Pathogenic Antibodies Research Articles

Acetylcholine Receptor Antibody Neutralization and Selective B-Cell Suppression Using Fc Fusion Protein

Myasthenia gravis (MG) is an autoimmune disease characterized by formation of autoantibodies against the nicotinic acetylcholine receptor (AChR). Some patients do not show sufficient improvement and develop adverse effects following administration of conventional immune therapy; therefore, the development of new treatments is important. Based on the concept of "selective removal of pathogenic antibodies and cells without suppression of normal immunity," we are developing a fusion protein referred to as AChR-Fc (composed of the AChR alpha subunit and Fc region of human immunoglobulin G1), which shows the following mechanisms of action: selective neutralization of AChR antibodies and cytotoxic activity against AChR antibody-producing pathogenic B cells. Treatment with AChR-Fc is a novel therapeutic approach that may be useful in the management of MG.

Neonatal Fc receptor : role and implications inmaternal fetal medicine

During pregnancy, the mother's IgG immunoglobulins cross the -placenta via the neonatal Fc receptor (FcRn), enabling the fetus to acquire passive immunity. In the presence of maternal allo- or auto-antibodies, placental transfer of these pathogenic antibodies mediated by FcRn can cause diseases in the fetus and/or the newborn. FcRn blockade therefore appears to be a therapeutic strategy in these high-risk pregnancies, firstly by reducing IgG recycling, -thereby reducing its concentration in the maternal circulation, and secondly by blocking placental transfer. The promising results of a recent trial testing nipocalimab, a monoclonal antibody targeting FcRn, in very severe erythrocyte alloimmunisation, has opened the way to new targeted therapeutic approaches for perinatal diseases mediated by maternal IgG.

Affinity Chromatography for Anti-Glucosylated Adhesin Antibody Purification: Depletion of Nonspecific Anti-Protein Antibodies and Antibody Recovery with Unconventional Elution Solutions.

Antibodies from sera of a multiple sclerosis (MS) patient subpopulation preferentially recognize the hyperglucosylated adhesin protein HMW1ct(Glc) of the pathogen Haemophilus influenzae. This protein is the first example of an N-glucosylated native antigen candidate, potentially triggering pathogenic antibodies in MS. Specific antibodies in patients' sera can be isolated exploiting their biospecific interaction with antigens by affinity chromatography. Herein, the proteins HMW1ct and HMW1ct(Glc) were first immobilized on appropriately functionalized supports and further used to purify antibodies directly from MS patients sera. We describe a protocol to obtain an antibody fraction specifically recognizing the glusosylated residues on the HMW1ct(Glc) adhesin protein depleting antibodies to the unglucosylated HMW1ct sequence. Different elution solutions have been tested to recover the purified antibody fraction, strongly bound to the immobilized HMW1ct(Glc) adhesin protein.

Neuromyelitis optic spectrum disorder following COVID- 19 (ChAdOx1-S) vaccine

Neuromyelitis optic spectrum disorder is an autoimmune demyelinating astro- cytopathy of central nervous system. It has emerged as a distinct clinical entity following the discovery of pathogenic antibody named AQP4-IgG against aquaporin-4 water channels in astrocytes. The clinical spectrum includes optic neuritis, acute myelitis, acute brain stem syndrome, area postrema syndrome, acute diencephalic syndrome and symptomatic cerebral syndrome.This case describes a young male patient who presented with subacute onset painful visual impairment of left eye along with bilateral lower limb weakness and numbness in the background of recent vaccination for covid-19 infection. Examination revealed bilateral papillitis and bilateral flaccid paraparesis with a sensory level at T4 level. Investigations revealed a longitudinally extensive myelitis spanning the entire length of the spinal cord, bilateral optic neuritis, positive serum AQP4-IgG antibodies with negative infective screening and autoimmune profile. Central nervous system demyelination has been observed with all types of covid-19 vaccines.

Role of Specific Autoantibodies in Neurodegenerative Diseases: Pathogenic Antibodies or Promising Biomarkers for Diagnosis.

Neurodegenerative diseases (NDDs) affect millions of people worldwide. They develop due to the pathological accumulation and aggregation of various misfolded proteins, axonal and synaptic loss and dysfunction, inflammation, cytoskeletal abnormalities, defects in DNA and RNA, and neuronal death. This leads to the activation of immune responses and the release of the antibodies against them. Recently, it has become clear that autoantibodies (Aabs) can contribute to demyelination, axonal loss, and brain and cognitive dysfunction. This has significantly changed the understanding of the participation of humoral autoimmunity in neurodegenerative disorders. It is crucial to understand how neuroinflammation is involved in neurodegeneration, to aid in improving the diagnostic and therapeutic value of Aabs in the future. This review aims to provide data on the immune system's role in NDDs, the pathogenic role of some specific Aabs against molecules associated with the most common NDDs, and their potential role as biomarkers for monitoring and diagnosing NDDs. It is suggested that the autoimmune aspects of NDDs will facilitate early diagnosis and help to elucidate previously unknown aspects of the pathobiology of these diseases.

Porphyromonas gingivalis with collagen immunization induces ACPA-positive rheumatoid arthritis in C3H mice

The pathogenic anti-citrullinated protein antibodies (ACPA) are thought to play a vital role in the initiation and immune maintenance of rheumatoid arthritis (RA). However, it is noteworthy that ACPA is not a salient characteristic of any conventional RA animal model. Porphyromonas gingivalis (Pg) is the first microorganism identified to induce citrullination and a target of autoantibodies in early rheumatoid arthritis (RA). Thus, we employed C3H mice with specific MHC types and combined Pg infection with collagen immunity to develop an animal model of ACPA-positive RA. The resulting model exhibited citrullination characteristics, as well as pathological and immune cell changes. 1) Mice showed a significant increase in ACPA levels, and various organs and tissues exhibited elevated levels of citrullinated protein. 2) The mice experienced heightened pain, inflammation, and bone destruction. 3) The spleen and lymph nodes of the mice showed a significant increase in the proportion of Tfh-GCB cell subpopulations responsible for regulating autoantibody production. In conclusion, the C3H mouse model of Pg infection with collagen immunity demonstrated significant alterations in ACPA levels, citrullinated protein expression, and immune cell subpopulations, which could be a crucial factor leading to increased pain, inflammation, and bone destruction.

Observations from Mass-Fix: Prevalence and Characteristics of Therapeutic Monoclonal Antibodies (t-mabs) Detected by Isotyping of Monoclonal Immunoglobulins via MALDI-TOF Mass Spectrometry

Abstract Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (“Mass-Fix”) has replaced serum immunofixation at our institution for detection of monoclonal immunoglobulins. One advantage of Mass-Fix is the ability to differentiate pathogenic endogenous monoclonal antibodies from therapeutic monoclonal antibodies (t-mabs), which are often the first-line treatment for multiple myeloma, by reviewing the light chain signature mass. The objective of this study was to describe the distribution of t-mabs and other key characteristics of clinical serum samples analyzed by Mass-Fix in 2022. Comprehensive results for each sample were extracted retrospectively from the laboratory information system. Of 66,710 total analyzed, 30,701 (46%) samples were reported as “positive” (i.e., contains a monoclonal protein), given the tertiary nature of our institution and reference laboratory services. The positive samples were further subdivided by t-mab (detected by Mass-Fix), with a total of 7,769 (25.3%) t-mabs reported. The top five IgG kappa t-mabs reported include daratumumab (n=6,537), elotuzumab or REGN5458/5459 (n=499), isatuximab (n=348), and rituximab (n=102). Rare observations included monoclonal proteins consistent with tixagevimab/ cilgavimab, for COVID-19 infection (n=7), obinutuzumab, an anti-CD20 t-mab for multiple sclerosis (n=1) and evolocumab, an IgG lambda anti-PCSK9 inhibitor (n=6). Of the 7,769 samples, the t-mab was the only reported clone in 47.7% of cases and was found along with the original disease-associated clone in remaining cases. When the original disease clone was present, it was an IgG kappa isotype 31% of the time and could have confounded the definition of complete therapy response for patients. 69% were non-IgG kappa original clones. Mass-Fix test orders have a multiple-choice prompt to provide any current therapy to aid in the patient’s result interpretation. For those samples where a t-mab was reported, the answer to this question regarding administered therapy was often discrepant from the report: only 38.5% of daratumumab-positive samples had an affirmation of daratumumab administration. The remaining 61.5% of daratumumab-positive samples reported an assortment of therapies, including bone marrow transplant, other t-mabs, no active therapy, or the field was left blank, highlighting poor compliance. Additionally, 154/36,009 samples where the provider affirmed daratumumab administration were reported as negative (i.e., no monoclonal protein observed on Mass-Fix). Often, the send-out laboratory does not have medical record access where treatment information is detailed. In conclusion, we reported the presence of a t-mab in 11.6% of all samples tested with Mass-Fix, suggesting that t-mabs have entered the clinical practice to stay, and accurately determining their presence in the challenging environment of poor compliance to prompt questions and evolving treatment regimens is something that cannot be ignored by clinical laboratories. Mass-Fix allows detection of a t-mab in the same routine run as other testing, without need of a second immunofixation to rule out the IgG kappa finding is not a t-mab.

Domain Specificity of Anti-Prothrombin Antibodies That Express Lupus Anticoagulant Activity

Background: The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by the persistent presence of antiphospholipid antibodies (aPL). aPL are a heterogeneous group of autoantibodies directed against negatively charged phospholipids or phospholipid-binding proteins. Some of these autoantibodies exert lupus anticoagulant (LAC) activity, a laboratory phenomenon characterized by the prolongation of phospholipid-dependent coagulation tests. Recently, our group identified that LAC-positive anti-prothrombin antibodies activate platelets via the platelet receptor, FcγRIIA. However, a lot is still unclear regarding the mechanism by which these antibodies cause LAC and why some, but not all, antibodies in this heterogeneous group express LAC activity. Furthermore, experimental data on the binding site of these pathogenic anti-prothrombin antibodies are lacking. Aims: In this study we aimed to investigate the binding domain of LAC-positive monoclonal anti-prothrombin antibodies and APS patient derived anti-prothrombin antibodies. Method: Recombinant fragments of prothrombin were designed and produced in Chinese hamster ovary cells. Prothrombin fragment 1, fragment 2 and fragment 1.2 were coated on ELISA plates to study epitope specificity. Two LAC-positive monoclonal anti-prothrombin antibodies (3B1 and 28F4), two LAC-negative anti-prothrombin monoclonal antibodies (11H2 and 9C6), isolated anti-prothrombin antibodies derived from APS patients and plasma from APS patients were studied. A modified dilute Russell's viper venom time (DRVVT) test was used to evaluate whether the recombinant prothrombin constructs could neutralize the effect of LAC-positive monoclonal and patient derived anti-prothrombin antibodies. Furthermore, a fragment 1.2 nanobody was produced by immunizing Llama's followed by phage display. The ability of this nanobody to compete with LAC-positive monoclonal and patient derived anti-prothrombin antibodies for the prothrombin binding site was tested using a modified DRVVT test. Results: LAC-positive monoclonal anti-prothrombin antibodies recognized prothrombin fragment 2. Interestingly, LAC-negative anti-prothrombin antibodies bound exclusively to prothrombin fragment 1. In addition, LAC-ratios of patient samples correlated significantly with anti-fragment 2 antibody levels ( p<0.05, ρ=0.33), but not with anti-fragment 1 antibody levels. Furthermore, incubation with the prothrombin F1.2 nanobody almost completely reversed the prolonged DRVVT clotting time in plasma containing monoclonal and patient derived LAC-positive anti-prothrombin antibodies, but not in plasma containing LAC-positive anti-β2GP1 antibodies. This neutralising effect was also observed when the monoclonal anti-prothrombin antibody was incubated with prothrombin fragment 2. In contrast, the LAC-activity of anti-prothrombin antibodies was not inhibited when incubated with prothrombin fragment 1. Conclusion: Our data show that LAC-positive anti-prothrombin antibodies react with fragment 2 of prothrombin. Furthermore, a novel nanobody directed against prothrombin fragment 1.2 and prothrombin fragment 2 were able to neutralize a prothrombin-dependent LAC.

Reverse Engineering of a Pathogenic Antibody Reveals the Molecular Mechanism of Vaccine-Induced Immune Thrombotic Thrombocytopenia.

The massive COVID-19 vaccine roll-out campaign illuminated a range of rare side effects, the most dangerous of which─vaccine-induced immune thrombotic thrombocytopenia (VITT)─is caused by adenoviral (Ad)-vectored vaccines. VITT occurrence had been linked to the production of pathogenic antibodies that recognize an endogenous chemokine, platelet factor 4 (PF4). Mass spectrometry (MS)-based evaluation of the ensemble of anti-PF4 antibodies obtained from a VITT patient's blood indicates that the major component is a monoclonal antibody. Structural characterization of this antibody reveals several unusual characteristics, such as the presence of an N-glycan in the Fab segment and high density of acidic amino acid residues in the complementarity-determining regions. A recombinant version of this antibody (RVT1) was generated by transient expression in mammalian cells based on the newly determined sequence. It captures the key properties of VITT antibodies such as their ability to activate platelets in a PF4 concentration-dependent fashion. Homology modeling of the Fab segment reveals a well-defined polyanionic paratope, and the docking studies indicate that the polycationic segment of PF4 readily accommodates two Fab segments, cross-linking the antibodies to yield polymerized immune complexes. Their existence was verified with native MS by detecting assemblies as large as (RVT1)3(PF4)2, pointing out at FcγRIIa-mediated platelet activation as the molecular mechanism underlying VITT clinical manifestations. In addition to the high PF4 affinity, RVT1 readily binds other polycationic targets, indicating a polyreactive nature of this antibody. This surprising promiscuity not only sheds light on VITT etiology but also opens up a range of opportunities to manage this pathology.

Cryo-EM Structure of Coagulation Factor VIII Bound to a Patient-Derived Anti-A2 Domain Antibody Inhibitor

Coagulation factor VIII (FVIII) promotes hemostasis by binding to activated factor IX on activated platelet surfaces to form the intrinsic tenase complex. The formation of inhibitory antibodies against FVIII occurs in approximately 30% of congenital hemophilia A patients receiving FVIII replacement therapy, significantly complicating treatments. By identifying the FVIII amino acids which bind to pathogenic inhibitory antibodies, FVIII replacement therapies can be engineered with reduced immunogenicity. Here, we aimed to structurally characterize FVIII bound to NB11B2, a patient-derived antibody inhibitor which binds to the A2 domain and disrupts tenase complex activity, using cryogenic electron microscopy (cryo-EM). The FVIII:NB11B2 structure was solved to a global resolution of 4.0 Å, representing the first structure of FVIII bound to an anti-A2 domain antibody inhibitor. Structural analysis revealed that NB11B2 binds to a discontinuous epitope encompassing FVIII residues S470-P472, R490-K493, and P502-E507. Furthermore, our structure identified a 20° swivel-like movement adopted by the C2 domain while the C1 domain remains immobile, consistent with a previous X-ray crystal structure of FVIII bound to an anti-C1 domain inhibitor. These results provide the structural basis for inhibition of FVIII by NB11B2 and elucidate a novel role for portions of the FVIII A2 domain in regulating tenase complex assembly and/or activity.

Inhibition of Bromodomain and Extraterminal Domain (BET) Proteins Alleviates Chronic Graft- Versus-Host-Disease Via Modulating the CXCR5/CXCL13 Axis

Introduction: Chronic graft-versus-host disease (cGVHD) is a severe complication of allogeneic hematopoietic cell transplant (allo-HCT) often leading to transplant-mediated mortality. Studies have shown that T follicular helper cells (Tfh) upregulate CXCR5 and migrate into the lymphoid organ follicles in response to CXCL13 promoting germinal center (GC) B cell proliferation and differentiation of plasma cells resulting in pathogenic antibody production and profibrotic pathways associated with cGVHD. In contrast, T follicular regulatory cells (Tfr) modulate the magnitude of the GC response; thus, the balance of Tfr/Tfh could be crucial to mitigating cGVHD.Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers influencing transcription of a multitude of inflammatory genes and our lab has previously reported pharmacological inhibition of BET, through OPN-51107, improves survivability and limits disease progression in murine models of acute GVHD that led to opening the first-in-human clinical trial of OPN-51107 in steroid refractory acute GVHD. We demonstrated BET proteins to be important in modulating acute GVHD pathogenesis, however, mechanisms of BET regulation within cGVHD are largely unknown. Here, using OPN-51107 we aim to further investigate mechanisms of cGVHD pathogenesis and identify successful BET inhibition-based therapeutic approaches. Methods: We used a Bronchiolitis Obliterans model (B6->B10.br) of cGVHD. Mice were administered 120mg/kg cyclophosphamide (D-3 and D-2) and 8Gy irradiation (D-1) as a conditioning regimen followed by B6 T-cell depleted bone marrow cells with and without CD45.1 B6 splenocytes. Mice that did not receive allogeneic splenocytes served as controls and did not develop GVHD. Recipients of allogeneic splenocytes were treated with a BET inhibitor - OPN-51107 at 10mg/kg 3x/weekly, or vehicle, from D28-54(+ 4) via oral gavage. Resting lung elastance and resistance was measured using the Flexivent system. At the time of lung function analysis, lung and liver tissue was harvested and stained via Hesson's Trichrome to identify and quantify collagen deposition. We performed immunofluorescence to quantify alveolar alloantibody deposition. Spleen and lung cells were stimulated with PMA/I and analyzed via spectral flow cytometry. An inflammatory murine cytokine array (#ARY006, R&D Systems) was used to analyze circulating plasma cytokine levels. Results: BET inhibition via OPN-51107 treatment decreased lung resistance (vehicle vs OPN-51107 =1.16Rrs vs 0.83Rrs p<0.05) and elastance (vehicle vs. OPN-51107=62Ers vs. 45Ers p<0.05). OPN-51107 treatment also significantly reduced alveolar antibody (Ig) deposition per area compared to vehicle (vehicle vs. OPN-51107= 4.91% vs. 3.3% p=0.01) and collagen deposition (vehicle vs. OPN-51107=19.0% vs. 12.9% p<0.05) and liver (vehicle vs. OPN-51107=19.3% vs. 2.35% p<0.0001). Congruently, BET inhibition decreased B220-CD19- CD138+mature plasma cells (vehicle vs. OPN-51107=15% vs 9.1% p<0.05) in the lung. Serum cytokine array analysis revealed a significant decrease in circulating CXCL13, relevant to Tfh migration as well as a decrease in other proinflammatory cytokines - CXCL9, MCP-1, IL3, RANTES and G-CSF important for macrophage activation and pathogenicity in OPN-51107 compared to vehicle treated mice. BET inhibition reduced splenic Tfh while increasing Tfr, thereby increasing Tfr:Tfh ratio (vehicle vs. OPN-51107= 5% vs. 9% p<0.05). Furthermore, total splenic CD19+ B-cells (vehicle vs. OPN51107= 70% vs. 33% p<0.001) as well as CXCR5 expression on B-cells were decreased in OPN-51107 treated mice (MFI vehicle vs. OPN-51107= 4744 vs. 571.5 p<0.0001). Conclusion: OPN-51107 improved lung function which correlated with significantly decreased lung fibrosis and antibody deposition in a BO model of cGVHD. BET inhibition reduces cGVHD immune pathology through decreased inflammatory cytokine production, promotion of GC Tfr cell population and modulates B-cell proliferation and/or trafficking. Significance: Using OPN-51107 in a BO model of cGVHD we demonstrate that BET inhibition is a feasible therapeutic strategy to reduce disease severity and improve lung function.

Increased binding of anti-dsDNA antibodies to short oligonucleotides modified with topoisomerase I reveals a potential new enzyme function independent from DNA relaxation

ObjectiveTopoisomerase I (topo I) is a highly conserved enzyme which is known to reduce torsional stress at double-stranded (ds) DNA. Torsional stress induced by supercoiling of dsDNA requires either very long dsDNA existing in genomic DNA or circulation as presented in plasmid DNA. To enable DNA relaxation, topo I induce a transient single-strand break followed by stress-relieving rotation of the released DNA strand. Our group found by serendipity that the topo I inhibitor irinotecan is able to suppress murine systemic lupus erythematosus (SLE), an autoimmune disease which is characterized by the existence of pathogenic anti-dsDNA antibodies (abs). As a possible mechanism we demonstrated in the absence of immunosuppression an increased binding of anti-dsDNA abs to long genomic or circulated plasmid dsDNA modified with topo I.ResultsHere we show that this effect requires active site tyrosine of topo I which is known to facilitate DNA relaxation activity. Moreover, topo I enhanced anti-dsDNA abs binding to short linear oligonucleotides down to a size of 42 bp. Since oligonucleotides of such length are devoid of torsional stress and relaxation respectively, our results suggest a new and unknown function for the enzyme topo I.

Expansion of extrafollicular B and T cell subsets in childhood-onset systemic lupus erythematosus.

Most childhood-onset SLE patients (cSLE) develop lupus nephritis (cLN), but only a small proportion achieve complete response to current therapies. The prognosis of children with LN and end-stage renal disease is particularly dire. Mortality rates within the first five years of renal replacement therapy may reach 22%. Thus, there is urgent need to decipher and target immune mechanisms that drive cLN. Despite the clear role of autoantibody production in SLE, targeted B cell therapies such as rituximab (anti-CD20) and belimumab (anti-BAFF) have shown only modest efficacy in cLN. While many studies have linked dysregulation of germinal center formation to SLE pathogenesis, other work supports a role for extrafollicular B cell activation in generation of pathogenic antibody secreting cells. However, whether extrafollicular B cell subsets and their T cell collaborators play a role in specific organ involvement in cLN and/or track with disease activity remains unknown. We analyzed high-dimensional mass cytometry and gene expression data from 24 treatment naïve cSLE patients at the time of diagnosis and longitudinally, applying novel computational tools to identify abnormalities associated with clinical manifestations (cLN) and disease activity (SLEDAI). cSLE patients have an extrafollicular B cell expansion signature, with increased frequency of i) DN2, ii) Bnd2, iii) plasmablasts, and iv) peripheral T helper cells. Most importantly, we discovered that this extrafollicular signature correlates with disease activity in cLN, supporting extrafollicular T/B interactions as a mechanism underlying pediatric renal pathogenesis. This study integrates established and emerging themes of extrafollicular B cell involvement in SLE by providing evidence for extrafollicular B and peripheral T helper cell expansion, along with elevated type 1 IFN activation, in a homogeneous cohort of treatment-naïve cSLE patients, a point at which they should display the most extreme state of their immune dysregulation.

Assessment of mimicking by EBV-CMV immunoglobulin M of anti-HLA antibodies

ObjectiveWe aimed to show the cross-reactivity that may occur between immunoglobulin (Ig) M antibodies that form against Cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV) and human leukocyte antigens (HLA). MethodsComplement-dependent cytotoxicity (CDC) cross-reactivity between serum samples of 57 patients with IgM positive CMV and/or EBV infections and T and B cells from 15 healthy donors were evaluated. Dithiothreitol was used to distinguish cross-reactivity caused by IgM antibodies from IgG. ResultsThe cross-reactivity ratio between pathogenic IgM antibodies with T cell of the 12th donor, and B cell of the 3rd, 4th, and 8th donors was significantly higher (p = 0.011, <0.001, <0.001 and 0.013, respectively). The ratio of B cell CDC cross-reactivity of all donors (26.4%) was higher than the ratio of T cell CDC cross-reactivity (5.2%) (p < 0.001). The ratio of T cell CDC cross-reactivity of sera containing both anti-CMV IgM and anti-EBV IgM antibodies was significantly higher than those of sera containing only anti-CMV IgM or only anti-EBV IgM antibodies (p = 0.002 and p < 0.001, respectively). There was no difference between B cell CDC cross-reactivity rates according to the presence of anti-CMV and/or anti-EBV IgM antibodies. ConclusionCross-reactivity may occur between anti-CMV and anti-EBV IgM antibodies with HLA molecules. Thus, in graft recipients, pathogenic IgMs can also act as de novo anti-HLA antibodies and aggravate the rejection process.

Pathogenic myelin-specific antibodies in multiple sclerosis target conformational proteolipid protein 1-anchored membrane domains.

B cell clonal expansion and cerebrospinal fluid (CSF) oligoclonal IgG bands are established features of the immune response in multiple sclerosis (MS). Clone-specific recombinant monoclonal IgG1 Abs (rAbs) derived from MS patient CSF plasmablasts bound to conformational proteolipid protein 1 (PLP1) membrane complexes and, when injected into mouse brain with human complement, recapitulated histologic features of MS pathology: oligodendrocyte cell loss, complement deposition, and CD68+ phagocyte infiltration. Conformational PLP1 membrane epitopes were complex and governed by the local cholesterol and glycolipid microenvironment. Abs against conformational PLP1 membrane complexes targeted multiple surface epitopes, were enriched within the CSF compartment, and were detected in most MS patients, but not in inflammatory and noninflammatory neurologic controls. CSF PLP1 complex Abs provide a pathogenic autoantibody biomarker specific for MS.

Guillain-Barré Syndrome.

This article summarizes the clinical features, diagnostic criteria, differential diagnosis, pathogenesis, and prognosis of Guillain-Barré syndrome (GBS), with insights into the current and future diagnostic and therapeutic interventions for this neuromuscular syndrome. GBS is an acute, inflammatory, immune-mediated polyradiculoneuropathy that encompasses many clinical variants and divergent pathogenic mechanisms that lead to axonal, demyelinating, or mixed findings on electrodiagnostic studies. The type of antecedent infection, the development of pathogenic cross-reactive antibodies via molecular mimicry, and the location of the target gangliosides affect the subtype and severity of the illness. The data from the International GBS Outcome Study have highlighted regional variances, provided new and internationally validated prognosis tools that are beneficial for counseling, and introduced a platform for discussion of GBS-related open questions. New research has been undertaken, including research on novel diagnostic and therapeutic biomarkers, which may lead to new therapies. GBS is among the most frequent life-threatening neuromuscular emergencies in the world. At least 20% of patients with GBS have a poor prognosis and significant residual deficits despite receiving available treatments. Research is ongoing to further understand the pathogenesis of the disorder, find new biomarkers, and develop more effective and specific treatments.

A role for pathogenic autoantibodies in small fiber neuropathy?

The immune system has a role in neuropathic pain which includes autoimmune mechanisms (e.g., autoantibodies). Clinical studies have identified a number of conditions where neuropathic pain is common and that are associated with autoantibodies targeting antigens within the nervous system. Interestingly sensory symptoms can be relieved with immunotherapies or plasma exchange, suggesting that pain in these patients is antibody-mediated. Recent preclinical studies have directly addressed this. For example, passive transfer of CASPR2 autoantibodies from patients cause increased pain sensitivity and enhanced sensory neuron excitability in mice confirming pathogenicity and demonstrating that patient autoantibodies are a mechanism to cause neuropathic pain. Small fiber neuropathy (SFN) exclusively affects small sensory fibers (typically nociceptors) and is characterized by severe neuropathic pain. Known causes include diabetes, B12 deficiency and rare variants in sodium channel genes, although around 50% of cases are idiopathic. SFN is associated with autoimmune conditions such as Sjorgen's syndrome, Sarcoidosis and Celiac disease and immunotherapy in the form of Intravenous immunoglobulin (IVIG) has proved an effective treatment. Autoantibodies have been identified and, in some cases, passive transfer of SFN patient IgG in mice can recapitulate neuropathic pain-like behavior. Here we will discuss clinical and preclinical data relating to the idea that pathogenic autoantibodies contribute to SNF. We discuss putative pathogenic antibodies, cellular targets and the molecular mechanisms by which they cause sensory neuron damage and the development of neuropathic pain. Finally, we will comment on future directions which may provide further insights into the mechanisms underlying SFN in patients.

Type 2 T-Cell Responses against Distinct Epitopes of the Desmoglein 3 Ectodomain in Pemphigus Vulgaris

Pemphigus vulgaris (PV) is an autoimmune blistering disorder of the skin and/or mucous membranes caused by IgG autoantibodies that predominantly target two transmembrane desmosomal cadherins: desmoglein (DSG)1 and DSG3. DSG-specific T cells play a central role in PV pathogenesis because they provide help to autoreactive B cells for autoantibody production. In this study, we characterized DSG3-specific peripheral T cells in a cohort of 52 patients with PV and 41 healthy controls with regard to cytokine profile and epitope specificity. By ELISpot analysis, type 2 T cells reactive with the DSG3 ectodomain were significantly increased in patients with PV compared with those in healthy controls. By dextramer analysis, CD4+ T cells specific for an epitope within the extracellular domain of DSG3, DSG3(206-220), were found at significantly higher frequencies in patients with PV than in HLA-matched healthy controls. T-cell recognition of two distinct DSG3 epitopes, that is, DSG3(206-220) and DSG3(378-392), correlated significantly, suggesting a synergistic effect in B-cell help. Immunization of HLA-DRB1∗04:02-transgenic mice with PV with the same set of DSG3 peptides induced pathogenic DSG3-specific IgG antibodies, which induced loss of keratinocyte adhesion invitro. Thus, DSG3 peptide-specific T cells are of particular interest as surrogate markers of disease activity and potential therapeutic targets in PV.

Identification of Rare Membrane Antigen Specific Human B Cells

The experimentally well supported model that MG pathology is caused by antibodies of the IgG class that bind to AChR at the neuromuscular junction, activate complement, and possibly cause internalization of receptors or their functional blockade has enabled the development of a range of reasonably effective treatments. A better understanding of which B cells are responsible for producing these pathogenic antibodies, and why such B cells develop would enable the development of more targeted therapies. Studies of antibodies isolated from single B cells from patients have provided some of this information that was not available from studies of polyclonal antibodies in sera, but perhaps future studies of the B cells themselves will provide deeper insight into the causes of the disease and thereby enable its prevention.

Impact of N-glycan mediated shielding of ADAMTS-13 on the binding of pathogenic antibodies in immune thrombotic thrombocytopenic purpura

BackgroundThrombotic thrombocytopenic purpura (TTP) is a rare thrombotic disorder, with 1.5 to 6.0 cases per million per year. The majority of patients with TTP develop inhibitory autoantibodies that predominantly target the spacer domain of ADAMTS-13. ADAMTS-13 is responsible for cleaving von Willebrand factor (VWF) multimers, thereby regulating platelet adhesion at sites of high-vascular shear stress. Inhibition and/or clearance of ADAMTS-13 by pathogenic autoantibodies results in accumulation of VWF multimers that promotes the formation of platelet-rich microthrombi. Previously, we have shown that insertion of a single N-glycan (NGLY) in the spacer domain prevents the binding of antispacer domain antibodies. ObjectivesTo explore whether NGLY mediated shielding of the ADAMTS-13 spacer domain effectively prevents binding of pathogenic antispacer autoantibodies in patients with immune-mediated TTP (iTTP). MethodsWe screened 5 NGLY-ADAMTS-13 variants (NGLY3, NGLY7, NGLY8, NGLY3+7, and NGLY3+8) for binding of autoantibodies and for their activity in the presence and absence of 50 samples derived from patients with iTTP. ResultsNGLY variants showed greatly reduced antibody binding, down to 27% of wild-type (wt) ADAMTS-13 binding. Moreover, NGLY variants of ADAMTS-13 remained more active in FRETS-VWF73 assay in the presence of the plasma samples from these 50 patients with acute phase iTTP when compared with wtADAMTS-13. On average, wtADAMTS-13 activity was reduced to 37% of regular levels in the presence of plasma, while NGLY3 and NGLY3+7 remained 69% and 81% active, respectively. ConclusionThese results reinforce our previous findings that NGLYs shield ADAMTS-13 from antibody binding and hence restore ADAMTS-13 activity in the presence of autoantibodies.