Polymerase chain reaction of pleural biopsy specimens for rapid diagnosis of tuberculous pleuritis.

SETTING: Government-administered regional teaching hospital. OBJECTIVE: To improve timeliness and sensitivity of laboratory diagnosis of tuberculous pleuritis. DESIGN: We applied polymerase chain reaction (PCR) to detect DNA (IS6110) specific for Mycobacterium tuberculosis complex in pleural biopsy specimens. RESULTS: Of 28 patients with pleural disease, 11 were diagnosed by microbiology (smear/culture of sputum or pleural fluid) with tuberculous pleuritis, eight were diagnosed with tuberculous pleuritis by histology (of pleural biopsies) and/or clinical presentation, and nine were diagnosed with carcinomatous pleuritis. Seventeen of the patients' pleural biopsies were PCR positive. Based on microbiological results, the sensitivity of the PCR assay was 100% (11/11). On the other hand, based on the results of the histological and clinical data, sensitivity and specificity of the PCR results were 89% (17/19) and 100% (9/9), respectively. CONCLUSION: PCR of pleural biopsy specimens can be a useful method when employed in combination with microbiological and histological examinations of pleural biopsy for rapid diagnosis of tuberculous pleuritis.

Pulmonary adenocarcinomas of the fetal lung type: a clinicopathologic study indicating differences in histology, epidemiology, and natural history of low-grade and high-grade forms.

Seven cases of high-grade adenocarcinoma of fetal lung type (H-FLAC) are compared with nine cases of pulmonary endodermal tumor resembling fetal lung or low-grade adenocarcinoma of fetal lung type (L-FLAC). Of the seven patients with of H-FLAC, four were men and three were women. All of the patients but one were in their 60s or 70s. Five patients were smokers. After resection of the tumor, three patients died of metastases, two patients are alive with no evidence of disease, and two patients died of a postoperative complication. Histologically, H-FLAC and L-FLAC have both complex glandular structures resembling fetal lung and neuroendocrine differentiation. Two cases of H-FLAC had stromal proliferation typical of biphasic pulmonary blastoma. The H-FLAC was distinguished from L-FLAC by the presence of disorganized glands, large vesicular nuclei, prominent nucleoli, pronounced anisonucleosis, absence of morules, transition to conventional adenocarcinoma, broad areas of necrosis, desmoplastic stroma, overexpression of p53 protein, and production of alpha-fetoprotein. High and low grades of FLAC explain discrepancies in previously reported clinicopathologic features of FLAC. The H-FLAC needs to be distinguished from L-FLAC. Both forms may have stromal components, so both have been referred to as blastomas. The H-FLAC represents the prototype of so-called pulmonary blastoma predominantly seen in the elderly, whereas L-FLAC and its biphasic form predominate in the middle-aged population.

Immunoexpression of the alpha subunit of a guanine nucleotide-binding protein (Go) in pulmonary neuroendocrine cells and neoplasms.

The alpha subunit of a GTP-binding protein, Go, was investigated in pulmonary neuroendocrine neoplasms and fetal tissues of the lung by an immunohistochemical method. Positive immunostaining for the alpha subunit of Go (Go alpha) was found predominantly on the cell membrane and found occasionally in the cytoplasm. Typical carcinoids were all positively stained (9/9), and small cell carcinoma showed weaker and less frequent staining (5 positive cases in 10). Atypical carcinoids were variously stained (3/4). The tendency for obvious neuroendocrine differentiation to be immunohistochemically determined in typical carcinoids and not in small cell carcinoma is also true of staining for neuron specific enolase (NSE), chromogranin A (CG-A) and synaptophysin. In the lung, Go alpha-immunostaining was positive not only in nerve tissues but also in the airway epithelium. In the fetal lung, serial sections immunostained for NSE, CG-A and Go alpha confirmed that Go alpha-immunoreactive cells belong to the neuroendocrine cell population. The biological significance of Go alpha is unclear in normal and neoplastic lung tissues, but Go alpha is a useful marker of neuroendocrine cells and neoplasma of the lung.

Atypical adenomatous hyperplasia and bronchoalveolar lung carcinoma. Analysis by morphometry and the expressions of p53 and carcinoembryonic antigen.

Atypical adenomatous hyperplasia (AAH) of the lung is a putative precursor of bronchoalveolar carcinoma (BAC). To define the steps in its development and to clarify at which stage critical cellular events occur, we studied 65 lesions of AAH, early BAC, and overt BAC by morphometric analysis and immunohistochemical evaluation of expression of p53 protein and carcinoembryonic antigen (CEA). Both the nuclear area and lesion size increased from AAH to early BAC and to overt BAC; the standardized variation of nuclear area was smallest in overt BAC. Discriminant analysis using these morphometric parameters revealed high accuracy rates for the respective categories. Analysis of distribution of lung lesions in terms of nuclear area and lesion size yielded effective, potentially diagnostic cutoff values for distinction between AAH and early BAC. Both p53 and CEA expression tended to increase with the advance of atypia grade. In particular, high-level p53 expression was strongly correlated with overt BAC. These findings indicate that our classification of lung lesions is reproducible and thus useful for analyzing the development of BAC. Furthermore, some kinds of p53 gene abnormalities that are correlated with high-level p53 expression likely play an important role in the progression of early to overt BAC.

Adenosine deaminase isozymes in tuberculous pleural effusion.

Total adenosine deaminase (ADA) activity and its isozyme (ADA1 and ADA2) activities were measured in pleural effusions and serum samples from patients with tuberculosis and in those from patients with lung cancer as controls. To analyze the cellular source of ADA isozymes in tuberculous pleural effusions, ADA isozyme activities in CD2+ T lymphocytes purified from tuberculous pleural effusions and cultured human cell lines derived from hematopoietic tumors were measured. Tuberculous pleural effusions had a much higher ADA activity than cancerous effusions, and high ADA activity mainly originated from the increase in ADA2 activity. Further, total ADA activity in tuberculous pleural effusions decreased after antituberculosis treatment, because of the decrease in ADA2 activity. On the other hand, measurement of ADA and ADA isozyme activities in T lymphocytes purified from tuberculous pleural effusions and human hematopoietic cell lines showed dominant expression of ADA1 in total ADA activity. In conclusion, we found that ADA2 is a dominant component of tuberculous pleural effusions and that ADA1 is a major component of lymphoid cells. These results suggest that elevation of ADA activity in tuberculous pleural effusions does not always reflect the activation of cell-mediated immunity.

In vivo growth and differentiation potential of tracheal basal cells of rabbits in vitamin A deficiency.

The growth and differentiation potential of rabbit tracheal basal cells were investigated in vitamin A deficient mice. Denuded rat tracheal grafts were xenotransplanted into nude mice made vitamin A deficient by feeding them retinol-free pellets from mid-gestation. Rabbit tracheal epithelial cells harvested enzymatically or cells derived from a basal-cell-rich fraction obtained by elutriation (purity 93.3%) had previously been inoculated into the grafts (n = 8, each). The grafts were implanted into the vitamin A deficient or control mice aged about 10 weeks. Four weeks later, the grafts were retrieved for histological examination. The graft epithelium established by either basal cells or un-fractionated cells in vitamin A deficient hosts (groups 1 and 2, respectively) was atrophic, whereas grafts repopulated with both cell types in the controls had pseudostratified columnar epithelium. Group 1 and 2 grafts both showed squamous metaplasia; 10 metaplastic foci in 32 tracheal rings in group 1 (P < 0.02 or 0.002, compared with values for group 2 or controls, respectively), and 2 foci in 35 rings in group 2 (no statistical difference compared with controls). In conclusion, during vitamin A deficiency, rabbit tracheal epithelial cells, including the progeny of highly-purified basal cells, lost their potential for establishing a mucociliary epithelium and rather appeared to undergo squamous metaplasia.

[A combined in vitro and in vivo system for culture of epithelial cells from distal human airways].

A system for combined in vitro and in vivo culture of epithelial cells from distal human airways was established. Lung tissues that appeared to be generally normal were obtained from lungs removed surgically from patients with lung cancer. Small pieces of peripheral lung tissue were placed on culture dishes and cultured in F-12 complete medium containing serum and various growth factors, to obtain outgrown cells. For in vivo culture, rat tracheal grafts were de-epithelialized by freezing and thawing and were then used as culture vessels. Outgrown cells were harvested after 4 weeks of in vitro culture, inoculated into the denuded tracheal grafts, and then implanted into nude mice. For comparative purposes, bronchial fragments were also cultured in vitro and in vivo, by the same method. In vitro efficiency of colony formation was about the same for cells derived from peripheral lung tissue and from bronchial tissue (14.8 +/- 8.9% and 16.0 +/- 4.7%, respectively). Four weeks after implantation, the grafts were retrieved and processed for morphologic evaluation. By that time, grafts in both groups had totally re-epithelialized. Therefore, the growth potential of the cells derived from peripheral lung tissue and from bronchi in vivo appeared to be almost the same. Newly formed epithelial cells in grafts showed the same well-developed pseudostratified columnar form in both groups at 4 weeks. The time course of epithelial cell differentiation was also studied, with outgrown cells from lungs. Two days after implantation, undifferentiated cells were attached to the inner surface of the grafts as a single cell layer, and at 4 days, small cell nests containing mitotic cells were observed. At 1 week, the grafts were totally covered with undifferentiated cells. Over 2 to 3 weeks, differentiated cells (ciliated, secretory, and basal cells) appeared, and the epithelia had become fully developed by 4 weeks. As reported previously, cells that outgrew from lung explants were considered to be derived from bronchioles. Therefore, this system may be useful for studies of growth and differentiation of human bronchiolar epithelial cells under various conditions.

Expression of tumor necrosis factor receptors in human lung cancer cells and normal lung tissues.

The purpose of this study was to investigate the expression of tumor necrosis factor (TNF) receptors for the control of the biologic action of TNF-alpha in lung cancer cells and normal lung tissues. Lung cancer specimens and normal lung tissues were freshly obtained in pairs from 15 patients who underwent surgery for lung cancer. Thirteen lung cancer specimens expressed the 55 kDa TNF receptor messenger RNA (mRNA), whereas only six lung cancer specimens expressed the 75 kDa TNF receptor mRNA by Northern blot analysis. The 55 kDa and 75 kDa TNF receptors mRNA were detected in all and 11 normal lung tissues, respectively. All four lung carcinoma cell lines examined expressed the 55 kDa TNF receptor mRNA, but only RERF-LC-MS (MS) expressed both the 55 kDa and 75 kDa TNF receptors mRNA. Immunohistochemical examination revealed that lung cancer cells expressed the 55 kDa TNF receptor, but not the 75 kDa TNF receptor at the protein level. In normal lung tissues, the 55 kDa TNF receptor was detected in alveolar macrophages, bronchioles, and some small vessels. The 75 kDa TNF receptor was detected in alveolar macrophages. All four lung carcinoma cell lines examined exhibited the only 55 kDa TNF receptor. TNF-mediated tumor cell lysis was observed in all lung carcinoma cell lines that exhibited the 55 kDa TNF receptor except A549, which is a TNF-insensitive cell line. In surface binding assays, specific surface binding of TNF-alpha to TNF-insensitive cell line A549 was observed to be about half that of TNF-sensitive cell lines. We demonstrated the expression of two distinct TNF receptors in human lung cancer and normal lung tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferative potential and p53 overexpression in precursor and early stage lesions of bronchioloalveolar lung carcinoma.

To elucidate the pathogenesis of bronchioloalveolar lung carcinoma (BAC), we evaluated the lesion size, growth fraction, and p53 overexpression of atypical adenomatous hyperplasia (AAH) and early stage BAC. AAH was classified as showing low grade or high grade atypia. AAH-like carcinoma, presumably very early stage BAC, was distinguished from AAH in that it exhibited remarkable atypia suggestive of malignant potential and from overt BAC in that it lacked unequivocal malignant features, including invasive/destructive growth. The growth fraction was determined immunohistochemically in terms of the Ki-67 labeling index. The overexpression of p53 was evaluated by assessing the nuclear accumulation of immunoreactive p53 protein. Both the lesion size and the growth fraction increased from low grade AAH, to high grade AAH, to AAH-like carcinoma, and to overt adenocarcinoma. The overexpression of p53 in AAH-like carcinoma was similar to that in overt adenocarcinoma and was more frequent than that in AAH. Our findings indicate that AAH, AAH-like carcinoma, and overt BAC represent different categories, although the cellular events occurring in these lesions presumably represent a continuous spectrum of the changes that are reflected in the cytomorphology and lesion size. The findings here suggest that AAH and AAH-like carcinomas constitute a population of heterogeneous lesions representing different steps toward overt BAC.

Effects of vitamin A on proliferation of human distal airway epithelial cells in culture.

Using a serum-free culture method, we investigated the effects of vitamin A on the proliferation of human distal airway epithelial cells. Outgrowth of epithelial cells from lung tissue explants was enhanced by treatment with all-trans retinol at concentrations of 10(-8) to 10(-7) M. The colony-forming activity of cells harvested from the primary culture and replated onto Swiss 3T3 fibroblastic feeders was, in contrast, significantly reduced by 10(-7) M to 10(-5) M retinol. When the primary cells were harvested and subcultured on Primaria plates, population expansion was also inhibited by retinol at 10(-10) to 10(-6) M. We further investigated the cells to determine whether there was any difference in sensitivity to the growth-inhibitory effects of vitamin A between cells from the primary culture incubated with and without retinol. The population increase in cells harvested from the primary culture was inhibited equally in retinol-treated and non-treated cells by subsequent treatment with retinol or retinoic acid, this inhibition being dose-dependent. DNA synthetic activity was also inhibited. Interestingly, both the growth rate and the colony-forming efficiency on feeders were greater in the subculture of cells from the retinol-treated primary culture than in those non-treated. When the cells in the secondary subculture were treated with retinoic acid and replated again, they showed a greater population increase rate than those non-treated. Our results showed that human distal airway epithelial cells isolated from lung tissue were sensitive to the growth-inhibitory effect of vitamin A, but the proliferative potential in some fraction of the epithelial cell population was possibly enhanced by vitamin A treatment.

Polymerase chain reaction for the differentiation of Mycobacterium intracellulare and Mycobacterium avium.

Polymerase chain reaction (PCR) amplification was performed using DNA purified from 15 mycobacterial type strains and from 21 specimens isolated from patients suspected to have non-tuberculous mycobacterial diseases. Using a primer set of MTB1-MTB2, 11 specimens out of 21 were Mycobacterium avium and 8 were M. intracellulare, which were verified by the Gen Probe Rapid Diagnostic System for the M. avium complex (MAC). One of the remaining 2 specimens which did not hybridize with the probe for the MAC was identified as M. kansasii and the other was not specifically identified by the conventional culture method. PCR amplification, using a primer set of TB1-TB3, was also performed for the specific identification of M. tuberculosis complex.

Encapsulated type II pneumocyte adenoma: a case report and review of the literature.

An unusual benign lung neoplasm, a papillary adenoma of type II pneumocytes, was resected from a 26-year-old man who showed no clinical symptoms. The tumor was 2.0 cm in diameter and was localized in the subpleural region of S7 of the right lung; the cut surface showed a spherical medullary mass encapsulated by a thin layer of connective tissue. Histologically, there were cuboidal to columnar epithelial cells with a little nuclear atypia showing a monotonous papillary pattern with a delicate stroma in most parts of the tumor. There was neither capsular invasion nor metastasis of tumor cells. Nuclear DNA analysis of the tumor cells showed a diploid pattern and a low S-phase fraction. The immunohistochemical study revealed that most tumor cells contained a large amount of surfactant apoprotein in the cytoplasm. Osmiophilic lamellar bodies characteristic of type II pneumocytes were frequently found by electron microscopy. These findings indicate that this was a benign adenoma of the lung arising from type II pneumocytes.

Preneoplastic changes of xenotransplanted human distal airway epithelium induced by systemic administration of 4-nitroquinoline-1-oxide to host nude mice.

To elucidate a possible role of hematogenously transported carcinogens in pathogenesis of peripheral lung carcinoma in humans, we investigated whether the bronchiolar and alveolar epithelial cells of adult human lung xenografts in nude mice could be a target for the chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) after its systemic administration to the host mice. Peripheral lung tissues from adult humans were transplanted s.c. into nude mice, and 4NQO (15 mg/kg) was administered s.c. to the host mice at a site distant from the xenografts at 2 and 3 weeks after transplantation. The human lung xenografts were maintained for from 20 to 52 weeks, and then serial sections were examined histologically and immunohistochemically. Three types of epithelial changes, i.e. epidermoid metaplasia, papillary hyperplasia of columnar and epidermoid cells, and atypical adenomatous hyperplasia, were induced in the 4NQO group, with a statistically significant difference for these combined epithelial lesions (P less than 0.01) and for epidermoid metaplasia (P less than 0.05) compared to the control group. Some epidermoid metaplasias showed significant nuclear atypia. In addition, almost all foci of epidermoid metaplasia and papillary hyperplasia contained cells positive for carcinoembryonic antigen, suggesting both types of the lesions were preneoplastic. The morphologic characteristics of the atypical adenomatous hyperplasia were very closely similar to those of the hitherto reported preneoplastic or putative neoplastic lesions in the human peripheral lung. Our results indicated that the alveolar and bronchiolar epithelial cells of human lung xenografts were affected by systemically applied 4NQO, and subsequently underwent transformation to a preneoplastic state.

Pulmonary blastoma with a topographic transition from blastic to more differentiated areas. An immunohistochemical assessment of its embryonic nature using stage-specific embryonic antigens.

In order to investigate the probable embryonic nature of pulmonary blastoma, immunohistochemical studies were performed using stage-specific embryonic antigens (Ley, Lex, sialyl Lex-i) in case of pulmonary blastoma with a very wide spectrum of morphological features. The tumour presented a topographic transition from primitive blastic and embryonic areas to more differentiated areas showing diverse differentiation. Blastic areas composed of extremely immature cells were found in most peripheral parts of the tumour. Inside the blastic areas there were "embryonic" areas which morphologically resembled human embryo lungs in the pseudoglandular and canalicular stages. Most central parts of the tumour showed more differentiated features including chondrosarcomatous, leiomyosarcomatous and rhabdomyosarcomatous elements and the common type of adenocarcinomatous element. Electron microscopic observation suggested the blastic and embryonic nature of these immature cell elements. Ley was expressed in the blastic and pseudoglandular areas. Lex was expressed in the canalicular areas. These antigens were not expressed in the more differentiated areas. The topographic gradient in the tumour of morphology and antigen expression from the peripheral blastic areas to the central more differentiated areas suggests that the primitive cells gradually differentiated into more mature cells of various directions as the tumour grew in size.

[Simultaneous bilateral spontaneous pneumothoraces].

Seven cases (1.9%) of simultaneous bilateral pneumothoraces were found in a retrospective study of 377 patients with spontaneous pneumothorax during the period from July, 1977 to June, 1989. Their symptoms were essentially those of unilateral pneumothorax, but with more severe dyspnea. All but two cases, both young, had underlying pulmonary diseases. Three (two lung cancers and one metastatic lung disease) had malignant pulmonary disease. During this period, five lung cancer patients were complicated with pneumothorax, and two of them had simultaneous bilateral pneumothoraces. Therefore the frequency of bilateral pneumothoraces in the lung cancer patients associated with pneumothorax is high. In these three patients with malignant disease, tube drainage was carried out but all died of respiratory failure. Two senile patients had small bilateral pneumothoraces. Bed rest without invasive treatment led to successful cure. Two younger patients without underlying pathology initially underwent tube drainage, followed by operation. We conclude that many patients with simultaneous bilateral spontaneous pneumothoraces have underlying pulmonary disease, the frequency of lung cancer being particularly high. Young patients without underlying disease should be operated on following alleviation of symptoms by tube drainage. Older patients and patients with malignancy should be treated with great care and individually.

Growth and differentiation of human distal airway epithelial cells in culture. Effects of small amounts of serum in defined medium.

We herein describe a new simple method for culturing primary epithelial cells derived from the distal airway of adult human lung. Peripheral lung tissue obtained from surgical materials was cultured as explants in Ham's F12 medium supplemented with hormones and growth factors. From days 10 to 14, outgrowth of epithelial cells started on the dish surface, and even after removal of explants at days 14 to 16, these cells continued to replicate during the 3rd and 4th week of culture, and eventually formed epithelial cell foci (10 to 20-fold increase in population). They then ceased to replicate and terminally differentiated in the 5th week. Addition of 1% serum to the culture medium enhanced the initial outgrowth of epithelial cells, whereas small amounts of serum had no effect on proliferation of cells after explant removal. On the other hand, serum modulated the differentiation phenotypes of epithelial cells. In the presence of 1% serum, numerous ciliated and secretory cells appeared, whereas the cells underwent epidermoid differentiation in the absence of serum. When replated onto 3T3 fibroblast feeder layers, the epithelial cells from earlier cultures showed a great replicative capability and formed colonies at higher frequencies (colony-forming efficiency, 8 to 27% at days 14 to 21), but the replicative capability was significantly reduced at the confluent stage (colony-forming efficiency, 0.44 to 2.0% at day 28). Morphologic examinations of explants strongly suggested that the primary cells were derived from the bronchiolar epithelium. We conclude that this new culture system provides an excellent model for studying the growth, differentiation, and function of human bronchiolar epithelial cells in vitro.

[A combined anti-emesis therapy of oral prednisolone and alprazolam for cisplatin-induced delayed emesis].

To evaluate the efficacy of a combined therapy of prednisolone and alprazolam for controlling cisplatin-induced delayed emesis, a study involving 14 non-small cell lung cancer patients receiving cisplatin (80 mg/m2) was conducted. Seven of these patients were given oral prednisolone at a dose of 30 mg/day for 5 days, then, 15 mg/day for 2 days, and with alprazolam at a dose 1.2 mg/day for 7 days. The other 7 patients were given an oral metoclopramide at a dose of 1 mg/kg/day for 5 days, and at 0.5 mg/kg/day for 2 days. All patients received 2 courses of chemotherapy and the same assigned regimen. Five of the 7 patients who had received prednisolone and alprazolam experienced no emesis, in contrast to only 1 patient who experienced no emesis from taking oral metoclopramide (p less than 0.05). Further, the duration of anorexia was shorter in those who received oral prednisolone with alprazolam. Thus, a combined therapy of oral prednisolone and alprazolam appears to be effective for controlling cisplatin-induced delayed emesis. Further evaluation and study, however, are necessary.

The mechanism of carbon tetrachloride induced pulmonary Clara cell damage: biochemical and morphologic studies.

In our present study, we have shown the different damages among pulmonary drug metabolizing systems and selective lesions of the pulmonary Clara cells in mice treated with carbon tetrachloride. Concerning with the inhibition degrees of pulmonary drug metabolizing systems, the inhibition of coumarin hydroxylase activity was the severest in this experiment. Moreover, at the in vitro carbon tetrachloride exposure study to estimate carbon tetrachloride metabolizing capacity, the severe degradation of coumarin hydroxylase activity was observed, while 7-ethoxycoumarin o-deethylase and NADPH-cytochrome c reductase activities only showed slight degradations by carbon tetrachloride treatment. From these results, it is suggested that cytochrome P-450 form which catalyzes coumarin hydroxylation may highly localize in the Clara cell and have carbon tetrachloride metabolizing activity.