Antibodies to Full-Length Agrin Protein in Chinese Patients With Myasthenia Gravis.

This study aimed to establish a cell-based assay (CBA) for the detection of agrin antibodies (Agrin-Ab) to explore the clinical features of agrin antibody-positive Chinese patients with myasthenia gravis (Agrin-MG). We developed a CBA based on the human full-length agrin protein expressed in HEK293T cells for the reliable and efficient detection of Agrin-Ab. Clinical data and serum samples were collected from 1948 MG patients in 26 provinces in China. The demographic and clinical features of Agrin-MG patients were compared with those of other MG patient subsets. Eighteen Agrin-MG cases were identified from 1948 MG patients. Nine patients were Agrin-Ab positive, and nine were AChR-Ab and Agrin-Ab double-positive (Agrin/AChR-MG). Eleven (61.11%) patients were males older than 40 years of age. The initial symptom in 13 (81.25%) cases was ocular weakness. Occasionally, the initial symptom was limb-girdle weakness (two cases) or bulbar muscle weakness (one case). Agrin-MG patients demonstrated slight improvement following treatment with either acetylcholinesterase inhibitor or prednisone; however, the combination of the two drugs could effectively relieve MG symptoms. In China, Agrin-MG demonstrated seropositivity rates of 0.92%. These patients were commonly middle-aged or elderly men. The patients usually presented weakness in the ocular, bulbar, and limb muscles, which may be combined with thymoma. These patients have more severe diseases, although the combination of pyridostigmine and prednisone was usually effective in relieving symptoms.

Clinical features of LRP4/agrin-antibody-positive myasthenia gravis: A multicenter study.

INTRODUCTION: Our aim in this study was to identify the prevalence and clinical characteristics of LRP4/agrin-antibody-positive double-seronegative myasthenia gravis (DNMG). METHODS: DNMG patients at 16 sites in the United States were tested for LRP4 and agrin antibodies, and the clinical data were collected. RESULTS: Of 181 DNMG patients, 27 (14.9%) were positive for either low-density lipoprotein receptor-related protein 4 (LRP4) or agrin antibodies. Twenty-three DNMG patients (12.7%) were positive for both antibodies. More antibody-positive patients presented with generalized symptoms (69%) compared with antibody-negative patients (43%) (P ≤ .02). Antibody-positive patients' maximum classification on the Myasthenia Gravis Foundation of America (MGFA) scale was significantly higher than that for antibody-negative patients (P ≤ .005). Seventy percent of antibody-positive patients were classified as MGFA class III, IV, or V compared with 39% of antibody-negative patients. Most LRP4- and agrin-antibody-positive patients (24 of 27, 89%) developed generalized myathenia gravis (MG), but with standard MG treatment 81.5% (22 of 27) improved to MGFA class I or II during a mean follow-up of 11 years. DISCUSSION: Antibody-positive patients had more severe clinical disease than antibody-negative patients. Most DNMG patients responded to standard therapy regardless of antibody status.

A Case of Triple-Negative Myasthenia Gravis Lambert-Eaton Overlap Syndrome With Negative Agrin and LRP-4 Antibodies.

A case of triple-negative myasthenia gravis Lambert-Eaton overlap syndrome with negative Agrin and LRP-4 antibodies. Myasthenia gravis (MG) is an autoimmune disorder that shares similar features with Lambert-Eaton myasthenic syndrome. The combined clinical and electrophysiological findings of MG and Lambert-Eaton myasthenic syndrome have been reported, these cases represent the so-called "myasthenia gravis Lambert-Eaton overlap syndrome" (MLOS). A total of 55 MLOS cases have been identified, 13 cases were reported before the acetylcholine receptor (AChR) antibody (ab) testing era, 14 during the AChR-ab era, 26 during the voltage-gated calcium channel (VGCC)-ab era, and 2 cases have been reported during the muscle-specific kinase (MuSK)-ab era, of these; only 1 patient tested negative for all 3 antibodies. New immunological markers have been identified in the study of MG [Agrin and the low-density lipopro-tein receptor-related protein 4 (LRP-4)]. We present a patient with MLOS who tested negative for all 5 (AChR, MuSK, VGCC, Agrin, and LRP-4) serologic markers.

Myasthenia Gravis: Pathogenic Effects of Autoantibodies on Neuromuscular Architecture.

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ). Autoantibodies target key molecules at the NMJ, such as the nicotinic acetylcholine receptor (AChR), muscle-specific kinase (MuSK), and low-density lipoprotein receptor-related protein 4 (Lrp4), that lead by a range of different pathogenic mechanisms to altered tissue architecture and reduced densities or functionality of AChRs, reduced neuromuscular transmission, and therefore a severe fatigable skeletal muscle weakness. In this review, we give an overview of the history and clinical aspects of MG, with a focus on the structure and function of myasthenic autoantigens at the NMJ and how they are affected by the autoantibodies' pathogenic mechanisms. Furthermore, we give a short overview of the cells that are implicated in the production of the autoantibodies and briefly discuss diagnostic challenges and treatment strategies.

Alleviating Sepsis-Induced Neuromuscular Dysfunction Linked With Acetylcholine Receptors by Agrin.

BACKGROUND: Abnormal expression and distribution of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle caused by sepsis can lead to neuromuscular dysfunction. Here, we asked whether neural agrin regulates nAChRs to ameliorate muscle function, which could be associated with the agrin/muscle-specific kinase pathway. METHODS: Rats were subjected to cecal ligation and puncture (CLP) group, sham group, or control group to observe the alteration caused by sepsis. To verify the effect of improving function, rats were injected with agrin or normal saline intramuscularly after CLP. Electromyogram was used to measure neuromuscular function. Cytokines levels of serum and the expression of related proteins and mRNA were tested after treatment. RESULTS: Compared with the rats in control or sham group, CLP-treated rats showed an acute inflammatory status and a reduction of neuromuscular dysfunction in tibialis anterior muscle, which was associated with abnormal expression in agrin/muscle-specific kinase pathway and increased expression of γ- and α7-nAChR. Exogenous agrin alleviated neuromuscular dysfunction and decreased the expression of γ- and α7-nAChR through agrin-related signaling pathway. CONCLUSIONS: The decreased expression of agrin may lead to skeletal muscle dysfunction. Early enhancement of intramuscular agrin levels after sepsis may be a potential strategy for the treatment of sepsis-induced muscle dysfunction.

Serological and experimental studies in different forms of myasthenia gravis.

Antibodies to the acetylcholine receptor (AChR) have been recognized for over 40 years and have been important in the diagnosis of myasthenia gravis (MG), and its recognition in patients of different ages and thymic pathologies. The 10-20% of patients who do not have AChR antibodies are now known to comprise different subgroups, the most commonly reported of which is patients with antibodies to muscle-specific kinase (MuSK). The use of cell-based assays has extended the repertoire of antibody tests to clustered AChRs, low-density lipoprotein receptor-related protein 4, and agrin. Autoantibodies against intracellular targets, namely cortactin, titin, and ryanodine receptor (the latter two being associated with the presence of thymoma), may also be helpful as biomarkers in some patients. IgG4 MuSK antibodies are clearly pathogenic, but the coexisting IgG1, IgG2, and IgG3 antibodies, collectively, have effects that question the dominance of IgG4 as the sole pathologic factor in MuSK MG. After a brief historical review, we define the different subgroups and summarize the antibody characteristics. Experiments to demonstrate the in vitro and in vivo pathogenic roles of MuSK antibodies are discussed.

Agrin and LRP4 antibodies as new biomarkers of myasthenia gravis.

Myasthenia gravis (MG) is a common disorder that affects the neuromuscular junction. It is caused by antibodies against acetylcholine receptor and muscle-specific tyrosine kinase; however, some MG patients do not have antibodies against either of the proteins. Recent studies have revealed antibodies against agrin and its receptor LRP4-both critical for neuromuscular junction formation and maintenance-in MG patients from various populations. Results from experimental autoimmune MG animal models indicate that anti-LRP4 antibodies are causal to MG. Clinical studies have begun to reveal the significance of the new biomarkers. With their identification, MG appears to be a complex disease entity that can be classified into different subtypes with different etiology, each with unique symptoms. Future systematic studies of large cohorts of well-diagnosed MG patients are needed to determine whether each subtype of patients would respond to different therapeutic strategies. Results should contribute to the goal of precision medicine for MG patients. Anti-agrin and anti-LRP4 antibodies are also detectable in some patients with amyotrophic lateral sclerosis or Lou Gehrig's disease; however, whether they are a cause or response to the disorder remains unclear.

Induction of Anti-agrin Antibodies Causes Myasthenia Gravis in Mice.

Myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction (NMJ). Most cases of MG are caused by autoantibodies against the acetylcholine receptor (AChR), muscle-specific kinase (MuSK) and low-density lipoprotein receptor-related protein 4 (LRP4). Recent studies have identified anti-agrin antibodies in MG patients lacking these three antibodies (i.e., triple negative MG). Agrin is a basal lamina protein that has two isoforms. Neural agrin (N-agrin) binds to LRP4 to activate MuSK to induce AChR clusters and is thus critical for NMJ formation. We demonstrate that mice immunized with N-agrin showed MG-associated symptoms including muscle weakness, fragmented and distorted NMJs. These effects were not observed in mice injected with muscle agrin (M-agrin), an isoform that is inactive in inducing AChR clusters. Treatment with anti-N-agrin, but not anti-M-agrin, antibodies reduced agrin-induced AChR clusters in muscle cells. Together, these observations suggest that agrin antibodies may be play a role in MG pathogenesis.

Screening for lipoprotein receptor-related protein 4-, agrin-, and titin-antibodies and exploring the autoimmune spectrum in myasthenia gravis.

In autoimmune myasthenia gravis (MG), the identification of antibodies and characterization of serological subgroups is of great importance for diagnosis and management of the disease. Our aims were to study the frequency of antibodies against lipoprotein-related protein 4 (LRP4), agrin, and titin using the most recent techniques, and to characterize corresponding clinical features and autoimmune diseases (AID) in 100 MG-patients. The antibody frequencies in the 55 AChR-antibody positive patients were 7% LRP4, 5% agrin, 53% titin, and in the 45 AChR-antibody negative patients 2% MuSK, 2% LRP4, 2% agrin, and 27% titin. LRP4-MG presented late-onset age, mild symptoms, good therapeutic response, and no thymic changes. Agrin-MG showed early onset age, mild-to-severe symptoms, and moderate treatment response. The phenotype of titin-MG depended on AChR-antibodies: AChR-antibody negative patients presented with mostly mild limb muscle weakness, whereas AChR-antibody positive patients showed more frequently severe symptoms, including myasthenic crisis, bulbar predominance, and thymoma. Additional AID were detected in 32% of MG-patients, most frequently Hashimoto's thyroiditis (21%). Based on our data, we recommend the detection of LRP4-antibodies for at least AChR-antibody negative MG-patients and titin-antibodies for all MG-patients. We propose taking an accurate medical history for typical symptoms of Hashimoto's thyroiditis in MG-patients.

Glomerular basement membrane heparan sulfate in health and disease: A regulator of local complement activation.

The glomerular basement membrane (GBM) is an essential component of the glomerular filtration barrier. Heparan sulfate proteoglycans such as agrin are major components of the GBM, along with α345(IV) collagen, laminin-521 and nidogen. A loss of GBM heparan sulfate chains is associated with proteinuria in several glomerular diseases and may contribute to the underlying pathology. As the major determinants of the anionic charge of the GBM, heparan sulfate chains have been thought to impart charge selectivity to the glomerular filtration, a view challenged by the negligible albuminuria in mice that lack heparan sulfate in the GBM. Recent studies provide increasing evidence that heparan sulfate chains modulate local complement activation by recruiting complement regulatory protein factor H, the major inhibitor of the alternative pathway in plasma. Factor H selectively inactivates C3b bound to surfaces bearing host-specific polyanions such as heparan sulfate, thus limiting complement activation on self surfaces such as the GBM, which are not protected by cell-bound complement regulators. We discuss mechanisms whereby the acquired loss of GBM heparan sulfate can impair the local regulation of the alternative pathway, exacerbating complement activation and glomerular injury in immune-mediated kidney diseases such as membranous nephropathy and lupus nephritis.

Agrin and low-density lipoprotein-related receptor protein 4 antibodies in amyotrophic lateral sclerosis patients.

INTRODUCTION: The prevalence and characteristics of agrin and low-density lipoprotein-related receptor protein 4 (LRP4) antibody-positive amyotrophic lateral sclerosis (ALS) patients were studied. METHODS: We tested 82 ALS patients and 59 controls for agrin and LRP4 antibodies using enzyme-linked immunoassay (ELISA). RESULTS: We found that 13.8% of ALS patients had agrin antibodies, and 9.8% had LRP4 antibodies. Women with ALS are twice as likely as men to have antibodies. Agrin-positive ALS patients are younger than agrin-negative ALS patients. CONCLUSIONS: Antibodies to agrin and LRP4 are found in ALS patients. It must be determined whether these antibodies are pathogenic. Because antibody-positive patients have upper as well as lower motor neuron findings, the antibodies' effects cannot be explained solely by their actions at the neuromuscular junction. A breakdown in interneuronal signaling may be the cause of ALS. Further research is needed to resolve this question. Muscle Nerve, 2016 Muscle Nerve 55: 430-432, 2017.

Anti-agrin autoantibodies in myasthenia gravis.

OBJECTIVE: Because the extracellular matrix protein agrin is essential for neuromuscular junction formation and maintenance, we tested the hypothesis that autoantibodies against agrin are present in sera from patients with myasthenia gravis (MG). METHODS: We determined the presence of anti-agrin antibodies in 54 sera from patients with generalized MG using a solid-phase ELISA with purified mini-agrin protein. Thirty of the 54 sera were seronegative for antibodies against the acetylcholine receptor (AChR) or muscle-specific tyrosine kinase (MuSK), 15 had elevated levels of anti-MuSK, and 9 had elevated levels of anti-AChR autoantibodies. Sixteen sera from healthy volunteers served as control. RESULTS: Five sera with elevated levels of anti-agrin antibodies were identified. The concentration of the antibodies ranged between 0.04 and 0.12 nM. Four of the 5 agrin-positive sera were also positive for anti-MuSK, one was positive for anti-AChR, and 2 had elevated levels of anti-low-density lipoprotein receptor-related protein 4 (LRP4) autoantibodies. Some of the sera stained adult mouse neuromuscular junctions and reacted with native mini-agrin expressed in 293HEK cells. CONCLUSIONS: The results provide evidence for agrin as a novel target protein for autoantibodies in patients with MG. Anti-agrin antibodies were always detected in combination with autoantibodies against MuSK, LRP4, or AChRs, indicating a high incidence of autoantibodies against several neuromuscular proteins in the agrin-positive MG cases.

Autoantibodies to agrin in myasthenia gravis patients.

To determine if patients with myasthenia gravis (MG) have antibodies to agrin, a proteoglycan released by motor neurons and is critical for neuromuscular junction (NMJ) formation, we collected serum samples from 93 patients with MG with known status of antibodies to acetylcholine receptor (AChR), muscle specific kinase (MuSK) and lipoprotein-related 4 (LRP4) and samples from control subjects (healthy individuals and individuals with other diseases). Sera were assayed for antibodies to agrin. We found antibodies to agrin in 7 serum samples of MG patients. None of the 25 healthy controls and none of the 55 control neurological patients had agrin antibodies. Two of the four triple negative MG patients (i.e., no detectable AChR, MuSK or LRP4 antibodies, AChR-/MuSK-/LRP4-) had antibodies against agrin. In addition, agrin antibodies were detected in 5 out of 83 AChR+/MuSK-/LRP4- patients but were not found in the 6 patients with MuSK antibodies (AChR-/MuSK+/LRP4-). Sera from MG patients with agrin antibodies were able to recognize recombinant agrin in conditioned media and in transfected HEK293 cells. These sera also inhibited the agrin-induced MuSK phosphorylation and AChR clustering in muscle cells. Together, these observations indicate that agrin is another autoantigen in patients with MG and agrin autoantibodies may be pathogenic through inhibition of agrin/LRP4/MuSK signaling at the NMJ.

MuSK IgG4 autoantibodies cause myasthenia gravis by inhibiting binding between MuSK and Lrp4.

Myasthenia gravis (MG) is a severely debilitating autoimmune disease that is due to a decrease in the efficiency of synaptic transmission at neuromuscular synapses. MG is caused by antibodies against postsynaptic proteins, including (i) acetylcholine receptors, the neurotransmitter receptor, (ii) muscle-specific kinase (MuSK), a receptor tyrosine kinase essential for the formation and maintenance of neuromuscular synapses, and (iii) low-density lipoprotein receptor-related protein 4 (Lrp4), which responds to neural Agrin by binding and stimulating MuSK. Passive transfer studies in mice have shown that IgG4 antibodies from MuSK MG patients cause disease without requiring complement or other immune components, suggesting that these MuSK antibodies cause disease by directly interfering with MuSK function. Here we show that pathogenic IgG4 antibodies to MuSK bind to a structural epitope in the first Ig-like domain of MuSK, prevent binding between MuSK and Lrp4, and inhibit Agrin-stimulated MuSK phosphorylation. In contrast, these IgG4 antibodies have no direct effect on MuSK dimerization or MuSK internalization. These results provide insight into the unique pathogenesis of MuSK MG and provide clues toward development of specific treatment options.

Extracellular matrix of secondary lymphoid organs impacts on B-cell fate and survival.

We describe a unique extracellular matrix (ECM) niche in the spleen, the marginal zone (MZ), characterized by the basement membrane glycoproteins, laminin α5 and agrin, that promotes formation of a specialized population of MZ B lymphocytes that respond rapidly to blood-borne antigens. Mice with reduced laminin α5 expression show reduced MZ B cells and increased numbers of newly formed (NF) transitional B cells that migrate from the bone marrow, without changes in other immune or stromal cell compartments. Transient integrin α6ß1-mediated interaction of NF B cells with laminin α5 in the MZ supports the MZ B-cell population, their long-term survival, and antibody response. Data suggest that the unique 3D structure and biochemical composition of the ECM of lymphoid organs impacts on immune cell fate.

Distinct localization of T cell Agrin during antigen presentation--evidence for the expression of Agrin receptor(s) in antigen-presenting cells.

Agrin is over-expressed by activated and autoimmune T cells, and synergizes with the T cell receptor (TCR) to augment cell activation. In the present study, we show that Agrin accumulates to distinct areas of the plasma membrane and that cell activation causes its redistribution. During antigen presentation, Agrin primarily accumulates to the periphery of the mature immunological synapse, mostly in lamellipodia-like protrusions that wrap around the antigen-presenting cell and, conversely, anti-Agrin sera induced a significant redistribution of TCR at the plasma membrane. We also provide evidence for the expression of Agrin receptors in peripheral blood monocytes, dendritic cells and a fraction of B cells. Interestingly, interferon-α treatment, which induces the expression of Agrin in T cells, also augmented Agrin binding to monocytes. Stimulation of monocytes with recombinant Agrin induced the clustering of surface receptors, including major histocompatibility complex class II, activation of intracellular signalling cascades, as well as enhanced dsRNA-induced expression of pro-inflammatory cytokines interleukin-6 and tumour necrosis factor-α. Collectively, these results confirm the location of Agrin at the immunological synapse between T cells and antigen-presenting cells and justify further characterization of its receptors in the immune system.

[Selective deposition of agrin in the microvasculature of hepatocellular carcinoma: aspects in pathogenesis and differential diagnosis]. / Az agrin szelektív megjelenése a hepatocellularis carcinoma erezetében: kórkeletkezési és elkülöníto diagnosztikai vonatkozások.

Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancers and is the fifth most common malignancy worldwide. HCC typically develops in the cirrhotic liver. Our preliminary results indicated that agrin, a heparan sulfate proteoglycan (HSPG) detected by us for the first time in the liver, accumulates in the basement membranes (BMs) of the cirrhotic liver and HCC. This novel finding prompted us to investigate the role of agrin in the pathogenesis and differential diagnosis of HCC. First, the previously unspecified monoclonal antibody anti-HSPG clone 7E12 was verified as anti-agrin, using mass spectrometry. Our subsequent experiments were carried out on specimens from 131 patients with chronic liver disease and 18 individuals with healthy liver, from 4 rats subjected to cirrhosis/HCC induction and 1 untreated control rat, as well as from cultured cells. In both human and rats, significantly increased expression of agrin in cirrhosis and HCC was demonstrated by immunohistochemistry (IHC), Western blot, and quantitative RT-PCR. By double immunofluorescent studies, agrin was localized to the muscular layer of blood vessel walls, the BM of bile ducts and ductular reaction, the microvessel walls of HCC, and occasionally the BM of hepatocellular tumor cells. Colocalization, gene expression, and mRNA in situ hybridization experiments suggested that the sources of agrin include vascular smooth muscle cells, epithelial cells of bile ducts and ductules, activated mesenchymal cells in the stroma of hepatocellular tumors, and occasionally tumor hepatocytes. Agrin in the BMs of bile ducts and blood vessels is thought to play an important role in the survival of bile duct epithelium and vascular endothelium, respectively. Thus, agrin may contribute to the formation of ductular reaction and HCC neovessels. As opposed to HCC neovessels that were consistently found agrin-positive, normal and cirrhotic sinusoids were always devoid of agrin, raising the possibility that agrin IHC might be useful in the differential diagnosis of benign versus malignant hepatocellular lesions. Agrin IHC was performed on 68 benign lesions (8 large regenerative nodules, 23 low-grade and 7 high-grade dysplastic nodules, 30 liver adenomas) and 29 malignant lesions (8 small HCC, 21 HCC), and was evaluated semi-quantitatively. Based on the results of IHC for agrin as well as CD34, a decision algorithm was devised that differentiated benign and malignant parenchymal lesions with a sensitivity of 93.1% and a specificity of 92.6%. Hence, we propose that agrin IHC might help distinguish between malignant hepatocellular lesions and their benign mimickers.