Magnetic resonance imaging approaches for studying mouse models of multiple sclerosis: A mini review.

Given that multiple sclerosis (MS) is a complex disease with an unclear etiology, a single animal model is unlikely to accurately represent all aspects of pathology and clinical features of the human condition. However, the availability of three major types of murine models of MS, that is, experimental autoimmune encephalomyelitis (EAE), viral models, and toxic models, enables studies of several relevant features of this debilitating disease. Researchers have recently begun to combine magnetic resonance imaging (MRI) technologies with other experimental strategies to acquire complementary information, for example, anatomical and functional, and study the effect of experimental manipulations longitudinally in a noninvasive way. This review summarizes the latest MRI studies investigating critical aspects of MS, such as atrophy, demyelination, neuroaxonal damage, and neuroinflammation, in mouse models of MS. Advanced techniques will be briefly discussed, providing references to specialized literature for the readers. Thus, this review aims to describe different imaging protocols used to study critical aspects of MS in a research laboratory, discussing the main related findings in the most significant murine models of the disease.

High throughput method for detecting murine brain atrophy using a clinical 3T MRI.

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.

The CXCL13 Index as a Predictive Biomarker for Activity in Clinically Isolated Syndrome.

Multiple sclerosis (MS) is a clinically heterogenous disease. Currently, we cannot identify patients with more active disease who may potentially benefit from earlier interventions. Previous data from our lab identified the CXCL13 index (ICXCL13), a measure of intrathecal production of CXCL13, as a potential biomarker to predict future disease activity in MS patients two years after diagnosis. Patients with clinically isolated syndrome (CIS) or radiologically isolated syndrome (RIS) underwent a lumbar puncture and blood draw, and the ICXCL13 was determined. They were then followed for at least 5 years for MS activity. Patients with high ICXCL13 were more likely to convert to clinically definite MS (82.4%) compared to those with low ICXCL13 (10.0%). The data presented below demonstrate that this predictive ability holds true in CIS and RIS patients, and for at least five years compared to our initial two-year follow-up study. These data support the concept that ICXCL13 has the potential to be used to guide immunomodulatory therapy in MS.

Differential neuro-immune patterns in two clinically relevant murine models of multiple sclerosis.

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.

Sleep Loss Promotes Astrocytic Phagocytosis and Microglial Activation in Mouse Cerebral Cortex.

We previously found that Mertk and its ligand Gas6, astrocytic genes involved in phagocytosis, are upregulated after acute sleep deprivation. These results suggested that astrocytes may engage in phagocytic activity during extended wake, but direct evidence was lacking. Studies in humans and rodents also found that sleep loss increases peripheral markers of inflammation, but whether these changes are associated with neuroinflammation and/or activation of microglia, the brain's resident innate immune cells, was unknown. Here we used serial block-face scanning electron microscopy to obtain 3D volume measurements of synapses and surrounding astrocytic processes in mouse frontal cortex after 6-8 h of sleep, spontaneous wake, or sleep deprivation (SD) and after chronic (∼5 d) sleep restriction (CSR). Astrocytic phagocytosis, mainly of presynaptic components of large synapses, increased after both acute and chronic sleep loss relative to sleep and wake. MERTK expression and lipid peroxidation in synaptoneurosomes also increased to a similar extent after short and long sleep loss, suggesting that astrocytic phagocytosis may represent the brain's response to the increase in synaptic activity associated with prolonged wake, clearing worn components of heavily used synapses. Using confocal microscopy, we then found that CSR but not SD mice show morphological signs of microglial activation and enhanced microglial phagocytosis of synaptic elements, without obvious signs of neuroinflammation in the CSF. Because low-level sustained microglia activation can lead to abnormal responses to a secondary insult, these results suggest that chronic sleep loss, through microglia priming, may predispose the brain to further damage.SIGNIFICANCE STATEMENT We find that astrocytic phagocytosis of synaptic elements, mostly of presynaptic origin and in large synapses, is upregulated already after a few hours of sleep deprivation and shows a further significant increase after prolonged and severe sleep loss, suggesting that it may promote the housekeeping of heavily used and strong synapses in response to the increased neuronal activity of extended wake. By contrast, chronic sleep restriction but not acute sleep loss activates microglia, promotes their phagocytic activity, and does so in the absence of overt signs of neuroinflammation, suggesting that like many other stressors, extended sleep disruption may lead to a state of sustained microglia activation, perhaps increasing the brain's susceptibility to other forms of damage.

Treatment of Theiler's virus-induced demyelinating disease with teriflunomide.

Teriflunomide is an oral therapy approved for the treatment of relapsing remitting multiple sclerosis (MS), showing both anti-inflammatory and antiviral properties. Currently, it is uncertain whether one or both of these properties may explain teriflunomide's beneficial effect in MS. Thus, to learn more about its mechanisms of action, we evaluated the effect of teriflunomide in the Theiler's encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) model, which is both a viral infection and an excellent model of the progressive disability of MS. We assessed the effects of the treatment on central nervous system (CNS) viral load, intrathecal immune response, and progressive neurological disability in mice intracranially infected with TMEV. In the TMEV-IDD model, we showed that teriflunomide has both anti-inflammatory and antiviral properties, but there seemed to be no impact on disability progression and intrathecal antibody production. Notably, benefits in TMEV-IDD were mostly mediated by effects on various cytokines produced in the CNS. Perhaps the most interesting result of the study has been teriflunomide's antiviral activity in the CNS, indicating it may have a role as an antiviral prophylactic and therapeutic compound for CNS viral infections.

The immune response in the CNS in Theiler's virus induced demyelinating disease switches from an early adaptive response to a chronic innate-like response.

Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is an important model of the progressive disability caused by irreversible CNS tissue injury, and provides an example of how a CNS pathogen can cause inflammation, demyelination, and neuronal damage. We were interested in which molecules, especially inflammatory mediators, might be upregulated in the CNS throughout TMEV-IDD. We quantitated by a real-time RT-PCR multi-gene system the expression of a pathway-focused panel of genes at 30 and 165 days post infection, characterizing both the early inflammatory and the late neurodegenerative stages of TMEV-IDD. Also, we measured 32 cytokines/chemokines by multiplex Luminex analysis in CSF specimens from early and late TMEV-IDD as well as sham-treated mice. Results indicate that, in the later stage of TMEV-IDD, activation of the innate immune response is most prominent: TLRs, type I IFN response genes, and innate immunity-associated cytokines were highly expressed in late TMEV-IDD compared to sham (p ≤ 0.0001) and early TMEV-IDD (p < 0.05). Conversely, several molecular mediators of adaptive immune response were highly expressed in early TMEV-IDD (all p ≤ 0.001). Protein detection in the CSF was broadly concordant with mRNA abundance of the corresponding gene measured by real-time RT-PCR in the spinal cord, since several cytokines/chemokines were increased in the CSF of TMEV-IDD mice. Results show a clear shift from adaptive to innate immunity from early to late TMEV-IDD, indicating that adaptive and innate immune pathways are likely involved in the development and progression of the disease to different extents. CSF provides an optimal source of biomarkers of CNS neuroinflammation.

Chemokine biomarkers in central nervous system tissue and cerebrospinal fluid in the Theiler's virus model mirror those in multiple sclerosis.

Chemokines have increasingly been implicated in inflammatory and infectious disease of the central nervous system, both as biomarkers and as molecules important in pathogenesis. Multiple sclerosis is a disabling disease of unknown etiology, and recently chemokines have been identified as being upregulated molecules in the disease. We were interested in how the chemokine expression patterns in the central nervous system of a viral model of multiple sclerosis, Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), compared to that in humans with multiple sclerosis. Cerebrospinal fluid and spinal cord tissue were analyzed for expression of a range of cytokines and chemokines. Three chemokines, CXCL10, CXCL9, and CCL5 were strongly and specifically upregulated in both the cerebrospinal fluid and spinal cord in chronic disease, a pattern identical to that in multiple sclerosis. These data, the first study of cytokines in central nervous system tissue and cerebrospinal fluid in TMEV-IDD, support the hypothesis that multiple sclerosis is caused by chronic infection with an as-yet unidentified pathogen, possibly a picornavirus.

Immunological shifts during early-stage Parkinson's disease identified with DNA methylation data on longitudinally collected blood samples.

Parkinson's disease (PD) is the second most common neurodegenerative disease in the United States. Decades before motor symptoms manifest, non-motor symptoms such as hyposmia and rapid eye movement (REM) sleep behavior disorder are highly predictive of PD. Previous immune profiling studies have identified alterations to the proportions of immune cells in the blood of clinically defined PD patients. However, it remains unclear if these phenotypes manifest before the clinical diagnosis of PD. We utilized longitudinal DNA methylation (DNAm) microarray data from the Parkinson's Progression Marker's Initiative (PPMI) to perform immune profiling in clinically defined PD and prodromal PD patients (Prod). We identified previously reported changes in neutrophil, monocyte, and T cell numbers in PD patients. Additionally, we noted previously unrecognized decreases in the naive B cell compartment in the defined PD and Prod patient group. Over time, we observed the proportion of innate immune cells in PD blood increased, but the proportion of adaptive immune cells decreased. We identified decreases in T and B cell subsets associated with REM sleep disturbances and early cognitive decline. Lastly, we identified increases in B memory cells associated with both genetic (LRRK2 genotype) and infectious (cytomegalovirus seropositivity) risk factors of PD. Our analysis shows that the peripheral immune system is dynamic as the disease progresses. The study provides a platform to understand how and when peripheral immune alterations occur in PD and whether intervention at particular stages may be therapeutically advantageous.

CXCL10/IgG1 Axis in Multiple Sclerosis as a Potential Predictive Biomarker of Disease Activity.

BACKGROUND AND OBJECTIVES: Multiple sclerosis (MS) is a heterogeneous disease, and its course is difficult to predict. Prediction models can be established by measuring intrathecally synthesized proteins involved in inflammation, glial activation, and CNS injury. METHODS: To determine how these intrathecal proteins relate to the short-term, i.e., 12 months, disease activity in relapsing-remitting MS (RRMS), we measured the intrathecal synthesis of 46 inflammatory mediators and 14 CNS injury or glial activation markers in matched serum and CSF samples from 47 patients with MS (pwMS), i.e., 23 RRMS and 24 clinically isolated syndrome (CIS), undergoing diagnostic lumbar puncture. Subsequently, all pwMS were followed for ≥12 months in a retrospective follow-up study and ultimately classified into "active", i.e., developing clinical and/or radiologic disease activity, n = 18) or "nonactive", i.e., not having disease activity, n = 29. Disease activity in patients with CIS corresponded to conversion to RRMS. Thus, patients with CIS were subclassified as "converters" or "nonconverters" based on their conversion status at the end of a 12-month follow-up. Twenty-seven patients with noninflammatory neurologic diseases were included as negative controls. Data were subjected to differential expression analysis and modeling techniques to define the connectivity arrangement (network) between neuroinflammation and CNS injury relevant to short-term disease activity in RRMS. RESULTS: Lower age and/or higher CXCL13 levels positively distinguished active/converting vs nonactive/nonconverting patients. Network analysis significantly improved the prediction of short-term disease activity because active/converting patients featured a stronger positive connection between IgG1 and CXCL10. Accordingly, analysis of disease activity-free survival demonstrated that pwMS, both RRMS and CIS, with a lower or negative IgG1-CXCL10 correlation, have a higher probability of activity-free survival than the patients with a significant correlation (p < 0.0001, HR ≥ 2.87). DISCUSSION: Findings indicate that a significant IgG1-CXCL10 positive correlation predicts the risk of short-term disease activity in patients with RRMS and CIS. Thus, the present results can be used to develop a predictive model for MS activity and conversion to RRMS.

Classification of individuals based on ex-vivo glatiramer acetate-induced interferon-γ and interleukin-4 response.

BACKGROUND: Glatiramer acetate (GA) in multiple sclerosis acts through the induction of GA-specific T-helper 2 cells. Nevertheless, the phenomenon is not universal in patients, explaining individual differences in clinical response. OBJECTIVE: The objective of this article was to categorize GA-treated patients. METHOD: An enhanced quantitative PCR assay was used for measuring ex-vivo GA-induced IFNγ and IL4 mRNA responses in mononuclear cells from 23 healthy donors, 27 untreated patients, 33 short-term (≤6 months) and 77 long-term (>6 months) GA-treated patients. Thresholds for IFNγ and IL4 transcriptional response were calculated by ROC analysis and long-term treated patients were compared in terms of prognostic stratification. RESULTS: Thresholds for IFNγ and IL4 transcriptional response were calculated at 5.36 and 1.41 relative expression (RE). Finally, 67% of long-term treated patients scored above both response thresholds. These patients had a higher proportion of relapse-free subjects (74% vs 40% when compare to patients who scored below both thresholds) and a significantly better relapse-free survival rate (p=0.006; CI 0.29-0.75). The negative predictive value to predict adverse clinical outcome was 79% (CI 0.63-0.90), meaning that by a positive response, there is a 79% chance that the patient will not experience a negative outcome at 3 years. CONCLUSIONS: Our enhanced quantitative PCR assay produced clinically significant results for GA-treated patients. As such, if patients have a positive response, it means they have less chance of a relapse, while patients with a negative response have a greater probability of a worse outcome.

Theiler's virus-induced demyelinating disease as an infectious model of progressive multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease of unknown etiology. However, several studies suggest that infectious agents, e.g., Human Herpes Viruses (HHV), may be involved in triggering the disease. Molecular mimicry, bystander effect, and epitope spreading are three mechanisms that can initiate immunoreactivity leading to CNS autoimmunity in MS. Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) is a pre-clinical model of MS in which intracerebral inoculation of TMEV results in a CNS autoimmune disease that causes demyelination, neuroaxonal damage, and progressive clinical disability. Given the spectra of different murine models used to study MS, this review highlights why TMEV-IDD represents a valuable tool for testing the viral hypotheses of MS. We initially describe how the main mechanisms of CNS autoimmunity have been identified across both MS and TMEV-IDD etiology. Next, we discuss how adaptive, innate, and CNS resident immune cells contribute to TMEV-IDD immunopathology and how this relates to MS. Lastly, we highlight the sexual dimorphism observed in TMEV-IDD and MS and how this may be tied to sexually dimorphic responses to viral infections. In summary, TMEV-IDD is an underutilized murine model that recapitulates many unique aspects of MS; as we learn more about the nature of viral infections in MS, TMEV-IDD will be critical in testing the future therapeutics that aim to intervene with disease onset and progression.

Divergent complement system activation in two clinically distinct murine models of multiple sclerosis.

Multiple sclerosis (MS) is a neurological disease featuring neuroinflammation and neurodegeneration in young adults. So far, most research has focused on the peripheral immune system, which appears to be the driver of acute relapses. Concurrently, the mechanisms underlying neurodegeneration in the progressive forms of the disease remain unclear. The complement system, a molecular component of the innate immunity, has been recently implicated in several neurological disorders, including MS. However, it is still unknown if the complement proteins detected in the central nervous system (CNS) are actively involved in perpetuating chronic inflammation and neurodegeneration. To address this knowledge gap, we compared two clinically distinct mouse models of MS: 1) proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (rEAE) resembling a relapsing-remitting disease course, and 2) Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) resembling a progressive disease. Real-time PCR was performed in the spinal cord of rEAE mice, TMEV-IDD mice and age-matched sham controls to quantify gene expression for a broad range of complement components. In both experimental models, we found significantly increased expression of complement factors, such as C1q, C3, CfB, and C3aR. We showed that the complement system, specifically the classical complement pathway, was associated with TMEV-IDD pathogenesis, as the expression of C1q, C3 and C3aR1 were all significantly correlated to a worse disease outcome (all P≤0.0168). In line with this finding, C1q and C3 deposition was observed in the spinal cord of TMEV-IDD mice. Furthermore, C1q deposition was detected in spinal cord regions characterized by inflammation, demyelination, and axonal damage. Conversely, activation of the classical complement cascade seemed to result in protection from rEAE (C1q: P=0.0307). Interestingly, the alternative pathway related to a worse disease outcome in rEAE (CFb: P=0.0006). Overall, these results indicate potential divergent roles for the complement system in MS. The chronic-progressive disease form is more reliant on the activation of the classic complement pathway, while protecting from acute relapses. Conversely, relapsing MS appears more likely affected by the alternative pathway. Understanding the functions of the complement system in MS is critical and can lead to better, more targeted therapies in the future.

Oxidized and aggregated recombinant human interferon beta is immunogenic in human interferon beta transgenic mice.

PURPOSE: To study the effect of oxidation on the structure of recombinant human interferon beta-1a (rhIFNß-1a) and its immunogenicity in wild-type and immune-tolerant transgenic mice. METHODS: Untreated rhIFNß-1a was degraded by metal-catalyzed oxidation, H(2)O(2)-mediated oxidation, and guanidine-mediated unfolding/refolding. Four rhIFNß-1a preparations with different levels of oxidation and aggregation were injected intraperitoneally in mice 15× during 3 weeks. Both binding and neutralizing antibodies were measured. RESULTS: All rhIFNß-1a preparations contained substantial amounts of aggregates. Metal-catalyzed oxidized rhIFNß-1a contained high levels of covalent aggregates as compared with untreated rhIFNß-1a. H(2)O(2)-treated rhIFNß-1a showed an increase in oligomer and unrecovered protein content by HP-SEC; RP-HPLC revealed protein oxidation. Guanidine-treated rhIFNß-1a mostly consisted of dimers and oligomers and some non-covalent aggregates smaller in size than those in untreated rhIFNß-1a. All degraded samples showed alterations in tertiary protein structure. Wild-type mice showed equally high antibody responses against all preparations. Transgenic mice were discriminative, showing elevated antibody responses against both metal-catalyzed oxidized and H(2)O(2)-treated rhIFNß-1a as compared to untreated and guanidine-treated rhIFNß-1a. CONCLUSIONS: Oxidation-mediated aggregation increased the immunogenicity of rhIFNß-1a in transgenic mice, whereas aggregated preparations devoid of measurable oxidation levels were hardly immunogenic.

Memory B Cells in Multiple Sclerosis: Emerging Players in Disease Pathogenesis.

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Once thought to be primarily driven by T cells, B cells are emerging as central players in MS immunopathogenesis. Interest in multiple B cell phenotypes in MS expanded following the efficacy of B cell-depleting agents targeting CD20 in relapsing-remitting MS and inflammatory primary progressive MS patients. Interestingly, these therapies primarily target non-antibody secreting cells. Emerging studies seek to explore B cell functions beyond antibody-mediated roles, including cytokine production, antigen presentation, and ectopic follicle-like aggregate formation. Importantly, memory B cells (Bmem) are rising as a key B cell phenotype to investigate in MS due to their antigen-experience, increased lifespan, and rapid response to stimulation. Bmem display diverse effector functions including cytokine production, antigen presentation, and serving as antigen-experienced precursors to antibody-secreting cells. In this review, we explore the cellular and molecular processes involved in Bmem development, Bmem phenotypes, and effector functions. We then examine how these concepts may be applied to the potential role(s) of Bmem in MS pathogenesis. We investigate Bmem both within the periphery and inside the CNS compartment, focusing on Bmem phenotypes and proposed functions in MS and its animal models. Finally, we review how current immunomodulatory therapies, including B cell-directed therapies and other immunomodulatory therapies, modify Bmem and how this knowledge may be harnessed to direct therapeutic strategies in MS.

Are CSF CXCL13 concentrations solely dependent on intrathecal production? A commentary on "Chemokine CXCL13 in serum, CSF, and blood-CSF barrier function".

Pilz et al. (Fluids Barriers CNS 17:7; 2020) investigated how CSF CXCL13 concentrations are influenced by CXCL13 serum concentrations and blood-CSF barrier (BCSFB) function, comparing the impact of serum CXCL13 levels and Qalbumin (CSF albumin/serum albumin) on CSF CXCL13 among patients with CNS inflammation categorized as CXCL13 negative, low, medium, or high. Among all CXCL13 groups, their results showed no correlation between CSF CXCL13 concentrations and serum CXCL13 or Qalbumin. The authors argue that, in contrast to other proteins, CXCL13 passage across the BCSFB does not occur, regardless of BCSFB function, and is instead solely influenced by intrathecal production. In contrast to the authors' findings, in our studies including both non-inflammatory neurological disorders (NIND; n = 62) and multiple sclerosis (MS) patients we observed a significant correlation between serum CXCL13 concentrations and CSF CXCL13 concentrations. We review several observations which may underlie these contrasting results, including (1) the impact of serum CXCL13 concentrations on CSF CXCL13 in patients with lower intrathecal CXCL13 production and thus lower CXCL13 concentrations (i.e. NIND and MS), (2) the proposed diffusion dynamics of the small molecule CXCL13 across the BCSFB, and (3) differing definitions of negative versus elevated CSF CXCL13 concentrations determined by an assay's relative sensitivity. In conclusion, we argue that for patients with moderately elevated CSF CXCL13 concentrations, serum CXCL13 concentrations influence CSF CXCL13 levels, and thus the appropriate corrections including incorporation of CSF/serum ratios and Qalbumin values should be utilized.

Acute-phase proteins investigation based on lectins affinity capture prior to 2-DE separation: application to serum from multiple sclerosis patients.

Plasma acute-phase proteins (APPs) glyco-isoforms are important biomarkers of inflammatory processes such as those occurring in multiple sclerosis (MS). Specific analysis of these proteins is often hampered by sample biochemical complexity. The aim of our study was to set up a method to accurately visualize, identify and quantify APPs glyco-isoforms in human serum. An enrichment strategy based on affinity chromatography using the carbohydrate-binding proteins concanavalin A (ConA) and erythrina cristagalli lectin (ECL) was applied to pooled serum samples from 15 patients and 9 healthy individuals. Image analysis of 2-DE detected 30 spots with a fold change higher than 1.5. A total of 14 were statistically significant (p value<0.05): 7 up-regulated and 7 down-regulated in MS samples. ESI LC-Nanospray IT mass spectrometry analysis confirmed that all of them were APPs isoforms supporting the idea that the accurate analysis of differential glycosylation profiles in these biomarkers is instrumental to distinguish between MS patients and healthy subjects. Additionally, overlaps in ConA/ECL maps protein patterns suggest how the used lectins are able to bind sugars harbored by the same oligosaccharide structure. Among identified proteins, the presence of complex and/or hybrid type N-linked sugar structures is well known. Performing galectin-3 binding and Western blotting, we were able to demonstrate a correlation between hybrid type glyco-isoforms of ß-haptoglobin and MS. In conclusion, although the patho-physiological role of the identified species still remains unclear and further validations are needed, these findings may have a relevant impact on disease-specific marker identification approaches.

Aggregated recombinant human interferon Beta induces antibodies but no memory in immune-tolerant transgenic mice.

PURPOSE: To study the influence of protein aggregation on the immunogenicity of recombinant human interferon beta (rhIFNbeta) in wild-type mice and transgenic, immune-tolerant mice, and to evaluate the induction of immunological memory. METHODS: RhIFNbeta-1b and three rhIFNbeta-1a preparations with different aggregate levels were injected intraperitoneally in mice 15x during 3 weeks, and the mice were rechallenged with rhIFNbeta-1a. The formation of binding (BABs) and neutralizing antibodies (NABs) was monitored. RESULTS: Bulk rhIFNbeta-1a contained large, mainly non-covalent aggregates and stressed rhIFNbeta-1a mainly covalent, homogeneous (ca. 100 nm) aggregates. Reformulated rhIFNbeta-1a was essentially aggregate-free. All products induced BABs and NABs in wild-type mice. Immunogenicity in the transgenic mice was product dependent. RhIFNbeta-1b showed the highest and reformulated rhIFNbeta-1a the lowest immunogenicity. In contrast with wild-type mice, transgenic mice did not show NABs, nor did they respond to the rechallenge. CONCLUSIONS: The immunogenicity of the products in transgenic mice, unlike in wild-type mice, varied. In the transgenic mice, neither NABs nor immunological memory developed. The immunogenicity of rhIFNbeta in a model reflecting the human immune system depends on the presence and the characteristics of aggregates.

Intrathecally produced CXCL13: A predictive biomarker in multiple sclerosis.

BACKGROUND: Clinicians caring for patients with Multiple Sclerosis (MS) need improved biomarkers to aid them in disease management. OBJECTIVE: We assessed the predictive value of the candidate biomarker CXCL13 index in comparison to oligoclonal bands (OCBs) and CSF neurofilament light (NfL) concentration, examining the ability of each biomarker to predict future disease activity in clinically and radiologically isolated syndromes, relapsing-remitting MS, and progressive MS. METHODS: Matched serum and CSF samples were obtained from 67 non-inflammatory neurologic disease patients and 67 MS patients. CSF and serum CXCL13 and CSF NfL were analyzed by Luminex and ELISA, respectively. CXCL13 data were also analyzed as CSF/serum ratios and indices. Electronic medical records were accessed to determine diagnosis, CSF profiles, and disease activity after the lumbar puncture. RESULTS: Among CXCL13 measures, CXCL13 index was the best predictor of future disease activity in MS patients (AUC = 0.82; CI = 0.69-0.95; p = 0.0002). CXCL13 index values were significantly elevated in activity-positive MS patients compared to activity-negative patients (p < 0.0001). As a single predictor, CXCL13 index outperformed both OCBs and CSF NfL in sensitivity, specificity, and positive and negative predictive value, for future disease activity in MS patients. Moreover, combining CXCL13 index and CSF NfL status improved sensitivity and predictive values for disease activity in MS patients. CONCLUSIONS: The CXCL13 index is an excellent candidate prognostic biomarker for disease activity in patients with MS.

SeXX Matters in Multiple Sclerosis.

Multiple sclerosis (MS) is the most common chronic inflammatory and neurodegenerative disease of the central nervous system (CNS). An interesting feature that this debilitating disease shares with many other inflammatory disorders is that susceptibility is higher in females than in males, with the risk of MS being three times higher in women compared to men. Nonetheless, while men have a decreased risk of developing MS, many studies suggest that males have a worse clinical outcome. MS exhibits an apparent sexual dimorphism in both the immune response and the pathophysiology of the CNS damage, ultimately affecting disease susceptibility and progression differently. Overall, women are predisposed to higher rates of inflammatory relapses than men, but men are more likely to manifest signs of disease progression and worse CNS damage. The observed sexual dimorphism in MS may be due to sex hormones and sex chromosomes, acting in parallel or combination. In this review, we outline current knowledge on the sexual dimorphism in MS and discuss the interplay of sex chromosomes, sex hormones, and the immune system in driving MS disease susceptibility and progression.