Effects of hypoxia and tumor necrosis factor-alpha on production of plasminogen activator inhibitor-1 in human proximal renal tubular cells

Chronic hypoxia has been newly proposed as a common mechanism of tubulointerstitial fibrosis in the progression of various chronic inflammatory renal diseases, where PAI-1 plays an important role in the accumulation of extracellular matrix (ECM) through inhibition of plasmin-dependent ECM degradation. In the present study, we investigated the presence of PAI-1 in renal tubular cells by immunostaining renal biopsy samples. We also closely examined the effects of hypoxia and TNF-alpha on PAI-1 expression in cultured human proximal renal tubular cells (HRCs). Confluent cells growth-arrested in DMEM for 24h were exposed to hypoxia (1% O2) and/or TNF-alpha at 10 ng/ml for 24 hours. Amounts of PAI-1 protein and mRNA after stimulation were measured by ELISA and TaqMan quantitative PCR, respectively and compared to those in cells incubated under control conditions (18% O2 without TNF-alpha). HIF-1alpha was demonstrated by immunoblot analysis. In crescentic glomerulonephritis, clusters of proximal tubules were specifically stained for PAI-1. Treatment of 24 hours with hypoxia, TNF-alpha and their combination induced a 2.7-fold, a 1.8-fold, and a 4.6-fold increase in PAI-1 protein secretion, and produced a 3.6-fold, a 3.3-fold, and a 12.1-fold increase at the PAI-1 mRNA level, respectively. Immunoblot analysis revealed that hypoxia-inducible factor-1alpha (HIF-1alpha) was markedly accumulated in the nuclear fraction after 16-hours exposure of HPTECs to hypoxia but not to TNF-alpha. In conclusion, hypoxia induces PAI-1 expression via remarkable nuclear accumulation of HIF-1alpha in HRCs. TNF-alpha can enhance this hypoxia-induced PAI-1 expression.     

Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar typhimurium

We evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains against Salmonella infection through infection studies with mice by using a Salmonella enterica serovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol. 184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 x 10(8) CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 10(3) to 10(4) CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate that Salmonella with a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.     

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.     

Systemic Administration of Immunostimulatory DNA Sequences Mediates Reversible Inhibition of Th2 Responses in a Mouse Model of Asthma

This study investigated whether immunostimulatory DNA sequences (ISS) induce a transient or sustained inhibition of Th2 responses to inhaled antigen. We sensitized mice with subcutaneous injections to develop a Th2 response to ovalbumin (ova) and then administered a dose of ISS prior to ova inhalation challenge. Mice were then rechallenged with ova by inhalation a second time at varying time points after the first ova inhalation (1 to 8 weeks later) to determine whether the ISS dose administered prior to the first ova inhalation protected against a subsequent second ova inhalation challenge. A single dose of ISS inhibited the Th2 response to the first inhalation of ova antigen, as well as 4 weeks later to the second inhalation of ova. However, ISS did not inhibit a Th2 response to the second inhalation of ova 8 weeks later. The reversible inhibition of Th2 responses at 8 weeks suggests the need for repeated ISS administration at monthly intervals.     

Microsatellite instability in primary and metastatic lung carcinomas

Fifty-seven primary lung carcinomas and 35 metastatic lung carcinomas were analyzed for microsatellite instability at 11 different chromosomal loci. Although no instability was detected in 37 small cell lung carcinomas (SCLC), it was frequently detected in non-small cell lung carcinomas (NSCLC) (16/55, 29%). In NSCLC, the incidence of replication errors (RERs) in metastatic tumors (12/22, 55%) was significantly higher than that in primary tumors (4/33, 12%) (P = 0.0021). Among 10 pairs of primary tumors and corresponding metastases, there were 4 cases which manifested the identical RER phenotypes in both primary and metastatic tumors. In two cases, RER phenotypes were detected in metastatic but not in primary tumors. Never was an RER phenotype found only in a primary tumor but not in the metastases. RERs were detected more frequently in stage III or IV tumors (3/8, 38%) than stage I or II tumors (1/25, 4%) (P = 0.0359). Tumor cells with allelic losses on chromosome arm 3p or 18q tended to have RER phenotypes (P = 0.0432 and P = 0.0187, respectively). The data suggest that microsatellite instability is common in NSCLC but not in SCLC, and that genomic instability appears late in tumor progression and plays an important role in the acquisition of more malignant phenotypes in NSCLC.     

Analysis of the dilution deviation in CA19-9 measurement

CA19-9 widely used as a tumor marker of the pancreas and a bile duct. There are a number of reports which describes the measured value discrepancies between RIA and non-RIA kits. RIA results also have shown lack of the linearity over 70 U/ml when the samples are diluted. The pH condition at assay reaction for RIA had been suggested as the major reason, it has been denied by the results from the same pH condition at assay reaction used by COBAS CORE CA19-9 EIA II. On the other hand, the lack of RIA antibody titer is indicated for the discordant results by changing the sample volume to reagent volume ratio in the reaction. Our further investigation also indicates that the specific Lewis blood type, i.e. Le (a-b+), shows the linearity issues by RIA. The discrepancies are not caused by the reaction pH, but the amount of the antibody used in the RIA kit is closely associated. Considering the CA19-9 antibody nature used in RIA kit, which covers broad molecular range, users need to pay more attention to setting up each laboratory's measuring range.     

Correlation between clinical pathologic factors and activity of 5-FU-metabolizing enzymes in colorectal cancer.

INTRODUCTION: Orotate phosphoribosyl transferase (OPRT), dihydropyrimidine dehydrogenase (DPD), and thymidylate synthase (TS) are initial key enzymes in the 5-fluorouracil (5-FU) metabolic pathway. The expression levels and activities of these three enzymes play important roles in the response of cancer patients to 5-FU-based chemotherapy. PURPOSE: The purpose of this study was to investigate the relationship between the activities of 5-FU metabolic enzymes and clinicopathologic factors in colorectal cancer. METHODS: We measured the activities of OPRT, DPD, and TS in colorectal cancer tissues. We also investigated the correlations between the activities of these three enzymes and clinicopathologic factors (histological type, depth of tumor invasion, extent of lymph node metastasis, Dukes' stage, lymphatic invasion, and vascular invasion). We examined 100 patients with surgically resected colorectal cancer. RESULTS: Poorly differentiated adenocarcinoma showed significantly higher DPD activities than did moderately differentiated or well-differentiated adenocarcinoma. In patients with lymph-node metastasis, OPRT activity was significantly lower than in patients without lymph-node metastasis. No significant relation was found between TS activity and histological type, depth of tumor invasion, extent of lymph node metastasis, Dukes' stage, lymphatic invasion, or vascular invasion. CONCLUSION: The response to 5-FU may be poor in patients with lymph-node metastasis, because of low OPRT activity, and in patients with poorly differentiated adenocarcinoma, because of high DPD activity.     

Identification of cryptochrome DASH from vertebrates

A new type of cryptochrome, CRY-DASH, has been recently identified. The CRY-DASH proteins constitute the fifth subfamily of the photolyase/cryptochrome family. CRY-DASHs have been identified from Synechocystis sp. PCC 6803, Vibrio cholerae, and Arabidopsis thaliana. The Synechocystis CRY-DASH was the first cryptochrome identified from bacteria, and its biochemical features and tertiary structure have been extensively investigated. To determine how broadly the subfamily is distributed within living organisms, we searched for new CRY-DASH candidates within several databases. We found five sequences as new CRY-DASH candidates, which are derived from four marine bacteria and Neurospora crassa. We also found many CRY-DASH candidates from the EST databases, which included sequences from fish and amphibians. We cloned and sequenced the cDNAs of the zebrafish and Xenopus laevis candidates, based on the EST sequences. The proteins encoded by the two genes were purified and characterized. Both proteins contained folate and flavin cofactors, and have a weak DNA photolyase activity. A phylogenetic analysis revealed that the seven candidates actually belong to the new type of cryptochrome subfamily. This is the first report of the CRY-DASH members from vertebrates and fungi.     

Large cell neuroendocrine carcinoma of the uterine cervix with cytogenetic analysis by comparative genomic hybridization: a case study

Large cell neuroendocrine carcinoma (LCNEC) of the uterine cervix is a newly introduced category of the revised World Health Organization classification. We reported a case of cervical LCNEC with cytogenetic analysis by comparative genomic hybridization (CGH). The cervical tumor showed moderately increased mitotic activity (8-14 mitotic figures per 10 high-power fields) and focal necrosis, which made it problematic to differentiate from atypical carcinoid. CGH analysis failed to detect chromosome 11q loss that has been reported to be characteristic of pulmonary atypical carcinoids. Furthermore, chromosome 3q amplification, which has been detected frequently in pulmonary small cell carcinomas and LCNECs but not in pulmonary typical and atypical carcinoids, was the most remarkable chromosomal aberration. Although CGH reports are extremely rare in neuroendocrine tumors of the uterine cervix, specific chromosomal aberrations may be useful in their distinction.