Ebrima Gibbs

Intermolecular transmission of superoxide dismutase 1 misfolding in living cells

Human wild-type superoxide dismutase-1 (wtSOD1) is known to coaggregate with mutant SOD1 in familial amyotrophic lateral sclerosis (FALS), in double transgenic models of FALS, and in cell culture systems, but the structural determinants of this process are unclear. Here we molecularly dissect the effects of intracellular and cell-free obligately misfolded SOD1 mutant proteins on natively structured wild-type SOD1. Expression of the enzymatically inactive, natural familial ALS SOD1 mutations G127X and G85R in human mesenchymal and neural cell lines induces misfolding of wild-type natively structured SOD1, as indicated by: acquisition of immunoreactivity with SOD1 misfolding-specific monoclonal antibodies; markedly enhanced protease sensitivity suggestive of structural loosening; and nonnative disulfide-linked oligomer and multimer formation. Expression of G127X and G85R in mouse cell lines did not induce misfolding of murine wtSOD1, and a species restriction element for human wtSOD1 conversion was mapped to a region of sequence divergence in loop II and β-strand 3 of the SOD1 β-barrel (residues 24–36), then further refined surprisingly to a single tryptophan residue at codon 32 (W32) in human SOD1. Time course experiments enabled by W32 restriction revealed that G127X and misfolded wtSOD1 can induce misfolding of cell-endogenous wtSOD1. Finally, aggregated recombinant G127X is capable of inducing misfolding and protease sensitivity of recombinant human wtSOD1 in a cell-free system containing reducing and chelating agents; cell-free wtSOD1 conversion was also restricted by W32. These observations demonstrate that misfolded SOD1 can induce misfolding of natively structured wtSOD1 in a physiological intracellular milieu, consistent with a direct protein–protein interaction.

Validating parameters of a luciferase reporter gene assay to measure neutralizing antibodies to IFNβ in multiple sclerosis patients

Neutralizing antibodies (NAbs) can occur in some multiple sclerosis (MS) patients receiving interferon beta (IFNbeta) therapy. NAbs reduce drug bioavailabity and high NAb titers reduce drug efficacy. We describe the validation of the R. Farrell and G. Giovannoni luciferase reporter gene assay to measure NAbs to INFbeta. We assayed 163 sera from IFNbeta treated MS patients with an optimized luciferase method and compared the results to those obtained with the reference cytopathic effect (CPE) method using A549 cells and an encephalomyocarditis virus (EMCV). Binding antibodies (BAbs) were measured using a capture ELISA as a screening test for NAbs in the CPE assay. NAb status measured by the luciferase and the ELISA/CPE method did not yield a significant difference. Log10 NAb titers obtained from the luciferase assay and the A549/EMCV CPE methods correlated very well. The inter-assay coefficient of variation for titers was between 17.8-29.3%, and the intra-assay coefficient of variation was between 6.3-15.2%. The luciferase assay is reliable, appropriately sensitive and requires less time than the currently available NAb methods.

Reduced effectiveness of long-term interferon-β treatment on relapses in neutralizing antibody-positive multiple sclerosis patients: a Canadian multiple sclerosis clinic-based study

Multiple sclerosis (MS) patients treated with interferon-beta (IFN-β) often form anti-IFN-β antibodies accompanied by a reduction in IFN-β bioavailability. The clinical effect of these antibodies remains controversial. MS patients in British Columbia, Canada, must be diagnosed and evaluated annually by neurologists in an MS clinic in order to be reimbursed for their IFN-β prescriptions. We have identified at the UBC MS clinic a cohort of 262 patients, each having been treated with a single IFN-β preparation more than three years, some for nearly a decade. Of 119 patients treated with Betaseron® (IFN-β1b), 18 (15.1%) were neutralizing antibody positive (NAb+) at the time of the study, whereas of 131 treated with subcutaneous Rebif® (IFN-β1a SC), 16 (12.2%) were NAb+, but none of 12 treated with intramuscular Avonex ® (IFN-β1a) had detectable neutralizing antibodies. During the first two years of treatment, the relapse rate was significantly reduced from pre-treatment rates (P < 0.001) and appeared to be unaffected by the subsequent NAb status. However, the relapse rates in the NAb+ patients were significantly greater than in the NAb— patients during years 3 (P < 0.010) and 4 (P < 0.027). Betaseron ® -treated NAb+ patients tended to have more relapses than NAb— patients during year 3 and this almost reached significance (P = 0.056) but their relapse rate did not differ in year 4 and later. In contrast, Rebif ® -treated NAb+ patients tended to have more relapses in year 3 than Rebif ® -treated NAb— patients (P = 0.074), but in year 4 they clearly (P = 0.009) had more relapses than Rebif ® -treated NAb— patients. There was no convincing effect on progression of disability in any group. Multiple Sclerosis 2007; 13: 1127—1137. http://msj.sagepub.com

During 3 years treatment of primary progressive multiple sclerosis with glatiramer acetate, specific antibodies switch from IgG1 to IgG4.

In this study we analyzed the humoral immune response to glatiramer acetate in 16 GA-treated primary progressive MS patients and 9 placebo patients from the PROMiSe study. We have demonstrated that all multiple sclerosis patients (n=16) continuously treated with GA for 3 years developed anti-GA antibodies that peaked at month 3 and remained elevated during the whole study. We have also demonstrated that initially GA-reactive antibodies of the IgG1 subclass predominate, peaking at month 9 of therapy, but after 9 months IgG1 decreases while anti-GA antibodies of the IgG4 subclass increase and remain high for the 3 years of follow-up. These results support a shift from Th1 to Th2 in the antibody response to glatiramer acetate treatment.

The prion protein binds thiamine

Although highly conserved throughout evolution, the exact biological function of the prion protein is still unclear. In an effort to identify the potential biological functions of the prion protein we conducted a small‐molecule screening assay using the Syrian hamster prion protein [shPrP(90–232)]. The screen was performed using a library of 149 water‐soluble metabolites that are known to pass through the blood–brain barrier. Using a combination of 1D NMR, fluorescence quenching and surface plasmon resonance we identified thiamine (vitamin B1) as a specific prion ligand with a binding constant of ∼ 60 μm. Subsequent studies showed that this interaction is evolutionarily conserved, with similar binding constants being seen for mouse, hamster and human prions. Various protein construct lengths, both with and without the unstructured N‐terminal region in the presence and absence of copper, were examined. This indicates that the N‐terminus has no influence on the protein’s ability to interact with thiamine. In addition to thiamine, the more biologically abundant forms of vitamin B1 (thiamine monophosphate and thiamine diphosphate) were also found to bind the prion protein with similar affinity. Heteronuclear NMR experiments were used to determine thiamine’s interaction site, which is located between helix 1 and the preceding loop. These data, in conjunction with computer‐aided docking and molecular dynamics, were used to model the thiamine‐binding pharmacophore and a comparison with other thiamine binding proteins was performed to reveal the common features of interaction.

A Biosensor-Based Characterization of the Affinity Maturation of the Immune Response Against Interferon-β and Correlations with Neutralizing Antibodies in Treated Multiple Sclerosis Patients

The study of antibodies against Interferon-beta (IFN-beta) in treated multiple sclerosis (MS) patients has focused primarily on detection and quantification, with no single method of detection providing a comprehensive characterization. We assessed serial samples of 18 MS patients, treated with IFN-beta for between 66 and 198 months, to characterize the affinity maturation of these antibodies using a biosensor-based approach (Biacore). Biacore utilizes the principles of surface plasmon resonance (SPR) with data from the dissociation phase of the antigen-antibody reaction being inversely proportional to relative antibody affinity. In patients with neutralizing antibodies (NAbs+), mean antibody dissociation rates decreased from 0.00118 +/- 0.00030 s(-1) at month 6 to 0.00021 +/- 0.00008 s(-1) at month 36, followed by a slight increase to 0.00027 +/- 0.00003 s(-1) at month 60. In NAb- patients, mean antibody dissociation rates decreased only very slightly from 0.00130 +/- 0.00025 s(-1) to 0.00105 +/- 0.00020 s(-1) at month 18, followed by a gradual increase to 0.00243 +/- 0.00099 s(-1) at month 60. Our study shows little improvement in antibody affinity in NAb- patients, in contrast to a marked increase in antibody affinity over time in NAb+ patients with a significant correlation between NAb titers and relative antibody dissociation rates (Spearmans correlation, R(2) = -0.374, p < 0.001). The evaluation of relative antibody affinities will contribute to a thorough understanding of the anti-IFN-beta antibody response.

The IgG subclass-specificities of anti-IFNβ antibodies change with time and differ between the IFNβ products in relapsing remitting multiple sclerosis patients

Anti-Interferon beta (IFNbeta) antibodies are frequently produced during treatment with IFNbeta in Multiple Sclerosis (MS) patients. In recent years, it has become clear that these antibodies cause a decrease in IFNbeta-induced biomarkers and in IFNbeta clinical efficacy. Anti-IFNbeta antibodies are mainly of the IgG isotype, which consists of 4 subclasses. In this study, we tested whether changes occurred in IgG subclasses over time. A series of sera from 21 IFNbeta-treated patients (11 IFNbeta-1a, 10 IFNbeta-1b) were analysed longitudinally using a capture ELISA. IFNbeta-1a treated patients had a restricted subclass distribution, whilst IFNbeta-1b-treated patients demonstrated a wider distribution. When compared to IFNbeta-1b-treated patients, IFNbeta-1a-treated patients had lower levels of total and subclass-specific IgGs against IFNbeta. In particular, antibody levels were markedly lower in the neutralizing antibody (NAb) negative (-) category of IFNbeta-1a-treated patients in comparison to the NAb-IFNbeta-1b-treated patients. The most striking observation of this study were the very low levels or complete absence of IgG3 subclass-specific antibodies to IFNbeta in IFNbeta-1a-treated patients. This difference in the levels of IgG3 may help to clarify the differences in the overall pattern of development of anti-IFNbeta antibodies in IFNbeta-1a-and IFNbeta-1b-treated patients.

Serial combination therapy: is immune modulation in multiple sclerosis enhanced by initial immune suppression?

Background Although the concept that an initial course of immune-suppression facilitates subsequent immune-modulation (such as Th1 to Th2 deviation) is attractive for several autoimmune diseases, such a mechanism for serial-combination therapy has never been formally demonstrated. Recently, brief mitoxantrone induction-chemotherapy followed by immune-modulation with glatiramer acetate (GA) was significantly more effective at reducing multiple sclerosis disease activity than with GA alone. Objective To examine whether the benefit of initial immune suppression with mitoxantrone before GA treatment is associated with more efficient immune modulation. Methods IgG1/IgG4 GA-reactive antibody profiles, previously established as markers of GA-induced Th2 immune-deviation, were prospectively measured in vivo in patients treated with GA alone or with mitoxantrone induction therapy followed by GA. Results Significant and sustained increase in IgG4 antibodies (and the anticipated reversal of the IgG1/IgG4 ratio) was seen in patients treated with GA alone. Combination therapy resulted in lesser IgG4 induction (and no reversal of IgG1/IgG4 ratio). Thus, the enhanced efficacy of mitoxantrone–GA combination regimen was associated with decreased, rather than increased, efficiency of shifting the GA-reactive IgG1/IgG4 antibody profile. Conclusion These results provide important insights into mechanisms of combination therapy and therapeutic strategies for autoimmune diseases.

Antibody dissociation rates are predictive of neutralizing antibody (NAb) course: A comparison of interferon beta-1b-treated Multiple Sclerosis (MS) patients with transient versus sustained NAbs

A proportion of multiple sclerosis (MS) patients treated with interferon-β (IFNβ) develop neutralizing antibodies (NAbs), which can reduce therapeutic efficacy. In the Betaseron/Betaferon in Newly Emerging MS for Initial Treatment (BENEFIT) study, 88/277 patients developed NAbs, 48 having transient positivity and 29 having sustained positivity. This study aimed to investigate the antibody binding characteristics of serial sera in a subset of these two patient groups. Using Biacore™, a surface plasmon resonance-based technology that monitors biomolecular interactions in real time, we immobilized pure IFNβ-1b and analyzed antibody binding responses and dissociation rates of these sera. NAb titers correlated directly with binding responses and inversely with dissociation rates, and sera from sustained NAb patients demonstrated significantly higher binding responses and slower dissociation rates than sera from transient NAb patients. Thus, transient and sustained NAbs are quantitatively and qualitatively different, and interestingly, binding responses and dissociation rates at month 12 could predict the NAb course.

High-density lipoproteins suppress Aβ-induced PBMC adhesion to human endothelial cells in bioengineered vessels and in monoculture

BackgroundAlzheimer’s Disease (AD), characterized by accumulation of beta-amyloid (Aβ) plaques in the brain, can be caused by age-related failures to clear Aβ from the brain through pathways that involve the cerebrovasculature. Vascular risk factors are known to increase AD risk, but less is known about potential protective factors. We hypothesize that high-density lipoproteins (HDL) may protect against AD, as HDL have vasoprotective properties that are well described for peripheral vessels. Epidemiological studies suggest that HDL is associated with reduced AD risk, and animal model studies support a beneficial role for HDL in selectively reducing cerebrovascular amyloid deposition and neuroinflammation. However, the mechanism by which HDL may protect the cerebrovascular endothelium in the context of AD is not understood.MethodsWe used peripheral blood mononuclear cell adhesion assays in both a highly novel three dimensional (3D) biomimetic model of the human vasculature composed of primary human endothelial cells (EC) and smooth muscle cells cultured under flow conditions, as well as in monolayer cultures of ECs, to study how HDL protects ECs from the detrimental effects of Aβ.ResultsFollowing Aβ addition to the abluminal (brain) side of the vessel, we demonstrate that HDL circulated within the lumen attenuates monocyte adhesion to ECs in this biofidelic vascular model. The mechanism by which HDL suppresses Aβ-mediated monocyte adhesion to ECs was investigated using monotypic EC cultures. We show that HDL reduces Aβ-induced PBMC adhesion to ECs independent of nitric oxide (NO) production, miR-233 and changes in adhesion molecule expression. Rather, HDL acts through scavenger receptor (SR)-BI to block Aβ uptake into ECs and, in cell-free assays, can maintain Aβ in a soluble state. We confirm the role of SR-BI in our bioengineered human vessel.ConclusionOur results define a novel activity of HDL that suppresses Aβ-mediated monocyte adhesion to the cerebrovascular endothelium.