Immunotherapies in MuSK-positive Myasthenia Gravis; an IgG4 antibody-mediated disease.

Muscle-specific kinase (MuSK) Myasthenia Gravis (MG) represents a prototypical antibody-mediated disease characterized by predominantly focal muscle weakness (neck, facial, and bulbar muscles) and fatigability. The pathogenic antibodies mostly belong to the immunoglobulin subclass (Ig)G4, a feature which attributes them their specific properties and pathogenic profile. On the other hand, acetylcholine receptor (AChR) MG, the most prevalent form of MG, is characterized by immunoglobulin (Ig)G1 and IgG3 antibodies to the AChR. IgG4 class autoantibodies are impotent to fix complement and only weakly bind Fc-receptors expressed on immune cells and exert their pathogenicity via interfering with the interaction between their targets and binding partners (e.g. between MuSK and LRP4). Cardinal differences between AChR and MuSK-MG are the thymus involvement (not prominent in MuSK-MG), the distinct HLA alleles, and core immunopathological patterns of pathology in neuromuscular junction, structure, and function. In MuSK-MG, classical treatment options are usually less effective (e.g. IVIG) with the need for prolonged and high doses of steroids difficult to be tapered to control symptoms. Exceptional clinical response to plasmapheresis and rituximab has been particularly observed in these patients. Reduction of antibody titers follows the clinical efficacy of anti-CD20 therapies, a feature implying the role of short-lived plasma cells (SLPB) in autoantibody production. Novel therapeutic monoclonal against B cells at different stages of their maturation (like plasmablasts), or against molecules involved in B cell activation, represent promising therapeutic targets. A revolution in autoantibody-mediated diseases is pharmacological interference with the neonatal Fc receptor, leading to a rapid reduction of circulating IgGs (including autoantibodies), an approach already suitable for AChR-MG and promising for MuSK-MG. New precision medicine approaches involve Chimeric autoantibody receptor T (CAAR-T) cells that are engineered to target antigen-specific B cells in MuSK-MG and represent a milestone in the development of targeted immunotherapies. This review aims to provide a detailed update on the pathomechanisms involved in MuSK-MG (cellular and humoral aberrations), fostering the understanding of the latest indications regarding the efficacy of different treatment strategies.

Thyroid autoimmunity following alemtuzumab treatment in multiple sclerosis patients: a prospective study.

Autoimmune thyroid disease (AITD) is the most common adverse effect in alemtuzumab (ALZ) treated relapsing-remitting (RR) multiple sclerosis (MS) patients. The objective of this prospective study was to analyze the occurrence, timing of onset, clinical course, and laboratory characteristics of AITD post-ALZ. We evaluated 35 RRMS patients treated with ALZ at a single academic MS center; clinical and laboratory data were collected before ALZ initiation and thereafter quarterly on follow-up with a median of 43.5 months. Seventeen out of 31 patients (54.8%) with no prior history of thyroid dysfunction developed AITD with a mean onset of 19.4 months ± 10.2 (SD) after the first ALZ cycle; Graves' disease (GD) (n = 9); hypothyroidism with positive stimulating thyrotropin receptor antibodies (TRAb) (n = 1); Hashimoto thyroiditis (HT) (n = 6); HT with hypothyroidism (n = 1). Interestingly, seven of nine (77.7%) GD patients showed a fluctuating course. Three out of four patients with preexisting thyroid disease remained stable, whereas one with prior HT and hypothyroidism developed fluctuating GD. All patients with GD commenced antithyroid drugs (ATDs); five continued on "block and replace" treatment; one required radioactive iodine, and one total thyroidectomy. Our analysis showed earlier onset of ALZ-induced AITD in comparison to most other ALZ cohorts; overall, these patients required complex therapeutic approaches of the AITD. We observed a higher rate of fluctuating GD, with earlier onset and lower remission rate than previously reported, which in the majority of patients required prolonged "block and replace" therapy in the minimum dose of each therapeutic agent or more definitive interventions.

Neuronal and Neuroaxonal Damage Cerebrospinal Fluid Biomarkers in Autoimmune Encephalitis Associated or Not with the Presence of Tumor.

The aim of this study was to evaluate the association of neuronal damage biomarkers (neurofilament light chain (NFL) and total tau protein (T-tau)) in the CSF of patients with autoimmune encephalitis (AE) with the presence of an underlying malignancy and to determine correlations with patient characteristics. The study comprised 21 patients with encephalitis associated with antibodies against intracellular (n = 11) and surface/synaptic antigens (extracellular, n = 10) and non-inflammatory disease controls (n = 10). Patients with AE associated with intracellular antigens had increased CSF-NFL (p = 0.003) but not T-tau levels compared to controls. When adjusted for age, CSF-NFL but not CSF-T-tau was higher in patients with encephalitis associated with intracellular antigens as compared to those with encephalitis associated with extracellular antigens (p = 0.032). Total tau and NFL levels were not significantly altered in patients with encephalitis associated with extracellular antigens compared to controls. NFL in the total cohort correlated with neurological signs of cerebellar dysfunction, peripheral neuropathy, presence of CV2 positivity, presence of an underlying tumor and a more detrimental clinical outcome. AE patients with abnormal MRI findings displayed higher NFL levels compared to those without, albeit with no statistical significance (p = 0.07). Using receiver operating characteristic curve analysis, CSF-NFL levels with a cut-off value of 969 pg/mL had a sensitivity and specificity of 100% and 76.19%, respectively, regarding the detection of underlying malignancies. Our findings suggest that neuronal integrity is preserved in autoimmune encephalitis associated with extracellular antigens and without the presence of tumor. However, highly increased NFL is observed in AE associated with intracellular antigens and presence of an underlying tumor. CSF-NFL could potentially be used as a diagnostic biomarker of underlying malignancies in the clinical setting of AE.

HLA-genotyping by next-generation-sequencing reveals shared and unique HLA alleles in two patients with coexisting neuromyelitis optica spectrum disorder and thymectomized myasthenia gravis: Immunological implications for mutual aetiopathogenesis?

The exact immunopathogenesis, genetic mechanisms and triggering factors underlying myasthenia gravis (MG) and neuromyelitis optica (NMO) remain unknown and the coexistence may underline an aetiopathogenetic link be- tween these two diseases. We report the cases of two thymectomized patients with acetylcholine receptor (AChR) antibody (Ab)-positive MG who eventually developed AQP4-NMO. Next-Generation Sequencing (NGS) analysis showed that patient-1 had two HLA alleles previously associated with MG, mainly HLA-A*01:01:01 and HLA-DRB1*03:01, present in a haplotype in Caucasian MG patients (HLA-A1-B8-DR3-DQ2). Patient-2, expressed HLA-C*07:01:01, a well characterized MG risk factor and HLA-DQB1*05:02:01, previously described both in MG and NMO patients. Finally, we observed two common alleles in patient 1 and 2, HLA-DQA1*05:01:01 and HLA-DPB1*04:02:01. We believe that this study provides clinical evidence of the role of specific HLA alleles in rare forms of combined human peripheral and CNS autoimmunity, a fact that enhances the aim towards tailor-made therapeutic decision making.

Immunopathology of Tumefactive Demyelinating Lesions-From Idiopathic to Drug-Related Cases.

Tumefactive demyelinating lesions (TDL) represent a diagnostic dilemma for clinicians, and in rare atypical cases a collaboration of a neuroradiologist, a neurologist, and a neuropathologist is warranted for accurate diagnosis. Recent advances in neuropathology have shown that TDL represent an umbrella under which many different diagnostic entities can be responsible. TDL can emerge not only as part of the spectrum of classic multiple sclerosis (MS) but also can represent an idiopathic monophasic disease, a relapsing disease with recurrent TDL, or could be part of the myelin oligodendrocyte glycoprotein (MOG)- and aquaporin-4 (AQP4)-associated disease. TDL can appear during the MS disease course, and increasingly cases arise showing an association with specific drug interventions. Although TDL share common features with classic MS lesions, they display some unique features, such as extensive and widespread demyelination, massive and intense parenchymal infiltration by macrophages along with lymphocytes (mainly T but also B cells), dystrophic changes in astrocytes, and the presence of Creutzfeldt cells. This article reviews the existent literature regarding the neuropathological findings of tumefactive demyelination in various disease processes to better facilitate the identification of disease signatures. Recent developments in immunopathology of central nervous system disease suggest that specific pathological immune features (type of demyelination, infiltrating cell type distribution, specific astrocyte pathology and complement deposition) can differentiate tumefactive lesions arising as part of MS, MOG-associated disease, and AQP4 antibody-positive neuromyelitis optica spectrum disorder. Lessons from immunopathology will help us not only stratify these lesions in disease entities but also to better organize treatment strategies. Improved advances in tissue biomarkers should pave the way for prompt and accurate diagnosis of TDL leading to better outcomes for patients.

Myasthenia gravis, atypical polyneuropathy and multiple autoimmune phenomena in the same patient, with HLA-immunogenetic profile expectable for Greek chronic inflammatory demyelinating polyneuropathy: a case report.

PURPOSE: The comorbidity of myasthenia gravis (MG), with other autoimmune disorders like systemic lupus erythematosus (SLE), is relatively frequent but the co-occurrence with chronic inflammatory demyelinating polyneuropathy (CIDP) along with various autoimmune manifestations in the absence of thymoma is of extreme rarity. Our aim is to report a case of a woman who presented the concomitant appearance of MG, axonal sensory-motor polyneuropathy and hepatitis that may indicate an underlying pathogenetic link among the different autoimmune disorders. MATERIALS AND METHODS/RESULTS: We present a case of a 54-year-old woman, with a generalized MG and a chronic sensory-motor polyneuropathy, hypothyroidism, anaemia, hepatitis, livedo reticularis and facial flush, of assumed autoimmune background, like SLE, although with persistent negative ANA antibodies, from the beginning and through the whole following years. The Human Leukocyte Antigen (HLA)-DRB1 genotyping showed a profile of alleles (DRB1*11:01/11:04) compatible with CIDP of mainly female gender in Greece and frequencies close to those of Sjogren's syndrome and scleroderma's in the Greek population. The diagnostic problems, the atypical clinical, electrophysiological and immunological features are discussed, along with the rarity of the case, with this exceptional combination of autoimmune manifestations, which could be truly associated under the clinical umbrella of a systemic disease, like SLE. However, our patient did not ever fulfil the SLE criteria. CONCLUSIONS: To raise awareness among clinicians about the exceptional combination of autoimmune manifestations driven by a specific HLA background.

Recurrent Fulminant Tumefactive Demyelination With Marburg-Like Features and Atypical Presentation: Therapeutic Dilemmas and Review of Literature.

Atypical forms of demyelinating diseases with tumor-like lesions and aggressive course represent a diagnostic and therapeutic challenge for neurologists. Herein, we describe a 50-year-old woman presenting with subacute onset of left hemiparesis, memory difficulties and headache. Brain MRI revealed a tumefactive right frontal-parietal lesion with perilesional edema, mass effect and homogenous post-contrast enhancement, along with other small atypical lesions in the white-matter. Brain biopsy of cerebral lesion ruled out lymphoma or any other neoplastic process and patient placed on corticosteroids with complete clinical/radiological remission. Two years after disease initiation, there was disease exacerbation with reappearance of the tumor-like mass. The patient initially responded to high doses of corticosteroids but soon became resistant. Plasma-exchange sessions were not able to limit disease burden. Resistance to therapeutic efforts led to a second biopsy that showed perivascular demyelination, predominantly consisting of macrophages, with a small number of T and B lymphocytes, and the presence of reactive astrocytes, typical of Creutzfeldt-Peters cells. The patient received high doses of cyclophosphamide with substantial clinical/radiological response but relapsed after 7-intensive cycles. She received 4-weekly doses of rituximab with disease exacerbation and brainstem involvement. She eventually died with complicated pneumonia. We present a very rare case of recurrent tumefactive demyelinating lesions, with atypical tumor-like characteristics, with initial response to corticosteroids and cyclophosphamide, but subsequent development of drug-resistance and unexpected exacerbation upon rituximab administration. Our clinical case raises therapeutic dilemmas and points to the need for immediate and appropriate immunosuppression in difficult to treat tumefactive CNS lesions with Marburg-like features.

Recurrent myelitis and asymptomatic hypophysitis in IgG4-related disease: case-based review.

IgG4-related disease (IgG4-RD) is a disorder with various clinical manifestations. Central nervous system (CNS) involvement is well recognized, with hypertrophic pachymeningitis and hypophysitis being the most common manifestations. Spinal cord involvement is an extremely rare manifestation. We present the first case of an IgG4-RD patient with spinal cord parenchymal disease and concurrent hypophysitis. We review also the current literature about CNS parenchymal involvement in the context of IgG4-RD. A young female presented with clinical symptoms of myelitis. Cervical spinal cord magnetic resonance imaging (MRI) displayed features of longitudinally extensive transverse myelitis (LETM). Brain MRI showed a small number of high-intensity lesions in the deep white matter and enlargement of hypophysis with homogeneous gadolinium enhancement (asymptomatic hypophysitis). Diagnostic workup revealed elevated IgG4 serum levels (146 mg/dL). Our patient fulfilled the organ-specific diagnostic criteria of IgG4-hypophysitis. Treatment with intravenous glucocorticoids led to rapid clinical response, and to the substantial resolution of imaging findings. Azathioprine was used as a maintenance treatment. One relapse occurred 2 years after the initial diagnosis and patient was re-treated with glucocorticoids. Three years after relapse, patient is in remission with azathioprine. We present the first case of myelitis with radiological features of LETM associated with increased IgG4 serum levels and the simultaneous presence of asymptomatic IgG4-related hypophysitis.

Cell-autonomous epithelial activation of AIM2 (absent in melanoma-2) inflammasome by cytoplasmic DNA accumulations in primary Sjögren's syndrome.

Primary Sjögren's syndrome (SS) is characterized by chronic periductal inflammatory infiltrates in the salivary glands. Several previous studies have indicated that the ductal epithelia of SS patients play a pro-inflammatory role and manifest an intrinsically activated status, as demonstrated in cultured non-neoplastic ductal salivary gland epithelial cell (SGEC) lines. Herein, we investigated the activation of inflammasomes in the salivary epithelia of SS patients and non-SS controls, using salivary biopsy tissues and SGEC lines. The ductal epithelial cells of SS patients were found to display significant activation of the AIM2 (absent in melanoma-2) inflammasome. Such activation occurred in a cell-autonomous manner, as it was illustrated by the constitutively high expression of AIM2 activation-related genes, the presence of cytoplasmic ASC specks and the increased spontaneous IL-1ß production observed in patients' SGEC lines. Since AIM2 activation is known to occur in response to cytoplasmic DNA, we further searched for the presence of undegraded extranuclear DNA in the SGEC lines and SG tissues of patients and controls. This investigation revealed marked cytoplasmic accumulations of damaged genomic DNA that co-localized with AIM2 in the specimens of SS patients (but not controls). The SGEC lines and the ductal tissues of SS patients were also found to manifest impaired DNase1 expression and activity, which possibly denotes defective cytoplasmic DNA degradation in patients' cells and AIM2 triggering thereof. In corroboration, DNase1-silencing in normal SGEC was shown to lead to high AIM2-related gene expression and IL-1ß production. Our findings indicate that the cell-intrinsic activation status of ductal epithelia in SS patients owes to persistent epithelial AIM2 activation by aberrant cytoplasmic DNA build-up.

Perturbation of transcriptome in non-neoplastic salivary gland epithelial cell lines derived from patients with primary Sjögren's syndrome.

The data presented here are related to the research article titled "Impaired anti-inflammatory activity of PPARγ in the salivary epithelia of Sjögren's syndrome patients imposed by intrinsic NF-κB activation" (Vakrakou et al., Journal of Autoimmunity, in press, 2017). In the cited manuscript, using comparative analyses of salivary gland biopsy specimens and ductal salivary gland epithelial cell (SGEC) lines from SS patients and disease controls, we have demonstrated that the ductal epithelia of SS patients display constitutively reduced PPARγ expression, transcriptional activity and anti-inflammatory function that were associated with cell-autonomously activated NF-κB and IL-1ß pathways in these cells. Herein, the comparative transcriptome analysis of SGEC lines is presented. We show that the ductal epithelia of SS patients with severe lymphoepithelial lesions manifest constitutive perturbation in various inflammation- and metabolism related signaling pathways.

Systemic activation of NLRP3 inflammasome in patients with severe primary Sjögren's syndrome fueled by inflammagenic DNA accumulations.

Sjögren's syndrome (SS) patients manifest high cell-free DNA (cf-DNA) levels in serum, associated with impaired DNaseI activity. Undegraded DNA may accumulate in tissues and act as an inflammasome-activating signal. Herein, we investigated the occurrence of aberrant DNA build-up in various biologic compartments of SS patients and its correlation with the activity of NLRP3 and AIM2 inflammasomes. For this purpose, we evaluated sera, PBMC, circulating monocytes and salivary glands (SG) from different SS patient subgroups and controls. We found that SS patients at high risk for lymphoma and those with established lymphoma display high serum cf-DNA levels, substantial extranuclear DNA accumulations in PBMC and SG tissues, a unique NLRP3 inflammasome gene signature in PBMC, and significantly increased serum IL-18 and ASC levels. In these patients, the circulating monocytes manifested NLRP3 inflammasome activation and increased response to NLRP3 stimuli, whereas SG-infiltrating macrophages exhibited signs of NLRP3 activation and pyroptosis. Cell-free nucleic acids isolated from patients' sera competently primed the activation of both NLRP3 and AIM2 inflammasomes in healthy monocytes. SS patients also manifested diminished DNaseI activity in serum and DNaseII expression in PBMC, which inversely correlated with indices of inflammasome activation. DNaseII gene-silencing in healthy monocytes led to cytoplasmic DNA deposition and activation of inflammasome-related genes and of caspase1. Our data reveal the occurrence of systemic NLRP3 inflammasome activation in severe SS, which is associated with widespread extranuclear accumulations of inflammagenic DNA and impaired DNA degradation. These findings can provide novel biomarkers and new therapeutic targets for the management of SS patients with adverse outcomes.

Impaired anti-inflammatory activity of PPARγ in the salivary epithelia of Sjögren's syndrome patients imposed by intrinsic NF-κB activation.

Sjögren's syndrome (SS) patients manifest inflammation in the salivary glands (SG) and evidence of persistent intrinsic activation of ductal SG epithelial cells (SGEC), demonstrable in non-neoplastic SGEC lines derived from patients (SS-SGEC). The peroxisome-proliferator-activated receptor-γ (PPARγ) mediates important anti-inflammatory activities in epithelial cells. Herein, the comparative analysis of SG biopsies and SGEC lines obtained from SS patients and controls had revealed constitutively reduced PPARγ expression, transcriptional activity and anti-inflammatory function in the ductal epithelia of SS patients that were associated with cell-autonomously activated NF-κB and IL-1ß pathways. Transcriptome profiling analysis revealed several differentially expressed proinflammatory and metabolism-related gene sets in SS-SGEC lines. These aberrations largely correlated with the severity of histopathologic lesions, the disease activity and the occurrence of adverse manifestations in SS patients studied, a fact which corroborates the key role of the persistently-activated epithelia in the pathogenesis of both local and systemic features of this disease. The treatment of control SGEC lines with PPARγ agonists was found to diminish the NF-κB activation and apoptosis induced by proinflammatory agents. In addition, the in-vitro application of PPARγ agonists and pharmacologic inhibitors of IL-1ß and NF-κB had significant beneficial effects on SS-SGEC lines, such as the restoration of PPARγ functions and the reduction of their intrinsic activation, a fact which may advocate the future clinical study of the above agents as therapeutic modalities for SS.

Decreased serum DNase1-activity in patients with autoimmune liver diseases.

Deoxyribonuclease1 (DNase1) is involved in chromatin degradation of apoptotic cells. Its deficiency results in accumulation of self-DNA, which in turn may induce inflammation and autoimmunity. We assessed for the first time serum DNase1-activity in a large consecutive cohort of treatment-naïve patients with autoimmune liver diseases (ALD). DNase1-activity was determined by single radial enzyme-diffusion (SRED) at diagnosis of 224 patients with autoimmune hepatitis (AIH), 249 with primary biliary cirrhosis (PBC) and 36 with primary sclerosing cholangitis (PSC). Sera from 146 patients with chronic hepatitis B or C, 140 with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) and 114 healthy individuals served as disease and healthy controls. Available serum samples during remission from 50 AIH and 39 PBC patients were also investigated by paired analyzes. DNase1-activity was significantly lower in AIH, PBC and PSC compared to viral hepatitis (p < 0.02, p < 0.001, p = 0.03), NAFLD/NASH (p < 0.001) and healthy (p < 0.001). No significant difference was found in between each specific ALD. In AIH, DNAse1-activity was positively correlated with aspartate aminotransferase (AST) (p < 0.02), bilirubin (p < 0.01) and increased IgG (>1400 mg/dl; p < 0.05); in PBC, with AST (p < 0.01), alanine aminotransferase (ALT) (p < 0.03) and anti-mitochondrial antibodies (AMA) (p = 0.008). In PSC, DNase1-activity was inversely associated with alkaline phosphatase (ALP) (p < 0.05). In AIH, complete responders were characterized by increased baseline DNase1-activity compared to partial responders, relapsers and non-responders (p < 0.02), whereas it was significantly increased after achievement of remission (p < 0.001). Serum DNase1-activity is significantly decreased in ALD patients, indicating its potential implication in their pathogenesis. Furthermore, DNase1-activity could be used as a new surrogate biomarker for predicting response to AIH treatment.

Impaired clearance of early apoptotic cells mediated by inhibitory IgG antibodies in patients with primary Sjögren's syndrome.

OBJECTIVES: Deficient efferocytosis (i.e. phagocytic clearance of apoptotic cells) has been frequently reported in systemic lupus erythematosus (SLE). Todate, patients with primary Sjögren's syndrome (SS) have not been assessed for phagocytosis of apoptotic cells (ApoCell-phagocytosis) and of particulate targets (microbeads, MB-phagocytosis). DESIGN: ApoCell-phagocytosis and MB-phagocytosis were comparatively assessed by flow cytometry in peripheral blood specimens and monocyte-derived macrophage (MDM) preparations from healthy blood donors (HBD) and consecutive SS, SLE and rheumatoid arthritis (RA) patients. Cross-admixture ApoCell-phagocytosis experiments were also performed using phagocytes from HBD or patients, and apoptotic cells pretreated with whole sera or purified serum IgG derived from patients or HBD. RESULTS: Compared to HBD, approximately half of SS and SLE patients studied (but not RA) manifested significantly reduced ApoCell-phagocytosis (p<0.001) and MB-phagocytosis (p<0.003) by blood-borne phagocytes that correlated inversely with disease activity (p≤0.004). In cross-admixture assays, healthy monocytes showed significantly reduced ApoCell-phagocytosis when fed with apoptotic cells that were pretreated with sera or purified serum IgG preparations from SS and SLE patients (p<0.0001, compared to those from HBD or RA). Such aberrant effect of the SS and SLE sera and IgG preparations correlated linearly with their content of IgG antibodies against apoptotic cells (p≤0.0001). Phagocytic dysfunction maybe also present in certain SS and SLE patients, as supported by deficient capacity of MDM for ApoCell-phagocytosis and MB-phagocytosis under patients' serum-free conditions. CONCLUSION: Similarly to SLE, efferocytosis is frequently impaired in SS and is primarily due to the presence of inhibitory IgG anti-ApoCell antibodies and secondarily to phagocytes' dysfunction.