重症肌无力患者CD4、CD8细胞表面Fas分子的变化

为探讨Fas分子、CD4、CD8分子在重症肌无力发生中的作用 ,从胸腺及外周血分离单个核细胞 ,用荧光标记的单克隆抗体 (Fas FITC、CD4 PE、CD8 Cy )和流式细胞仪检测细胞表面Fas、CD4、CD8表达情况。结果发现 ,(1)MG患者外周血单个核细胞 (PBMC )CD4 + CD8 细胞明显低于对照组 (P <0 0 2 ) ;(2 )MG患者Fas分子在PBMC的表达率明显低于对照组 (P<0 0 2 ) ,Fas+ CD4 + 细胞也低于对照组 (P <0 0 5 ) ;(3)在Fas+ 的胸腺细胞中 ,MG患者的双阳性细胞显著低于对照组 (P <0 0 1) ,而CD4 CD8+ 细胞则高于对照组 (P <0 0 5 )。说明重症肌无力患者淋巴细胞存在Fas表达异常 ,表现为外周CD4 + 细胞活化后的凋亡障碍和胸腺CD4 + CD8+ 细胞凋亡障碍 ,可能与重症肌无力患者自身反应性T细胞的形成及疾病的发生有关     

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.     

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.     

重症肌无力患者胸腺细胞凋亡障碍与Fas表达异常的关系

目的 :探讨重症肌无力 (MG)胸腺细胞凋亡异常、Fas表达异常在自身免疫应答形成中的作用。方法 :用手术切除的MG患者的胸腺 ,制备胸腺提取液和胸腺细胞悬液 ,用MTT比色法测定胸腺细胞的增殖 ;用DNA电泳及细胞形态学观察细胞凋亡 ;用流式细胞仪检测细胞表面Fas的表达 ,用RT PCR结合单链多态构象性分析 (SSCP) ,探讨Fas基因的转录及可能存在的Fas基因突变。结果 :MG患者的胸腺提取液 ,能抑制正常胸腺细胞增殖 ,但不能抑制MG患者胸腺细胞增殖。在地塞米松作用下 ,正常人及患者胸腺细胞的增殖均可被抑制 ,细胞增殖抑制率分别为 5 8.33%和 5 4 .2 6 %。MG患者胸腺细胞的DNA电泳可见梯状条带 ,并且胸腺细胞上Fas的表达百分率增加 [(2 0 .38± 6 .0 7) % ],与空白对照组 [(12 .4 3±4 .32 ) % ]相比较P <0 .0 5。对MG患者胸腺细胞FasmRNA进行RT PCR SSCP分析 ,部分MG患者出现异常电泳条带。结论 :MG患者胸腺细胞的凋亡及Fas分子表达均异常 ,而且存在Fas基因突变 ,提示与本病的发生发展有关。     

重症肌无力患者胸腺细胞基因突变的初步探讨

目的 探讨重症肌无力(MG)胸腺细胞异常增生的原因，揭示其发生机制。方法 利用患者手术摘除的胸腺组织，分离胸腺细胞，进行体外培养并用植物血凝素(PHA)或地塞米松、雌激素诱导48h，培养前后均从胸腺细胞提取RNA，反转录后，用三对引物分段扩增Fas cDNA，用银染法进行单链多态构象性分析(SSCP)。结果 正常人胸腺细胞诱导后均可见Fas mRNA表达，大约有25％的MG患者胸腺细胞体外诱导未见Fas mRNA表达，部分Fas mRNA表达与未诱导时电泳条带存在差异。结论 部分重症肌无力患者胸腺细胞Fas基因存在突变，这种突变可能是胸腺细胞异常增生的原因之一，与疾病的发生发展有关。     

Myasthenic and nonmyasthenic thymoma. An expansion of a minor cortical epithelial cell subset?

The authors report an immunohistologic study of primary thymomas from 23 cases with myasthenia gravis (MG) and 7 without. Typical T6+ cortical thymocytes were usually abundant. Most epithelial cells initially appeared to be of cortical type, too, though many bore subcapsular markers in most samples. However, two-color immunofluorescence revealed unexpected heterogeneity, numerous epithelial cells simultaneously expressing some or all of the markers of both these subsets (even in two pleural metastases). It is inferred that there is a common tumor stem cell whose normal counterpart may be related to the rare patches of similar phenotype in the cortex in control samples. The authors could detect no major differences in 5 of 7 samples from nonmyasthenics; thus, most thymoma cases may risk the development of MG. Finally, thymomas from 6 of 7 further MG cases pretreated with corticosteroids showed very few cortical thymocytes, and the (phenotypically similar) epithelium was more obvious.     

Detection of thymocyte antigens by monoclonal antibodies of the BL series

The reactivity of 4 monoclonal pan T cell antibodies and 6 pan leukocyte antibodies was detected with human thymocytes. The study was performed with single cell suspensions and cryostat sections using immunofluorescence and immunoperoxidase techniques. Monoclonal antibodies with T cell specificity react with different percentages of thymocytes. The antibody BL-T1, which is directed against the sheep erythrocyte receptor, reacts with 90% of thymocytes. These cells were found uniform in the thymus cortex and medulla. On the other hand, the antibodies e.g. BL-T2 and T3 react only with 20% of the thymocytes, identified mainly in the medulla. Pan leukocyte antibodies are also suitable for the detection of different thymocyte membrane antigens. In single cell suspensions, it was found a reactivity of the used antibodies with 20, or 40, or 95% of the cells. The immunohistological findings are described.     

Detection of antibodies directed against the cytoplasmic region of the human acetylcholine receptor in sera from myasthenia gravis patients

The nicotinic acetylcholine receptor (AChR) is the autoantigen in the human autoimmune disease myasthenia gravis (MG). Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore the determination of their specificities requires the use of native AChR. Antibody competition studies suggest that most MG antibodies are directed against the extracellular part of the molecule, whereas antibodies directed against the cytoplasmic region of the AChR have not been detected. To determine whether even small quantities of such antibodies exist in MG sera, we performed competition experiments based on the inhibition by MG sera of the binding of MoAbs to the human AChR, rather than inhibition by MoAbs of the binding of MG sera performed earlier. When MoAbs directed against cytoplasmic epitopes on the alpha or beta subunits (alpha 373-380 and beta 354-360) were used as test MoAbs, 17% or 9% of MG sera inhibited the binding of the anti-alpha or anti-beta subunit MoAbs, respectively, by > or = 50%. Non-specific inhibition was excluded. These results suggest the presence, in several MG sera, of antibodies directed against cytoplasmic regions of the AChR; yet these antibodies seemed to represent a relatively small proportion of the total anti-AChR antibodies. The corresponding epitopes may be involved in the inducing mechanisms in certain MG cases, and knowledge of the presence of such antibodies may be useful in understanding the autoimmune mechanism involved in MG.     

Autoantibodies in d-penicillamine-induced myasthenia gravis: A comparison with idiopathic myasthenia and rheumatoid arthritis

The distribution of autoantibodies was studied in patients with rheumatoid arthritis (RA) treated by d-penicillamine and who developed myasthenia gravis (MG). The anti-human acetylcholine receptor (AChR) antibodies were specifically associated with clinical symptoms of MG without any difference in the pattern of specificities in idiopathic (id-MG) or in induced MG (DPen-MG). Conversely, anti-nuclear antibodies were elevated in DPen-MG sera compared to id-MG sera (P < 0.001) but were also compared to patients with RA treated by d-penicillamine (or thiopronine) and who did not develop MG. Anti-denatured DNA antibodies were enhanced in sera from treated patients, whether they had presented or not a MG disease. Anti-histone antibodies were associated with RA. These observations suggest that the immunological imbalance in RA patients, can be increased by a drug treatment which may trigger the appearance of a second autoimmune disease such as MG, where anti-AChR antibodies are associated with anti-nuclear antibodies.     

Heart muscle antibodies in myasthenia gravis.

Myasthenia gravis (MG) may in some patients, especially in those with a thymoma, involve the heart. Using an ELISA, we measured heart muscle antibodies in sera from 75 MG patients and in 173 control sera. Heart muscle antibodies were detected in 29/30 thymoma MG patients, in none of 30 young onset MG patients with thymic hyperplasia and in 7/15 late onset MG patients with thymic atrophy. Among the controls, four sera had a very low concentration of heart muscle antibodies. Absorption studies showed that the heart muscle antibodies cross-react with skeletal muscle, but are unrelated to acetylcholine receptor antibodies.     

Differences in fine specificity of anti-acetylcholine receptor antibodies between subgroups of spontaneous myasthenia gravis of recent onset, and of penicillamine induced myasthenia

The antigenic specificity of anti-acetylcholine receptor antibodies (anti-AChR) from 70 recent onset myasthenia gravis (MG) and nine penicillamine MG patients was determined by inhibition experiments using monoclonal antibodies (m.abs) raised against human AChR. Differences were found between individuals and between the three clinical subgroups of idiopathic MG distinguished by age of onset, thymic pathology and HLA associations. Penicillamine-induced MG anti-AChR differed from that in age-matched MG controls but was similar to that in young-onset cases. The variable and heterogeneous antigenic specificity in MG suggests that AChR itself rather than a cross-reacting epitope is the primary antigen. Differences in specificity between MG subgroups may reflect a diversity of triggering factors or of immunoglobulin genes.     

Chicken thymocyte-specific antigen identified by monoclonal antibodies: ontogeny,tissue distribution and biochemical characterization

Two monoclonal antibodies, CT-1 (IgG₁,χ) and CT-1a (IgG₃,χ), were prepared against chicken thymocytes. The antigen identified by these antibodies was found to be a glycoprotein with a major polypeptide component having an apparent molecular weight of 63000 and a minor polypeptide component of approximately 103000. Immunoprecipitation and blocking experiments revealed that the two antibodies react with different antigenic determinants of the molecule. Ontogenic studies employing immunofluorescence failed to reveal the antigenic determinants on cells from the embryo or embryonic yolk sac on days 3 and 6 of incubation. The number of embryonic thymocytes bearing the molecules detected by these antibodies increased from 6% on day 12 to 54% on day 13; the frequency of thymocytes expressing this glycoprotein reached adult levels (> 90%) by the 15th day of embryonic age. In contrast, the CT-1 and CT-1a antibodies reacted with only 2-5% of blood and splenic cells and less than 0.05% of cells from bursa or bone marrow of young adult chickens. In the quail CT-1 reacted with cortical, but not medullary, thymocytes, while the CT-1a antibody was unreactive with quail thymocytes. The surface glycoproteins detected by these discriminating monoclonal antibodies may provide an important discriminating marker for thymic lymphocytes in the chicken and the quail.     

Autoimmunity against the ryanodine receptor in myasthenia gravis.

Some myasthenia gravis (MG) patients have antibodies against skeletal muscle antigens in addition to the acetylcholine receptor (AChR). A major antigen for these antibodies is the Ca2+ release channel of the sarcoplasmic reticulum the ryanodine receptor (RyR). These antibodies are found mainly in MG patients with a thymoma MG and correlate with severe MG symptoms. The antibodies recognize a region near the N-terminus on the RyR, which seems to be of importance for RyR regulation. The antibodies cause allosteric inhibition of RyR function in vitro, inhibiting Ca2+ release from sarcoplasmic reticulum.     

Induction of specific anti-guinea pig T cell sera in rabbits.

In an attempt to increase the specificity of antisera raised in rabbits against strain 2 guinea pig thymocytes and brain, the rabbits were screened for titres of natural antibodies to thymocytes and other lymphocytes. Although unimmunized rabbits commonly had moderate titres of cytotoxic antibodies to guinea pig thymocytes, occasional animals had low titres to thymocytes and moderate titres to bone marrow cells. Intravenous immunization of this latter group of rabbits with thymocytes led to the production of high titred anti-thymocyte sera which were easily made specific for thymus-derived lymphocytes (T cells) by absorption with L2 C lymphoma, a bone marrow-derived lymphoma of strain 2 guinea pigs. Sera raised against guinea pig brain in complete Freund's adjuvant which had high titres of antibodies to both thymocytes and bone marrow cells could be made specific for T cells only with great difficulty. The cytotoxic activity of the anti-T cell serum could be absorbed by strain 2 thymocytes and brain homogenates, while high dilutions of this serum inhibited the formation of spontaneous rosettes between guinea pig lymphoid cells and normal rabbit erythrocytes.     

Analysis of autoantibodies to glycolipids on thymocytes in New Zealand black mice.

Carbohydrate specificities of thymocyte cytotoxic autoantibodies in New Zealand Black (NZB) mice were examined using liposome immune lysis assay (LILA) system. NZB mice progressively produced antibodies to nine neutral glycolipids and two gangliosides tested by 10 months of age. Such increases were not detected in normal BALB/c and CBA/J mice. Among these antibodies, antibodies to ceramide monohexoside (CMH), ceramide dihexoside (CDH), paragloboside (PG), ceramide pentahexoside (CPH), asialo GM1 (GA1), asialo GM2 (GA2), GM4 and GM2 in the plasma of NZB mice were absorbed by single cell suspensions of autologous untreated as well as protease-treated thymocytes. In contrast, antibodies to ceramide trihexoside (CTH), globoside (Glo), and Forssman antigen (For) were not absorbed by these cells. Analysis of glycolipid compositions of young NZB thymocytes demonstrated that strict correlation was observed between the presence of glycolipids on thymocytes and absorbing pattern of anti-glycolipid antibodies by thymocytes. These data indicate that anti-carbohydrate antibodies with thymocyte binding capacity in NZB mice mainly recognize sugar portions of glycolipids rather than glycoproteins on thymocytes. The failure to find such carbohydrate specific antibodies in some normal strains of mice suggested that these antibodies may be important for subsequent immunologic abnormalities in NZB mice.     

In Vitro Cross-Reacting Antibodies from Rabbit Antisera to Rat Brain Synaptic Membranes and Thymocytes

Rabbit anti-rat brain synaptic membrane and anti-rat thymocyte antisera were used in cytotoxicity, immunofluorescence, and absorption assays to define the antigenic relationship between the rat brain and thymocytes, and the localizing properties of antibodies. Both antisera cross-reacted with brain tissue and thymocytes. However, anti-synaptic membrane and anti-thymocyte antisera also contained antibodies specific for neurons and thymocytes, respectively. Immunofluorescence showed that antibodies from anti-thymocyte antiserum reacted with antigenic determinants situated on the surface membrane of brain cells.     

Characterization of anti-acetylcholine receptor antibody activity in patients with anti-mitochondrial antibodies

Antibodies binding to the acetylcholine receptor of human skeletal muscle were found in patients with different kinds of anti-mitochondrial antibodies. In patients with primary biliary cirrhosis (PBC), the antibody activity was found in monomeric and pentameric IgM as well as in IgG, whereas patients with other kinds of anti-mitochondrial antibodies had anti-receptor antibodies of predominantly IgG class. Mice transfused with immunoglobulins from patients with PBC showed a reduction of skeletal muscle receptors comparable to that found in mice who had received immunoglobulins from myasthenia gravis (MG) patients. The antibody affinity was of the same order of magnitude in MG and PBC but antibodies with multiple affinities were more common in PBC. In PBC, the anti-receptor antibody associated idiotype repertoire was markedly different from that found in MG.     

Molecular identification of T cell-specific antigens on human T lymphocytes and thymocytes

We have identified membrane glycoproteins which carry T cell-specific antigens on human T lymphocytes and thymocytes. Purified cells were surface-labeled with NaB³H₄ after treatment with neuraminidase and galactose oxidase. Immunoprecipitations were performed with rabbit anti-human T cell-specific antibodies using co precipitation with protein A-containing staphylococci strain Cowan I. The labeled membrane glycoproteins and the precipitates were subjected to polyacrylamide slab gel electrophoresis and visualized by fluorography. The antibodies specifically precipitated 4 proteins called GP200, GP180, GP165 and GP160 (mol. wts. = 200000, 180000, 165000 and 160000) from surface-labeled T lymphocytes and low-density (medullary) thymocytes. The GP200 and GP180 were not labeled on high-density (cortical) thymocytes. A protein with a mol. wt. of 45000 was precipitated from thymocytes. Another glycoprotein on T lymphocytes and thymocytes with a mol. wt. similar to that of mouse and rat Thy-1 or Θ antigen (mol. wt. 25000) reacted with the antibodies.     

Antibodies to acetylcholinesterase cross-reacting with thyroglobulin in myasthenia gravis and Graves's disease.

In the present study we analysed by ELISA the ability of sera from 50 patients with myasthenia gravis (MG), 20 with Hashimoto's thyroiditis (HT), 53 with Graves' disease (GD) and 36 healthy controls (CR) to react with acetylcholinesterase (AChE) from Electrophorus electricus and human thyroglobulin (Tg). Significantly increased anti-AChE activity was exhibited by a high proportion of MG (IgG 36%) and GD (IgG 21%) sera, while increased anti-Tg activity was detected in all three patient groups (MG, IgG 26% and IgA 26%; HT, IgG 85% and IgA 40%; and GD, IgG 51%). Interestingly, a significant proportion of MG and GD sera exhibited both IgG anti-AChE and anti-Tg activities (MG, 18%; P < 0.001; and GD, 15%; P < 0.001, versus CR, 0%). This bi-reactivity was exhibited by anti-AChE antibodies cross-reacting with Tg (anti-AChE/Tg activity); (i) serum anti-AChE activity was effectively inhibited by soluble Tg, and (ii) affinity-purified anti-Tg antibodies cross-reacted with AChE. Cross-reactivity seems to be a property of pathological (auto)antibodies; induced (rabbit) antibodies to AChE or Tg were highly monospecific. Analysis of clinical data showed that increased IgG anti-AChE/Tg activity was well associated with: (i) overlapping GD in MG (P < 0.02), and (ii) ophthalmopathy in GD (P < 0.01). In contrast, no correlation was noted in MG between anti-AChE activity units and anti-Tg activity units or acetylcholine receptor antibody titres. The clinical significance of anti-AChE/Tg antibodies remains to be elucidated.     

Autoimmunity against the ryanodine receptor in myasthenia gravis

Some myasthenia gravis (MG) patients have antibodies against skeletal muscle antigens in addition to the acetylcholine receptor (AChR). A major antigen for these antibodies is the Ca²⁺ release channel of the sarcoplasmic reticulum the ryanodine receptor (RyR). These antibodies are found mainly in MG patients with a thymoma MG and correlate with severe MG symptoms. The antibodies recognize a region near the N‐terminus on the RyR, which seems to be of importance for RyR regulation. The antibodies cause allosteric inhibition of RyR function in vitro, inhibiting Ca²⁺ release from sarcoplasmic reticulum.