The MOG antibody non-P42 epitope is predictive of a relapsing course in MOG antibody-associated disease.

BACKGROUND: Myelin oligodendrocyte glycoprotein (MOG) IgG seropositivity is a prerequisite for MOG antibody-associated disease (MOGAD) diagnosis. While a significant proportion of patients experience a relapsing disease, there is currently no biomarker predictive of disease course. We aim to determine whether MOG-IgG epitopes can predict a relapsing course in MOGAD patients. METHODS: MOG-IgG-seropositive confirmed adult MOGAD patients were included (n=202). Serum MOG-IgG and epitope binding were determined by validated flow cytometry live cell-based assays. Associations between epitopes, disease course, clinical phenotype, Expanded Disability Status Scale and Visual Functional System Score at onset and last review were evaluated. RESULTS: Of 202 MOGAD patients, 150 (74%) patients had MOG-IgG that recognised the immunodominant proline42 (P42) epitope and 115 (57%) recognised histidine103/serine104 (H103/S104). Fifty-two (26%) patients had non-P42 MOG-IgG and showed an increased risk of a relapsing course (HR 1.7; 95% CI 1.15 to 2.60, p=0.009). Relapse-freedom was shorter in patients with non-P42 MOG-IgG (p=0.0079). Non-P42 MOG-IgG epitope status remained unchanged from onset throughout the disease course and was a strong predictor of a relapsing course in patients with unilateral optic neuritis (HR 2.7, 95% CI 1.06 to 6.98, p=0.038), with high specificity (95%, 95% CI 77% to 100%) and positive predictive value (85%, 95% CI 45% to 98%). CONCLUSIONS: Non-P42 MOG-IgG predicts a relapsing course in a significant subgroup of MOGAD patients. Patients with unilateral optic neuritis, the most frequent MOGAD phenotype, can reliably be tested at onset, regardless of age and sex. Early detection and specialised management in these patients could minimise disability and improve long-term outcomes.

Seedling performance covaries with dormancy thresholds: maintaining cryptic seed heteromorphism in a fire-prone system.

The production of morphologically different seeds or fruits by the same individual plant is known as seed heteromorphism. Such variation is expected to be selected for in disturbance-prone environments to allow germination into inherently variable regeneration niches. However, there are few demonstrations that heteromorphic seed characteristics should be favored by selection or how they may be maintained. In fire-prone ecosystems, seed heteromorphism is found in the temperatures needed to break physical dormancy, with seeds responding to high or low temperatures, ensuring emergence under variable fire-regime-related soil heating. Because of the relationship between dormancy-breaking temperature thresholds and fire severity, we hypothesize that different post-fire resource conditions have selected for covarying seedling traits, which contribute to maintenance of such heteromorphism. Seeds with low thresholds emerge into competitive conditions, either after low-severity fire or in vegetation gaps, and are therefore likely to experience selection for seedling characteristics that make them good competitors. On the other hand, high-temperature-threshold seeds would emerge into less competitive environments, indicative of stand-clearing high-severity fires, and would not experience the same selective forces. We identified high and low-threshold seed morphs via dormancy-breaking heat treatments and germination trials for two study species and compared seed mass and other morphological characteristics between morphs. We then grew seedlings from the two different morphs, with and without competition, and measured growth and biomass allocation as indicators of seedling performance. Seedlings from low-threshold seeds of both species performed better than their high-threshold counterparts, growing more quickly under competitive conditions, confirming that different performance can result from this seed characteristic. Seed mass or appearance did not differ between morphs, indicating that dormancy-breaking temperature threshold variation is a form of cryptic heteromorphism. The potential shown for the selective influence of different post-fire environmental conditions on seedling performance provides evidence of a mechanism for the maintenance of heteromorphic variation in dormancy-breaking temperature thresholds.

Intra-population level variation in thresholds for physical dormancy-breaking temperature.

BACKGROUND AND AIMS: Intra-population variation in seed dormancy is an advantage for population persistence in unpredictable environments. The important role played by physically dormant species in these habitats makes understanding the level of variation in their dormancy a key ecological question. Heat produced in the soil is the major dormancy-breaking stimulus and, in fire prone ecosystems, soil temperatures generated by fire may vary spatially and over time. While many studies have investigated variation in initial dormancy, a measure that is of little value in fire-prone ecosystems, where initial dormancy levels are uniformly high, intra-population variation in dormancy-breaking temperature thresholds has never been quantified. This study predicted that species would display variation in dormancy-breaking temperature thresholds within populations, and investigated whether this variation occurred between individual plants from the same maternal environment. METHODS: The intra-population variation in dormancy-breaking thresholds of five common physically dormant shrub species (family Fabaceae) from fire-prone vegetation in south-eastern Australia was assessed using heat treatments and germination trials. Replicate batches of seeds from each of four maternal plants of Dillwynia floribunda, Viminaria juncea, Bossiaea heterophylla, Aotus ericoides and Acacia linifolia were treated at 40, 60, 80, 100 and 120 °C. KEY RESULTS: Dormancy-breaking response to heat treatments varied significantly among individual plants for all species, with some individuals able to germinate after heating at low temperatures and others restricting germination to temperatures that only occur as a result of high-severity fires. Germination rate (T50) varied among individuals of three species. CONCLUSIONS: Variation detected among individuals that were in close proximity to each other indicates that strong differences in dormancy-breaking temperature thresholds occur throughout the broader population. Differences found at the individual plant level could contribute to subsequent variation within the seed bank, providing a bet-hedging strategy, and represent a mechanism for increasing the probability of population persistence in the face of fire regime variability.

Targeting B cell dysregulation with emerging therapies in autoimmune demyelinating disorders.

The depletion of B cells has proven to be beneficial in the treatment of autoimmune demyelinating disorders. The high efficacy of these therapies has highlighted the importance of B cells in autoimmunity and prompted investigations into specific B cell subsets that may be aberrant. Recently, a rise in the trialling of alternative B cell-targeting therapies that inhibit targets such as Bruton's tyrosine kinase, interleukin-6 receptor and fragment crystallisable neonatal receptor has also been observed. These agents interfere with specific dysregulated functions of B cells in contrast to the broad removal of many B cell subsets with depletion agents. The therapeutic benefit of these emerging agents will help delineate the contributions of B cells in demyelinating disorders and holds great potential for future treatment.

Validation of a Flow Cytometry Live Cell-Based Assay to Detect Myelin Oligodendrocyte Glycoprotein Antibodies for Clinical Diagnostics.

BACKGROUND: Myelin oligodendrocyte glycoprotein antibodies (MOG Ab) are essential in the diagnosis of MOG Ab-associated disease (MOGAD). Live cell-based assays (CBAs) are the gold standard for MOG Ab detection with improved sensitivity and specificity over fixed CBAs. A number of testing centers have used flow cytometry for its high throughput and quantitative utility. Presently, there is increasing demand to translate these research-based methods into an accredited routine diagnostic setting. METHODS: A flow cytometry live CBA was used to detect MOG Ab in patients with demyelination. Serostatuses were compared between a research-based assay and a streamlined diagnostic assay. Inter-laboratory validation of the streamlined assay was performed in an accredited diagnostic laboratory. Further streamlining was performed by introducing a borderline serostatus range and reducing the number of controls used to determine the positivity threshold. RESULTS: High serostatus agreement (98%-100%) was observed between streamlined and research-based assays. Intra- and inter-assay imprecision was improved in the streamlined assay (mean intra- and inter-assay CV = 7.3% and 27.8%, respectively) compared to the research-based assay (mean intra- and inter-assay CV = 11.8% and 33.6%, respectively). Borderline positive and clear positive serostatuses were associated with confirmed phenotypes typical of MOGAD. Compared to using 24 controls, robust serostatus classification was observed when using 13 controls without compromising analytical performance (93%-98.5% agreement). CONCLUSIONS: Flow cytometry live CBAs show robust utility in determining MOG Ab serostatus. Streamlining and standardizing use of this assay for diagnostics would improve the accuracy and reliability of routine testing to aid diagnosis and treatment of patients with demyelination.

Pro-inflammatory dopamine-2 receptor-specific T cells in paediatric movement and psychiatric disorders.

OBJECTIVES: A dysregulated inflammatory response against the dopamine-2 receptor (D2R) has been implicated in movement and psychiatric disorders. D2R antibodies were previously reported in a subset of these patients; however, the role of T cells in these disorders remains unknown. Our objective was to identify and characterise pro-inflammatory D2R-specific T cells in movement and psychiatric disorders. METHODS: Blood from paediatric patients with movement and psychiatric disorders of suspected autoimmune and neurodevelopmental aetiology (n = 24) and controls (n = 16) was cultured in vitro with a human D2R peptide library, and D2R-specific T cells were identified by flow cytometric quantification of CD4+CD25+CD134+ T cells. Cytokine secretion was analysed using a cytometric bead array and ELISA. HLA genotypes were examined in D2R-specific T-cell-positive patients. D2R antibody seropositivity was determined using a flow cytometry live cell-based assay. RESULTS: Three immunodominant regions of D2R, amino acid (aa)121-131, aa171-181 and aa396-416, specifically activated CD4+ T cells in 8/24 patients. Peptides corresponding to these regions were predicted to bind with high affinity to the HLA of the eight positive patients and had also elicited the secretion of pro-inflammatory cytokines IL-2, IFN- γ, TNF, IL-6, IL-17A and IL-17F. All eight patients were seronegative for D2R antibodies. CONCLUSION: Autoreactive D2R-specific T cells and a pro-inflammatory Th1 and Th17 cytokine profile characterise a subset of paediatric patients with movement and psychiatric disorders, further underpinning the theory of immune dysregulation in these disorders. These findings offer new perspectives into the neuroinflammatory mechanisms of movement and psychiatric disorders and can influence patient diagnosis and treatment.

Effects of the Positive Threshold and Data Analysis on Human MOG Antibody Detection by Live Flow Cytometry.

Human autoantibodies targeting myelin oligodendrocyte glycoprotein (MOG Ab) have become a useful clinical biomarker for the diagnosis of a spectrum of inflammatory demyelinating disorders. Live cell-based assays that detect MOG Ab against conformational MOG are currently the gold standard. Flow cytometry, in which serum binding to MOG-expressing cells and control cells are quantitively evaluated, is a widely used observer-independent, precise, and reliable detection method. However, there is currently no consensus on data analysis; for example, seropositive thresholds have been reported using varying standard deviations above a control cohort. Herein, we used a large cohort of 482 sera including samples from patients with monophasic or relapsing demyelination phenotypes consistent with MOG antibody-associated demyelination and other neurological diseases, as well as healthy controls, and applied a series of published analyses involving a background subtraction (delta) or a division (ratio). Loss of seropositivity and reduced detection sensitivity were observed when MOG ratio analyses or when 10 standard deviation (SD) or an arbitrary number was used to establish the threshold. Background binding and MOG ratio value were negatively correlated, in which patients seronegative by MOG ratio had high non-specific binding, a characteristic of serum that must be acknowledged. Most MOG Ab serostatuses were similar across analyses when optimal thresholds obtained by ROC analyses were used, demonstrating the robust nature and high discriminatory power of flow cytometry cell-based assays. With increased demand to identify MOG Ab-positive patients, a consensus on analysis is vital to improve patient diagnosis and for cross-study comparisons to ultimately define MOG Ab-associated disorders.