A comparative study of the spatial distribution of mast cells and microvessels in the foetal, adult human thymus and thymoma

Mast cells (MCs) are widely distributed in human and animal tissues and have been shown to play an important role in angiogenesis in normal and pathological conditions. Few data are available about the relationship between MCs and blood vessels in the normal human thymus, and there are virtually no data about their distribution and significance in thymoma. The aim of this study was to analyse the spatial distribution of MCs and microvessels in the normal foetal and adult thymus and thymoma. Twenty biopsy specimens of human thymus, including foetal and adult normal thymus and thymoma were analysed. Double staining with CD34 and mast cell tryptase was used to count both mast cells and microvessels in the same fields. Computer-assisted image analysis was performed to characterize the spatial distribution of MCs and blood vessels in selected specimens. Results demonstrated that MCs were localized exclusively to the medulla. Their number was significantly higher in thymoma specimens as compared with adult and foetal normal specimens respectively. In contrast the microvessel area was unchanged. The analysis of the spatial distribution and relationship between MCs and microvessels revealed that only in the thymoma specimens was there a significant spatial association between MCs and microvessels. Overall, these data suggest that MCs do not contribute significantly to the development of the vascular network in foetal and adult thymus, whereas in thymoma they show a close relationship to blood vessels. This could be an expression of their involvement not only in endothelial cells but also in tumour cell proliferation.     

Platelet-derived growth factor and platelet-derived growth factor receptor-α expression in the normal human thymus and thymoma

Platelet-derived growth factor (PDGF) and its receptors (PDGFRs) are strongly involved in the normal development of several organs, tumour angiogenesis and malignant progression and metastasis. Few studies concerning their expression, distribution and role in normal and pathological human thymus are available in the literature. The aim of this study has been to analyse the immunohistochemical expression of PDGF and PDGFR-α in prenatal and postnatal normal human thymus and thymomal biopsy specimens. The results demonstrated immunoreactivity to both PDGF and PDGFR-α in all specimens, but the intensity, distribution and number of positive cells were different in normal thymus and thymomas, and also among different tumour types. PDGF and PDGFR-α were weakly expressed in foetal and postnatal humans with a different distribution between cortex and medulla in both blood vessels and epithelial cells, whereas they were overexpressed in thymoma, especially in type B2 and B3, in the tumour epithelial cells. Overall, these data suggest that PDGF and PDGFR-α may be involved in the pathophysiology of the human thymus.     

Immunopathological study related to myoglobin in myasthenic and non-myasthenic thymuses

Twelve biopsied thymuses taken from 4 cases with myasthenia gravis (MG group) and 8 cases without myasthenia gravis (control group) including 2 thymoma cases in each group were immunopathologically investigated in relation to myoglobin (Mb). Mb positive cells of various degrees were detected in all thymuses of both groups, and immunoelectron microscopical examination disclosed that Mb positive cells corresponded to interdigitating reticulum cells and myoid cells in non-neoplastic thymuses, and neoplastic epithelial reticular cells in thymomas. Anti-Mb antibody staining by direct immunoperoxidase technique revealed positive localization to the lymphoid cells in the thymuses of 2 cases of MG group with thymoma. In addition, indirect immunofluorescent study with the serum of each case which was applied to the normal human skeletal muscle, showed positive staining of the sarcoplasm in 3 cases of MG group, including 2 thymoma cases, and using peroxidase labeled serum IgG F (ab')2 of the same patients this anti-muscle antibodies were proved to be against both postsynaptic cytosol and sarcoplasm of the extraocular muscle of the guinea pig. From these results, it was suggested that Mb may conduct itself as a homologous antigen between the thymus and the skeletal muscle in the myasthenic patient with thymoma, and in the thymus the interdigitating reticulum cell, the myoid cell, or the neoplastic epithelial reticular cell may retain or produce Mb as an antigen-presenting cell.     

IMMUNOPATHOLOGICAL STUDY RELATED TO MYOGLOBIN IN MYASTHENIC AND NON-MYASTHENIC THYMUSES

Twelve biopsied thymuses taken from 4 cases with myasthenia gravis (MG group) and 8 cases without myasthenia gravis (control group) including 2 thymoma cases in each group were immunopathologically investgated in relation to myoglobin (Mb). Mb positive cells of various degrees were detected in all thymuses of both groups, and immunoelectron microscopical examination disclosed that Mb positive cells corresponded to interdigitating reticulum cells and myoid cells in non-neoplastic thymuses, and neoplastic epithelial reticular cells in thymomas. Anti-Mb antibody staining by direct immunoperoxidase technique revealed positive localization to the lymphoid cells in the thymuses of 2 cases of MG group with thymoma. In addition, indirect immunofluorescent study with the serum of each case which was applied to the normal human skeletal muscle, showed positive staining of the sarcoplasm in 3 cases of MG group, including 2 thymoma cases, and using peroxidase labeled serum IgG F(ab')₂ of the same patients this anti-muscle antibodies were proved to be against both postsynaptic cytosol and sarcoplasm of the extraocular muscle of the guinea pig. From these results, it was suggested that Mb may conduct itself as a homologous antigen between the thymus and the skeletal muscle in the myasthenic patient with thymoma, and in the thymus the interdigitating reticulum cell, the myoid cell, or the neoplastic epithelial reticular cell may retain or produce Mb as an antigen-presenting cell.     

Differential expression of LAMPs and ubiquitin in human thymus

Lysosome‐associated membrane proteins 1 and 2 (LAMP‐1 and LAMP‐2) are implicated in a variety of normal and pathological processes. LAMP‐2 is proposed to participate in chaperone‐mediated autophagy. Autophagy regulates T‐lymphocyte homeostasis by promoting both survival and proliferation. The biological importance of this process in the thymic gland and especially the involvement of LAMPs are far from being elucidated. The aim of the study was to examine the parallel expression of LAMPs and ubiquitin, a key molecule in autophagy, in normal human thymic glands and thymomas. The immunohistochemical expression of both markers was compared with that of cyclin D1 – an important regulator of cell cycle progression. Novel evidence for differential expression of LAMPs and ubiquitin is presented. Most Hassal's corpuscules in thymoma were negative for LAMPs, but positive in normal thymus. Both lymphocytes and epithelial cells in pathological thymus showed higher intensity for LAMP‐2 compared with LAMP‐1. In thymoma, ubiquitin was more intensively positive in these cell types compared with the normal thymus, suggesting activated autophagy in the course of this pathological state. A deregulation in cyclin D1 expression in thymoma is also reported. The functional importance of these molecules in autoghagy accompanying normal and pathological processes in the thymic gland is reviewed.     

Human thymoma lymphocyte mitogenesis: glucocorticoid inhibitory capacity as a function of the size of the more mature T cell subset

The relationship between the expression of T3 and T6 antigens and the capacity to respond to phytohaemagglutinin (PHA) has been studied in the lymphocyte component of nine human thymomas. It appears that there is a positive correlation between the mitogen responsiveness of thymoma lymphocytes and the relative proportion of T3 positive (T3+), T6 negative (T6-), peanut receptor negative and IgM-Fc receptor positive cells. Moreover we have comparatively investigated the dexamethasone (Dex) inhibitory effect on the mitogenesis of T lymphocytes from thymoma, normal thymus and peripheral blood. In the presence of the macrophage product interleukin 1, the capacity of Dex to inhibit the mitogenesis of peripheral blood purified T cells (PBT) is inversely correlated with the PHA concentration used. Conversely, Dex completely (greater than 90%) inhibits thymocyte mitogenesis irrespective of PHA concentration. The entity of Dex inhibitory effect on thymoma lymphocyte mitogenesis shows a great variability ranging between that observed in normal thymocytes and that observed in PBT. In each thymoma case the degree of inhibition appears to be dependent on the size of the more mature thymocyte pool being positively correlated with the number of T6+ but negatively with that of T3+ cells. Our data demonstrate a high degree of variability among thymomas relative to the phenotypic and functional properties of their lymphocytic component. On the other hand, in some thymomas the phenotypic and functional characteristics of lymphocyte component seem to represent only a narrow span of normal thymocyte maturative pathway. In this respect, human thymoma may constitute a profitable tool for studying the intrathymic T lymphocyte maturative steps.     

Immunohistochemical expression of vascular endothelial growth factor A (VEGF), and its receptors (VEGFR1, 2) in normal and pathologic conditions of the human thymus.

Vascular endothelial growth factor A (VEGF-A) is an angiogenic growth factor that is a primary stimulant of the vascularization of solid tumors. In the tumor microenvironment, an upregulation of both VEGF and its receptors occurs, leading to a high concentration of occupied receptors on tumor vascular endothelium. Also, VEGF is involved in the development of the normal vascular network of the thymus. Little is known about VEGF expression in normal and malignant thymic tissue. Our purpose was to study the pattern and localization of VEGF expression in benign conditions of the thymus and thymoma to determine a possible correlation with VEGF receptors VEGFR1, VEGFR2 and microvascular density. All cases were positive for VEGF and VEGFR1, 2 in the epithelial cells, in a cytoplasmic, granular pattern. In the normal thymus, there were positive epithelial cells with subcapsular distribution and Hassall's corpuscle epithelial cells. In acute thymic involution, the positive fields were correlated with dilation and stasis of blood vessels and lymphocyte depletion. Rare positive cells were found in other types of involution; the myasthenic thymus showed an intense and diffuse reaction in lymphoid follicles of the medulla. A strong reaction for VEGF was observed in type B3 thymomas in neoplastic epithelial cells, normal endothelial cells, plasma within the blood vessels and focally in the stroma adjacent to the tumor. Receptors for VEGF were positive in neoplastic epithelial cells and endothelium. We hypothesized that VEGF acts as an immunoregulatory factor in the normal thymus and as proangiogenic and autocrine factor in thymomas.     

Expression of p63 in thymomas and normal thymus

The p63 gene, a member of the p53 family, is an epithelial marker expressed in embryonic ectoderm, breast myoepithelium, prostate, oral epithelium, epidermis, and urothelium. The DeltaN-p63 isoforms of p63, which are believed to behave as oncogenes, are expressed in squamous cell carcinoma, basal cell carcinoma, and transitional cell carcinoma. Only a few authors have looked for p63 expression in thymomas and normal thymus. We, therefore, thought of undergoing such a search by taking advantage of our archival material. We studied 66 cases of thymoma (1 type A, 8 type AB, 12 type B1, 19 type B2, 12 type B3, and 14 type C/thymic carcinoma) and 10 specimens of normal human thymus arranged in tissue microarrays. All thymomas (including thymic carcinomas) were positive for p63 regardless of type. Most of the epithelial cells of the normal thymus were also positive for this marker.     

Demonstration of phenotypic abnormalities of thymic epithelium in thymoma including two cases with abundant Langerhans cells.

A panel of monoclonal antibodies that phenotypically define stages of normal human thymic epithelial (TE) cell maturation was used to compare thymic epithelium of nine thymomas with hyperplastic thymic epithelium in myasthenia gravis (MG) and thymic epithelium of normal thymuses. It has been shown previously that normal thymic epithelial cells express antigens of early TE cell maturation (A2B5, TE-4) throughout thymic ontogeny and acquire antigens 12/1-2, TE8, and TE-15 at 14 to 16 weeks of fetal gestation. Hyperplastic MG thymic epithelial cells expressed TE antigens in phenotypic patterns similar to that seen in normal postnatal thymus, ie, TE in subcapsular cortex and medulla was TE4+, A2B5+, and 12/1 - 2+ and Hassall's bodies were reactive with antibodies TE8 and TE15. In contrast, thymic epithelium in primary mediastinal thymomas was TE4+, A2B5+, TE8-, and greater than 75% of thymoma epithelium was 12/1 - 2-, a thymic epithelial phenotype similar to that seen on normal fetal thymic epithelium at 14 to 16 weeks fetal gestation. In one subject with a mature epithelial histologic pattern, thymoma epithelium was found to be strongly TE8+, a phenotype suggestive of a later stage of TE maturation. Lymphocytes in five of seven thymomas with immature thymic epithelial cells predominantly expressed immature thymocyte phenotype while two thymomas with immature epithelial phenotype showed a predominance of Langerhans cells and surrounding lymphocytes expressing a mature phenotype. Lymphocytes in the thymoma with differentiated epithelial cells expressed a mature thymocyte phenotype. Thus, in thymomas of varying histologic types, phenotypic abnormalities of thymic epithelium are present; these phenotypic abnormalities may reflect abnormal thymic epithelial maturation.     

Adenine nucleotide content of thymoma-derived lymphocytes.

Purine and pyrimidine metabolites are essential substances for cells. We have measured the adenine nucleotide (AN) contents of thymocytes from 15 human thymomas, 11 adjacent non-neoplastic thymuses and 3 children's thymuses. There was no significant difference in AN content of thymocytes between thymoma and children's thymus. But, AN content of adjacent non-neoplastic thymuses was significantly lower than that of thymoma or children's thymus. In mixed type thymoma, ATP content in thymocytes was significantly higher than that in lymphocytic type thymoma. These data indicate that thymocytes in thymoma may show further T cell maturation probably associated with the functional microenvironment of the neoplastic epithelial cells just like the children's thymus.     

Acetylcholine receptor alpha-subunit isoforms are differentially expressed in thymuses from myasthenic patients.

A central role of the thymus in autosensitization to the acetylcholine receptor has been proposed to explain the immunopathogenetic processes in myasthenia gravis (MG). Two isoforms of the alpha-subunit of the acetylcholine receptor are known; they differ by a 25-amino-acid insertion coded by the P3A exon. We investigated the expression of the P3A exon by RNA polymerase chain reaction in fetal and adult human myoblasts and TE671 cells; both isoforms were expressed. Muscle biopsies from patients with MG, Duchenne muscular dystrophy, and polymyositis were also studied and it was again found that both isoforms were expressed, indicating that the P3A exon is not associated with autoimmune, degenerative, and inflammatory muscle diseases. When P3A expression was studied in thymus samples from normal subjects and from thymectomized MG patients, the P3A+ subunit was absent in 75% of patients with involuted thymus and in all patients with thymomas but was present in normal thymuses and in patients with hyperplasia. Differential expression of the alpha-subunit isoforms of the acetylcholine receptor within the thymus may play a role in the immune pathogenesis of MG.     

Thymomatous epithelial cells and skeletal muscle share a common epitope defined by a monoclonal antibody.

Monoclonal antibodies have been raised against thymomatous epithelial cells with the use of fragments of a human thymoma as source of antigen. These monoclonals, which do not react with cultured epithelial cells or sections from normal thymus (except for some cells of Hassall's corpuscles), label a large number of cells in thymoma sections. In addition, they recognize cross-striations from skeletal muscle cells. Immunoblotting studies reveal that the proteins recognized on thymoma and muscle are in the same molecular weight range, suggesting that these proteins, which share a common epitope, are identical. These findings indicate that the production of circulating anti-cross-striational autoantibodies observed in most patients with thymoma could derive from immunization against a protein abnormally expressed by neoplastic epithelial cells.     

Thymoma with abundant L26-positive 'asteroid' cells. A case report with an analysis of normal thymus and thymoma specimens.

A case of thymoma is presented that was referred for consultation with the differential diagnosis of thymoma and non-Hodgkin's lymphoma. Immunoperoxidase studies performed on fixed, paraffin-embedded sections demonstrated the presence of numerous epithelial cells, supporting the diagnosis of thymoma. However, the pan-B-cell antibody L26 also demonstrated abundant staining, an unexpected finding that may be a potential source of diagnostic confusion. The L26 antibody stained cells with elongate cell processes that interdigitated between and surrounded thymocytes. We pursued this observation by performing immunoperoxidase studies on three thymoma and seven normal thymus specimens using fixed sections. Each thymoma had occasional cells or small clusters of L26-positive cells scattered throughout the neoplasm. In sections of normal thymus, L26-positive cells were also found, almost exclusively in the medullary regions. These cells tended to congregate around Hassall's corpuscles and had elongate cell processes that often surrounded medullary lymphocytes. Occasional small lymphocytes also appeared to be positive for L26. Our results demonstrate that cell populations that express B-cell antigens are consistently found in the thymic medulla and that these cells may be numerous in occasional thymomas. The presence of many L26-positive cells in a mediastinal mass should not dissuade one from making the diagnosis of thymoma if all other findings are consistent with that interpretation.     

凋亡基因bcl-2在胸腺增生性疾病中的表达

目的：探讨bcl-2的表达与胸腺增生性疾病发生的关系。方法：33例胸腺增生性疾病组织中，28例胸腺瘤按M-H标准分类，并与Salyer标准作一比较。采用微波处理二步法进行bcl-2的免疫组化染色。结果：bcl-2主要在胸腺的上皮细胞成分中表达。髓质型胸腺瘤中bcl-2的阳性率高，其余胸腺瘤中阳性表达率则较低或阴性。在重症肌无力胸腺的髓质中bcl-2的表达远高于对照组胸腺。结论：bcl-2在髓质型胸腺瘤上皮中的强表达可作为区分髓质型胸腺瘤和皮质型胸腺瘤的一个手段。过表达的bcl-2可能与MG胸腺中修复的凋亡细胞的死亡有关。     

An attempt to produce “pre-T” cell hybridomas and to identify their antigens

With the aim of identifying some of the stages in the development of pre-T cells (cells of the T cell lineage before they enter the thymus), we have produced a large number of hybridomas by the fusion of BALB/c bone marrow cells, bone marrow cells from BALB/c-nu/nu mice, BALB/c fetal liver cells and BALB/c fetal thymocytes with the AKR thymoma BW5147. The hybridomas were selected for the expression of the Thy-1.2 antigen of th3 normal celldonor and for their ability t9produce interleukin 2 (IL 2) upon co-culture with irradiated normal spleen cells. A set of these hybridomas is described in this communication. The hybridomas were then used to immunize rats and to generate monoclonal antibody-producing B cell hybridomas. Most, if not all, of the immunizing hybridomas were derived from pre-T cells as evidenced by the fact that they produce IL 2, and express some of the T cell markers (the Thy-1.2, Ly-1, Ly-2 or L3T4 antigens). The monoclonal antibodies were tested on a panel of pre-T cell hybridomas and on normal cells obtained from spleen, lymph nodes, thymus and bone marrow. The testing was carried out by the microcytotoxicity assay and flow cytometric analysis. Three groups of antibodies could be distinguished. Some antibodies were broadly reactive, being positive with virtually all the clones in the pre-T cell panel and with a substantial fraction of normal lymphoid cells. The identity of the antigens detected by these antibodies remains unknown but they do not seem to correspond to any of the known cell surface markers. Other antibodies reacted only with some of the pre-T cell clones and did not react at all with normal lymphoid cells obtained from adult animals. Finally, other antibodies still reacted only with a minor subpopulation of thymocytes or of thymocytes and bone marrow cells, as well as some of the pre-T cell clones; they did not react with spleen and lymph node cells. These antibodies might be specific for cells in the prethymic phase of the T cell differentiation pathway. They should prove useful for the identification of pre-T cell markers and hence for the isolation of pre-T cells and their functional analysis.     

成年大鼠最后区神经干细胞的检测及鉴定

目的 观察性别决定基因高迁移率组蛋白( SOX2)和巢蛋白(Nestin)阳性表达细胞及溴脱氧尿密啶核苷（BrdU）阳性标记细胞在最后区的分布。方法 成年雄性SD大鼠12只,6~8周龄,随机分为两组,每组6只。一组大鼠按照50mg/kg（0.3ml）腹腔注射BrdU,连续3d给药,每天2次;另一组注射等量生理盐水。4d后灌注大鼠,行免疫组织化学及免疫荧光检测。 结果 免疫组织化学染色显示,SOX2阳性表达细胞在最后区的腹侧部呈明显的V字形分布,背侧部呈带状分布,中央部散在分布。Nestin阳性表达细胞在最后区的腹侧部呈明显的V字形强阳性分布,中央部呈弱阳性表达。在最后区可见少量阳性BrdU标记细胞。SOX2/BrdU荧光双标染色显示,SOX2阳性表达细胞较密集分布,可见少量SOX2/BrdU双阳性细胞。SOX2/Nestin荧光双标染色显示,SOX2阳性表达细胞较密集分布,可见少量SOX2/Nestin双阳性表达细胞。结论 最后区SOX2及Nestin阳性细胞密集表达,呈明显的区域分布;在最后区内存在少量BrdU阳性标记细胞及SOX2/BrdU和SOX2/Nestin双阳性细胞;最后区可能存在神经干细胞/祖细胞。     

T-cell development in human thymoma.

Human thymoma is derived from thymic epithelial cells and often associated with a large number of cortical thymocytes. Since thymic epithelial cells play key roles in T-cell development in the normal thymus, we hypothesized that the neoplastic epithelial cells of thymoma may support T-cell differentiation. We attempted to reconstitute the T-cell development in vitro by using neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on a proportion of CD4-CD8- cells in thymoma. These CD34+CD4-CD8- cells also expressed both IL-7R alpha-chain and common gamma-chain. Purified CD4-CD8- cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4+CD8+ cells via CD4 single positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. The CD34+CD4-CD8- cells purified from a normal thymus also differentiated to CD4+CD8+ cells in an allogeneic co-culture with the neoplastic epithelial cells of thymoma. In addition, a pleural dissemination from thymoma contained a large amount of cortical thymocytes. These results suggest that the neoplastic epithelial cells retain the function of thymic epithelium and can support T-cell development in thymomas.     

Immunohistological evidences of cortical and medullary differentiation in thymoma

Summary The phenotypical characteristics of human epithelial and lymphoid cells have been studied with immunohistochemical methods on frozen sections of 12 thymomas. On the basis of the cytohistological characteristics of thymoma epithelial cells (EC) the thymomas were divided in cortical, medullary and mixed types, according to recently developed light microscopical criteria. When tested with a series of monoclonal antibodies, thymoma EC were all stained by the antibody Ki-M3 (as in the thymus), but reacted with anti-HLA-DR, anti-HLA-A,B,C and with a new monoclonal antibody to cortical EC,21A6, to a lesser extent and with weaker, variable intensity in comparison with the normal thymus. Cortical type thymomas were most reactive and the medullary type almost negative. Thymomas, like normal thymus showed different immunoreactivity patterns with antibodies to prekeratins of different specificities. Cortical type thymomas and areas in mixed thymoma showed an EC staining with the antibody to non-squamous type keratin (35H11) whereas medullary type thymomas and areas showed staining with antibodies to squamous-type keratin (34E12-IV/82) in addition. Lymphoidcellswithcortical(OKT6+,Leu 1 weakly+,Leu2a+,Leu3a+) or mature medullary (OKT6-, Leu 1 strongly+, Leu 2a or Leu 3a+) phenotype were found to colonize tumours with diferent EC types. These immunohistochemical findings largely confirm our earlier cytological distinction of thymoma EC. In addition important differences have been observed in neoplastic cortical EC concerning the HLA-DR and 21A6 immunoreactivity that may be intimately related to the neoplastic process and paraneoplastic immune phenomena.This work has been supported by the Deutsche Forschungsgemeinschaft, SFB 111, project CN5     

Lymphocyte subpopulation in neoplastic and non-neoplastic thymus and in blood of patients with Myasthenia gravis.

The lymphocyte subpopulations in the thymus and in the blood were investigated in ten myasthenic patients who had been thymectomized. Histologically, the thymuses tested comprised three cases of thymoma including two cases with malignant characteristics, five cases of hyperplastic thymus with lymph follicles and germinal centres, and two cases of persistent thymus without lymph follicles. Virtually all lymphoid cells in the three thymomas formed spontaneous rosettes with sheep red blood cells as did normal thymocytes from non-myasthenic patients. There was no significant proportion of immunoglobulin Ig-bearing lymphocytes. While the majority consisted of cells forming spontaneous rosettes with sheep red blood cells, there was a certain proportion (2-17%) of Ig-bearing lymphocytes in four of five hyperplastic thymuses, in one of two persistent thymuses, and in a residual atrophic thymus of a thymoma. The myasthenic patients tested were for the most part normal, as compared with healthy individuals, in the proportion of rosette-forming lymphocytes and Ig-bearing lymphocytes in the blood collected immediately before and one to three months after thymectomy. The presence of Ig-bearing lymphocytes in the thymus was not necessarily related to the appearance of circulating antibody to striated muscle. The antibody to striated muscle was demonstrated in all myasthenic patients with thymoma.