Regulation of acetylcholine receptor alpha subunit variants in human myasthenia gravis. Quantification of steady-state levels of messenger RNA in muscle biopsy using the polymerase chain reaction.

Myasthenia gravis (MG) is an autoimmune disease mediated by auto-antibodies that attack the nicotinic acetylcholine receptor (AChR). To elucidate the molecular mechanisms underlying the decrease in AChR levels at the neuromuscular junction, we investigated the regulation of AChR expression by analyzing mRNA of the two AChR alpha subunit isoforms (P3A+ and P3A-) in muscle samples from myasthenic patients relative to controls. We applied a quantitative method based on reverse transcription of total RNA followed by polymerase chain reaction (PCR), using an internal standard we constructed by site-directed mutagenesis. An increased expression of mRNA coding for the alpha subunit of the AChR isoforms was observed in severely affected patients (P < 0.003 versus controls) but not in moderately affected patients, independently of the anti-AChR antibody titer. Study of mRNA precursor levels indicates a higher expression in severely affected patients compared to controls, suggesting an enhanced rate of transcription of the message coding for the alpha subunit isoforms in these patients. We have also reported that mRNA encoding both isoforms are expressed at an approximate 1:1 ratio in controls and in patients. We have thus identified a new biological parameter correlated with disease severity, and provide evidence of a compensatory mechanism to balance the loss of AChR in human myasthenia gravis, which is probably triggered only above a certain degree of AChR loss.

Thymoma epithelial cells secrete thymic hormone but do not express class II antigens of the major histocompatibility complex.

17 thymomas were studied by indirect immunofluorescence for the presence of thymic hormones and antigens of the major histocompatibility complex (MHC). The thymoma epithelial cells (specifically identified by their keratin content) contained thymic hormones (thymulin and thymosin alpha 1), a finding corroborated by the observation of elevated thymulin serum levels. In contrast with normal or hyperplastic thymuses, thymoma epithelial cells did not express HLA-DR and HLA-DC antigens as assessed by immunofluorescence as well as immunoblot analyses. Conversely, MHC class I antigens (HLA-ABC) were normally expressed. Thus, we conclude that thymoma epithelial cells are endocrinologically active but are defective for the expression of some MHC products (class II molecules) known to play an essential role in intrathymic T cell differentiation.

Regulation of acetylcholine receptor gene expression in human myasthenia gravis muscles. Evidences for a compensatory mechanism triggered by receptor loss.

Myasthenia gravis (MG) is a neuromuscular disorder mediated by antibodies directed against the acetylcholine receptor (nAChR) resulting in a functional nAChR loss. To analyze the molecular mechanisms involved at the muscular target site, we studied the expression of nAChR subunits in muscle biopsy specimens from MG patients. By using quantitative PCR with an internal standard for each subunit, we found that the levels of beta-, delta-, and epsilon-subunit mRNA coding for the adult nAChR were increased in severely affected MG patients, matching our previous data on the alpha-subunit. Messenger levels were highly variable in MG patients but not in controls, pointing to individual factors involved in the regulation of nAChR genes. The fetal subunit (gamma-chain) transcripts were almost undetectable in the extrajunctional region of MG muscle, suggesting that gene regulation in MG differs from that in the denervation model, in which nAChR gamma-subunit mRNA is reexpressed. Nicotinic AChR loss mediated by monoclonal anti-nAChR antibodies in both the TE671 muscle cell line and cultured normal human myotubes induces a similar increase in beta- alphand delta-subunit mRNA levels, suggesting the existence of a new muscular signaling pathway system coupled to nAChR internalization and independent of muscle electrical activity. These data demonstrate the existence of a compensatory mechanism regulating the expression of the genes coding for the adult nAChR in patients with MG.

The effect of aminocandin (HMR 3270) on the in-vitro adherence of Candida albicans to polystyrene surfaces coated with extracellular matrix proteins or fibronectin.

Aminocandin is a new representative of the echinocandins that could potentially affect the cellular morphology and metabolic status of Candida albicans cells within biofilms. This study investigated the influence of a sub-inhibitory concentration (MIC/2) of aminocandin on in-vitro growth of C. albicans and subsequent fungal adherence to plastic surfaces coated with fibronectin or extracellular matrix (ECM) proteins. Eleven strains of C. albicans were studied, of which six were susceptible and five were resistant to fluconazole. All 11 strains were susceptible to aminocandin in vitro, regardless of the culture medium used for the microdilution method. Aminocandin induced a significant (p <0.005) decrease in adherence when polystyrene was coated with ECM gel (ten strains) or fibronectin (seven strains). Growth in medium containing aminocandin (MIC/2) decreased the adherence of five (ECM gel) or three (fibronectin) of the six strains susceptible to fluconazole, and inhibition was observed for all five (ECM gel) or four (fibronectin) of the five fluconazole-resistant strains. Overall, the study demonstrated the anti-adherent properties of aminocandin with fluconazole-susceptible strains, and suggested that this activity was at least equivalent with fluconazole-resistant strains. Thus, the ability of aminocandin to inhibit the first step in the development of C. albicans biofilms appeared to be independent of the in-vitro resistance of C. albicans to fluconazole.

Defective priming of the phagocyte oxidative burst in a child with recurrent intracellular infections.

Human phagocytes (polymorphonuclear neutrophils and monocytes) play a critical role in host defense against invading microorganisms. Recent studies reported that circulating phagocytes undergo a final maturation process, in particular in terms of oxidative burst, during extravasation and migration to local sites of inflammation. This process is known as priming. We report here on a nine-year-old boy with successive disseminated infections due to intracellular microorganisms (Mycobacterium bovis, BCG, and Salmonella typhimurium). No T- or B-cell quantitative or qualitative defects were found. Polymorphonuclear neutrophil (PMN) migration and NADPH oxidase in PMNs and monocytes stimulated with various agents at optimal concentrations were normal, ruling out a leukocyte adhesion deficiency syndrome, a Chediak Higashi syndrome, and a chronic granulomatous disease. Nevertheless, the patient's PMNs and monocytes showed defective priming capacity, as measured by H(2)O(2) production after pretreatment with LPS (5 microg/mL for 30 min), TNFalpha (100 units/mL for 30 min), or IL-8 (50 ng/mL for 30 min) in response to bacterial N-formyl peptides (fMLP 10(-6) M for 5 min). In these conditions, H(2)O(2) production of PMNs and monocytes from the patient did not exceed that of the samples treated with fMLP or LPS alone, while the controls strongly produced H(2)O(2). Moreover, monocytes from the patient showed an impaired capacity to kill S. typhimurium in vitro. Such an impairment could be related at least in part to the priming deficiency of phagocyte oxidative burst. This case suggests, for the first time, that in vivo priming processes are critical in host defence against intracellular pathogens.

Cellular aspects of myasthenia gravis.

Several cellular aspects were investigated in a large series of patients with MG. First, non-Ag-specific proliferation was tested by measuring the response to r-IL2. Thymocytes from most MG patients showed hyperactivity to r-IL2. Peripheral blood lymphocytes (PBL) from some patients also showed a high response to r-IL2. These responding patients were generally those tested before thymectomy, presenting a high anti-AChR Ab titer and a severe form of the disease. Second, Ag-specific proliferation of MG PBL was assayed using 8 synthetic peptides corresponding to selected domains of torpedo or human AChR. Only 2 peptides gave a positive response in a significant number of patients, essentially in those presenting high anti-AChR Ab titer. The first is located near the alpha-bungarotoxin binding site and the second is in a cytoplasmic domain, according to models predicting the AChR transmembrane orientation. The positive results were essentially obtained with the human peptides; the corresponding torpedo peptides were positive in very few patients. Both human and torpedo peptides which include a part of the alpha-bungarotoxin binding site were negative. Finally, although morphological abnormalities were clearly visible in thymic hyperplasia, no correlation could be established between the thymus type and the cellular proliferation either to r-IL2, or to the peptides. Overall, our data indicate that cell-dependent mechanisms participate in the pathogenesis of MG, but the level of their involvement deserves further investigation.

Expression of ciliary neurotrophic factor receptor in myasthenia gravis.

In myasthenia gravis, high anti-nicotinic receptor (AChR) antibody titers are not always associated with severity of the disease, suggesting the involvement of other pathological effectors. We showed that ciliary neurotrophic factor receptor (CNTFR) gene expression was higher in muscle biopsy tissue from severely affected MG patients regardless of anti-nAChR antibody titer. This increase was also triggered in vitro by a seric factor from MG patients. CNTFR protein expression was decreased in muscles from seropositive MG patients only.Altogether, our data indicate that the alteration of CNTFR expression in some MG patients could contribute to the severity of the disease in a subgroup of patients.

Evidence of enhanced recombinant interleukin-2 sensitivity in thymic lymphocytes from patients with myasthenia gravis: possible role in autoimmune pathogenesis.

We evaluated the activation state of thymic lymphocytes in patients with myasthenia gravis (MG) by cytofluorographic analysis of CD25 expression and by testing their sensitivity to recombinant interleukin-2 (rIL-2) in the absence of any known previous stimulation. We detected no phenotypic signs of activation in fresh MG thymic lymphocyte suspensions, while functional signs of activation were reflected in a significantly higher sensitivity to rIL-2 in MG patients than in controls. The responses to rIL-2 were time- and dose-dependent, were inhibited by a blocking anti-IL-2 receptor antibody, and were associated with an increase in CD25+ T cells in both patients and controls. The T cells with functional signs of previous activation may represent autoreactive cells involved in the autoimmune process and confirm thymus gland hyperactivity in MG. These cells could result from primary autosensitization against the thymic acetylcholine receptor (AChR)-like molecule or from altered migration of peripheral activated cells into an abnormal thymic environment. Our results also provide a clue for understanding the effect of thymectomy in myasthenia gravis.

Results and prognostic factors in resections of primary tracheal tumors: a multicenter retrospective study. The French Society of Cardiovascular Surgery.

To determine long-term survival and prognostic factors, 208 patients with primary tracheal tumors were evaluated in a retrospective multicenter study including 26 centers. Ninety-four patients had squamous cell carcinoma, four had adenocarcinoma, 65 had adenoid cystic carcinoma, and 45 patients had miscellaneous tumors. The following resections were performed: tracheal resection with primary anastomosis, 165; carinal resection, 24; and laryngotracheal resection, 19. Postoperative mortality rate was 10.5% and correlated with the length of the resection, the need for a laryngeal release, the type of resection, and the histologic type of the cancer. Fifty-nine percent of patients with tracheal cancer and 43% of patients with adenoid cystic carcinomas had postoperative radiotherapy. The 5- and 10-year survivals, respectively, were 73% and 57% for adenoid cystic carcinomas and 47% and 36% for tracheal cancers (p < 0.05). Among patients with tracheal cancers, survival was significantly longer for those with complete resections than for those with incomplete resections. On the other hand, the presence of positive lymph nodes did not seem to decrease survival. Postoperative radiotherapy increased survival only in the case of incompletely resected tracheal cancers. Long-term prognosis was worsened by the occurrence of second primary malignancies in patients with tracheal cancers and by the occurrence of late pulmonary metastases in patients with adenoid cystic carcinomas.

Replacement of the superior vena cava with polytetrafluoroethylene grafts combined with resection of mediastinal-pulmonary malignant tumors. Report of thirteen cases.

From 1979 to 1986, we replaced the superior vena cava with polytetrafluoroethylene grafts in 13 patients to enable resection of malignant mediastinal or pulmonary tumors. Five prostheses could be interposed between a tumor-free proximal superior vena cava and right atrium while eight required separate innominate anastomoses (Y grafts constructed intraoperatively). There were no deaths. Phlebograms obtained 15 to 30 days after the operation demonstrated patency in 12 of 13 patients. Long-term (average 24 months) patency was evident in eight. Five patients are alive at 1, 3, 40, 49, and 60 months, a cumulative survival rate of 27% at 3 years.

Congenital respiratory-esophageal fistula in the adult. Report of nine cases and review of the literature.

Congenital tracheoesophageal or bronchoesophageal fistulas, if not associated with esophageal atresia, may not appear initially until adult life. Nine such cases (two tracheoesophageal and seven bronchoesophageal) are reported. The chief presenting symptoms were recurrent bouts of coughing, after drinking, and hemoptysis. In the majority of cases the duration of symptoms exceeded 15 years. The diagnosis was confirmed in seven patients by esophagography, in one patient by bronchoscopy, and in one patient the fistula was discovered incidentally during thoracotomy. The esophageal opening of the fistula was in the lower third in seven patients and in the middle third in two. Bronchoesophageal fistulas communicated with a segmental bronchus in four patients and with a main or lobar bronchus in three. Treatment involved excision of the fistula (five patients) or division and suturing (four patients). Postoperative follow-up revealed no long-term sequelae except persistent chronic respiratory failure in one patient. The respiratory failure had developed before treatment of the fistula. The analysis of this series and a review of the literature underline the high index of suspicion required in all cases of chronic cough and lung suppuration, to diagnose this benign condition before life-threatening complications occur.

Lung resection after high doses of mediastinal radiotherapy (sixty grays or more). Reinforcement of bronchial healing with thoracic muscle flaps in nine cases.

Mediastinal radiotherapy of more than 60 Gy highly compromises bronchial and wound healing after lung resection. Nine patients with primary lung cancers underwent radical resection after high radiation doses. Eight patients had primary lung cancer previously treated by radiotherapy alone (n = 2) or associated with chemotherapy (n = 6). One patient had a tracheal cancer involving the carina that was previously treated by radiotherapy. Seven patients underwent pneumonectomy and one patient underwent lobectomy with reinforcement of bronchial stump closure with use of the serratus anterior muscle. One patient underwent a sleeve lobectomy with bronchial reconstruction wrapped with an intercostal pedicle flap. Five patients had no postoperative complications and four patients had empyema, one associated with a small bronchial fistula. All except one patient were successfully treated by thoracostomy and immediate or secondary transposition of the pectoralis major muscle and the omentum to fill the cavity. These results show that lung resections can be done after high doses of radiotherapy without a high rate of bronchial fistula by using thoracic muscle flaps to reinforce bronchial stumps and anastomoses. In this procedure, surgical dissection is more time-consuming and increases the postoperative empyema rate (4/9). However, the higher long-term survival may justify this choice in selected cases.

Bronchioloalveolar lung carcinoma: results of surgical treatment and prognostic factors.

STUDY DESIGN: To determine the long-term results after surgical treatment of bronchioloalveolar lung carcinoma (BALC) and to identify prognostic factors. PATIENTS AND METHODS: A retrospective study of 70 patients (49 men, 21 women), mean age 61+/-10 years, was carried out. Their carcinomas were classified into three clinicopathologic types: nodular or tumoral, pneumonic, and diffuse types. All the diagnosed BALC cases were reviewed and were classified into histologic types: mucinous, nonmucinous (including fibrotic center), and mixed tumors. Univariate and multivariate analyses were carried out. RESULTS: The nodular or tumoral type was identified in 42 patients, pneumonic in 21, and diffuse in seven. Histologically, there were 36 mucinous, 25 nonmucinous, and nine mixed tumors. Resection was complete in 61 instances (87%) and incomplete in five. The 5-year survival rate was 34% in patients with curative resections. Five prognostic factors were identified by univariate analysis, but in multivariate analysis, only three factors remained significant: the absence of symptoms, the TNM stage, and completeness of resection. Thirty-six patients with curative resection (59%) developed recurrences (in the lung in 26 patients; mediastinal lymph nodes, four; distant metastases, nine). The frequency of recurrence was significantly greater in patients with pneumonic-type BALC than in nodular or tumoral types (p<0.01), and pulmonary recurrences were significantly more frequent in pneumonic than in tumoral types (p<0.02). CONCLUSIONS: This study confirmed that the overall prognosis of BALC is not significantly different from that of the other non-small cell lung cancers. We found that the lungs are the predominant site of recurrence in BALC, especially in the pneumonic types. The complete surgical resection of localized BALC offers the best chances of long-term survival.

Completion pneumonectomy: experience in eighty patients.

OBJECTIVE: Because completion pneumonectomy is a procedure reputed to place patients at risk, we reviewed our results with the objective of identifying factors that influence complications and survival. METHODS: In a 25-year period, 80 completion pneumonectomies were performed after first operations for 17 cases of benign disease and 63 cases of lung cancer (89% stages I and II), with 7 of the latter patients receiving postoperative radiotherapy. Completion pneumonectomy was performed in 18 cases of benign disease and 62 cases of lung cancer: 28 second primary cancers, 26 recurrent cancers, 3 metastases, and 5 primary cancers in patients previously operated on for benign disease. RESULTS: No intraoperative deaths occurred. Postoperative mortality rates were 5% for the entire series, 6.4% for patients operated on for cancer, and 0% for patients operated on for benign diseases. Patients previously irradiated and those operated on for infectious disease were at risk for postoperative empyema and fistula formation. In the cancer treatment group the actuarial 5-year survival was 36%, without significant difference between patients with recurrent and second primary lung cancers. The actuarial 5-year survivals were 51% for patients with stage I disease, 42% for patients with stage II disease, and 18% for patients with stage IIIA disease (P <.05). CONCLUSIONS: Completion pneumonectomy can be performed with an acceptable operative mortality rate and offers a second chance for cure to patients with cancer. Patients previously irradiated and those requiring completion pneumonectomy for infectious benign disease are at risk for postoperative complications.

Open window thoracostomy followed by intrathoracic flap transposition in the treatment of empyema complicating pulmonary resection.

OBJECTIVE: Successful treatment of postoperative empyema remains a challenge for thoracic surgeons. We report herein our 12-year experience in the management of this condition by means of open window thoracostomy. METHODS: Open window thoracostomy was used in the treatment of 46 patients with empyema complicating pulmonary resection. A bronchopleural fistula was associated in 39 of 46 cases. Previous operations included pneumonectomy (n = 30), bilobectomy (n = 5), lobectomy (n = 9), and wedge resection (n = 2) performed for benign (n = 10) or malignant (n = 36) disease. In 10 patients open window thoracostomy was definitive because of patient death (n = 2), concomitant major illness (n = 2), tumor recurrence (n = 4), spontaneous closure (n = 1), or patient choice (n = 1). In 36 cases intrathoracic flap transposition was eventually performed. Muscular (n = 29), omental (n = 5), or combined muscular and omental (n = 2) flaps were used to obliterate the thoracostomy cavity and to close a possibly associated bronchopleural fistula. In 9 patients with postpneumonectomy cavities too wide to be filled by the available flaps, a limited thoracoplasty represented an intermediate step. RESULTS: Among patients treated with definitive open window thoracostomy, local control of the infection was achieved in all the survivors (8/8). After open window thoracostomy and subsequent flap transposition, success (definitive closure of the thoracostomy and, if present, of the bronchopleural fistula) was achieved in 27 (75. 0%) of 36 patients. Four initial failures could be salvaged by means of reoperation (initial reopening of thoracostomy and subsequent muscular or omental transposition). CONCLUSION: Open window thoracostomy followed by intrathoracic muscle or omental transposition represents a valid therapeutic option in patients with empyema complicating pulmonary resections.

Prognostic factors and long-term results after thymoma resection: a series of 307 patients.

Three hundred seven cases of patients who underwent operation for thymoma (196 of whom had myasthenia gravis) were analyzed to assess the prognostic values of Masaoka clinical staging, completeness of resection, histologic classification, history of myasthenia gravis, and postoperative radiotherapy. According to the Masaoka staging system, 135 thymomas were stage I, 70 were stage II, 83 were stage III, and 19 were stage IV. According to the Verley and Hollmann histologic classification system, 67 thymomas were type 1, 77 were type 2, 139 were type 3, and 24 were type 4. Two hundred sixty patients underwent complete resection, 30 underwent incomplete resection, and 17 underwent biopsy. Postoperative radiotherapy was performed mainly in cases of invasive or metastatic thymoma. Mean follow-up was 8 years; eight patients were unavailable for follow-up. The overall 10- and 15-year survivals were 67% and 57%, respectively. In univariate analysis, three prognostic factors were established: completeness of resection, Masaoka clinical staging, and histologic classification. Furthermore, among patients with stage III thymomas, survival was significantly higher for patients with complete resection than for patients with incomplete resection (p < 0.001). Completeness of resection should therefore be taken into account in clinical-pathologic staging. We did not find any significant difference with respect to disease-free survival between patients who had postoperative radiotherapy and those who did not. In multivariate analysis, the sole significant prognostic factor was completeness of resection. On the basis of these findings, a new clinical-pathologic staging system is proposed.

Primary mediastinal germ cell tumors. Results of a French retrospective study.

Eighty-seven patients with primary mediastinal germ cell tumors treated between 1983 and 1990 were studied. Among the 23 patients classified as pure seminoma, eight (35 percent) underwent surgery followed by radiotherapy (n = 6), radiotherapy and/or chemotherapy (n = 2); two patients underwent radiotherapy; 13 patients (57 percent) underwent induction cisplatin-based chemotherapy (ten complete responses) followed by radiotherapy (n = 9), second line chemotherapy (n = 2) and surgical resection of residual tumor (n = 2). On completion of treatment, 22 patients (96 percent) with seminoma were free of disease. The two-year Kaplan-Meier survival rate of these patients was 86 percent. Among the 64 patients with nonseminomatous germ cell tumor, 19 patients (30 percent) underwent surgery as first treatment (ten complete resections) followed by chemotherapy (n = 17) and radiotherapy (n = 5). On completion of treatment, 12 of 19 patients were disease free. Forty-five patients (70 percent) underwent induction cisplatin-based chemotherapy (ten complete responses), and 22 of them underwent resection of residual tumor (19 complete resections). Twenty-three patients were treated with first line chemotherapy without postchemotherapy surgery (three complete responses). In summary, 33 patients (52 percent) with nonseminomatous germ cell tumors became free of disease, and seven patients (21 percent) relapsed after achieving a complete response. The two-year Kaplan-Meier survival rate of the nonseminomatous germ cell tumor patients was 53 percent (87 percent if a complete response), with a median survival of 28 months. Despite a worse prognosis than nonseminomaous tumors from other primary sites, this series of mediastinal germ cell tumors has confirmed the efficacy of therapy.

Congenital tracheobiliary fistula.

Congenital tracheobiliary fistulas are uncommon and almost always are diagnosed in the first weeks or months of life. Untreated congenital tracheobiliary fistulas cause intractable pneumonia and are revealed by a persistent cough. Medical therapy is not effective, and surgical repair is needed. A 22-year-old woman was treated successfully by resection of a fistula communicating between the tracheobronchial tree and the liver.

Management of empyema complicating lobectomy with superior vena cava replacement.

We present the case of a 49-year-old man with right upper lobe adenocarcinoma invading the right brachiocephalic vein and the origin of the superior vena cava. En bloc resection of right upper lobe with the involved venous segments was carried out through a median sternotomy. Venous pathway was reestablished with a Gore-Tex (W.L. Gore & Assoc, Flagstaff, AZ) prosthesis. Postoperative course was marked by right pneumonia complicated by empyema. The patient underwent thoracotomy with completion pneumonectomy and latissimus dorsi transposition to cover both the prosthesis and the bronchial stump, as well as to fill the cavity. A favorable outcome was observed and long-term survival achieved.

Results of re-resection for recurrent thymomas.

BACKGROUND: The treatment of recurrent thymomas remains controversial. PATIENTS: The place for re-resection was retrospectively studied in 28 consecutive patients operated on during the last 40 years. The initial Masaoka staging of the thymoma was stage I, 4; stage II, 8; stage III, 11; and stage IVa, 3. Postoperatively, 14 have had radiation therapy, 1 chemotherapy, and 13 no adjuvant treatment. Seven patients had development of recurrences, 15 had pleuropulmonary metastases, 5 had both, and 1 had thoracotomy scar recurrence. Nineteen patients had a complete resection and 9 an incomplete one. RESULTS: Most local recurrences appeared after resection of stage I or II thymomas. On the other hand, in patients with stage III or IV thymomas pleural or pulmonary metastases mainly developed. No local recurrence occurred in patients who initially received postoperative radiation therapy. Five-year and 10-year survival rates were 51% and 43%, respectively, for the overall population. Among the 19 patients with complete resection, only 3 patients had a subsequent recurrence; 1 of them could be reoperated on and is still alive and free of disease. CONCLUSIONS: Thymoma recurrences often appear as a locoregional rather than a hematogenous spread. Reresection can be recommended in selected patients.