Unveiling the proteome-wide autoreactome enables enhanced evaluation of emerging CAR T cell therapies in autoimmunity.

Given the global surge in autoimmune diseases, it is critical to evaluate emerging therapeutic interventions. Despite numerous new targeted immunomodulatory therapies, comprehensive approaches to apply and evaluate the effects of these treatments longitudinally are lacking. Here, we leveraged advances in programmable-phage immunoprecipitation methodology to explore the modulation, or lack thereof, of autoantibody profiles, proteome-wide, in both health and disease. Using a custom set of over 730,000 human-derived peptides, we demonstrated that each individual, regardless of disease state, possesses a distinct and complex constellation of autoreactive antibodies. For each individual, the set of resulting autoreactivites constituted a unique immunological fingerprint, or "autoreactome," that was remarkably stable over years. Using the autoreactome as a primary output, we evaluated the relative effectiveness of various immunomodulatory therapies in altering autoantibody repertoires. We found that therapies targeting B cell maturation antigen (BCMA) profoundly altered an individual's autoreactome, while anti-CD19 and anti-CD20 therapies had minimal effects. These data both confirm that the autoreactome comprises autoantibodies secreted by plasma cells and strongly suggest that BCMA or other plasma cell-targeting therapies may be highly effective in treating currently refractory autoantibody-mediated diseases.

Individual myasthenia gravis autoantibody clones can efficiently mediate multiple mechanisms of pathology.

Serum autoantibodies targeting the nicotinic acetylcholine receptor (AChR) in patients with autoimmune myasthenia gravis (MG) can mediate pathology via three distinct molecular mechanisms: complement activation, receptor blockade, and antigenic modulation. However, it is unclear whether multi-pathogenicity is mediated by individual or multiple autoantibody clones. Using an unbiased B cell culture screening approach, we generated a library of 11 human-derived AChR-specific recombinant monoclonal autoantibodies (mAb) and assessed their binding properties and pathogenic profiles using specialized cell-based assays. Five mAbs activated complement, three blocked α-bungarotoxin binding to the receptor, and seven induced antigenic modulation. Furthermore, two clonally related mAbs derived from one patient were each highly efficient at more than one of these mechanisms, demonstrating that pathogenic mechanisms are not mutually exclusive at the monoclonal level. Using novel Jurkat cell lines that individually express each monomeric AChR subunit (α2ßδε), these two mAbs with multi-pathogenic capacity were determined to exclusively bind the α-subunit of AChR, demonstrating an association between mAb specificity and pathogenic capacity. These findings provide new insight into the immunopathology of MG, demonstrating that single autoreactive clones can efficiently mediate multiple modes of pathology. Current therapeutic approaches targeting only one autoantibody-mediated pathogenic mechanism may be evaded by autoantibodies with multifaceted capacity.

A score that predicts aquaporin-4 IgG positivity in patients with longitudinally extensive transverse myelitis.

BACKGROUND AND PURPOSE: Longitudinally extensive transverse myelitis (LETM) associated with aquaporin-4 autoantibodies (AQP4-IgG) can cause severe disability. Early diagnosis and prompt treatment are critical to prevent relapses. A novel score is described based on clinical and neuroimaging characteristics that predicts AQP4-IgG positivity in patients with LETM. METHODS: Patients were enrolled both retrospectively and prospectively from multiple Italian centers. Clinical and neuroimaging characteristics of AQP4-IgG positive and negative patients were compared through univariate and multivariate analysis. RESULTS: Sixty-six patients were included. Twenty-seven (41%) were AQP4-IgG positive and median age at onset was 45.5 years (range 19-81, interquartile range 24). Female sex (odds ratio [OR] 17.9, 95% confidence interval [CI] 2.6-381.9; p = 0.014), tonic spasms (OR 45.6, 95% CI 3.1-2197; p = 0.017) and lesion hypointensity on T1-weighted images (OR 52.9, 95% CI 6.8-1375; p = 0.002) were independently associated with AQP4-IgG positivity. The AQP4-IgG positivity in myelitis (AIM) score predicted AQP4-IgG positivity with 85% sensitivity and 95% specificity. Positive and negative likelihood ratios were 16.6 and 0.2 respectively. The inter-rater and intra-rater agreement in the score application were both excellent. CONCLUSIONS: The AIM score predicts AQP4-IgG positivity with good sensitivity and specificity in patients with a first episode of LETM. The score may assist clinicians in early diagnosis and treatment of AQP4-IgG positive LETM.

Unearthing the Rosetta Stone of public antibody responses.

Comprehensive profiling of humoral responses to viruses reveals that germline-encoded V gene motifs govern the emergence of recurrent antibody epitopes across individuals.

Clinicoserological insights into patients with immune checkpoint inhibitor-induced myasthenia gravis.

To compare the immunopathology of immune checkpoint inhibitor-induced myasthenia gravis (ICI-MG) and idiopathic MG, we profiled the respective AChR autoantibody pathogenic properties. Of three ICI-MG patients with AChR autoantibodies, only one showed complement activation and modulation/blocking potency, resembling idiopathic MG. In contrast, AChR autoantibody-mediated effector functions were not detected in the other two patients, questioning the role of their AChR autoantibodies as key mediators of pathology. The contrasting properties of AChR autoantibodies in these cases challenge the accuracy of serological testing in establishing definite ICI-MG diagnoses and underscore the importance of a thorough clinical assessment when evaluating ICI-related adverse events.

Unveiling the autoreactome: Proteome-wide immunological fingerprints reveal the promise of plasma cell depleting therapy.

The prevalence and burden of autoimmune and autoantibody mediated disease is increasing worldwide, yet most disease etiologies remain unclear. Despite numerous new targeted immunomodulatory therapies, comprehensive approaches to apply and evaluate the effects of these treatments longitudinally are lacking. Here, we leverage advances in programmable-phage immunoprecipitation (PhIP-Seq) methodology to explore the modulation, or lack thereof, of proteome-wide autoantibody profiles in both health and disease. We demonstrate that each individual, regardless of disease state, possesses a distinct set of autoreactivities constituting a unique immunological fingerprint, or "autoreactome", that is remarkably stable over years. In addition to uncovering important new biology, the autoreactome can be used to better evaluate the relative effectiveness of various therapies in altering autoantibody repertoires. We find that therapies targeting B-Cell Maturation Antigen (BCMA) profoundly alter an individual's autoreactome, while anti-CD19 and CD-20 therapies have minimal effects, strongly suggesting a rationale for BCMA or other plasma cell targeted therapies in autoantibody mediated diseases.

Early Basal Leaf Removal at Different Sides of the Canopy Improves the Quality of Aglianico Wine.

It is well known that the early removal of basal leaves is a viticultural practice adopted to improve the exposure of clusters to direct sunlight and UV radiation and increase the phenolic compounds and anthocyanin concentration in the berries. The aim of this work was to study the influence of early basal leaf removal on Aglianico wines produced in the Apulia region (southern Italy) during three consecutive seasons. Three vine treatments were carried out, where 100% of the cluster-zone leaves on the north, south and both sides of the canopy were removed. Undefoliated plants were used as a control. The effect of the treatments on the basic chemical parameters, phenol content and volatile composition of wines was investigated using WineScan FT-MIR, spectrophotometry, HPLC-DAD and SPME-GC/MS. Early defoliation increased the amounts of flavonoids (+35-40%), anthocyanins (+15-18%), total polyphenols (+10%), antioxidant activity (+8-14%) and colour intensity (+10%), especially when leaf removal was applied on the south side. Moreover, leaf removal led to a 40% increase in free anthocyanins when applied on the south side of the canopy, while a 24% increase was observed when applied to the north side and 21% when applied to the north and south sides. A negative effect was observed on volatile compounds, which decreased by about 18, 14 and 13% when the treatment was applied on the north, north-south and south sides of the canopy, respectively. In conclusion, early leaf removal treatments allow for the modulation of the phenolic and volatile concentrations of Aglianico wines.

Novel pathophysiological insights in autoimmune myasthenia gravis.

PURPOSE OF REVIEW: This review summarizes recent insights into the immunopathogenesis of autoimmune myasthenia gravis (MG). Mechanistic understanding is presented according to MG disease subtypes and by leveraging the knowledge gained through the use of immunomodulating biological therapeutics. RECENT FINDINGS: The past two years of research on MG have led to a more accurate definition of the mechanisms through which muscle-specific tyrosine kinase (MuSK) autoantibodies induce pathology. Novel insights have also emerged from the collection of stronger evidence on the pathogenic capacity of low-density lipoprotein receptor-related protein 4 autoantibodies. Clinical observations have revealed a new MG phenotype triggered by cancer immunotherapy, but the underlying immunobiology remains undetermined. From a therapeutic perspective, MG patients can now benefit from a wider spectrum of treatment options. Such therapies have uncovered profound differences in clinical responses between and within the acetylcholine receptor and MuSK MG subtypes. Diverse mechanisms of immunopathology between the two subtypes, as well as qualitative nuances in the autoantibody repertoire of each patient, likely underpin the variability in therapeutic outcomes. Although predictive biomarkers of clinical response are lacking, these observations have ignited the development of assays that might assist clinicians in the choice of specific therapeutic strategies. SUMMARY: Recent advances in the understanding of autoantibody functionalities are bringing neuroimmunologists closer to a more detailed appreciation of the mechanisms that govern MG pathology. Future investigations on the immunological heterogeneity among MG patients will be key to developing effective, individually tailored therapies.

The clinical need for clustered AChR cell-based assay testing of seronegative MG.

Trial eligibility in myasthenia gravis (MG) remains largely dependent on a positive autoantibody serostatus. This significantly hinders seronegative MG (SNMG) patients from receiving potentially beneficial new treatments. In a subset of SNMG patients, acetylcholine receptor (AChR) autoantibodies are detectable by a clustered AChR cell-based assay (CBA). Of 99 SNMG patients from two academic U.S. centers, 18 (18.2%) tested positive by this assay. Autoantibody positivity was further validated in 17/18 patients. In a complementary experiment, circulating AChR-specific B cells were identified in a CBA-positive SNMG patient. These findings corroborate the clinical need for clustered AChR CBA testing when evaluating SNMG patients.

Clinical features and outcome of patients with autoimmune cerebellar ataxia evaluated with the Scale for the Assessment and Rating of Ataxia.

BACKGROUND AND PURPOSE: This study was undertaken to assess the long-term outcome of patients with paraneoplastic and non paraneoplastic autoimmune cerebellar ataxia (ACA) using the Scale for the Assessment and Rating of Ataxia (SARA). METHODS: Patients with subacute cerebellar ataxia admitted to our institution between September 2012 and April 2020 were prospectively recruited. Serum and/or cerebrospinal fluid was tested for neural autoantibodies by indirect immunofluorescence on mouse brain, cell-based assays, and radioimmunoassay. SARA and modified Rankin Scale (mRS) score were employed to assess patients' outcome. RESULTS: Fifty-five patients were recruited, of whom 23 (42%) met the criteria for cerebellar ataxia of autoimmune etiology. Neural autoantibodies were detected in 22 of 23 patients (Yo-immunoglobulin G [IgG], n = 6; glutamic acid decarboxylase 65-IgG, n = 3; metabotropic glutamate receptor 1-IgG, n = 2; voltage-gated calcium channel P/Q type-IgG, n = 2; Hu-IgG, n = 1; glial fibrillary acidic protein-IgG, n = 1; IgG-binding unclassified antigens, n = 7). Thirteen patients were diagnosed with paraneoplastic cerebellar syndrome (PCS) and 10 with idiopathic ACA. All patients received immunotherapy. Median SARA score was higher in the PCS group at all time points (p = 0.0002), while it decreased significantly within the ACA group (p = 0.049) after immunotherapy. Patients with good outcome (mRS ≤ 2) had less neurological disability (SARA < 15) at disease nadir (p = 0.039) and presented less frequently with paraneoplastic neurological syndrome (p = 0.0028). The univariate linear regression model revealed a good correlation between mRS and SARA score both at disease onset (p < 0.0001) and at last follow-up (p < 0.0001). SARA score < 11 identified patients with good outcome. CONCLUSIONS: Patients with idiopathic ACA significantly improved after immunotherapy. SARA score accurately reflects patients' clinical status and may be a suitable outcome measure for patients with ACA.

GABA-cadabra: autoantibodies trick neurotransmitter receptors and induce seizures.

Epileptic seizures can be mediated by patient autoantibodies targeting ion channels expressed in the brain.

Assessing the role of a tissue-based assay in the diagnostic algorithm of autoimmune encephalitis.

Tissue-based assay (TBA) is a widely-used method to detect neural autoantibodies, but the diagnostic accuracy for autoimmune encephalitis (AE) has not yet been adequately measured. We retrospectively evaluated the sensitivity and specificity of an indirect immunofluorescence TBA (IIF-TBA) in 159 patients with suspected AE. Serum and cerebrospinal fluid (CSF) specimens were collected and tested from December 2012 to September 2020. In the paired sample analysis, serum testing showed higher sensitivity than CSF, while the latter had higher specificity. Based on these results, we clarify the advantages of using a TBA as the principal screening method for patients with suspected AE.

Effect of pre-harvest inactivated yeast treatment on the anthocyanin content and quality of table grapes.

Due to the global warming, more and more often the red-pink grape varieties grown in the Mediterranean basin reveal an insufficient accumulation of anthocyanins and thus a scarce coloration. Nowadays, this is becoming an important technological issue, which may result in the reduction of the fresh market value of table grape. This study aimed at assessing the effect of the pre-harvest treatment by specific inactivated yeasts (YE) on the qualitative parameters and anthocyanin pattern of three red table grape varieties, which typically present poor and/or incomplete coloration during warm years. An increment of anthocyanins level up to almost seven folds corresponding to an improvement of red skin appearance was observed in all the treated table grapes, which was preserved after cold storage, too. While, no significant influence on chemical composition and berry texture and dimensions was found, meaning that YE did not cause appreciable taste changes in grapes.

Effect of two exogenous plant growth regulators on the color and quality parameters of seedless table grape berries.

Some red-pink table grape varieties, cultivated in warm climates, can fail in achieving the right level of anthocyanins responsible for the intense and uniform red color of berries. Nowadays, this is becoming an important technological issue in the Mediterranean area, which may result in decreasing market acceptance and potential economic value of table grape. Usually, plant growth regulators or phytohormones, such as S-ABA, can overcome this problem because they drive the accumulation of anthocyanins over the ripening season. Harpin proteins (HrP), which enhance the plant disease resistance, may be supposed to stimulate the anthocyanins biosynthesis in grape skin if applied close to veraison. Therefore, this research aimed at comparing the effect of HrP and S-ABA over the anthocyanin and color improvement of Crimson Seedless table grape grown in Southern Italy. For the first time, the exogenous treatment with HrP showed as effective as the less sustainable S-ABA one in favoring the anthocyanin accumulation, leading to peonidin-3-O-glucoside, cyanidin-3-O-glucoside, and malvidin-3-O-glucoside values up to 4 folds higher than control grapes and giving rise to a greater concentration of the more stable acylated anthocyanins. Overall, the color of berries was improved but keeping high the other quality characteristics.

Paraneoplastic neurological syndromes.

Paraneoplastic neurological syndromes (PNS) constitute a heterogeneous group of cancer-related disorders that can affect any level of the central and peripheral nervous system. There is compelling evidence that PNS are caused by an immune response directed against neural antigens that are abnormally expressed by the tumour. PNS are frequently associated with neural-specific autoantibodies whose characterization has direct implications for diagnostic workup, treatment and outcome. The last decade has seen a dramatic rise in the discovery of novel autoantibodies associated with PNS, which has led to more accurate diagnoses and earlier treatments, potentially resulting in better outcomes. The latest advancements in the field of autoimmune neurology have paved the way to a more comprehensive understanding of PNS; yet, many aspects of their immunopathogenesis remain to be elucidated and patient-tailored treatment strategies still need to be optimized.

Acute cerebellar stroke and middle cerebral artery stroke exert distinctive modifications on functional cortical connectivity: A comparative study via EEG graph theory.

OBJECTIVE: We tested whether acute cerebellar stroke may determine changes in brain network architecture as defined by cortical sources of EEG rhythms. METHODS: Graph parameters of 41 consecutive stroke patients (<5 days from the event) were studied using eLORETA EEG sources. Network rearrangements of stroke patients were investigated in delta, alpha 2, beta 2 and gamma bands in comparison with healthy subjects. RESULTS: The delta network remodeling was similar in cerebellar and middle cerebral artery strokes, with a reduction of small-worldness. Beta 2 and gamma small-worldness, in the right hemisphere of patients with cerebellar stroke, increase respect to healthy subjects, while alpha 2 small-worldness increases only among patients with a middle cerebral artery stroke. CONCLUSIONS: The network remodeling characteristics are independent on the size of the ischemic lesion. In the early post-acute stages cerebellar stroke differs from the middle cerebral artery one because it does not cause alpha 2 network remodeling while it determines a high frequency network reorganization in beta 2 and gamma bands with an increase of small-worldness characteristics. SIGNIFICANCE: These findings demonstrate changes in the balance of local segregation and global integration induced by cerebellar acute stroke in high EEG frequency bands. They need to be integrated with appropriate follow-up to explore whether further network changes are attained during post-stroke outcome stabilization.