Tadashi Hanafusa

Selective delivery of beta cell antigen to dendritic cells in vivo leads to deletion and tolerance of autoreactive CD8+ T cells in NOD mice.

Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet β cells. Cytotoxic CD8+ T cells, reactive to β cell antigens, are required for T1D development in the NOD mouse model of the disease, and CD8+ T cells specific for β cell antigens can be detected in the peripheral blood of T1D patients. It has been evident that in nonautoimmune-prone mice, dendritic cells (DCs) present model antigens in a tolerogenic manner in the steady state, e.g., in the absence of infection, and cause T cells to proliferate initially but then to be deleted or rendered unresponsive. However, this fundamental concept has not been evaluated in the setting of a spontaneous autoimmune disease. To do so, we delivered a mimotope peptide, recognized by the diabetogenic CD8+ T cell clone AI4, to DCs in NOD mice via the endocytic receptor DEC-205. Proliferation of transferred antigen-specific T cells was initially observed, but this was followed by deletion. Tolerance was achieved because rechallenge of mice with the mimotope peptide in adjuvant did not induce an immune response. Thus, targeting of DCs with β cell antigens leads to deletion of autoreactive CD8+ T cells even in the context of ongoing autoimmunity in NOD mice with known tolerance defects. Our results provide support for the development of DC targeting of self antigens for treatment of chronic T cell-mediated autoimmune diseases.

Serum gamma-interferon-inducing factor (IL-18) and IL-10 levels in patients with acute hepatitis and fulminant hepatic failure.

Background and Aims: The aim was to determine the role of T‐helper (Th)1/Th2 cytokine responses in the clinical outcome of patients with acute liver injury.

Aberrant promoter methylation of insulin‐like growth factor binding protein‐3 gene in human cancers

Insulin‐like growth factor binding protein‐3 (IGFBP‐3) is postulated to be a mediator of growth suppression signals. Here, we examined the methylation status of IGFBP‐3 to correlate to clinicopathological factors in human cancers. The methylation status of IGFBP‐3 was determined by bisulfite DNA sequencing and was correlated with expression semi‐quantified by real‐time RT‐PCR to develop a methylation‐specific PCR (MSP) assay for IGFBP‐3. Using the MSP assay, we examined the methylation status of IGFBP‐3 in gastric cancer (GC), colorectal cancer (CRC), breast cancer (BC) and malignant mesothelioma (MM). IGFBP‐3 methylation was detected in 6 of 13 (46%) and 16 of 24 (67%) GC cell lines and tumors, respectively; 4 of 8 (50%) and 15 of 26 (58%) CRC cell lines and tumors, respectively; 3 of 11 (27%) and 7 of 39 (18%) BC cell lines and tumors, respectively and 1 of 5 (20%) and 18 of 56 (32%) MM cell lines and tumors, respectively. Interestingly, the methylation status of MM specimens from Japanese patients (75%, 12 out of 16 patients) was significantly higher than those from the USA (15%, 6 out of 40 patients) (p < 0.0001), suggesting the presence of ethnic differences in the IGFBP‐3 methylation status. We also found that IGFBP‐3 methylation was preferentially present in GCs arising in the lower‐third of the stomach (p = 0.079). In summary, our results showed that IGFBP‐3 methylation played an important role in the silencing of its expression, suggesting that IGFBP‐3 may act as a tumor suppressor gene in several human cancers examined.

Altered expression of vascular endothelial growth factor, fibroblast growth factor-2 and endostatin in patients with hepatocellular carcinoma

Background: Advanced hepatocellular carcinoma (HCC) in humans is characterized by hypervascularity. In the present study, the expressions of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF‐2) and endostatin were analyzed in patients with chronic liver disease to clarify the effect of these major angiogenic factors.

Decreased expression of B7 costimulatory molecules and major histocompatibility complex class-I in human hepatocellular carcinoma

Background and Aim:  We analyzed the expression of antigen‐processing and antigen‐presenting molecules in surgically resected fresh samples of human hepatocellular carcinoma (HCC) tissue to elucidate a mechanism of immune escape. We also examined the expression of interleukin (IL)‐10 protein, which might act to downregulate expression of antigen‐processing and antigen‐presenting molecules.

Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation

BackgroundInsulin-like growth factor binding protein (IGFBP)-3 functions as a carrier of insulin-like growth factors (IGFs) in circulation and a mediator of the growth suppression signal in cells. There are two reported p53 regulatory regions in the IGFBP3 gene; one upstream of the promoter and one intronic. We previously reported a hot spot of promoter hypermethylation of IGFBP-3 in human hepatocellular carcinomas and derivative cell lines. As the hot spot locates at the putative upstream p53 consensus sequences, these p53 consensus sequences are really functional is a question to be answered.MethodsIn this study, we examined the p53 consensus sequences upstream of the IGFBP-3 promoter for the p53 induced expression of IGFBP-3. Deletion, mutagenesis, and methylation constructs of IGFBP-3 promoter were assessed in the human hepatoblastoma cell line HepG2 for promoter activity.ResultsDeletions and mutations of these sequences completely abolished the expression of IGFBP-3 in the presence of p53 overexpression. In vitro methylation of these p53 consensus sequences also suppressed IGFBP-3 expression. In contrast, the expression of IGFBP-3 was not affected in the absence of p53 overexpression. Further, we observed by electrophoresis mobility shift assay that p53 binding to the promoter region was diminished when methylated.ConclusionFrom these observations, we conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells.

Telomerase reverse transcriptase gene amplification in hepatocellular carcinoma.

Background and Aim:  Telomerase activation is essential for the immortality of cancer cells. The expression of telomerase reverse transcriptase (hTERT), the catalytic component of the telomerase complex, regulates telomerase activity in human cancers. Amplification of the hTERT gene, located at chromosome 5p, is thought to be a potential genetic event contributing to telomerase activation in sporadic tumors.

Estimation of soil-to-plant transfer factors of radiocesium in 99 wild plant species grown in arable lands 1 year after the Fukushima 1 Nuclear Power Plant accident.

One year after the deposition of radionuclides from the Fukushima 1 Nuclear Power Plant (A formal name is Fukushima Daiichi Nuclear Power Station) in March 2011, radiocesium (134Cs, 137Cs) concentrations ([Cs]) were comprehensively investigated in the wild plants of 99 species most of which were annual or summer green perennial herbs and started to grow from April 2012 at the heavily contaminated fields of paddy (three study sites) and upland (one study site) in Fukushima Prefecture. The survey was conducted three times (April, July and October) in the year. In each site, soils (soil cores of 5-cm depth) and plants (aerial shoots) were collected for determination of [Cs] on a dry weight basis, and then the transfer factor (TF) of radiocesium from soil to plant ([Cs]plant/[Cs]soil) was estimated in each species. The [Cs] values of both soils and plants largely varied. However, some species exhibited relatively high TF values (more than 0.4) (e.g., Athyrium yokoscense, Dryopteris tokyoensis, and Cyperus brevifolius), while others exhibited almost negligible values (less than 0.01) (e.g., Salix miyabeana, Humulus scandens, and Elymus tsukushiensis). In addition, judging from the 11 species grown in both paddy and upland fields, TF values were generally higher in the paddy fields. The estimation of phytoextraction efficiency of soil radiocesium by weed communities in the paddy fields suggests that the weed community is not a practical candidate for phytoremediation technique.

Identification of the antigens predominantly reacted with serum from patients with hepatocellular carcinoma

To identify antigens specifically recognized by the immune surveillance system in patients with hepatocellular carcinoma (HCC), the authors examined two complementary DNA (cDNA) libraries of moderately differentiated HCC by serologic analysis of recombinant cDNA expression libraries (SEREX).

Isolation and characterization of Escherichia coli hag operator mutants whose hag48 expression has become repressible by a Salmonella H1 repressor

SummaryThe expression of an Escherichia coli K12 flagellin gene, hagA48, is insensitive to the Salmonella H1 repressor (rh1−).By selecting merodiploid cells H2-rh1on-off/F′hag48 for motility in the presence of anti-H48 serum, mutants which had escaped from inhibition by the serum because of repression of their hag48 expression by rh1+ were isolated. Their nucleotide sequences were examined in the region containing the promoter, the position of which was confirmed by S1 nuclease analysis of the transcriptional initiation site. The two independently isolated mutants had the same heptamer insertion AGACGAT at a site overlapping with the promoter sequence, creating a putative operator sequence homologous to Salmonella H1, but not to H2. Other candidates for operator mutants had reduced flagellar synthesis because of mutations between the transcriptional and translational initiation sites or in the structural gene. The sequence analysis also revealed a repetitive extragenic palindrome (REP) consensus sequence and a transcriptional terminator of hag48 in a small, functionally unknown open reading frame (ORF).