relative to patients without NAbs. For the preserved bioactivity group, the enhancing lesion/scan ratio decreased from 7.6 in the pretreatment period to 2.6 in the posttreatment period, a 66% decrease. For the lost bioactivity group, the decrease was 8.5 to 5.8, only a 32% decrease. Thus, lost bioactivity from high levels of NAbs resulted in reduced therapeutic efficacy of IFNbeta as manifested by diminished reductions in enhancing lesions on MRI.
High levels of anti-interferon beta (IFNbeta) antibodies, which result in diminished bioactivity, are correlated with reduced therapeutic efficacy of IFNbeta.
Multiple sclerosis is (MS) a T-cell autoimmune disease characterized by a relapsing-remitting followed by a progressive phase. Relapses are driven by the adaptive immune system and involve waves of T helper cell 1 (Th1), Th17, and CD8 cells that infiltrate the nervous system and provoke a attack. These cells are modulated by regulatory T and B cells. Infiltration of T cells into the nervous system initiates a complex immunological cascade consisting of epitope spreading, which triggers new attacks, and activation of the innate immune system (microglia, dendritic cells, astrocytes, B cells), which leads to chronic inflammation. The secondary progressive phase is due to neurodegeneration triggered by inflammation and is driven by the innate immune system. Why a shift to the progressive stage occurs and how to prevent it is a central question in MS. Effective treatment of MS must affect multiple disease pathways: suppression of proinflammatory T cells, induction of regulatory T cells, altering traffic of cells into the nervous system, protecting axons and myelin, and controlling innate immune responses. Without biomarkers, the clinical and pathological heterogeneity of MS makes treatment difficult. Treatment is further hampered by untoward adverse effects caused by immune suppression. Nonetheless, major progress has been made in the understanding and treatment of MS. There are three definitions of cure as it applies to MS: (1) halt progression of disease, (2) reverse neurological deficits, and (3) prevent MS. Although the pathways to each of these cures are linked, each requires a unique strategy.
Advanced MRI studies demonstrated several diffuse non-lesional features in multiple sclerosis, including changes detectable in gray matter areas. Standard T2 weighted MRI scans of deep gray matter structures, including the thalamus, caudate, putamen, dentate nuclei often demonstrate hypointensity. T2 hypointensity has been shown to correlate with cognitive, neuropsychiatric and motor dysfunction. The exact pathogenesis of this MRI phenomenon remains unknown. In this manuscript, we demonstrate the first known MS animal model of deep gray matter T2 hypointensity. In TMEV infected SJL/J mice, gradual development of thalamic T2 hypointensity was noted over the disease course. Quantitative analysis of the hypointensity demonstrated a strong correlation between the degree of T2 hypointensity and rotarod detectable disability. We propose that this model will allow mechanistic studies investigating the pathogenesis and significance of deep gray matter T2 hypointensity in MS.
An unusual biphasic central nervous system disease developed in 3-week-old Swiss outbred mice after intracerebral inoculation of the DA strain of Theiler's murine encephalomyelitis virus. Nine to 20 days postinfection 86% of mice became paralyzed, and approximately one-half of these animals survived. During this period neuronal necrosis and microglial proliferation were seen in thalamus, brainstem, and spinal cord. There was an initial phase of virus growth in spinal cord followed by persistent infection at a lower concentration. Virus antigen was readily found in the cytoplasm of neurons by immunofluorescent staining early in the course of infection, whereas after 30 days there was a paucity of cells containing virus antigen which were present only in the spinal cord white matter. Between 1 and 5 months, an intense mononuclear inflammatory cell lesion evolved in the spinal cord leptomeninges and white matter, which coincided with a mild gait disturbance in some surviving mice, and patchy demyelination was found in areas of inflammation. The acute gray matter pathology would appear to be the result of direct virus lytic effect. Although the late white matter lesion culminating in demyelination probably represents a cytocidal infection similar to the situation that exists in certain picornavirus carrier culture systems, a virus-induced immunopathological process merits further study.
Experimental Theiler's mouse encephalomyelitis virus (TMEV) infection in mice is atypical of most other picornavirus infections because virus persists in the host. It was shown previously that low levels of infectious virus are readily detectable in the central nervous system (CNS) despite the presence of substantial titers of serum neutralizing antibody. In this study antibody assays were performed on CNS tissue homogenates, and neutralizing antibody was regularly found in the CNS of TMEV-infected mice. That neutralization by infected CNS extracts was due to antibody was demonstrated by the specificity of neutralization for TMEV and by elimination or marked reduction of neutralization by in vitro treatment with goat antiserum to mouse IgG. In addition, immunofluorescent staining consistently revealed IgG- but not IgM-containing cells in perivascular cuffs and parenchymal lesions of the brains of infected animals. Evidence of local antibody formation in the CNS was found in the actual reversal of the serum-CNS antibody ratio in about one-third of infected mice after three weeks. In contrast, normal mice had a mean serum-CNS antibody ratio of approximately 100:1 after passive transfer of antibody. Possible reasons for the fact that TMEV is not neutralized by antibody and chronic infection is not aborted include the formation of complexes of infectious virus and antibody in the CNS and the production of antibodies with low affinity for TMEV.
We performed yearly MRI analyses on 327 of the total 372 patients in a multicenter, randomized, double-blind, placebo-controlled trial of interferon beta-1b (IFNB). Clinical results are presented in the preceding companion paper. Baseline MRI characteristics were the same in all treatment groups. Fifty-two patients at one center formed a cohort for frequent MRIs (one every 6 weeks) for analysis of disease activity. The MRI results support the clinical results in showing a significant reduction in disease activity as measured by numbers of active scans (median 80% reduction, p = 0.0082) and appearance of new lesions. In addition, there was an equally significant reduction in MRI-detected burden of disease in the treatment as compared with placebo groups (mean group difference of 23%, p = 0.001). These results demonstrate that IFNB has made a significant impact on the natural history of MS in these patients.
This report provides results of CSF analyses done in a subset of relapsing remitting MS patients participating in a placebo-controlled, double-blind, phase III clinical trial of IFNbeta-Studies supported by the National Multiple Sclerosis Society (grants RG2019, RG2827),a (Avonex , Biogen). The clinical trial demonstrated that IFNbeta-1a treatment resulted in significantly reduced disability progression, annual relapse rate, and new brain lesions visualized by cranial magnetic resonance imaging. The objectives of the current study were to determine: (a) whether CSF abnormalities in MS patients correlated with disease or MRI characteristics, and (b) effects of IFNbeta-1a therapy on these CSF abnormalities.
CSF was analyzed from 262 (87%) of the 301 study subjects at entry into the clinical trial, and a second CSF sample was analyzed from 137 of these 262 subjects after 2 years of therapy. CSF cell counts, oligoclonal bands (OCB), IgG index, and free kappa light chains were measured using standard assays. Baseline CSF results were compared with demographic, disease, and MRI parameters. Differences in on-study relapse rate, gadolinium enhancement, and EDSS change according to baseline CSF status was used to determine the predictive value of CSF for subsequent clinical and MRI disease activity. Change in CSF parameters after 104 weeks were used to determine the effects of treatment.
(1) At study baseline, 37% of the subjects had abnormal CSF WBC counts, 61% had abnormal levels of CSF free kappa light chains, 84% had abnormal IgG index values, and 90% were positive for OCB. (2) Baseline IgG index, kappa light chains, and OCB showed weakly positive, statistically significant correlations with Gd-enhanced lesion volume and T2 lesion volume. WBC showed a statistically significant correlation with Gd-enhancing lesion volume but was uncorrelated with T2 lesion volume. (3) There was an associated between baseline CSF WBC counts and on-study clinical and MRI disease activity in placebo recipients. (4) IFNbeta-1a treatment resulted in significantly reduced CSF WBC counts, but there was no treatment-related change in CSF IgG index, kappa light chains, or OCB, which remained relatively stable over time in both patient groups.
The current study documents significant reductions in CSF WBC counts in patients treated with IFNbeta-1a for 104 weeks. This finding is considered relevant to the therapeutic response, since CSF WBC counts were found to be positively correlated with subsequent clinical and MRI disease activity in placebo-treated relapsing MS patients.
Interferon therapies suffer from a relatively short half-life of the products in circulation. To address this issue we investigated the effects of polyethylene glycol modification (PEGylation) on the pharmacokinetic properties of human interferon (IFN)-beta-1a. PEGylation with a linear 20-kDa PEG targeted at a single site on the N-terminal amine had no deleterious effect on its specific activity in an in vitro antiviral assay. In monkeys, PEG IFN-beta-1a treatment induced neopterin and beta2-microglobulin expression (pharmacodynamic markers of activity). Systemic clearance values in monkeys, rats, and mice decreased, respectively, from 232, 261, and 247 ml/h/kg for the unmodified IFN-beta-1a to 30.5, 19.2, and 18.7 ml/h/kg for the PEGylated form, while volume of distribution values decreased from 427, 280, and 328 ml/kg to 284, 173, and 150 ml/kg. The decreased clearance and volume of distribution resulted in higher serum antiviral activity in the PEG IFN-beta-1a-treated animals. In the rat, a more extensive set of dosing routes was investigated, including intraperitoneal, intratracheal, and oral administration. Bioavailability for the PEG IFN-beta-1a was similar to the unmodified protein for each of the extravascular routes examined. For the intraperitoneal route, bioavailability was almost 100%, whereas for the oral and intratracheal routes absorption was low (<5%). In rats, subcutaneous bioavailability was moderate (28%), whereas in monkeys it was approximately 100%. In all instances an improved pharmacokinetic profile for the PEGylated IFN-beta-1a was observed. These findings demonstrate that PEGylation greatly alters the pharmacokinetic properties of IFN-beta-1a, resulting in an increase in systemic exposure following diverse routes of administration.
To examine MRI changes resulting from treatment of secondary progressive MS (SPMS) with two doses of interferon-beta-1a (Rebif).
Interferon-beta (IFN-beta) reduces relapses and delays progression in relapsing-remitting MS, but there are conflicting results on its clinical benefit in SPMS.
In a double-blind, randomized, multicenter, placebo-controlled study (SPECTRIMS), 618 patients received IFN-beta-1a 22 microg, 44 microg, or placebo subcutaneously three times weekly for 3 years. T2 activity and burden of disease (BOD) were measured in 617 patients by using semiannual proton density/T2-weighted (PD/T2) MRI scans. A cohort of 283 patients also had 11 monthly PD/T2 and T1-weighted gadolinium-enhanced (T1-Gd) scans at study start.
Treatment reduced median numbers of active lesions per patient per scan (semiannual T2 activity: 0.17, 0.20 and 0.67 for the high dose, low dose, and placebo, p < 0.0001; monthly combined unique activity [T1+T2]: 0.11, 0.22, and 1.00, p < 0.0001) and accumulation of BOD (percent change from baseline to month 36: -1.3, -0.5, and 10.0 for the high dose, low dose, and placebo, respectively; p = 0.0001). MRI benefit was most evident in the subgroup of patients who reported relapses in the 2 years before the study. Neutralizing antibody development was associated with reduction in treatment effect: antibody-positive patients did not show significant differences from placebo at either dose.
Interferon-beta-1a used in SPMS showed significant effects on all MRI measures, particularly in patients with relapses in the 2 years before the study.
We study the power of IgG synthesis value as a marker of disease activity in multiple sclerosis (MS).
Link index was calculated in 202 MS patients. Time between first, second and third attack and progression index (PI) were compared in patient with normal (NLI) high (HL) or very high Link index (VHLI).
Secondary progressive (SP) patients had a higher LI than relapsing-remitting (RR) and primary progressive (PP) courses (1.10 +/- 0.5 for SP vs 0.86 +/- 0.5 for RR and 0.81 +/- 0.5 for PP, P=0.01 and 0.03, respectively). Having a HLI in MS RR and SP patients has no time effect in the development of the second and third attack. PI was higher in patients with VHIL (0.67 +/- 0.7) vs patients with NLI (0.42 +/- 0.4, P=0.008) and with HLI (0.39 +/- 0.3, P=0.001).
This study confirmed that LI is a good marker of subsequent progression of MS.
Infection of mice by low-neurovirulence Theiler's murine encephalomyelitis virus (TMEV), such as BeAn and DA viruses, provides a relevant experimental animal model for multiple sclerosis (MS). As a step toward determining the kinetics of a persistent central nervous system (CNS) infection that leads to chronic demyelination, we adapted a rapid, accurate and highly specific real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection and quantitation of BeAn virus RNA copy equivalents in mouse tissues. The assay enabled detection of as few as 20-30 copies of BeAn virus RNA per microg of total RNA from infected mouse tissues and results for spinal cord revealed the same high levels of BeAn RNA as detected by Northern hybridization during the first 4 months of the persistent infection, but also was able to detect virus RNA copies as late as 1 year post-infection. Real-time RT-PCR analysis of BeAn virus RNA copy equivalents in different parts of the CNS, analyses not possible by Northern hybridization, revealed the following cline of virus persistence: spinal cord>brainstem/cerebellum>cerebrospinal fluid (CSF)>cerebral hemispheres. Systemic organs, including heart, intestine and mesenteric lymph nodes of infected mice, showed no evidence of viral persistence at 4 months post-infection.
This non-systematic review identified four randomised trials that have tested the efficacy of interferon beta in secondary progressive multiple sclerosis (SPMS). Two were trials of interferon beta 1a (IFNb1a) and two of interferon beta 1b (IFNb1b). All have shown significant reductions in relapse rates and accumulation of new magnetic resonance imaging (MRI) lesions, but only one trial (of IFNb1b) showed significant slowing of disability progression. Post hoc analyses of these trials suggest that the differences in outcomes might be partly explained by the differences between the trials in the proportions of patients with relapsing disease. In one of the trials of IFNb1a (the SPECTRIMS trial), the hazard ratio for progression in the treated relapsing patients with relapses in the two pre-study years was 0.74 compared to placebo patients with pre-study relapses and 1.01 in the treated patients compared to the placebo patients without pre-study relapses. In the same trial, the treatment effects on MRI parameters were more marked in the patients who had recent pre-study relapses compared with those who had not. These observations have led to the recommendation in national guidelines that prescribing of IFNb in SPMS be limited to those patients who have had disabling relapses in the last 2 years. These conclusions should be reviewed when the full results of all four trials have been published.
Japanese encephalitis virus (JEV), although confined to Asia, causes about 35000-50000 cases and 10000 deaths every year, and is the most important cause of encephalitis worldwide. There is no known antiviral treatment for any flavivirus. Results from in-vitro studies and work in animals have shown inteferon alfa has antiviral activity on Japanese encephalitis and other flaviviruses; therefore, we aimed to assess the efficacy of inteferon alfa-2a in Japanese encephalitis.
We did a randomised double-blind placebo-controlled trial of interferon alfa-2a (10 million units/m2, daily for 7 days) in 112 Vietnamese children with suspected Japanese encephalitis, 87 of whom had serologically confirmed infections. Our primary endpoints were hospital death or severe sequelae at discharge. Analysis was by intention to treat.
Overall, 21 children (19%) died, and 17 (15%) had severe sequelae. Outcome at discharge and 3 months did not differ between the two treatment groups; 20 children in the interferon group had a poor outcome (death or severe sequelae), compared with 18 in the placebo group (p=0.85, difference 0.1%, 95% CI -17.5 to 17.6%), there were no long-term side effects of interferon.
The doses of interferon alfa-2a given in this regimen did not improve the outcome of patients with Japanese encephalitis.
Theiler's murine encephalitis virus (TMEV) infection in mice is an established model of CNS demyelinating diseases. The aim of the study was to determine the chronological pattern of lesion development in this model of monophasic fulminant demyelinating disease. We followed six highly susceptible interferon-gamma receptor knockout mice with serial in vivo brain magnetic resonance imaging (MRI) studies to determine changes in overall T2 lesion load and gadolinium enhancement. Altogether, 163 individual lesions were followed over 52 days. The number of lesions increased linearly with time. Four chronological patterns of lesion development were seen: (a) expanding lesions (48.5% of all lesions, 54.05% volume contribution); (b) expanding-retracting lesions (20.85% of all lesions, 15.03% volume contribution); (c) fluctuating lesions (16.6% of all lesions, 28.8% volume contribution); (d) stable lesions (14.05% of all lesions, 2.12% volume contribution). Gadolinium enhancement was not seen in the evolution of every lesion. Enhancement was both time- and lesion type-dependent. Early in the disease course (<43 days after infection), enhancement was almost always seen, later on (>43 days after infection) it was only seen in 8% of new lesions. All of fluctuating, 85.3% of expanding, 83.5% of expanding-retracting, and 56.5% of stable lesions were associated with gadolinium enhancement. We conclude that the MRI features of TMEV-induced demyelination in this model showed four unique chronological patterns, and inconsistent gadolinium enhancement. These novel findings may provide new insights into the pathogenesis of acute fulminant multiple sclerosis (MS).