Distant failure after treatment of postoperative locoregional recurrence of non-small cell lung cancer

BACKGROUND: The standard treatment for patients with locoregional recurrence of non-small cell lung cancer (NSCLC) after complete resection has not been established. The aim of this study was to evaluate clinicopathologic characteristics, type of locoregional recurrence, pattern of subsequent failure, and survival after the recurrence. METHODS: Of 743 patients undergoing pulmonary resection for NSCLC in the National Cancer Center Hospital between 1990 and 1995, we retrospectively reviewed the medical charts of the 43 patients (5.8 %) found to have locoregional recurrence without distant metastasis or pleural or pericardial involvement. RESULTS: The median time to locoregional recurrence was 13.6 months (range: 1.6 - 85.8 months). The most frequent site of recurrence was the mediastinal nodes in 21 of 43 patients (49 %). 33 patients (77 %) received further treatment for the recurrence: thoracic irradiation in 26, surgery in two, systemic chemotherapy in two, and a combination of the above in 3 patients. Subsequent distant failure was detected in 26 (68 %) of the 38 patients assessable for the analysis of failure pattern: lung in 11, brain in 6, bone in 5, and others in 13. The median interval from the recurrence to distant failure was 8.4 months (range: 1.7-56.4 months). The median survival time after diagnosis of the locoregional recurrence was 10.5 months (range: 0-74.0 months). A multivariate analysis showed that local therapy for the locoregional recurrence had no significant impact on postrecurrent survival or distant failure-free survival. CONCLUSIONS: Many patients with postoperative locoregional recurrence developed distant metastases early after the first recurrence. Systemic chemotherapy in addition to local therapy may be of benefit in this population.     

Comparison of HCV-associated gene expression and cell signaling pathways in cells with or without HCV replicon and in replicon-cured cells

Background

Hepatitis C virus (HCV) replication is affected by several host factors. Here, we screened host genes and molecular pathways that are involved in HCV replication by comprehensive analyses using two genotypes of HCV replicon-expressing cells, their cured cells and naïve Huh7 cells.

Methods

Huh7 cell lines that stably expressed HCV genotype 1b or 2a replicon were used. The cured cells were established by treating HCV replicon cells with interferon-alpha. Expression of 54,675 cellular genes was analyzed by GeneChip DNA microarray. The data were analyzed by using the KEGG Pathway database.

Results

Hierarchical clustering analysis showed that the gene-expression profiles of each cell group constituted clear clusters of naïve, HCV replicon-expressed, and cured cell lines. The pathway process analysis between the replicon-expressing and the cured cell lines identified significantly altered pathways, including MAPK, steroid biosynthesis and TGF-beta signaling pathways, suggesting that these pathways were affected directly by HCV replication. Comparison of cured and naïve Huh7 cells identified pathways, including steroid biosynthesis and sphingolipid metabolism, suggesting that these pathways were required for efficient HCV replication. Cytoplasmic lipid droplets were obviously increased in replicon-expressing and cured cells as compared to naïve cells. HCV replication was significantly suppressed by peroxisome proliferator-activated receptor (PPAR)-alpha agonists but augmented by PPAR-gamma agonists.

Conclusion

Comprehensive gene expression and pathway analyses show that lipid biosynthesis pathways are crucial to support proficient virus replication. These metabolic pathways could constitute novel antiviral targets against HCV.     

Fasting concentrations of nesfatin‐1 are negatively correlated with body mass index in non‐obese males

Background We recently identified a novel anorexigenic protein, nesfatin‐1, which is processed from nesfatin/nucleobindin‐2 (NUCB2). However, the clinical importance of this protein has not been determined. Objective To investigate its clinical significance in humans, we have established a new specific enzyme‐linked immunosorbent assay (ELISA) for human nesfatin‐1 in peripheral blood and measured its circulating concentration in healthy subjects. Design The new sandwich‐type ELISA method was validated and then used to measure nesfatin‐1 levels in plasma samples, under overnight fasting conditions, followed by oral glucose tolerance and meal tests. Patients and measurements A total of 43 nonobese males (age: 24·5 ± 0·6 , body mass index (BMI); 21·1 ± 0·3 kg/m²) were recruited to the study for evaluating fasting concentrations of nesfatin‐1. In those, fifteen subjects underwent a 75‐ g oral glucose tolerance test (OGTT) and another 15 underwent a meal test. In addition, fasting concentrations of nesfatin‐1 were measured in nine males with high BMI (age: 32·4 ± 3·7 , BMI; 37·3 ± 3·8 kg/m²). Results Peripheral concentrations of nesfatin‐1 showed a significant negative correlation with BMI, percentage body fat, body fat weight and blood glucose (P < 0·05). Nesfatin‐1 concentrations were not significantly changed during OGTT and meal tests. Fasting nesfatin‐1 levels were significantly lower in subjects with high BMI compared to nonobese subjects (P < 0·05). Conclusions A new specific and sensitive ELISA for nesfatin‐1 was established. Further accumulation of clinical observations is necessary to clarify the role of circulating nesfatin‐1 in various metabolic disorders.     

Endotoxin contamination in wound dressings made of natural biomaterials

Contamination by endotoxin of nine kinds of wound dressings made of natural biomaterials (calcium alginate, collagen, chitin, and poly-L-leucine) was examined with the use of water extracts. By applying the Limulus amoebocyte lysate (LAL) test, high concentrations of endotoxin were detected in extracts from three kinds of products made of calcium alginate. These extracts evoked fever in rabbits and induced the release of a proinflammatory (pyrogenic) cytokine, interleukin-6 (IL-6), from human monocytic cells (MM6-CA8). The effects disappeared when the extracts were treated with endotoxin-removing gel column chromatography or with an endotoxin antagonist, B464, confirming that the contaminating pyrogen was endotoxin. A noteworthy finding was that one of the endotoxin-containing extracts showed very weak IL-6-inducibility in human monocytic cells in contrast to its high pyrogenicity to rabbits. The discrepancy could be explained based on differences between humans and rabbits in sensitivity to the endotoxin, because the extract showed higher proinflammatory-cytokine (TNF-alpha)-inducibility in rabbit whole-blood cells (WBCs) than human WBCs. The results suggest that the LAL test is a useful method of detecting endotoxin contamination in wound dressings and the MM6-CA8 assay is a good supplement to the LAL test for evaluating pyrogenicity in humans accurately.     

Importance of controlling drug-resistant Pseudomonas aeruginosa infection: experience from lung transplantation in a cystic fibrosis case

Cystic fibrosis (CF) is rare in Japan. We encountered a CF case with drug-resistant Pseudomonas aeruginosa infection and successfully performed lung transplant from living related donors. A combination of beta-lactams and aminoglycosides for drug-resistant P. aeruginosa infection was administered before lung transplantation. Intravenous colistin was also used immediately before and after transplant surgery. Gram staining of respiratory specimens was performed every day after surgery and it was useful in monitoring infection status. Strict monitoring of infections by the Gram staining and culture of respiratory specimens is considered to be effective in preventing lower respiratory infection in lung transplantation.     

A set of genes selectively expressed in murine dendritic cells: utility of related cis-acting sequences for lentiviral gene transfer

Professional antigen presenting cells such as dendritic cells (DC) and macrophages (Mphi) share similar characteristics; however, they differ in their ability to initiate an immune response. DCs are much more potent in priming and stimulating nai;ve T-cells. Thus, DCs are good targets for the expression of foreign genes to elicit and specifically modify immune responses. To identify DC markers cDNA subtraction was performed using murine MHC class II(high), B7(high) bone marrow derived DCs as tester and interferon-gamma/E. coli lipopolysaccaride (LPS) treated bone marrow derived macrophages as driver. Analysis of 114 resulting clones revealed a diverse pattern of DC selective (DC(DeltaMphi)) gene expression including known genes whose expression in DCs had not been previously demonstrated as well as multiple novel genes. For several identified DC(DeltaMphi) genes, proximal promoter elements were isolated and incorporated into self-inactivating lentiviral GFP reporter vectors. Promoter activity was measured in bone marrow derived macrophages or dendritic cells. Of the promoters analyzed those for B7-DC and CCL17 drove strong GFP expression in DCs but not in resting or activated macrophages. The CCL17 promoter offered the highest level of expression in DCs and was further activated by culture with LPS or interleukin-4 (IL-4). In contrast, the B7-DC promoter was induced by IL-4 but not by LPS. Endogenous CCL17 and B7-DC mRNAs were increased similarly in IL-4 cultured DCs but only CCL17 was induced by LPS. Additionally, IL-4 increased cell surface expression of B7-DC in both immature and mature DCs.     

SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen,mediates uptake of the polysaccharide dextran

The marginal zone macrophages of the spleen are implicated in the clearance of polysaccharides, but underlying mechanisms need to be pinpointed. SIGN-R1 is one of five recently identified mouse genes that are homologous to human DC-SIGN and encode a single, external, C-terminal C-type lectin domain. We find that a polyclonal antibody to a specific SIGN-R1 peptide reacts primarily and strongly with a subset of macrophages in the marginal zone of spleen and lymph node medulla. In both sites, SIGN-R1 exists primarily in an aggregated form, resistant to dissociation into monomers upon boiling in SDS under reducing conditions. Upon transfection into three different cell lines, high-mol.-wt forms bearing SIGN-R1 are expressed, as well as reactivity with ER-TR9, a mAb previously described to react selectively with marginal zone macrophages. SIGN-R1-expressing macrophages preferentially sequester dextrans following i.v. injection. Likewise, when phagocytic cells are enriched from spleen and tested in culture, dextran is selectively endocytosed by a subset of very large SIGN-R1(+) cells representing approximately 5% of total released macrophages. Uptake of FITC-dextran by these macrophages in vivo and in vitro is blocked by ER-TR9 and polyclonal anti-SIGN-R1 antibodies. Following transfection with SIGN-R1, cell lines become competent to endocytose dextrans. The dextran localizes primarily to compartments lacking transferrin receptor and the LAMP-1 CD107a panlysosomal antigen. Therefore, SIGN-R1 mediates the uptake of dextran polysaccharides, and it is predominantly expressed in the macrophages of the splenic marginal zone and lymph node medulla.     

Association of Fcgamma receptor IIb and IIIb polymorphisms with susceptibility to systemic lupus erythematosus in Thais

We recently reported association of a newly identified polymorphism of Fcgamma receptor (FcgammaR) IIb, I232T, with systemic lupus erythematosus (SLE) in Japanese. To date, information on FcgammaR genotypes and their association with SLE is limited in South-east Asian populations. To gain further insight into the role of FcgammaR polymorphisms in the genetic predisposition of SLE, association of FcgammaRIIa-H131R, IIb-I232T, IIIa-F176V and IIIb-NA1/NA2 (HNA-1a/1b) polymorphisms with SLE was analyzed in the Thai population, using case-control association analysis. FcgammaRIIb-232T/T and IIIb-NA2/NA2 genotypes were associated with SLE with the odds ratio of 2.55. Genotype relative risk analysis revealed significant association of IIb-232T/T and IIIb-NA2/NA2, and a tendency of association of the IIIa-176F/F genotype. Moreover, carriers of FcgammaRIIa-131R were significantly increased in patients with lupus nephritis. Significant linkage disequilibrium was present among FcgammaRIIb, IIIa and IIIb, and two-locus analyses suggested that the tendency of association of FcgammaRIIIa could derive from linkage disequilibrium with IIb and IIIb. These results provided evidence that FcgammaR polymorphisms may be an important predisposing factor also in Thais in a complex manner.     

<Emphasis Type="Italic">Rice stripe virus</Emphasis> 23.9 K protein aggregates and forms inclusion bodies in cultured insect cells and virus-infected plant cells

Summary. The genome of Rice stripe virus (RSV, genus Tenuivirus) contains seven open reading frames (ORFs). Little is known about the products of four of these ORFs, including the 23.9K protein encoded by the virus-sense ORF of RNA3. Western blotting revealed that the 23.9K protein was synthesized in the host plant and also in the planthopper vector of RSV. Using a baculovirus vector, the 23.9K protein was expressed, both unfused and fused with red-shifted green fluorescent protein, in Spodoptera frugiperda cells. Inclusion bodies were observed by light microscope in cells expressing fused or unfused proteins. Inclusion bodies in cells expressing the fused protein fluoresced under blue light. By immunoelectron microscopy, electron-dense inclusion bodies in cells expressing the unfused protein were specifically labeled with 23.9K protein antiserum. Moreover, electron-dense masses labeled with 23.9K protein antiserum were observed in virus-infected wheat tissue by electron microscopy. This paper thus demonstrates that RSV 23.9K protein can aggregate in vivo and form inclusion bodies in infected plant tissue.Received December 24, 2003; accepted June 11, 2003 Published online August 7, 2003     

Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo

We have established stable, bright green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma clones. These cell lines showed similar cell proliferation rates and high-frequency experimental lung metastasis. The HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. HT-1080 cells were transduced with retroviral vectors containing GFP or RFP and the neomycin resistance gene. Stable transformants were selected stepwise with G418 up to 800 μl/ml. Subsequently, high GFP- or RFP-expressing clones, HT-1080-GFP or HT-1080-RFP, respectively, were selected. 3×10⁶ cells from each clone were mixed and injected into the tail vein of SCID mice. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies with different patterns visualized directly on excised lungs. The lung metastases were also visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Lung metastases were observed on the lung surface of all mice. SCID mice well tolerated multiple surgical procedures for direct-view imaging via skin-flap windows. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies described in this report gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of gene of interest. This revised version was published online in July 2006 with corrections to the Cover Date.