Marc H. De Baets

Anti-Inflammatory Activity of Human IgG4 Antibodies by Dynamic Fab Arm Exchange

Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.

Proteasome Inhibition with Bortezomib Depletes Plasma Cells and Autoantibodies in Experimental Autoimmune Myasthenia Gravis

Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.

Perilipin 2 Improves Insulin Sensitivity in Skeletal Muscle Despite Elevated Intramuscular Lipid Levels

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)–coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation–related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet–induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance.

Antibody effector mechanisms in myasthenia gravis-pathogenesis at the neuromuscular junction

Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies that are either directed to the muscle nicotinic acetylcholine receptor (AChR) or to the muscle-specific tyrosine kinase (MuSK). These autoantibodies define two distinct subforms of the disease—AChR-MG and MuSK-MG. Both AChR and MuSK are expressed on the postsynaptic membrane of the neuromuscular junction (NMJ), which is a highly specialized region of the muscle dedicated to receive and process signals from the motor nerve. Autoantibody binding to proteins of the postsynaptic membrane leads to impaired neuromuscular transmission and muscle weakness. Pro-inflammatory antibodies of the human IgG1 and IgG3 subclass modulate the AChR, cause complement activation, and attract lymphocytes; together acting to decrease levels of the AChR and AChR-associated proteins and to reduce postsynaptic folding. In patients with anti-MuSK antibodies, there is no evidence of loss of junctional folds and no apparent loss of AChR density. Anti-MuSK antibodies are predominantly of the IgG4 isotype, which functionally differs from other IgG subclasses in its anti-inflammatory activity. Moreover, IgG4 undergoes a posttranslational modification termed Fab arm exchange that prevents cross-linking of antigens. These findings suggest that MuSK-MG may be different in etiological and pathological mechanisms from AChR-MG. The effector functions of IgG subclasses on synapse structure and function are discussed in this review.

Effects of cytokines on acetylcholine receptor expression: implications for myasthenia gravis.

Myasthenia gravis is an autoimmune disease associated with thymic pathologies, including hyperplasia. In this study, we investigated the processes that may lead to thymic overexpression of the triggering Ag, the acetylcholine receptor (AChR). Using microarray technology, we found that IFN-regulated genes are more highly expressed in these pathological thymic tissues compared with age- and sex-matched normal thymus controls. Therefore, we investigated whether proinflammatory cytokines could locally modify AChR expression in myoid and thymic epithelial cells. We found that AChR transcripts are up-regulated by IFN-γ, and even more so by IFN-γ and TNF-α, as assessed by real-time RT-PCR, with the α-AChR subunit being the most sensitive to this regulation. The expression of AChR protein was increased at the cytoplasmic level in thymic epithelial cells and at the membrane in myoid cells. To examine whether IFN-γ could influence AChR expression in vivo, we analyzed AChR transcripts in IFN-γ gene knock-out mice, and found a significant decrease in AChR transcript levels in the thymus but not in the muscle, compared with wild-type mice. However, up-regulation of AChR protein expression was found in the muscles of animals with myasthenic symptoms treated with TNF-α. Altogether, these results indicate that proinflammatory cytokines influence the expression of AChR in vitro and in vivo. Because proinflammatory cytokine activity is evidenced in the thymus of myasthenia gravis patients, it could influence AChR expression and thereby contribute to the initiation of the autoimmune anti-AChR response.

Pathophysiology of myasthenia gravis with antibodies to the acetylcholine receptor, muscle-specific kinase and low-density lipoprotein receptor-related protein 4.

Myasthenia gravis is caused by antibodies to the acetylcholine receptor, muscle-specific kinase, low-density lipoprotein receptor-related protein 4, or possibly yet unidentified antibodies. The mechanisms by which these antibodies interfere with the function of postsynaptic proteins include complement activation, antigenic modulation by crosslinking of the target proteins, competition with ligand binding sites, or steric hindrance which inhibits conformational changes or binding to associated proteins. Screening for auto-antibodies to different postsynaptic targets, and also for low-affinity antibodies, is contributing to a more accurate diagnosis of MG patients. Further studies into the specific pathophysiological pathways of the several MG subforms might help to develop new, more antigen specific, therapies.

Implication of Double-Stranded RNA Signaling in the Etiology of Autoimmune Myasthenia Gravis

Myasthenia gravis (MG) is an autoimmune disease mediated mainly by anti–acetylcholine receptor (AChR) antibodies. The thymus plays a primary role in MG pathogenesis. As we recently showed an inflammatory and antiviral signature in MG thymuses, we investigated whether pathogen‐sensing molecules could contribute to an anti‐AChR response.

In vivo electroporation of the central nervous system: a non-viral approach for targeted gene delivery.

Electroporation is a widely used technique for enhancing the efficiency of DNA delivery into cells. Application of electric pulses after local injection of DNA temporarily opens cell membranes and facilitates DNA uptake. Delivery of plasmid DNA by electroporation to alter gene expression in tissue has also been explored in vivo. This approach may constitute an alternative to viral gene transfer, or to transgenic or knock-out animals. Among the most frequently electroporated target tissues are skin, muscle, eye, and tumors. Moreover, different regions in the central nervous system (CNS), including the developing neural tube and the spinal cord, as well as prenatal and postnatal brain have been successfully electroporated. Here, we present a comprehensive review of the literature describing electroporation of the CNS with a focus on the adult brain. In addition, the mechanism of electroporation, different ways of delivering the electric pulses, and the risk of damaging the target tissue are highlighted. Electroporation has been successfully used in humans to enhance gene transfer in vaccination or cancer therapy with several clinical trials currently ongoing. Improving the knowledge about in vivo electroporation will pave the way for electroporation-enhanced gene therapy to treat brain carcinomas, as well as CNS disorders such as Alzheimers disease, Parkinsons disease, and depression.

The auto-antigen repertoire in myasthenia gravis

Myasthenia Gravis (MG) is an antibody-mediated autoimmune disorder affecting the postsynaptic membrane of the neuromuscular junction (NMJ). MG is characterized by an impaired signal transmission between the motor neuron and the skeletal muscle cell, caused by auto-antibodies directed against NMJ proteins. The auto-antibodies target the nicotinic acetylcholine receptor (nAChR) in about 90% of MG patients. In approximately 5% of MG patients, the muscle specific kinase (MuSK) is the auto-antigen. In the remaining 5% of MG patients, however, antibodies against the nAChR or MuSK are not detectable (idiopathic MG, iMG). Although only the anti-nAChR and anti-MuSK auto-antibodies have been demonstrated to be pathogenic, several other antibodies recognizing self-antigens can also be found in MG patients. Various auto-antibodies associated with thymic abnormalities have been reported, as well as many non-MG-specific auto-antibodies. However, their contribution to the cause, pathology and severity of the disease is still poorly understood. Here, we comprehensively review the reported auto-antibodies in MG patients and discuss their role in the pathology of this autoimmune disease.

Main immunogenic region structure promotes binding of conformation-dependent myasthenia gravis autoantibodies, nicotinic acetylcholine receptor conformation maturation, and agonist sensitivity.

The main immunogenic region (MIR) is a conformation-dependent region at the extracellular apex of α1 subunits of muscle nicotinic acetylcholine receptor (AChR) that is the target of half or more of the autoantibodies to muscle AChRs in human myasthenia gravis and rat experimental autoimmune myasthenia gravis. By making chimeras of human α1 subunits with α7 subunits, both MIR epitopes recognized by rat mAbs and by the patient-derived human mAb 637 to the MIR were determined to consist of two discontiguous sequences, which are adjacent only in the native conformation. The MIR, including loop α1 67–76 in combination with the N-terminal α helix α1 1–14, conferred high-affinity binding for most rat mAbs to the MIR. However, an additional sequence corresponding to α1 15–32 was required for high-affinity binding of human mAb 637. A water soluble chimera of Aplysia acetylcholine binding protein with the same α1 MIR sequences substituted was recognized by a majority of human, feline, and canine myasthenia gravis sera. The presence of the α1 MIR sequences in α1/α7 chimeras greatly promoted AChR expression and significantly altered the sensitivity to activation. This reveals a structural and functional, as well as antigenic, significance of the MIR.