Association of B-cell activating factor gene variants with serum anti-JCV antibody positivity in male patients with multiple sclerosis under natalizumab treatment: Implications for progressive multifocal leukoencephalopathy risk stratification.

INTRODUCTION: Progressive multifocal leukoencephalopathy (PML) is a potentially life-threatening complication among Multiple Sclerosis (MS) patients under natalizumab treatment, with serum anti-JCV antibody titers being used for stratification risk. Given the critical role of interferon (IFN)/B-cell activating factor (BAFF) axis in humoral immune responses against viruses, we explored whether it is involved in the generation of serum anti-JCV antibodies among these patients. METHODS: 162 consecutive patients with relapsing-remitting MS under natalizumab treatment were included. Serum anti-JCV antibodies were measured at baseline, as well as 12 and 24 months after treatment initiation. Type I and II IFN-inducible genes and BAFF expression were quantitated in peripheral blood by qRT-PCR. Moreover, BAFF rs9514828, rs1041569, and rs9514827 gene variants were assessed by RFLP-PCR. RESULTS: While type I and II IFN inducible gene expression were not associated with anti-JCV serum titers, the latter were significantly correlated with BAFF gene expression. Of interest, the TTT haplotype of the studied BAFF variants was more frequently detected in male, but not female anti-JCV (+) MS patients compared to anti-JCV (-) counterparts at baseline, as well as at 12 months and 24 months of natalizumab treatment. Measures of clinical validity/utility for the BAFF TTT haplotype showed 88% specificity, 45%, positive predictive value, and sensitivity of 70% for the discrimination of anti-JCV (+) male MS patients after 24 months of treatment. CONCLUSIONS: Our study suggests an implication of the BAFF axis in the production of serum anti-JCV antibodies. Additionally, the BAFF TTT haplotype derived from the rs9514828, rs1041569, and rs9514827 variants may represent a novel risk factor for anti-JCV seropositivity and indirectly for PML development among male MS patients treated with natalizumab.

B-cell activating factor gene variants in multiple sclerosis: Possible associations with disease susceptibility among females.

Although B cells and B cell activating factor (BAFF) have been previously implicated in MS pathogenesis, data regarding the genetic influence of BAFF polymorphisms on MS susceptibility are limited. Here we aim to explore whether BAFF polymorphisms could contribute to MS susceptibility. 156 RRMS patients fulfilling the revised McDonald criteria for MS diagnosis and 220 HCs were enrolled. Clinical, laboratory, and imaging characteristics were recorded. BAFF rs9514827, rs1041569, and rs9514828 polymorphisms were assessed by RFLP-PCR in DNA samples extracted from whole peripheral blood. The BAFF rs1041569 TT genotype along with the CTT and TTC haplotypes were associated with significantly increased risk for MS development in female MS patients compared to healthy female counterparts. These findings were not confirmed in males. The rs1041569 BAFF variant together with the CTT and TTC BAFF haplotypes derived from the BAFF rs9514827, rs1041569, and rs9514828 polymorphisms may represent novel genetic contributors to the development of MS in females.

Multiple faces of multiple sclerosis in the era of highly efficient treatment modalities: Lymphopenia and switching treatment options challenges daily practice.

The expanded treatment landscape in relapsing-remitting multiple sclerosis (MS) has resulted in highly effective treatment options and complexity in managing disease- or drug-related events during disease progression. Proper decision-making requires thorough knowledge of the immunobiology of MS itself and an understanding of the main principles behind the mechanisms that lead to secondary autoimmunity affecting organs other than the central nervous system as well as opportunistic infections. The immune system is highly adapted to both environmental and disease-modifying agents. Immune reconstitution following cell depletion or cell entrapment therapies eliminates pathogenic aspects of the disease but can also lead to distorted immune responses with harmful effects. Atypical relapses occur with second-line treatments or after their discontinuation and require appropriate clinical decisions. Lymphopenia is a result of the mechanism of action of many drugs used to treat MS. However, persistent lymphopenia and cell-specific lymphopenia could result in disease exacerbation, secondary autoimmunity, or the emergence of opportunistic infections. Clinicians treating patients with MS should be aware of the multiple faces of MS under novel, efficient treatment modalities and understand the intricate brain-immune cell interactions in the context of an altered immune system. MS relapses and disease progression still occur despite the current treatment modalities and are mediated either by failure to control effector mechanisms inherent to MS pathophysiology or by new drug-related mechanisms. The multiple faces of MS due to the highly adapted immune system of patients impose the need for appropriate switching therapies that safeguard disease remission and further clinical improvement.

Stiff-Leg Syndrome Associated with Autoimmune Retinopathy and Its Treatment with IVIg-A Case Report and Review of the Literature.

Antibodies to glutamic acid decarboxylase (GAD) have been predominantly associated with stiff-person syndrome (SPS), which is often accompanied by organ-specific autoimmune diseases, such as late-onset type 1 diabetes. Autoimmune retinal pathology in SPS has recently been suggested to coexist in patients suffering from this disease; however, evidence reporting potential treatment options for the neurological and visual symptoms these patients experience remains scarce. We provide a review of the relevant literature, presenting a rare case of a middle-aged woman with autoimmune retinopathy (AIR) followed by stiff-leg syndrome who responded to intravenous immune globulin treatment (IVIg). Our report adds to previously reported data supporting the efficacy of IVIg in SPS spectrum disorders while also proposing the potential effect of IVIg in treating SPS spectrum patients with coexisting AIR.

An Updated Evaluation of Intrathecal IgG Synthesis Markers in Relation to Oligoclonal Bands.

The aim was to evaluate the performance of the latest quantitative marker for intrathecal IgG synthesis and to compare it with other established markers used for the same purpose. We retrospectively applied Auer's and Reiber's intrathecal IgG synthesis formulae in a cohort of 372 patients under investigation for central nervous system demyelination who had undergone lumbar puncture and oligoclonal bands (OCBs) detection for demonstrating intrathecal IgG synthesis. A ROC analysis revealed Auer's formula had lower sensitivity (68%) compared to Reiber's formula (83%) and IgG index (89%), in our cohort of patients that exhibited normal to mildly elevated albumin quotients (4.48 ± 3.93). By excluding possible sources of errors, we assume that Auer's formula is less sensitive than other established tools for the "prediction" of the detection of OCBs in routine cerebrospinal fluid (CSF) analyses due to the mathematical model used. Given the ability of Reiber's hyperbolic formula to describe the blood-CSF IgG distribution across a wide range of blood-brain barrier functionality, its use and the use of similar formulae are recommended for the discrimination between CNS-derived and blood-derived molecules in clinical laboratories.

Specific myeloid signatures in peripheral blood differentiate active and rare clinical phenotypes of multiple sclerosis.

Current understanding of Multiple Sclerosis (MS) pathophysiology implicates perturbations in adaptive cellular immune responses, predominantly T cells, in Relapsing-Remitting forms (RRMS). Nevertheless, from a clinical perspective MS is a heterogeneous disease reflecting the heterogeneity of involved biological systems. This complexity requires advanced analysis tools at the single-cell level to discover biomarkers for better patient-group stratification. We designed a novel 44-parameter mass cytometry panel to interrogate predominantly the role of effector and regulatory subpopulations of peripheral blood myeloid subsets along with B and T-cells (excluding granulocytes) in MS, assessing three different patient cohorts: RRMS, PPMS (Primary Progressive) and Tumefactive MS patients (TMS) (n=10, 8, 14 respectively). We further subgrouped our cohort into inactive or active disease stages to capture the early underlying events in disease pathophysiology. Peripheral blood analysis showed that TMS cases belonged to the spectrum of RRMS, whereas PPMS cases displayed different features. In particular, TMS patients during a relapse stage were characterized by a specific subset of CD11c+CD14+ CD33+, CD192+, CD172+-myeloid cells with an alternative phenotype of monocyte-derived macrophages (high arginase-1, CD38, HLA-DR-low and endogenous TNF-a production). Moreover, TMS patients in relapse displayed a selective CD4 T-cell lymphopenia of cells with a Th2-like polarised phenotype. PPMS patients did not display substantial differences from healthy controls, apart from a trend toward higher expansion of NK cell subsets. Importantly, we found that myeloid cell populations are reshaped under effective disease-modifying therapy predominantly with glatiramer acetate and to a lesser extent with anti-CD20, suggesting that the identified cell signature represents a specific therapeutic target in TMS. The expanded myeloid signature in TMS patients was also confirmed by flow cytometry. Serum neurofilament light-chain levels confirmed the correlation of this myeloid cell signature with indices of axonal injury. More in-depth analysis of myeloid subsets revealed an increase of a subset of highly cytolytic and terminally differentiated NK cells in PPMS patients with leptomeningeal enhancement (active-PPMS), compared to those without (inactive-PPMS). We have identified previously uncharacterized subsets of circulating myeloid cells and shown them to correlate with distinct disease forms of MS as well as with specific disease states (relapse/remission).

The role of type I IFN in autoimmune and autoinflammatory diseases with CNS involvement.

Type I interferons (IFNs) are major mediators of innate immunity, with well-known antiviral, antiproliferative, and immunomodulatory properties. A growing body of evidence suggests the involvement of type I IFNs in the pathogenesis of central nervous system (CNS) manifestations in the setting of chronic autoimmune and autoinflammatory disorders, while IFN-ß has been for years, a well-established therapeutic modality for multiple sclerosis (MS). In the present review, we summarize the current evidence on the mechanisms of type I IFN production by CNS cellular populations as well as its local effects on the CNS. Additionally, the beneficial effects of IFN-ß in the pathophysiology of MS are discussed, along with the contributory role of type I IFNs in the pathogenesis of neuropsychiatric lupus erythematosus and type I interferonopathies.

Cholesterol levels in plasma and cerebrospinal fluid in patients with clinically isolated syndrome and relapsing-remitting multiple sclerosis.

Cholesterol is essential for myelin formation, but may also modulate mechanisms involved in adaptive immune responses. It is unclear whether lack of remyelination in multiple sclerosis (MS) results from an insufficient capacity of oligodendrocyte precursor cells to differentiate or from cholesterol insufficiency. Several studies have assessed the potential association of lipid profile and its metabolism with demyelination, disability, and disease progression. The aim of the present study was to measure cholesterol levels in plasma and cerebrospinal fluid (CSF) of patients with relapsing remitting MS (RRMS) or with clinically isolated syndrome (CIS), and to investigate whether there is an association between cholesterol levels and disease characteristics. Sixty-two patients with CIS and 46 patients with RRMS were included in the study. All patients had low EDSS and were medication-free at assessment. Forty-eight subjects within the same age range served as controls. Cholesterol concentrations were measured in plasma and in CSF by the same enzymatic - colorimetric method, and were related to clinical status, disease activity in magnetic resonance imaging (MRI) and presence of oligoclonal bands in CSF (OBs). Significantly lower levels of plasma and CSF cholesterol were found in patients compared to controls. Patients with OBs showed significantly lower levels of CSF cholesterol but not plasma cholesterol compared to OBs-negative patients. A positive correlation of plasma cholesterol with age and of CSF cholesterol with EDSS was found. Our results suggest that low CSF cholesterol in MS patients with positive OBs might reflect extensive demyelination and a more aggressive disease course associated with an increased humoral immune response against membrane components.

Differentiating central nervous system demyelinating disorders: The role of clinical, laboratory, imaging characteristics and peripheral blood type I interferon activity.

Objective: While multiple sclerosis (MS) is considered the cornerstone of autoimmune demyelinating CNS disorders, systemic autoimmune diseases (SADs) are important MS mimickers. We sought to explore whether distinct clinical, laboratory, and imaging characteristics along with quantitation of peripheral blood type I interferon (IFN) activity could aid in differentiating between them. Methods: A total of 193 consecutive patients with imaging features suggesting the presence of CNS demyelinating disease with or without relevant clinical manifestations underwent full clinical, laboratory, and imaging evaluation, including testing for specific antibodies against 15 cellular antigens. Expression analysis of type I IFN-inducible genes (MX-1, IFIT-1, and IFI44) was performed by real-time PCR, and a type I IFN score, reflecting type I IFN peripheral activity, was calculated. After joint neurological/rheumatological evaluation and 1 year of follow-up, patients were classified into MS spectrum and CNS autoimmune disorders. Results: While 66.3% (n = 128) of the patients were diagnosed with MS spectrum disorders (predominantly relapsing-remitting MS), 24.9% (n = 48) were included in the CNS autoimmune group, and out of those, one-fourth met the criteria for SAD (6.7% of the cohort, n = 13); the rest (18.1% of the cohort, n = 35), despite showing evidence of systemic autoimmunity, did not fulfill SAD criteria and comprised the "demyelinating disease with autoimmune features" (DAF) subgroup. Compared to the MS spectrum, CNS autoimmune patients were older, more frequently females, with increased rates of hypertension/hyperlipidemia, family history of autoimmunity, cortical dysfunction, anti-nuclear antibody titers ≥1/320, anticardiolipin IgM positivity, and atypical for MS magnetic resonance imaging lesions. Conversely, lower rates of infratentorial and callosal MRI lesions, CSF T2 oligoclonal bands, and IgG-index positivity were observed in CNS autoimmune patients. Patients fulfilling SAD criteria, but not the DAF group, had significantly higher peripheral blood type I IFN scores at baseline compared to MS spectrum [median (IQR)]: 50.18 (152.50) vs. -0.64 (6.75), p-value: 0.0001. Conclusion: Our study suggests that underlying systemic autoimmunity is not uncommon in patients evaluated for possible CNS demyelination. Distinct clinical, imaging and laboratory characteristics can aid in early differentiation between MS and CNS-involving systemic autoimmunity allowing for optimal therapeutic strategies. Activated type I IFN pathway could represent a key mediator among MS-like-presenting SADs and therefore a potential therapeutic target.

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.

Genetic Variants of the BAFF Gene and Risk of Fatigue Among Patients With Primary Sjögren's Syndrome.

Background/Purpose: Primary Sjögren's Syndrome (SS) is characterized by B lymphocyte hyperactivity with B cell activating factor (BAFF) acting as an important regulator. Single Nucleotide Polymorphisms (SNPs) of the BAFF gene have been implicated in the pathogenesis of several autoimmune diseases characterized by heightened fatigue levels, including primary SS. We aimed to explore potential associations between BAFF SNPs and fatigue status of primary SS patients. Methods: Fatigue status was assessed in 199 consecutive primary SS patients (Greek cohort) using the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) scale. Clinical, histological, laboratory, psychometric and personality data were also collected. DNA extracted from peripheral blood of all patients underwent evaluation for the presence of five BAFF SNPs (rs9514827, rs1041569, rs9514828, rs1224141, rs12583006) by PCR. To confirm our findings, an independent replicative cohort of 62 primary SS patients (Dutch cohort) was implemented. Finally, 52 multiple sclerosis (MS) patients were served as disease controls (MS cohort). Analysis of BAFF SNPs in association with fatigue levels was performed by the online platforms SNPStats and SHEsis and the SPSS 26 and Graph Pad Prism 8.00 software. Results: TT genotype of the rs9514828 BAFF polymorphism was significantly less frequent in the fatigued primary SS patients of the Greek cohort compared to the non-fatigued (14.1% vs 33.3%). The corresponding ORs [95%CI] in the dominant and overdominant models were 0.33 [0.15-0.72], p=0.003 and 0.42 [0.23-0.78], p=0.005 respectively. The association remained significant after adjustment for the variables contributing to fatigue in the univariate analysis (OR [95% CI]: 0.3 [0.1-0.9], p=0.026). Accordingly, in the Dutch cohort, there was a trend of lower mental fatigue among patients carrying the TT rs9514828 BAFF genotype compared to their CC counterparts (4.1 ± 2.4 vs 6.0 ± 2.2 respectively, p=0.06). The rs9514828 BAFF SNP was not significantly associated with fatigue in the MS cohort. Conclusions: We report a novel association between genetic makeup and primary SS-associated fatigue with the rs9514828 TT genotype decreasing the likelihood of fatigue development among these patients. These findings need validation in multi-center studies.

Fingolimod as a first- or second-line treatment in a mini-series of young Hellenic patients with adolescent-onset multiple sclerosis: focus on immunological data.

BACKGROUND: Pediatric onset multiple sclerosis(POMS) is characterized by a highly active profile, often warranting treatment with high efficacy disease-modulating therapies (DMTs). Fingolimod, an oral sphingosine-1-phosphate receptor modulator, is the first Food and Drug Administration (FDA)- and European Medicines Agency (EMA)-approved DMT for the treatment of POMS. OBJECT: Our aim is to present real-world data of seven fingolimod-treated POMS-patients, recruited in a single MS center in Greece. METHODS: Clinical and imaging/laboratory data from 7 Hellenic patients fulfilling the International Pediatric Multiple Sclerosis Study Group (IPMSSG) criteria for POMS diagnosis, who have received fingolimod treatment, were selected. Human leukocyte antigen (HLA) genotyping was performed with standard low-resolution sequence-specific oligonucleotide techniques. RESULTS: Three patients were treatment-naïve adolescents who received fingolimod as first-line treatment. Two experienced ongoing clinical and radiological disease activity and have been switched to natalizumab. The remaining cases were post-adolescent adults with POMS, where the vast majority experienced total/near-total disease remission. Fingolimod was generally well-tolerated. Two patients with high disease activity carried the HLA-DRB1*03 allele, while five patients were carriers of at least one of the HLA-DRB1*04, HLA-DRB1*13, and HLA-DRB1*14 alleles, which when not combined with HLA-DRB1*03 showed a trend towards a more favorable clinical course. Fingolimod responders showed a trend towards increased CD(16-56)+NK cell counts in immunophenotyping assays. CONCLUSIONS: Our preliminary results support that response of POMS patients to fingolimod may be partially dependent on age and previous DMT, with younger and treatment-naïve patients presenting worse outcomes. The role of immunogenetics and immunophenotyping in personalized treatment warrants investigation in larger and more diverse populations.

Methylprednisolone stimulated gene expression (GILZ, MCL-1) and basal cortisol levels in multiple sclerosis patients in relapse are associated with clinical response.

Glucocorticoids (GCs) are the main treatment of relapse in multiple sclerosis (MS). Decreased sensitivity to GCs in MS patients has been associated with lack of the suppressive effect of GCs on inflammatory molecules as well as increased resistance to apoptosis. We investigated GC-sensitivity by measuring the effect of intravenous methylprednisolone (IVMP) treatment on transactivation of anti-inflammatory and apoptotic genes (GILZ, MCL-1 and NOXA respectively), in accordance to clinical outcome. Thirty nine MS patients were studied: 15 with clinically isolated syndrome (CIS), 12 with relapsing remitting (RRMS) and 12 with secondary progressive (SPMS) under relapse. Patients underwent treatment with IVMP for 5 days. Blood was drawn before IVMP treatment on day 1 and 1 h after IVMP treatment on days 1 and 5. GIlZ, MCL-1 and NOXA were determined by qPCR. The Expanded Disability Status was evaluated and patients were divided according to their clinical response to IVMP. GILZ and MCL-1 gene expression were significantly higher following first IVMP treatment in responders, compared to non-responders. Furthermore, serum basal cortisol and 1,25-OH Vitamin D levels were significantly higher in clinical-responders as compared to non-clinical responders. Our findings suggest that the differential GILZ and MCL-1 gene expression between clinical-responders and non-clinical responders may implicate the importance of GILZ and MCL-1 as possible markers for predicting glucocorticoid sensitivity and response to GC-therapy in MS patients following first IVMP injection.

Type I interferon detection in autoimmune diseases: challenges and clinical applications.

INTRODUCTION: Accumulating data highlights that the dysregulation of type I interferon (IFN) pathways plays a central role in the pathogenesis of several systemic and organ-specific autoimmune diseases. Advances in understanding the role of type I IFNs in these disorders can lead to targeted drug development as well as establishing potential disease biomarkers. AREAS COVERED: Here, we summarize current knowledge regarding the role of type I IFNs in the major systemic, as well as organ-specific, autoimmune disorders, including prominent inflammatory CNS disorders like multiple sclerosis. EXPERT OPINION: Type I IFN involvement and its clinical associations in a wide spectrum of autoimmune diseases represents a promising area for research aiming to unveil common pathogenetic pathways in systemic and organ-specific autoimmunity.

New Strategies in Neurogenic Heterotopic Ossification.

The term neurogenic heterotopic ossification (NHO) is used to describe the pathological bone formation in soft tissues, due to spinal cord or brain injury. Commonly is presented with pain and stiffness of the affected joint. NHO affects the quality of life of these patients, delays their rehabilitation and therefore increases morbidity. The aim of this article is to emphasize pathophysiology mechanism and review new molecular treatments of heterotopic ossification (HO). It was demonstrated that potent treatment strategies are based on understanding the molecular mechanisms and aiming to inhibit the pathological process of the HO in various stages. New treatments are targeting several factors such as bone morphogenetic proteins (BMPs), retinoic acid receptors (RARs), hypoxic inhibitors (Hif1-inhibitors, rapamycin), free radical scavengers and immunological agents (imatinib). The endogenous pathways that lead to HO at molecular and cellular levels have been the aim of many studies in recent years. New treatment options for HO should be recommended due to the ineffectiveness of traditional older options, such as anti-inflammatory drugs and radiation, especially in the case of NHO.