HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Salakou, S. Articles by Dougenis, D. Search for Related Content PubMed PubMed Citation Articles by Salakou, S. Articles by Dougenis, D. Related Collections Mediastinum Molecular biology

Eur J Cardiothorac Surg 2001;20:712-721
© 2001 Elsevier Science NL

The potential role of bcl-2, bax, and Ki67 expression in thymus of patients with myasthenia gravis, and their correlation with clinicopathologic parameters

^a Department of Neurology, University of Patras, School of Medicine, Patras, Greece
^b Department of Pathology, University of Patras, School of Medicine, Patras, Greece
^c Department of Cardiothoracic Surgery, University of Patras, School of Medicine, Patras, Greece

Received 9 October 2000; received in revised form 12 April 2001; accepted 27 April 2001.

Corresponding author. Tel.: +3061-999299; fax: +3061-993984
e-mail: ddougen{at}med.upatras.gr

	Abstract

Top Abstract 1. Introduction 2. Material and methods 3. Results 4. Discussion References

 
Objective: The aim of this study was to evaluate bcl-2, bax (apoptotic-oncoproteins), and Ki67 (cell proliferation-marker) expression in thymus of patients with myasthenia gravis (MG) and to determine the potential correlation with clinicopathologic parameters. Methods: The study was done on 38 patients (16 males, 22 females; mean age 38±10 years) with MG who underwent modified maximal thymectomy (MMT). Clinical staging (Osserman classification) included stage I in three, IIA in 19, IIB in 13 and III in three. Microscopic examination of thymus showed thymic hyperplasia in 19, atrophy in eight, thymoma in nine and thymic carcinoma in two. On paraffin sections, the streptavidin–biotin technique, using antibodies to bcl-2, bax, and Ki67, was employed, and in situ hybridization with digoxigenin-labeled probes to bcl-2 and bax was performed. In addition, the apoptotic body index (ABI) was assessed via the TUNEL method. Staining results were correlated with clinicopathologic parameters. Results: Bcl-2 expression was higher in hyperplasia and thymoma cases, compared to thymic carcinomas (P<0.001). Higher expression in carcinomas, compared to hyperplasia and thymomas, was observed for bax (P<0.001), Ki67 (P<0.001) and ABI (P<0.001). Statistical analysis demonstrated: (A) positive correlation of bax(+) cells with MG stage (P<0.001), ABI and %Ki67(+) cells with MG stage (P<0.001, respectively), %Ki67(+) and %bax(+) cells with ABI (P<0.05); and (B) reverse correlation between %bcl-2(+) cells and MG stage (P<0.05). Conclusions: In patients with MG who underwent MMT, bcl-2, bax, and Ki67 expression correlates positively or reversibly with the microscopic findings of thymus. Increased apoptosis and proliferation accompany advanced disease stage and possible worse prognosis.

Key Words: Myasthenia gravis • Thymectomy • Apoptosis • Gene expression • Bcl-2 • Bax • Ki67 • Prognosis

	1. Introduction

Top Abstract 1. Introduction 2. Material and methods 3. Results 4. Discussion References

 
Myasthenia gravis (MG) is a term that refers to an autoimmune disorder of neuromuscular transmission, which is characterized by increase in autoantibodies directed against the nicotinic acetylcholine receptor [1]. MG is associated with thymic hyperplasia in 65% and thymoma in 10% of cases. Thymomas are associated with the presence of MG in 30–45% of the cases [2]. It has been reported that development of MG in patients with thymomas is probably a result of sensitization of developing T cells against autologous muscle-like antigens, perhaps including the acetylcholine receptor, expressed by the neoplastic epithelia [3]. Among several therapeutic modalities employed in the treatment of MG, thymectomy is the most efficient one in the integrated management of the disease [4–6].

Today it is accepted that the development of non-neoplastic (hyperplastic) and neoplastic abnormalities of the organs result from alterations of genetic actions on normal cell growth and differentiation. Proto-oncogenes and suppressor genes host the most common specific gene changes. In the past years, disregulation of growth was explained largely in terms of increased cell population. One of the commonly used methods for estimating cell proliferating activity is the identification of proliferating cells using immunohistochemical detection of the Ki67 antigen [7]. This antigen recognizes an epitope on a nuclear antigen in cell cycling and labels cells that are in phase S of the cell cycle. The proportion of Ki67-labeled cells in a given cell population (Ki67-index) provides a measure of the growth fraction [7]. In patients with thymomas with or without associated MG, Ki67 shows enhanced expression and is correlated with thymus size [3].

However, tissue growth (especially during tumor development) is not only a result of cell proliferation but also of enhanced cell survival (through inhibition of apoptosis) or it results from combination of both mechanisms [8]. Apoptosis is a morphologically distinct, gene-directed form of cell death characterized by cytoplasmic fragmentation and nuclear condensation that contributes to both physiological and pathological processes [9]. Previous studies reported that apoptosis plays a critical role in T-cell development within thymus and probably is the mechanism that underlies deletion of autoreactive T-cell precursors [10]. Furthermore, a high proportion of thymocytes undergo apoptosis and they are cleared by thymic macrophages [11].

Bcl-2 is a gene located at chromosome 18q21 and encodes a 26-kD protein localized mainly in the mitochondrial membrane, but also in the nuclear envelope, perinuclear membrane, and endoplasmic reticulum, and blocks programmed cell death without affecting cellular proliferation [12]. It was first described in studies regarding t chromosome translocation in B-cell leukemia [13] and later on, it was detected in other malignant tumors such as breast and non-small cell lung carcinomas where it has been associated with a more favorable clinical outcome [14]. In the normal thymus, bcl-2 has been localized mainly in medullary and less in cortex thymocytes but not in epithelial cells [15]. In the diseased thymus and especially in patients with thymomas, with or without accompanied MG, bcl-2 is expressed in thymocytes but not in neoplastic cells [3]. In addition, an upregulation of bcl-2 oncoprotein has been described in myasthenic thymus [15].

Bax protein is a homologue of bcl-2 that promotes apoptosis [16]. Bax may bind to bcl-2, forming bax/bcl-2 heterodimers, or may bind to itself forming bax/bax homodimers [16]. The ratio of bax to bcl-2 determines the susceptibility of a cell to apoptosis. Thus, in cells with bax overexpression, bax homodimers predominate, and the susceptibility of such cells to apoptotic stimuli is enhanced. Bax/bcl-2 heterodimers predominate in cells that overexpress bcl-2, and the susceptibility of these cells to apoptosis is reduced [16,17]. Bax mRNA appears to be present in a variety of tissues, including lung, stomach, kidney, and thymus [16], whereas bax protein has been detected by immunohistochemistry in mouse and human tissues. In the thymus, bax is located in the thymic medulla and much of its expression is associated with the thymic epithelial cells and less with the medullary or cortical thymocytes [18,19]. To the best of our knowledge, bax expression in the thymus of patients with MG has not been investigated.

The aim of this study was to evaluate bcl-2 and bax oncoproteins and Ki67 antigen expressions in the thymus of patients subjected to thymectomy for MG and to determine the potential correlation with clinicopathologic parameters.

	2. Material and methods

Top Abstract 1. Introduction 2. Material and methods 3. Results 4. Discussion References

 
2.1. Patients
The study included 38 patients (16 males and 22 females aged 16–82 (mean 38±10) years) who underwent modified maximal thymectomy (MMT) for MG and five controls. Diagnosis of MG was confirmed on clinical and electromyography grounds. Preoperative investigation included, among others, computed tomography of the chest in all and magnetic resonance imaging of the chest in six patients. Table 1 shows the clinical, and histological features of all patients at the time of the thymectomy. Concomitant total thyroidectomy was performed in three patients for Hashimoto's thyroiditis (n=2) and cystic thyroid goiter (n=1). For each patient, the slides and pathology report were reviewed to confirm the pathologic diagnosis. Original histologic diagnoses of the 38 thymectomy specimens included thymic hyperplasia in 19, thymic atrophy in eight, thymoma in nine and well-differentiated thymic carcinoma in two. All nine thymomas belonged to the predominantly cortical type according to M-H classification [20]. Thymic tissues obtained from five aborted embryos (aged 28–30 weeks) were used as controls as mentioned previously [3].

A full median sternotomy is used. After entering the mediastinum, the left branchiocephalic vein is carefully identified and mobilized free using diathermy, and the thymic veins, occasionally including the internal mammary vein branches, are ligated and divided. Subsequently, all the soft perithymic tissue and mediastinal, pericardial, and cervical fat are en block resected with the thymus, including both mediastinal pleuras. Particular care is taken while dissecting the fat tissue at the aortopulmonary window, where the possibility of damaging the left phrenic nerve and the recurrent laryngeal nerve is increased. In the presence of a thymoma, a similar extended resection is carried out including the involved lung parenchyma and/or pericardium, as well as the phrenic nerve and branchiocephalic vein when implicated. Before closing, two drains are inserted, draining both pleural cavities and mediastinum.

2.3. Immunohistochemistry
Immunohistochemistry for the detection of bcl-2 and bax proteins and Ki67 antigen was performed on 4-µm-thick paraffin sections from one selected block from each case. After standard deparaffinization, hydration, and blocking of endogenous peroxidase, sections were processed in a microwave oven twice (5 min each) at high power. Subsequently, a standard streptavidin–biotin-peroxidase technique was applied to detect the antigens. Sections were incubated with anti-bcl-2 (dilution 1:40, DAKO, USA), anti-bax (dilution 1:1500, SantaCruz, UK) and anti-Ki67 (dilution 1:40, DAKO, USA) for 30 min at room temperature. Diaminobenzidine (Sigma Fast DAB) was used as the chromogen. As positive controls, we used sections from human tonsils. For negative control purposes, the same streptavidin–biotin technique was used in tissue sections where 1% BSA in PBS replaced the primary antibody. Cytoplasmic staining for bcl-2 and bax and nuclear staining for Ki67 were considered as positive.

2.4. In situ labeling of fragmented DNA for the detection of apoptotic cells
On paraffin sections, a standard TUNEL method was employed to detect the fragmented nuclear DNA associated with apoptosis. For this purpose, the in situ cell death detection kit, POD (Roche, USA), was used according to the manufacturer's instructions. After standard deparaffinization, hydration, incubation with proteinase K, and blocking of endogenous peroxidase, tissue sections (4 µm thick) were incubated: (a) with terminal deoxynucleotidyl transferase (TdT) and digoxigenin-dUTP (DIG-dUTP) (TUNEL reaction mixture) at 37°C for 60 min; and (b) with peroxidase converter anti-fluorescein antibody at 37°C for 30 min. Diaminobenzidine (Sigma Fast DAB) was used as the chromogen. For physiological positive controls, sections of rat small intestine were subjected to the same procedure. For negative controls, some slides were incubated with label solution that did not contain TdT.

2.5. In situ hybridization for bcl-2 and bax mRNA detection in paraffin sections
For the detection of bcl-2 and bax mRNA, a standard non-radioactive in situ hybridization method (ISH) was performed using the hybridization/detection complete system (MBI, USA). Briefly, 4-µm-thick sections were dewaxed, hydrated, incubated with 1x proteinase K solution for 30 min at 37°C, dehydrated, and dried at 37°C for 5 min. Thereafter, sections were incubated with DIG-labeled riboprobes (for bcl-2 and bax, in a 10-fold dilution, MBI, CA, USA) at 70°C for 10 min (RNA secondary structure denaturation) and subsequently at 37°C for 4 h to complete hybridization. Thereafter, the specific protocol (hybridization/detection complete system (MBI, USA)) was performed according to the manufacturer's instructions. Nuclear Fast Red (DAKO, USA) was used as the chromogen. Cytoplasmic staining for bcl-2 and bax was considered as positive. For positive control tissues, we used human tonsils. To confirm that positive stain was specific, slides were processed identically and hybridized with probes known to be complementary to sequences in the test sections; these were similar in length and GC content to bcl-2 and bax probes. For negative control purposes, the slides were processed in the same way but hybridized with heterologous probes, which were not complementary to any sequence in the test tissues. These negative control probes were similar in length and GC content to bcl-2 and bax probes.

2.6. Morphometric analysis
Quantitative analysis of the %cells that displayed (i) positive immunohistochemical stain for bcl-2 and bax proteins and Ki67 antigen, (ii) positive ISH stain for bcl-2 and bax mRNA, and (iii) positive TUNEL reaction for fragmented DNA was performed in a way similar to that described earlier [15]. All tissue sections were scanned under low power and areas with positive stain were chosen. Thereafter, a 10x10 microscope grid at 400x magnification was applied on the sections and both the number of positive stained cells and the total number of cells (at least 500) was determined by visual inspection of six different fields per section. Calculations were made separately for the thymic medulla and cortex (three fields for each). For each field, a %value for each marker was obtained by dividing the positive stained cells by the total number of cells included in the grid. It should be noted that the variance in cell counts from field to field in the same section was <10%. The average of these scores was then taken. The %TUNEL⁺ cells gave the apoptotic body index (ABI) for each case. The sections were scored independently by two of the authors (S.S., A.C.T.) blinded to the clinical profile of the patients. When major discrepancies occurred, a consensus score was reached.

2.7. Statistical analysis
Results were reported as mean±SD, median, and range. Intergroup comparisons – with regard to correlation of thymus pathology and MG stage with staining results – were performed using one-way analysis of variance (ANOVA). When the equal variance test or normality test failed, the Kruskall–Wallis non-parametric test was applied. In order to address the problem of multiple comparisons, these tests (ANOVA, Kruskall–Wallis) were followed by a posthoc Bonferroni test. Spearman's rank correlation was used to examine the possible associations between the immunohistochemical or ISH results for bcl-2 and bax, the immunohistochemical results for Ki67, and the results for TUNEL stain, on the one hand, and the thymus weight and size, on the other. Spearman's rank correlation was also used to detect any relationships between bcl-2, bax, Ki67, and ABI. Data were analyzed using the SigmaStat (Jandel Scientific, USA). Significance was defined as P<0.05.

	3. Results

Top Abstract 1. Introduction 2. Material and methods 3. Results 4. Discussion References

 
3.1. Early patients' outcome
Thirty-three patients were extubated within hours following surgery, while five underwent elective overnight ventilation. There were no deaths. Morbidity included four patients who developed postoperative respiratory complications, three of whom required assisted mechanical ventilation and one plasmapheresis in addition, one patient with transient laryngeal nerve injury, and one with deep venous thrombosis. Only two patients were transfused, both having invasive thymomas that needed extensive resection and reconstruction of the branchiocephalic vein and/or superior vena cava. At discharge, five patients were free of pyridostigmine or prendisone.

3.2. Bcl-2, bax, and Ki67expression
3.2.1. Bcl-2
All the results for bcl-2 are summarized in Table 2. Immunohistochemistry for bcl-2 protein and ISH for bcl-2 mRNA displayed a similar staining pattern. In normal control cases, bcl-2 was mainly detected in the medulla and less in the cortex (Fig. 1A ). Epithelial cells were negative and germinal centers contained few bcl-2(+) cells. These results are in agreement with those of a previous study [15]. In cases with MG that accompanied hyperplasia and atrophy, bcl-2 oncoprotein and mRNA were expressed mainly in the thymic medulla. The pattern of the staining was cytoplasmic and perinuclear for the protein (Fig. 1B) and cytoplasmic for mRNA (Fig. 1D). Fewer bcl-2(+) cells were detected in the cortex. Germinal centers contained only scattered bcl-2(+) cells, whereas epithelial cells showed rare active production of bcl-2 (few cells positive for bcl-2 mRNA). In patients with MG associated with thymoma, bcl-2 protein and mRNA were expressed mainly in thymocytes and less in epithelial cells. In the surroundings of the tumor tissues, bcl-2 was present in a similar fashion to that described for hyperplastic and atrophic thymuses. The two cases of thymic carcinomas displayed fewer cells positive for bcl-2 protein (Fig. 1C) or bcl-2 mRNA. In these two instances also the surroundings of the tumor tissues showed similar bcl-2 expression to the instances of hyperplasia and atrophy. Bcl-2 expression in medulla was lower in cases of carcinoma compared to thymoma and hyperplasia (P<0.001). In addition, bcl-2 expression in medulla was higher in patients with MG compared to controls (P<0.001). Bcl-2 expression, mainly in the medulla (and less in the cortex), is correlated reversibly with MG stage (P<0.001) (Table 2). Spearman's rank correlation did not reveal any relationship between bcl-2 expression and thymus weight and size.

View larger version (138K): [in this window] [in a new window]   Fig. 1. (A) Bcl-2 expression in normal thymus. M, medulla; C, cortex (Strept–biotin 40x). (B) Numerous bcl-2-positive cells in a hyperplastic thymus (Strept–biotin 250x). (C) Few bcl-2-positive cells in a thymic carcinoma (arrows – Strept–biotin 400x). (D) Bcl-2 mRNA expression in a hyperplastic thymus (in situ hybridization 400x).

View larger version (149K): [in this window] [in a new window]   Fig. 2. (A) Bax expression in normal thymus. M, medulla; C, cortex (Strept–biotin 40x). (B) Few bax positive cells in a hyperplastic thymus (Strept–biotin 250x). (C) Numerous bax positive cells in a thymic carcinoma (arrows – Strept–biotin 400x). (D) Bax mRNA expression in a thymic carcinoma (in situ hybridization 400x).

View larger version (117K): [in this window] [in a new window]   Fig. 3. (A) Ki67 expression in a normal thymus. M, medulla; C, cortex (Strept–biotin 40x). (B) Ki67 expression in a hyperplastic thymus. M, medulla; C, cortex (Strept–biotin 40x). (C) Ki67 expression in a thymic carcinoma. Note the rare cytoplasmic expression (arrow – Strept–biotin 400x).

View larger version (169K): [in this window] [in a new window]   Fig. 4. (A) Apoptotic bodies in a hyperplastic thymus (TUNEL-peroxidase 100x). (B) Apoptotic bodies in a thymic carcinoma (TUNEL-peroxidase 400x).

	4. Discussion

Top Abstract 1. Introduction 2. Material and methods 3. Results 4. Discussion References

Bcl-2 oncogene inhibits apoptosis by affecting several cell pathways. Overexpression of bcl-2 can prevent or reduce cell death induced by a wide variety of stimuli [21]. Thus, this oncogene may mediate the mechanism that desensitizes thymocytes to apoptotic triggering. Previous studies have demonstrated that during the development of the thymus, bcl-2 is expressed mainly from precursor cells and mature thymocytes rather than intermediate thymocytes [22,23]. We found that bcl-2 expression was higher in patients with MG compared to control thymuses. In addition, bcl-2 was mainly present in the medulla rather than in the cortex. Our results are in agreement with previous reports [15]. We are tempted to speculate that these findings implicate bcl-2 for the impairment of autoreactive thymocyte apoptosis in patients with MG. Alternatively, the presence of bcl-2 mainly in the medulla could be related with the presence of inflammatory cells and thus the thymic medulla, in cases of MG, may mimic the inflammatory lymph nodes [24]. The heterogeneous bcl-2 expression observed confirms the broad spectrum of maturation that occurs in thymoma lymphocytes, as well as in normal thymocytes. Additionally, we found that the number of bcl-2-positive thymocytes in the medulla was significantly greater in the hyperplastic thymic tissue from MG patients compared to the control thymic tissue. Other studies have reported a significant increase in the percentage of CD4(+)/CD8(-) cells in the myasthenic thymus [25]. Therefore, upregulation of bcl-2 in the MG thymus might reflect an increase in mature-type thymic cells expressing bcl-2.

Despite the small number of thymic carcinomas evaluated in this study, bcl-2 protein or mRNA was more frequently expressed in cases of hyperplasia/atrophy and thymoma rather than carcinoma. Although the role of the bcl-2 gene in the development or progression of the thymomas accompanied by MG has not been yet elucidated, the significant loss of bcl-2 expression in the two cases of thymic carcinoma compared to thymoma and hyperplasia, may suggest that bcl-2 expression is an early event in oncogenesis in thymus. This implies that downregulation of the gene may play a role in thymic cell differentiation and turnover by committing them to apoptosis and suggests that abnormal expression of bcl-2 may lead to the accumulation of long-living cells and finally to tumor development. A deregulated expression of the bcl-2 gene in neoplastic cells may transform them and increase their resistance to apoptosis.

In our study, loss of bcl-2 expression was evident in advanced MG stages. A possible explanation is that bcl-2 prolongs cell survival of autoreactive thymocytes by inhibiting apoptosis and affects cellular growth at a slower rate compared to growth induced by oncogenes, which act via cell proliferation. All these observations (regarding the loss of bcl-2 expression in the thymic carcinomas or its gradual decrease as the severity of MG progresses) may favor an abnormal bcl-2 expression in the early stages of 0MG only. Alternatively, bcl-2 expression could be an epiphenomenon without any biological significance. These discrepancies could be explained by the cooperation of different oncogenes and tumor suppressor genes with a tissue-specific role in cell transformation and thymoma progression. The present study did not demonstrate any correlation of bcl-2 presence with proliferation activity as this was expressed by Ki67 detection. This supports the hypothesis that bcl-2 does not play a significant role in the progress or late stages of MG. However, a larger sample and further studies are warranted to confirm the above hypothesis.

In this study, bax expression in the control thymus was similar to that described previously [18,19] and somehow displayed an opposite pattern to that of bcl-2 presence. It was expressed in the medulla, but it was more present within germinal centers; the latter, in general, displays high apoptotic indices. This difference could be explained in two possible ways: (a) bax may act as a cell death effector protein, since it is a dominant inhibitor of bcl-2, and thus bcl-2 could regulate bax as has been implied in previous studies [18]; or (b) other oncogenes, members of the bcl-2 family (i.e. bcl-x) may be co-produced with bax and oppose its apoptotic process, in the areas where bax is present but bcl-2 is absent. Further study is required to elucidate the aforementioned hypotheses.

We have demonstrated, for the first time, that bax is actively produced (bax mRNA detection) and also expressed (bax protein presence) by epithelial cells and thymocytes in MG patients. In addition, bax expression was higher in cases of thymic carcinoma compared to hyperplasia and thymoma despite the small number of thymic carcinomas included in the study. The significant higher expression of bax in thymic carcinomas, compared to thymomas and hyperplasia, suggests that this oncogene may play an important role in the late stages of thymic oncogenesis. This implies that upregulation of the gene may commit thymic epithelial cells to apoptosis, leading finally to progress of the disease. Furthermore, there was a trend for increased bax protein or bax mRNA expression toward advanced MG stages. A non-mutual exclusive explanation could be that bax enhances apoptosis of autoreactive thymocytes and subsequently leads to increased cellular proliferation and growth and finally to advanced stage disease. All these observations favor a late abnormal bax expression in late stages of MG.

A higher cell proliferation rate was found in cases of thymic carcinomas compared to thymomas and hyperplasia. In agreement with a previous study [3], we showed that Ki67 expression was directly correlated to the size of the thymus and also to the weight of the organ. Furthermore, our study showed an increase of ABI in advanced MG stages and in MG instances accompanied by thymic carcinomas compared to those that were associated with hyperplasia or thymoma. In addition, a positive relationship was observed between apoptosis and proliferation determined as the expression of Ki67 antigen. The role and importance of apoptotic mechanisms in the progression, prognosis, and treatment of MG has not yet been fully clarified. Inhibition of apoptosis leads to enhanced cell survival and thus may be one of the mechanisms through which autoreactive cell promoters exert their effect. The progress of MG and development of advanced stages could be attributed to possible higher activation or lower inhibition of apoptosis.

In conclusion, this study suggested that in patients with MG who underwent MMT, bcl-2, bax, and Ki67 expression correlates positively or reversibly with the microscopic findings of thymus. Increased apoptosis and proliferation accompany advanced disease stage and, therefore, possible worse prognosis. In view of the recently raised interest and ‘call to arms’ for MG [6], we believe these markers presented here may help in our further understanding of the etiopathology of this disease and might provide additional information in terms of prognosis of patients after thymectomy. However, clinical correlation studies, as well as molecular research, are required to further identify the role of these specific markers in determining the long-term effects of surgical treatment in MG.

	Acknowledgments

 
The authors would like to thank a team of anesthetists and intensivists, headed by Professor S. Lacoumenta, for their superb perioperative management.

	Footnotes

 
Presented at the 14th Annual Meeting of the European Association for Cardio-thoracic Surgery, Frankfurt, Germany, October 7–11, 2000.

	References

Top Abstract 1. Introduction 2. Material and methods 3. Results 4. Discussion References

 

1. Vincent A. Immunology of acetylcholine receptors in relation to myasthenia gravis. Physiol Rev 1990;60:756-824.
2. Lewis J.E., Wick M.R., Scheithauer B.W., Bernatz P.E., Taylor W.F. Thymoma. A clinicopathologic review. Cancer 1987;60:2727-2743.[Medline]
3. Gilhus N.E., Jones M., Turley H., Gatter K.C., Nagvekar N., Newsom-Davis J., Willcox N. Oncogene proteins and proliferation antigens in thymomas: increased expression of epidermal growth factor receptor and Ki67 antigen. J Clin Pathol 1995;48:447-455.[Abstract/Free Full Text]
4. Jaretzki A., Wolff M. Maximal thymectomy for myasthenia gravis. J Thorac Cardiovasc Surg 1988;96:711-716.[Abstract]
5. Bulkey G.B., Bass K.N., Stephenson R., Deiner-West M., George S., Reilly P.A., Baker R.R., Drachman D.B. Extended cervicomediastinal thymectomy in the integrated management of myasthenia gravis. Ann Surg 1997;226:324-335.[Medline]
6. Jaretzki A., Barohn R.B., Ernstoff R., Kaminski H.R., Keesey J.C., Penn A.S., Sanders D.B. Myasthenia gravis: recommendation for clinical research standards. Ann Thorac Surg 2000;70:327-334.[Free Full Text]
7. Kruger S., Muller H. Correlation of morphometry, nucleolar organizer regions, proliferating cell nuclear antigen and Ki67 antigen expression with grading and staging in urinary bladder carcinomas. Br J Urol 1995;75:480-484.[Medline]
8. Hannun Y.A. Apoptosis and the dilemma of cancer chemotherapy. Blood 1997;89:1845-1853.[Free Full Text]
9. Cummings M.C., Winterford C.M., Walker N.I. Apoptosis. Am J Surg Pathol 1997;21:88-101.[Medline]
10. Von Boehmer H. Developmental biology of T cells in T cell-receptor transgenic mice. Annu Rev Immunol 1990;8:531-556.[Medline]
11. Surh C.D., Sprent J. T-cell apoptosis detected in situ during positive and negative selection in the thymus. Nature 1994;372:100-103.[Medline]
12. Hockenbery D., Nunez G., Milligan C., Schreiber R.D., Korsmeyer S.J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990;348:33-336.
13. Cleary M.L., Sklar J. Nucleotide sequence of a t (14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a break-point cluster region near a transcriptionally active locus on chromosome 18. Proc Natl Acad Sci USA 1985;82:7439-7443.[Abstract/Free Full Text]
14. Doglioni C., Dei Tos A.P., Laurino L., Chiarelli C., Barbareschi M., Viale G. The prevalence of bcl-2 immunoreactivity in breast carcinomas and its clinicopathological correlates, with particular reference to estrogen receptor status. Virchows Archiv 1994;424:47-51.[Medline]
15. Onodera J., Nakamura S., Nagano I., Tobita M., Yoshioka M., Takeda A., Oouchi M., Itoyama Y. Upregulation of Bcl-2 Protein in the myasthenic thymus. Ann Neurol 1996;39:521-528.[Medline]
16. Oltvai Z., Milliman C., Korsmeyer S.J. Bcl-2 heterodimerizes in vivo with a conversed homolog, Bax, that accelerates programmed cell death. Cell 1993;74:609-619.[Medline]
17. Yang E., Korsmeyer S. Molecular thanatopics: a discourse on the BCL2 family and cell death. Blood 1996;88:386-401.[Free Full Text]
18. Krajewski S., Krajewska M., Shabaik A., Miyashita T., Wang H.G., Reed J.C. Immunohistochemical determination of in vivo distribution of bax, a dominant inhibitor of bcl-2. Am J Pathol 1994;145:1323-1336.[Abstract]
19. Penault-Llorca, Bouabdallah R., Devilard E., Charton-Bain M.C., Hassoun J., Birg F., Xerri L. Analysis of bax expression in human tissues using the anti-bax 4F11 monoclonal antibody on paraffin sections. Pathol Res Pract 1998;194:457-464.[Medline]
20. Kirchner T., Muller-Hermelink H.K. New approaches in the diagnosis of thymic epithelial tumors. Prog Surg Pathol 1989;10:167-190.
21. Sentman C.L., Shutter J.R., Hockenbery D., Kanagawa O., Korsmeyer S.J. Bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell 1991;67:879-888.[Medline]
22. Gratiot-Deans J., Ding L., Turka L.A., Nunez G. bcl-2 proto-oncogene expression during human T cell development: evidence for biphasic regulation. J Immunol 1993;151:83-91.[Abstract]
23. Moore N.C., Anderson G., Williams G.T., Qwen J.J., Jenkinson E.J. Developmental regulation of bcl-2 expression in the thymus. Immunology 1994;81:115-119.[Medline]
24. Kornstein M.J., Brooks J.J., Anderson A.O., Levinson A.I., Lisak R.P., Zweiman B. The immunohistology of the thymus in myasthenia gravis. Am J Pathol 1984;117:184-194.[Abstract]
25. Fujii N., Itoyama Y., Goto I. Increase in differentiated type of T lineage cells in the myasthenic thymus: two-color fluorocytometric analysis. Ann Neurol 1990;27:642-646.[Medline]

This article has been cited by other articles:

				A. Gilboa-Geffen, P. P. Lacoste, L. Soreq, G. Cizeron-Clairac, R. Le Panse, F. Truffault, I. Shaked, H. Soreq, and S. Berrih-Aknin The thymic theme of acetylcholinesterase splice variants in myasthenia gravis Blood, May 15, 2007; 109(10): 4383 - 4391. [Abstract] [Full Text] [PDF]

				H. Ishibashi, T. Suzuki, S. Suzuki, T. Moriya, C. Kaneko, T. Nakata, M. Sunamori, M. Handa, T. Kondo, and H. Sasano Estrogen Inhibits Cell Proliferation through In situ Production in Human Thymoma Clin. Cancer Res., September 15, 2005; 11(18): 6495 - 6504. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Salakou, S. Articles by Dougenis, D. Search for Related Content PubMed PubMed Citation Articles by Salakou, S. Articles by Dougenis, D. Related Collections Mediastinum Molecular biology