The immunohistology of the thymus in myasthenia gravis.

We have investigated cell subpopulations in frozen sections of thymus tissue obtained from myasthenic (MG) and control subjects. With the use of an avidin-biotin immunoperoxidase system with monoclonal antibodies, the following cell surface antigens were studied on frozen sections (12 MG and 3 control thymus); T11, T4, T6, T8, IgM, IgD, and Ia. The pattern of T cell phenotypes in MG thymus is similar to that of normal control thymus when examined by immunohistologic techniques. MG cortical thymocytes are virtually all T11+, T4+, T8+, and T6+. In the medulla, at least 45% of thymocytes are T11+, with T4+ cells predominating over T8+ cells. Approximately 10% of medullary thymocytes are T6+. Scattered medullary cells expressing surface IgM and IgD are identified in both MG and normal thymuses. However, unlike the normal thymus, the MG thymus has numerous secondary follicles containing IgM- and IgD-bearing cells. This finding supports the hypothesis that the MG thymus microenvironment is aberrant. The Ia antigen is found in similar tissue section localization patterns in MG and control thymus. Ultramicroscopic studies show the Ia antigen predominantly on epithelial and interdigitating dendritic cells. By immunoperoxidase techniques, numerous keratin-positive cells are demonstrated in MG and control thymus. This suggests that thymic epithelial cells, like epithelial cells elsewhere, contain keratin. Because these data differ in degree from our previous findings in suspensions of MG thymocytes, this study emphasizes the importance of examining tissue sections as well as cell suspensions when one is studying lymphocyte surface markers.     

The intrathymic pathogenesis of myasthenia gravis

The thymus is considered to play an important role in the pathogenesis of Myasthenia gravis, an autoimmune disease characterized by antibody-mediated skeletal muscle weakness. However, its role is yet to be defined. The studies described herein summarize our efforts to determine how intrathymic expression of the neuromuscular type of acetylcholine (ACh) receptors is involved in the immunopathogenesis of this autoimmune disease. We review the work characterizing the expression of neuromuscular ACh receptors in the thymus and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.     

Microenvironments in the normal thymus and the thymus in myasthenia gravis.

The disposition of epithelial cells and extracellular matrix, in the thymus of 8 cases of myasthenia gravis (MG) and in controls (over a wide age range) was studied. In the controls, the subcapsular epithelium was strongly Leu-7-positive in the fetus, negative in childhood, and positive again in adults. Another antibody, RFD4, also labeled the subcapsular epithelium in childhood and adults, but not fetal samples. The samples from MG cases showed the same staining pattern as adult control samples. The medullary epithelium was also RFD4+, and at all ages. The most striking changes in the advanced cases of MG were the unusual arrangement and hypertrophic appearance of medullary epithelial cell areas, separated by laminin-positive basement membranes from the alternating multiple bands of peripheral lymph-node-like areas. The latter had regions resembling the paracortex of lymph nodes as well as germinal centers (GCs). The T-cell zones contained heavy deposits of fibronectin. These T-cell zones were unique to the thymus in MG and were absent in the two normal thymic samples with isolated GCs. In MG the laminin-containing basement membrane, which separated the medullary epithelial and peripheral lymph-node-like areas, was fenestrated at circumscribed points closest to the GCs, thus apparently permitting communication among the medullary epithelium, the T-cell zones, the GCs and the associated antigen-presenting cells. Large numbers of interdigitating cells and some lymphocytes of cortical thymocyte phenotype were also found at these special sites, where opportunities for autosensitization may persist in MG.     

The pathogenesis of myasthenia gravis — A hypothesis

We have suggested that a thymic factor plays a critical role in the pathogenesis of myasthenia gravis. The presence of specific ACh R antibodies in more than 90% of patients with myasthenia gravis has provided us with a marker for this disorder and has greatly increased our understanding of the pathophysiology. These antibodies can induce physiological parameters similar to those seen in myasthenia gravis following passive transfer to experimental animals and can accelerate degradation of ACh R in myotube culture. However, clinical studies suggest that additional factors of thymic origin are necessary for the development of the clinical features of myasthenia gravis. While the nature of the thymic factor(s) is speculative, this hypothesis has a significant clinical implication as it argues for an early, total thymectomy as the treatment of choice for myasthenia gravis.     

Histiocytosis X of the thymus in association with myasthenia gravis

A patient with myasthenia gravis underwent thymectomy following the failure of medical treatment. The thymus was normal except for four nodules, which, on histologic and electron microscopic examination, were found to consist of proliferative clusters of Langerhans' cells characteristic of histiocytosis X. This patient therefore had two disease processes in which thymic abnormalities are frequently encountered, but their simultaneous occurrence in the same patient has not hitherto been documented. The case is briefly discussed in the context of current concepts of the pathogenesis of histiocytosis X and myasthenia gravis.     

Myasthenia gravis patients with thymus hyperplasia and myasthenia gravis patients with thymoma display different HLA associations

Thirty myasthenia gravis (MG) patients (9 with thymoma, 12 with thymus hyperplasia and 9 with thymic atrophy) and 181 Norwegian healthy controls were serologically typed for HLA-A, -B and -DR antigens and genomically typed for HLA-DQA1 and HLA-DQB1 alleles by probing in vitro amplified DNA with sequence-specific oligonucleotides. In patients with thymus hyperplasia the frequency of the DQB1*0201 allele was increased compared to controls (RR = 3.5, p less than 0.05), whereas among the patients with thymoma this allele was not observed (RR = 0.06, p less than 0.01). The frequencies of HLA-B8, -DR3 and -DQA1*0501, which are in strong linkage disequilibrium with DQB1*0201, were increased in patients with hyperplasia and reduced in patients with thymoma. The data suggest that different HLA genes predispose to two different forms of MG.     

Epithelial cells and macrophages in myasthenia gravis thymus culture

Monolayer culture of thymic nonlymphoid cells derived from female patients with myasthenia gravis (MG) and individuals who underwent heart surgery was established to investigate the cellular composition of the thymic microenvironment and the interaction of nonlymphoid cells with autologous thymocytes. Thymic epithelial cells were identified by immunoperoxidase staining using monoclonal antibodies (mAbs) specific for cytokeratin and MR6 and MR19 antigens expressed on cortical and medullary epithelial cells, respectively. Macrophages were characterized by determination of alpha-naphthyl acetate esterase activity and detection of M1 antigen by mAb. It was demonstrated that in MG thymus cultures the number of cortical MR6+ epithelial cells is significantly reduced, and the ability of the remaining MR6+ cells to bind autologous thymocytes is markedly affected. On the other hand, the number of macrophages and the interaction of those cells with thymocytes were similar in MG and control thymus cultures. Since MR6+ epithelial cells are numerically and functionally affected in MG, maturational events of T cells occurring in the inner cortex may be altered. The mechanisms underlying the induction and expansion of T helper clones in MG are discussed.     

Human foamy virus genome in the thymus of myasthenia gravis patients

The etiological relationship of human foamy virus (HFV), which is a spumaretrovirus, with human diseases is not clear. We analyzed thymus specimens from four patients with myasthenia gravis for the presence of HFV proviral genome by polymerase chain reaction (PCR). The results showed the presence of both 257 base pair (bp) and 299 bp DNA fragments representing a part of gag and bel-2 sequences, respectively, in all four thymuses. Their specificity was confirmed by Southern blot hybridization with the corresponding probes. This was also confirmed by sequence analysis, although there were some point mutations. We confirmed the presence of gag related sequence, a 1353 bp Xba I-cleaved DNA fragment in all four thymus samples, a 693 bp fragment in two (#3 and #4) and a 4300 bp Hind III-cleaved DNA fragment in another two (#1 and #4), indicating possible chromosomal integration of the HFV partial genome. To our knowledge, this is the first report on the presence of HFV genome in thymus tissues of myasthenia gravis patients. Our efforts to isolate the infectious HFV by cultivation of the tissues were not successful. Low titers of neutralizing antibody were detected in all four patients' serum samples. The possible role of the HFV in this autoimmune disease needs further investigation.     

Toll样受体在重症肌无力患者胸腺组织中的表达及其临床意义

目的 研究Toll样受体(Toll-like receptor,TLRs)在重症肌无力(myasthenia gravis,MG)患者胸腺组织中的表达,分析其与MG发生、发展的关系.方法 收集我科2007年7月至2008年2月手术治疗的36例MG患者胸腺标本,分为胸腺瘤组、瘤旁胸腺组、非瘤MG胸腺组,以无MG的正常胸腺21例为对照(正常胸腺组),采用逆转录-PCR技术,检测TLR2、TLR3、TLR4在胸腺组织中的mRNA转录表达水平,进一步采用实时定量逆转录-PCR检测TLR4 mRNA转录表达水平以及与MG临床特点间的关系.结果 逆转录-PCR结果显示,TLR2 mRNA和TLR3 mRNA在4组之间的差别没有统计学意义,TLR4 mRNA的表达在4组之间的差别有统计学意义.进一步的实时定量逆转录.PCR检测TLR4 mRNA转录表达的结果显示,瘤旁胸腺组的TLR4 mRNA的表达比胸腺瘤组有明显的增加(0.8214±0.1019 vs 0.7101±0.0916,P=0.005),非瘤MG胸腺组TLR4 mRNA的表达量比正常胸腺组明显增加(0.8544±0.1200 vs 0.6851±0.1524,P=0.018),瘤旁胸腺组和非瘤MG胸腺组TLR4 mRNA的表达量之间的差别没有统计学意义,且都比正常胸腺组明显增加;TLR4 mRNA的表达与患者的Osserman分型呈正相关(R=0.609;P=0.004).结论 TLR4异常表达可能在MG发病中有重要作用,MG合并胸腺瘤患者瘤旁胸腺组织中TLR4的表达状态对胸腺瘤患者是否并发MG可能有重要影响.     

细胞因子在重症肌无力发病机制中的研究进展

重症肌无力(MG)主要是由乙酰胆碱受体抗体(AChR-Ab)介导,依赖细胞免疫和补体参与的使神经肌肉接头处突触后膜上乙酰胆碱受体受损的自身免疫性疾病.近年来的研究表明,细胞因子间的平衡紊乱与MG的发病有重要关系.因而深入了解不同细胞因子在MG中的作用可以更加明确MG的发病机制及对其治疗提出新的方案.     

重症肌无力患者胸腺内microRNA-29表达水平的探究

目的 检测重症肌无力(myasthenia gravis,MG)患者胸腺组织中microRNA-29的表达水平并探讨其与MG发病的相关性.方法 22例经手术治疗的MG患者胸腺组织作为实验组,22例经手术治疗的非MG伴胸腺异常患者胸腺组织作为对照组.通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)的方法检测实验组及对照组胸腺组织中microRNA-29表达水平,采用秩和检验(Mann-Whitney U test)分析实验组与对照组中microRNA-29表达水平.采用Spearman秩相关分析实验组胸腺中microRNA-29表达水平与定量重症肌无力(quantitative myasthenia gravis score,QMG)之间的相关性.结果 MG患者胸腺组织中microRNA-29表达水平较对照组显著增高(P=0.032);依据MG患者性别、年龄、临床特点、胸腺病理结果将实验组分成不同临床亚组,microRNA-29在各组间表达差异无统计学意义(P值分别为0.614、0.471、0.267、0.329);microRNA-29表达水平与QMG呈显著正相关性(r=0.689,P=0.000a).结论 microRNA-29在MG患者胸腺组织中表达增高,其表达水平与肌无力严重程度呈正相关,而与患者性别、年龄、临床类型、胸腺病理结果无明显关联.     

The thymus in myasthenia gravis. Changes typical for the human disease are absent in experimental autoimmune myasthenia gravis of the Lewis rat.

In human myasthenia gravis (MG) formation of autoantibodies against acetylcholine receptor (AChR) is commonly associated with thymic changes termed lymphofollicular hyperplasia (LFH). To learn whether the thymic lesions of human MG are primary changes in the autoimmune pathogenesis, or rather secondary events caused by peripheral autoimmunization, the authors compared the pathologic changes of MG thymuses with the thymuses of Lewis rats with experimental autoimmune myasthenia gravis (EAMG). EAMG was induced either actively by immunization with AChR, or transferred passively with monoclonal antibodies (mAb) binding to AChR. The clinical diagnosis of EAMG was confirmed by electromyography. Germinal centers, which are typical for human MG thymuses, were not detectable in the thymus of EAMG rats. Scattered B cells were seen as normal components of the thymic medulla. In EAMG their number was not augmented, nor were they accumulated focally. The perivascular spaces (PVS) were not distended and the amount of reticulin was not increased. Thymic myoid cells were identified in EAMG as well as in control thymuses; their cellular microenvironment was inconspicuous. Both in normal and in EAMG thymuses, a subpopulation of myoid cells expressed the main immunogenic region of the AChR. Heavily affected rats showed a severe cortical involution, but no specific changes of the medulla. The fact that none of the thymic lesions characteristic for human MG was found in EAMG is compatible with the concept that the thymic changes in MG are primary events in the autoimmune pathogenesis of this disease.     

A molecular hypothesis concerning the pathogenesis of myasthenia gravis

The purine enzyme, adenosine deaminase, is essential for the maturation of lymphocytes, cell growth and normal immune function. Since adenosine deaminase has the highest activity in the thymus and in T lymphocytes, it is hypothesized that a defective or altered enzyme may be a cause of myasthenia gravis, a lymphoid dyscrasia. It is proposed that the alteration is on the non-catalytic portion of adenosine deaminase concerned with the normal immune function of T lymphocytes. Lymphocytes, particularly suppressor T lymphocytes containing a defective adenosine deaminase will function improperly. They will lose their normal immune regulatory function, allowing immunoglobulin-producing B lymphocytes to produce autoantibodies against the nicotinic acetylcholine receptor, with resultant induction and perpetuation of the autoimmune state. In an attempt to compensate for the defect, there may be hypertrophy of the thymus and lymphoid system, with overproduction of a defective adenosine deaminase. Since many of the functions of thymosin, the alleged active principle in thymus are identical to those of adenosine deaminase, it is postulated that thymosin may be a subunit of adenosine deaminase.     

Basement membrane proteins and reticulin in a normal thymus and the thymus in myasthenia gravis

Summary The distribution of basement membrane (BM) proteins, laminin and type IV collagen were studied immunohistochemically in a series of 12 normal thymuses representing different age groups (0–52 years) and in 10 cases of myasthenia gravis (age 7–53 years). The staining pattern was compared with that of conventional reticulin staining. BM proteins were present at the capsule-parenchyma interface and scantily distributed in the medullary stroma, where they were closely associated with reticulin fibres. The extrathymic perivascular space was effectively vizualized by the staining of the BM's marginal to it. The fiber network present in this space stained with reticulin stain and, less continuously, in BM stainings. Lymph node like tissue with germinal centers was occasionally present in the perivascular spaces in normal thymuses and commonly in the myasthenia gravis cases, where the perivascular spaces were often dilated. The BM's of the perivascular space were mostly continuous in normal cases, but discontinuities were observed in cases of myasthenia gravis, especially in the spaces which were widely dilated. Immunohistochemical detection of BM proteins seems to be useful in the study of thymic structure, particularly in the demonstration of the characteristic changes of the perivascular space in myasthenia gravis. It is suggested that the reticulin fibres present in the medulla and in the perivascular space contain laminin and type IV collagen.     

Lymphocyte subpopulations in non-neoplastic thymus from myasthenia gravis patients.

Lymphocyte populations in non-neoplastic thymuses from fifteen patients with myasthenia gravis (MG) were examined. As in normal subjects, the great majority of thymic lymphocytes of MG patients are T cells. When MG thymuses were compared to normal glands, lower percentages of lymphocytes able to form E rosettes resistant to incubation at 37 degrees C (stable E rosettes) were found in MG thymuses. A negligible B cell content was detected in eight normal and in eight MG thymuses with absent or rare lymph follicles; but there was a substantial B cell presence in the thymuses of seven MG cases with thymic hyperplasia containing many germinal centres. Normal and MG thymuses contain the same percentage of lymphocytes bearing receptors for the Fc portion of IgM (TM). Moreover, the IgM Fc receptor was found mostly on cells which did not form stable E rosettes and did not bear surface immunoglobulin. The possible significance of these findings is discussed.     

Characterization of the thymus in Lrp4 myasthenia gravis: Four cases

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction. Most patients have pathogenic autoantibodies against the acetylcholine receptor (AChR). In the last years a novel subpopulation of MG patients has been described that harbors antibodies against low-density lipoprotein receptor-related protein 4 (Lrp4), another postsynaptic neuromuscular antigen. In early-onset AChR MG (EOMG), the thymus plays an important role in immunopathogenesis, and early thymectomy is beneficial. It is still unknown if the thymus plays any role in Lrp4-MG. In this pilot study, we compared thymus samples from four patients with Lrp4-MG (one pre-treated with immunosuppressive drugs), four non-MG controls and five EOMG patients (not pretreated with immunosuppressive drugs). Immunohistochemistry of the Lrp4-MG thymi revealed normal architecture, with normal numbers and distribution of B-cells, lymphoid follicles and Hassall's corpuscles. Primary CD23⁺ lymphoid follicles were similarly infrequent in Lrp4-MG and control thymic sections. In none of the control or Lrp4-MG thymi did we find secondary follicles with CD10⁺ germinal centers. These were evident in 2 of the 5 EOMG thymi, where primary lymphoid follicles were also more frequent on average, thus showing considerable heterogeneity between patients. Even if characteristic pathological thymic changes were not observed in the Lrp4 subgroup, we cannot exclude a role for the thymus in Lrp4-MG pathogenesis, since one Lrp4-MG patient went into clinical remission after thymectomy alone (at one year follow-up) and one more improved after thymectomy in combination with immunosuppressive therapy.