Acidic Activated Charcoal Prevents Obesity and Insulin Resistance in High-Fat Diet-Fed Mice.

Obesity is becoming a major public health problem worldwide. Making charcoal from wood ("Sumi-yaki") has been a traditional activity in the southern part of Nagano Prefecture for centuries, with activated charcoal having reported detoxifying effects. However, it is unclear whether activated charcoal also possesses anti-obesity properties. Additionally, since activated charcoal is usually alkaline and might be affected by gastric juice, we evaluated the effect of acidic activated charcoal on high-fat diet (HFD)-induced obesity. This study demonstrated that co-treatment of acidic activated charcoal with a HFD significantly improved obesity and insulin resistance in mice in a dose-dependent manner. Metabolomic analysis of cecal contents revealed that neutral lipids, cholesterol, and bile acids were excreted at markedly higher levels in feces with charcoal treatment. Moreover, the hepatic expressions of genes encoding cholesterol 7 alpha-hydroxylase and hydroxymethylglutaryl-CoA reductase/synthase 1 were up-regulated by activated charcoal, likely reflecting the enhanced excretions from the intestine and the enterohepatic circulation of cholesterol and bile acids. No damage or abnormalities were detected in the gastrointestinal tract, liver, pancreas, and lung. In conclusion, acidic activated charcoal may be able to attenuate HFD-induced weight gain and insulin resistance without serious adverse effects. These findings indicate a novel function of charcoal to prevent obesity, metabolic syndrome, and related diseases.

Application of surface plasmon resonance imaging to monitoring G protein-coupled receptor signaling and its modulation in a heterologous expression system.

BACKGROUND: G protein-coupled receptors (GPCRs) are ubiquitous surface proteins mediating various biological responses and thus, important targets for therapeutic drugs. GPCRs individually produce their own signaling as well as modulate the signaling of other GPCRs. Real-time observation of GPCR signaling and modulation in living cells is key to molecular study of biological responses and pharmaceutical development. However, fluorescence imaging, the technique widely used for this purpose, requires a fluorescent dye which may inhibit biological responses or a fluorescent-tagged target protein created through time-consuming genetic manipulation. In this study, we applied two-dimensional surface plasmon resonance (SPR) imaging to monitoring the translocation of protein kinase C (PKC), a major GPCR-coupled signaling molecule in the widely used HEK293 cell lines and examined whether the signaling of, and, modulation between heterologously expressed GPCRs can be measured without fluorescent labeling. RESULTS: We cultured HEK293 cells on the gold-plated slide glass and evoked SPR at the interface between the cell's plasma membrane and the gold surface with incident light. The translocation of activated native PKC to the plasma membrane is expected to alter the incident angle-SPR extent relation, and this could be detected as a change in the intensity of light reflection from the specimen illuminated at a fixed incident angle. Direct activation of PKC with 12-O-tetradecanoylphorbol-13-acetate increased the reflection intensity. This increase indeed reported PKC translocation because it was reduced by a pre-treatment with bisindolylmaleimide-1, a PKC inhibitor. We further applied this technique to a stable HEK293 cell line heterologously expressing the GPCRs type-1 metabotropic glutamate receptor (mGluR1) and adenosine A1 receptor (A1R). (RS)-3,5-dihydroxyphenylglycine, a mGluR1 agonist, increased the reflection intensity, and the PKC inhibitor reduced this increase. A pre-treatment with (R)-N(6)-phenylisopropyladenosine, an A1R-selective agonist suppressed mGluR1-mediated reflection increase. These results suggest that our technique can detect PKC translocation initiated by ligand binding to mGluR1 and its modulation by A1R. CONCLUSIONS: SPR imaging turned out to be utilizable for monitoring GPCR-mediated PKC translocation and its modulation by a different GPCR in a heterologous expression system. This technique provides a powerful yet easy-to-use tool for molecular study of biological responses and pharmaceutical development.

Community analysis of picocyanobacteria in an oligotrophic lake by cloning 16S rRNA gene and 16S rRNA gene amplicon sequencing.

In this study, the picocyanobacterial species composition of Lake Miyagase was examined by analyzing the 16S rRNA gene in a clone library and by amplicon sequencing using a benchtop next-generation sequencer. Five separate samples were analyzed from different days over a ten-month period. In the picocyanobacterial lineage, 9 and 12 OTUs were identified from a clone library and by amplicon sequencing, respectively. Both analyses suggested that a picocyanobacterium related to Synechococcus sp. MW6B4 was dominant in Lake Miyagase. Our findings suggest that 16S rRNA gene amplicon sequencing enables detailed evaluation of picocyanobacteria composition. One OTU identified was found to be a novel cluster that does not group with any of the known freshwater picocyanobacteria.

Altered Actions of Memantine and NMDA-Induced Currents in a New Grid2-Deleted Mouse Line.

Memantine is a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, and is an approved drug for the treatment of moderate-to-severe Alzheimer's disease. We identified a mouse strain with a naturally occurring mutation and an ataxic phenotype that presents with severe leg cramps. To investigate the phenotypes of these mutant mice, we screened several phenotype-modulating drugs and found that memantine (10 mg/kg) disrupted the sense of balance in the mutants. Moreover, the mutant mice showed an attenuated optokinetic response (OKR) and impaired OKR learning, which was also observed in wild-type mice treated with memantine. Microsatellite analyses indicated that the Grid2 gene-deletion is responsible for these phenotypes. Patch-clamp analysis showed a relatively small change in NMDA-dependent current in cultured granule cells from Grid2 gene-deleted mice, suggesting that GRID2 is important for correct NMDA receptor function. In general, NMDA receptors are activated after the activation of non-NMDA receptors, such as AMPA receptors, and AMPA receptor dysregulation also occurs in Grid2 mutant mice. Indeed, the AMPA treatment enhanced memantine susceptibility in wild-type mice, which was indicated by balance sense and OKR impairments. The present study explores a new role for GRID2 and highlights the adverse effects of memantine in different genetic backgrounds.

Characterization of a novel mutant KCNQ1 channel subunit lacking a large part of the C-terminal domain.

A mutation of KCNQ1 gene encoding the alpha subunit of the channel mediating the slow delayed rectifier K(+) current in cardiomyocytes may cause severe arrhythmic disorders. We identified KCNQ1(Y461X), a novel mutant gene encoding KCNQ1 subunit whose C-terminal domain is truncated at tyrosine 461 from a man with a mild QT interval prolongation. We made whole-cell voltage-clamp recordings from HEK-293T cells transfected with either of wild-type KCNQ1 [KCNQ1(WT)], KCNQ1(Y461X), or their mixture plus KCNE1 auxiliary subunit gene. The KCNQ1(Y461X)-transfected cells showed no delayed rectifying current. The cells transfected with both KCNQ1(WT) and KCNQ1(Y461X) showed the delayed rectifying current that is thought to be mediated largely by homomeric channel consisting of KCNQ1(WT) subunit because its voltage-dependence of activation, activation rate, and deactivation rate were similar to the current in the KCNQ1(WT)-transfected cells. The immunoblots of HEK-293T cell-derived lysates showed that KCNQ1(Y461X) subunit cannot form channel tetramers by itself or with KCNQ1(WT) subunit. Moreover, immunocytochemical analysis in HEK-293T cells showed that the surface expression level of KCNQ1(Y461X) subunit was very low with or without KCNQ1(WT) subunit. These findings suggest that the massive loss of the C-terminal domain of KCNQ1 subunit impairs the assembly, trafficking, and function of the mutant subunit-containing channels, whereas the mutant subunit does not interfere with the functional expression of the homomeric wild-type channel. Therefore, the homozygous but not heterozygous inheritance of KCNQ1(Y461X) might cause major arrhythmic disorders. This study provides a new insight into the structure-function relation of KCNQ1 channel and treatments of cardiac channelopathies.

Thymic carcinoma with dissemination: a retrospective analysis of ten patients.

OBJECTIVE: Thymic carcinoma is a rare mediastinal neoplasm with frequent pleural or pericardial dissemination. We retrospectively studied ten such cases and analyzed factors that influenced the survival of the patients. METHODS: Ten thymic carcinoma patients with dissemination have been treated since 1987. The clinical and pathological data were retrospectively reviewed. RESULTS: Pretreatment tumor biopsy was performed and demonstrated squamous cell carcinomas in nine and small cell carcinoma in one. In six of ten patients pleural or pericardial dissemination was clinically evident (cT4). These patients were basically regarded as inoperable and treated with chemotherapy and/or radiotherapy. Four other patients were diagnosed as cT3 preoperatively but were found to have dissemination at the time of thoracotomy. They underwent total resection of the thymic tumor and all visible pleural dissemination but without pericardial dissemination. Radiotherapy was performed pre-or postoperatively with or without chemotherapy. The 5-year survival rate in all patients was 42.0%. The patients with Masaoka stage IVa showed significantly better prognosis than the patients with stage IVb (MST, 69.7 months vs. 14.5 months; 5-year survival rate, 64.3% vs. 0%) (P = 0.03). The patients with cT3 disease showed significantly better prognosis (P = 0.016) than the patients with cT4 disease (MST, 69.7 months vs. 14.5 months; 5-year survival rate, 100% vs. 16.7%). CONCLUSIONS: Among thymic carcinoma patients with pleural or pericardial dissemination, there seem to be some patients who show good prognosis. These candidates are patients who underwent subtotal resection with disseminations that were identified only at the operation and without hematogenous or lymphogenous metastasis.

EGFR polymorphism of the kinase domain in Japanese lung cancer.

BACKGROUND: Mutations of the epidermal growth factor receptor (EGFR) gene at kinase domain have been reported in non-small-cell lung cancer (NSCLC), and some common somatic mutations in EGFR have been examined for their ability to predict sensitivity to gefitinib or erlotinib. However, EGFR mutations at exon 20 have been reported to predict resistance to gefitinib therapy. MATERIALS AND METHODS: We investigated the EGFR mutations and/or polymorphism statuses at kinase domain in 303 surgically treated non-small cell lung cancer (NSCLC) cases. One hundred ninety-four adenocarcinoma cases were included. The presence or absence of EGFR polymorphism of kinase domains was analyzed by direct sequences. We have also investigated EGFR polymorphism status at exon 20 for 23 NSCLC patients who had undergone surgery followed by treatment with gefitinib at the National Hospital Organization, Kinki-chuo Chest Medical Center. RESULTS: EGFR mutations at kinase domain were found in 75 of 303 lung cancer patients. During sequencing of EGFR tyrosine kinase domain in tumors, 86 EGFR polymorphism (G2607A) cases were identified at exon 20. G2067A polymorphism was significantly higher in nonadenocarcinomas (37.4%) than in adenocarcinoma (25.3%, P = 0.0415). The polymorphism status did not correlate with gender, smoking (never smoker versus smoker), and EGFR mutations. In 46 total gefitinib treated NSCLC patients, there was a tendency toward better prognosis in EGFR wild type (GG) patients than AG + AA patients. EGFR polymorphism in Japanese lung cancers seemed to be less frequent than Caucasian lung cancers. CONCLUSIONS: EGFR-tyrosine kinase polymorphism might be associated with clinicopathological background of lung cancers.

Expression of excision repair cross-complementation group 1 and class III beta-tubulin predict survival after chemotherapy for completely resected non-small cell lung cancer.

In this study, we examined the expression of excision repair cross-complementation group 1 (ERCC1) protein in 90 completely resected lung cancer samples from patients who received adjuvant or neo-adjuvant platinum-based chemotherapy. Epidermal growth factor receptor (EGFR) was also studied in these samples. We also examined class III beta-tubulin protein expression in 50 patients treated with a platinum-based drug plus paclitaxel. Among 90 patients treated with platinum-based chemotherapy, the loss of ERCC1 protein expression was associated with a better prognosis (p=0.0068). The effect of ERCC1 expression on survival was not seen in a separate set of 59 patients who underwent curative resection but did not receive adjuvant chemotherapy. Among 50 patients treated with a platinum-based drug plus paclitaxel, loss of class III beta-tubulin protein expression was also associated with a better prognosis (p=0.0303). When combined, patients with a tumor that was negative for both ERCC1 and class III beta-tubulin had a significantly longer overall survival than those with a tumor that expressed either ERCC1 or class III beta-tubulin (p=0.0230). There was no relationship between the presence of an EGFR mutation and the patients' survival after the platinum-based chemotherapy. In conclusion, we found that the loss of ERCC1 and class III beta-tubulin protein expression were predictors of better survival in patients who received a platinum-based plus taxane chemotherapy.

Thymic carcinoma: 30 cases at a single institution.

INTRODUCTION: Thymic carcinoma is a rare and invasive mediastinal neoplasm that often metastasizes. It constitutes a heterogeneous group of tumors that displays different biologic behavior and prognosis. The clinical prognostic factors and treatment of thymic carcinoma are not yet standardized. METHODS: Thirty patients with thymic carcinoma have been treated at Nagoya City University Hospital since 1983. The clinical and pathologic data of these patients were retrospectively reviewed. Thirteen cases were considered to be unresectable or inoperable and received chemotherapy or chemoradiotherapy. Seventeen cases underwent resection; total in 7 cases and subtotal in 10 cases. Postoperative irradiation was added as adjuvant therapy in the tolerable cases. The most recent five cases received induction chemotherapy. RESULTS: In 17 of the 30 cases, the patients died. The survival periods in the death cases were from 2.4 to 78.1 months (mean, 32.4 months; median, 21.0 months). The observation periods in the 13 live cases were 6.3 to 232 months (average follow-up, 64.6 months). The 5-year survival rate was 47.5%, and median survival time (MST) was 49.0 months. Cases that underwent total resection showed significantly better prognosis than cases with subtotal resection (p = 0.011) and inoperable cases (p = 0.002). The cases that underwent subtotal resection showed significantly better prognosis than the inoperable cases (p = 0.050). The cases with hematogenous metastasis demonstrated significantly poorer prognosis (p = 0.021), but lymphogenous metastasis was not a significant predictor of poor prognosis. Only resectability was a significant prognostic factor in multivariate Cox regression analysis, and the hazard ratio was 5.123. CONCLUSIONS: Resectability was the only prognostic factor in thymic carcinoma. We suggest the importance of preoperative precise evaluation to exclude unresectable Masaoka stage IVb disease and expect preoperative chemotherapy or chemoradiotherapy to improve the respectability.

Epidermal growth factor receptor gene amplification and gefitinib sensitivity in patients with recurrent lung cancer.

To evaluate the epidermal growth factor receptor (EGFR) protein expression, gene mutations and amplification as predictors of clinical outcome in patients with non-small-cell lung cancer (NSCLC) receiving gefitinib, we have performed fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). We investigated the EGFR amplification and EGFR protein expression statuses in 27 surgically treated non-small-cell lung cancer (NSCLC) cases. These patients experienced relapse after surgery and received gefitinib 250 mg/day. The presence or absence of EGFR mutations of kinase domains was analyzed by genotyping analysis and sequences, and already reported. EGFR mutations were found from 15/27 lung cancer patients. EGFR mutation status was significantly correlated with better prognosis (log-rank test P=0.0023). Smoking status (never smoker vs. smoker, P=0.0032), and pathological subtypes (adenocarcinoma vs. non-adenocarcinoma, P=0.0011), but not EGFR amplification (P=0.1278), were correlated with survival of lung cancers. EGFR IHC results were correlated with FISH results (P=0.0125), but not correlated with prognosis (P=0.7921). Thus, the EGFR gene amplification or protein expression is not a predictor of gefitinib efficacy in Japanese patients with NSCLC. We have also evaluated the EGFR mutation status and clinico-pathological features for 27 NSCLC patients who had undergone surgery followed by treatment with gefitinib at the National Hospital Organization, Kinki-chuo Chest Medical Center. The EGFR mutation status, especially exon19 mutation was correlated with good response to gefitinib than exon 21 point mutation.

CCND1 messenger RNA expression is correlated with EGFR mutation status in lung cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) stimulation markedly increases cyclin D1 protein expression. Recently, it has been reported that cyclin D1 expression was increased in EGFR mutant cell lines; however, the expression status of CCND1 in EGFR mutant lung cancer tissues has not been reported. PATIENTS AND METHODS: We have investigated the CCND1 messenger RNA (mRNA) levels and other clinicopathologic data in 74 lung cancers. The CCND1 mRNA levels were quantified by real-time reverse-transcriptase polymerase chain reaction using LightCycler. RESULTS: The CCND1/GAPDH mRNA levels were significantly higher in adenocarcinoma (35.125 +/- 37.387) than in non-adenocarcinoma (15.2 +/- 24.699; P = .0158), and CCND1/GAPDH mRNA levels were not significantly different among smoking status, sex, or pathologic stage. The CCND1/GAPDH mRNA levels were significantly higher in lung cancer with EGFR mutation (39.713 +/- 41.265) than in lung cancer without EGFR mutation (21.805 +/- 29.152; P = .0338). CCND/GAPDH mRNA expression did not correlate with prognosis of lung cancer. CONCLUSION: Using the LightCycler real-time reverse-transcriptase polymerase chain reaction assay, CCND1 gene expression might correlate with EGFR mutation in lung cancer. However, further studies are needed to confirm the impact of cyclin D1 for a molecular target of lung cancer.

EGFR exon 20 insertion mutation in Japanese lung cancer.

Mutations of the epidermal growth factor receptor (EGFR) gene have been reported in non-small cell lung cancer (NSCLC), especially in female, never smoker patients with adenocarcinoma. Some common somatic mutations in EGFR, including deletion mutations in exon 19 and leucine to arginine substitution at amino acid position 858 (L858R) in exon 21, have been examined for their ability to predict sensitivity to gefitinib or erlotinib. On the other hand, previous report has shown that the insertion mutation at exon 20 is related to gefitinib resistance. We investigated the exon 20 EGFR mutation statuses in 322 surgically treated non-small cell lung cancer cases. Two hundred and five adenocarcinoma cases were included. The presence or absence of EGFR mutations of kinase domains was analyzed by direct sequences. EGFR insertion mutations at exon 20 were found from 7 of 322 (2.17%) lung cancer patients. We also detected the 18 deletion type mutations in exon 19, and 25 L858R type mutations in exon 21. There was a tendency towards higher exon 20 insertion ratio in never smoker (never smoker 4.4% versus smoker 1.3%, p=0.0996) and female (female 4.5% versus male 1.3%, p=0.0917). Two exon 20 insertion cases were treated with gefitinib and failed to response. EGFR insertion mutation in exon 20 could not be ignored from Japanese lung cancers.

Nras and Kras mutation in Japanese lung cancer patients: Genotyping analysis using LightCycler.

Activating mutations of Ras gene families have been found in a variety of human malignancies, including lung cancer, suggesting their dominant role in tumorigenesis. Many studies have showed that the Kras gene is activated by point mutations in approximately 15-20% of non-small cell lung cancers (NSCLCs), however, there are only a few reports on Nras mutations in NSCLC. We have genotyped Nras mutation status (n=195) and Kras mutation status (n=190) in surgically treated lung adenocarcinoma cases. The presence or absence of Nras and Kras mutations was analyzed by real-time quantitative polymerase chain reaction (PCR) with mutation-specific sensor and anchor probes. EGFR mutation status at kinase domain has already been reported. Nras mutation was found in 1 of 195 patients. This mutation was a G-to-T transversion, involving the substitution of the normal glycine (GGT) with cystein (TGT) and thought to be a somatic mutation. The patient was male and a smoker. Kras mutant patients (11.1%; 21/190) had a significantly worse prognosis than wild-type patients (p=0.0013). Eighty-two EGFR mutations at kinase domain had exclusively Nras or Kras mutations. Although Nras gene mutation might be one of the mechanisms of oncogenesis of lung adenocarcinoma, this was a very rare event. Further studies are needed to confirm the mechanisms of Nras mutations for the sensitivity of molecular target therapy for lung cancer.

ErbB4 expression and mutation in Japanese patients with lung cancer.

BACKGROUND: Much evidence has accumulated that the epidermal growth factor receptor (EGFR) and its family members are strongly implicated in the development and progression of lung cancers. Recently, erbB4 kinase domain mutations were reported in Korean patients with lung cancer. We hypothesized that erbB4 mutations correlate with clinicopathologic features of lung cancers. PATIENTS AND METHODS: The presence or absence of erbB4 kinase domain mutations was analyzed by reverse-transcription polymerase chain reaction amplification and direct sequencing in 105 surgically treated non-small-cell lung cancer cases from Nagoya City University Hospital. Sixty-three adenocarcinoma cases were included. The EGFR mutation status for these 105 samples were already reported. We have investigated erbB4 expression status by immunohistochemistry in 40 non-small cell lung cancer cases. RESULTS: ErbB4 mutation was not found in 105 patients with lung cancer. The EGFR mutation status was significantly correlated with sex (women, 74.2% vs. men, 9.5%; P < 0.0001), smoking status (never-smokers, 68.8% vs. smokers, 11%; P < 0.0001), pathologic subtype (adenocarcinoma, 44.4% vs. non-adenocarcinoma, 4.8%; P < 0.0001), and differentiation status of the lung cancer (well-differentiated, 47.4% vs. others, 14.8%; P = 0.0004). We detected ErbB4 protein positivity in 20 samples. The ErbB4 protein status was not significantly correlated with sex (women, 28.6% vs. men, 54.5%; P = 0.4075), smoking status (never-smokers, 69.4% vs. smokers, 16.9%; P < 0.0001), pathologic subtype (adenocarcinoma, 46.2% vs. nonadenocarcinoma, 51.9%; P > 0.9999), or differentiation status of the lung cancer (well-differentiated, 54.5% vs. others, 48.3%; P > 0.9999). CONCLUSION: Thus, erbB4 mutations are rare in Japanese people with lung cancer and of limited value for molecular-targeted therapy.

PIK3CA gene amplification in Japanese non-small cell lung cancer.

We have investigated 92 non-small cell lung cancer tissues and found 11 PIK3CA amplification. PIK3CA amplification incidence was significantly higher in male, smoker and squamous cell carcinoma patients. Among 11 patients with PIK3CA amplification, two patients harbored a PIK3CA mutation. There was significant difference in survival between the patients with PIK3CA normal copy number and the patients with PIK3CA amplification.

Flow-cytometric diagnosis of thymoma using needle biopsy specimens.

INTRODUCTION: Thymoma is a neoplasm of thymic epithelial cells which is characteristically associated with a large number of non-neoplastic T lymphocytes. These T cells are induced by epithelial cells and show a unique phenotype of CD4(+)8(+) double positive (DP), when studied by flow cytometry. This DP phenotype can be used as one of the diagnostic criteria to indicate a thymoma. The preoperative diagnosis of thymoma and other thymic tumors is an important problem because the treatment differs according to the diagnosis. METHODS: A flow-cytometric analysis of needle biopsy specimens was performed on ten thymic tumors. The results were then compared with the findings of a histological diagnosis using conventional hematoxylin-eosin (H&E) staining. RESULTS: Six cases with high frequencies of DP cells were diagnosed as thymoma by a flow-cytometric analysis, and were confirmed by a histological analysis of the resected specimen. Flow cytometry did not suggest a thymoma in the other four cases with few DP cells. The final diagnoses of the resected specimen of these four cases were: one thymic carcinoma, one malignant lymphoma, and two germ-cell tumors. The accuracy and specificity of diagnosis of thymoma using a fluorescence-activated cell sorting analysis from needle biopsy specimens were 6/6 (100%). On the other hand, the specificity of H&E staining from needle biopsy specimens for the diagnosis of thymoma was 6/6 (100%), but the accuracy was only 6/9 (66.7%). DISCUSSION: Flow cytometry can be applied to needle biopsy specimens and thus is considered to offer useful information for the preoperative diagnosis of thymic tumors.

Diagnosis of visceral pleural invasion by lung cancer using intraoperative touch cytology.

BACKGROUND: Invasion to the visceral pleura is an important component of lung cancer staging and an independent prognostic factor. However, the accuracy of pathologic examination depends on how the sections are made, and the pathologist may miss the most invaded part of the pleura. Therefore, we have designed "touch" cytology in an effort to more accurately diagnose the pleural invasion by lung cancer. METHODS: Immediately after thoracotomy, the surface of the visceral pleura just above the tumor was gently touched by a glass slide without scrubbing in 100 patients who simultaneously underwent pleural lavage cytology or cytology of the subclinical pleural effusion. RESULTS: Seventeen percent of the tumors were diagnosed as invading the visceral pleura by touch cytology. Lavage cytology was found to be positive in 7%. In reference to the pathologic examination of the tumor specimen, touch cytology was found to be positive in all of p3, 5 out of 6 of p2, 5 out of 30 of p1, and 5 out of 62 of p0 cases. Touch cytology correctly diagnosed all the positive cases detected by lavage or effusion cytology. CONCLUSIONS: This study suggests that our method is useful in detecting the visceral pleural invasion and raises a possibility that pathologic p0 and p1 lung cancers include a subset of patients with tumor cells exposed on the pleural surface.

The expression of Bcl-2 family proteins (Bcl-2, Bcl-x, Bax, Bak and Bim) in human lymphocytes.

Bcl-2 family proteins regulate programmed cell death, and may play an important role in the selection of lymphocytes. We investigated the expression of Bcl-2, Bcl-x, Bax, Bak and Bim in human lymphocytes using flow-cytometry. Bcl-2 was down-regulated in CD4(+)8(+) (DP) thymocytes and CD19(+)38(+) tonsillar lymphocytes (GC B cells). Among DP thymocytes, cells co-expressing CD69 up-regulated Bcl-2, suggesting that the role of Bcl-2 is promoting survival of positively selected DP cells. Unexpectedly, the expression level of Bcl-x was higher in DP cells than in Single Positive (SP) cells and in CD69(+) DP thymocytes it was lower than in CD69(+) DP thymocytes. Expression of Bim was low in DP thymocytes but high in a subset of GC B cells. Bim and Bax were expressed more highly in SP than in DP thymocytes. Among peripheral blood lymphocytes (PBL), CD8(+) T cells expressed an approximately ten-fold higher level of Bcl-x than CD4(+) T cells while both subsets expressed similar levels of Bcl-2. Bak expression was low and Bim expression was absent in PBL. These results suggest that not only Bcl-2 but other members of the Bcl-2 family are involved in T cell development in the thymus and affinity maturation of B cells in the germinal center.

Developmentally regulated expression of survivin in the human thymus.

Survivin is a member of the inhibitor-of-apoptosis (IAP) family. It has been reported to be expressed during development, but not in differentiated normal tissue. However, its expression has been reported to be high in the thymus. To assess the role of survivin in human thymocyte development, we investigated the expression of survivin using reverse-transcriptase-polymerase chain reaction, flow cytometry, and immunohistochemistry in freshly isolated human thymocytes. Survivin was expressed in all thymocyte subsets but its expression level was developmentally regulated. Its expression was low in the double negative (DN) thymocytes, upregulated in double positive (DP) thymocytes, and was highest in the T-cell receptor(high), late DP thymocytes; it was then downregulated in the single positive thymocytes and negative in the peripheral blood T cells. Moreover, there was a positive correlation between the expression of survivin and that of CD69 and Bcl-2 in DP thymocytes. These results suggest that survivin may play an important role in the T-cell development in the human thymus.