Hiroshi Hiai

Development of Lupus-like Autoimmune Diseases by Disruption of the PD-1 Gene Encoding an ITIM Motif-Carrying Immunoreceptor

PD-1, a 55 kDa transmembrane protein containing an immunoreceptor tyrosine-based inhibitory motif, is induced in lymphocytes and monocytic cells following activation. Aged C57BL/6(B6)-PD-1(-/-) congenic mice spontaneously developed characteristic lupus-like proliferative arthritis and glomerulonephritis with predominant IgG3 deposition, which were markedly accelerated by introduction of a Fas mutation (lpr). Introduction of a PD-1 null mutation into the 2C-TCR (anti-H-2Ld) transgenic mice of the H-2(b/d) background resulted in the chronic and systemic graft-versus-host-like disease. Furthermore, CD8+ 2C-TCR+ PD-1(-/-) T cells exhibited markedly augmented proliferation in vitro in response to H-2d allogenic cells. Collectively, it is suggested that PD-1 is involved in the maintenance of peripheral self-tolerance by serving as a negative regulator of immune responses.

Persistent oxidative stress in cancer

DNA of cancers such as renal cell carcinoma and mammary invasive ductal carcinoma, is persistently exposed to more oxidative stress than that of adjacent nornal tissue. We suggest that the concept of ‘persistent oxidative stress in cancer’ may open up a new research area, explaining part of the characteristic tumor biology of cancer such as activated transcription factors and proto‐oncogenes, genomic instability, chemotherapy‐resistance, invasion and metastasis.

Inhibition of gastric inhibitory polypeptide signaling prevents obesity

Secretion of gastric inhibitory polypeptide (GIP), a duodenal hormone, is primarily induced by absorption of ingested fat. Here we describe a novel pathway of obesity promotion via GIP. Wild-type mice fed a high-fat diet exhibited both hypersecretion of GIP and extreme visceral and subcutaneous fat deposition with insulin resistance. In contrast, mice lacking the GIP receptor (Gipr−/−) fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Moreover, double-homozygous mice (Gipr−/−, Lepob/Lepob) generated by crossbreeding Gipr−/− and obese ob/ob (Lepob/Lepob) mice gained less weight and had lower adiposity than Lepob/Lepob mice. The Gipr−/− mice had a lower respiratory quotient and used fat as the preferred energy substrate, and were thus resistant to obesity. Therefore, GIP directly links overnutrition to obesity and it is a potential target for anti-obesity drugs.

Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas.

It has been reported that the rho genes, which consist of a ras-related small GTPase protein family, regulate cytoskeletal structures and have the potential to transform cultured cells. To investigate the biological relevance of the rho genes in pancreatic carcinogenesis, we examined expressions of the rhoA, B and C genes by polymerase chain reaction after reverse transcription (RT-PCR) in 33 cases of ductal adenocarcinoma of the pancreas. In addition, mutations of the K-ras, rhoA, B and C genes were studied in the same series of tumour tissues to correlate with rho gene expressions. The expression levels of the rhoC gene were significantly higher in tumours than in non-malignant portions (P < 0.001). Metastatic lesions overexpressed the rhoC gene compared with primary tumours (P < 0.05). Carcinoma tissues with perineural invasion and lymph node metastasis exhibited significantly higher expressions of the rhoC gene than tumours without these manifestations (P < 0.001 and P < 0.05 respectively). Overexpression of the rhoC gene significantly correlated with poorer prognosis of patients with pancreatic adenocarcinoma (P < 0.05). In contrast, the expression levels of the rhoA and B genes showed no significant relationship with clinicopathological findings. Mutation was not found either in the rhoA, B or C gene sequences examined. K-ras gene mutation, detected in 27 out of 33 (81.8%) cases, did not affect the expression levels in any of the rho genes. These suggest that elevated expression of the rhoC gene may be involved in the progression of pancreatic carcinoma independent of K-ras gene activation.

Candidate markers for stem and early progenitor cells, Musashi‐1 and Hes1, are expressed in crypt base columnar cells of mouse small intestine

Musashi‐1, a neural RNA‐binding protein, is important for maintaining neural stem cells. Both Musashi‐1 and Hes1, a transcriptional factor regulated by Musashi‐1, are expressed in the small intestine. Here we show that Musashi‐1 is present in a few epithelial cells just above the Paneth cells in the small intestinal crypt, the putative position of stem cells, whereas Hes1 is expressed in lower crypt cells just above the Paneth cells, including Musashi‐1‐positive cells. Musashi‐1 and Hes1 were not expressed in Paneth cells. Notably, Musashi‐1 and Hes1 were coexpressed in the crypt base columnar cells located between the Paneth cells. These findings suggest that not only the cells just above Paneth cells but also the crypt base columnar cells between the Paneth cells have stem cell characteristics.

Biomarker evidence of DNA oxidation in lung cancer patients: association of urinary 8‐hydroxy‐2′‐deoxyguanosine excretion with radiotherapy, chemotherapy, and response to treatment

Ratios of urinary 8‐hydroxy‐2′‐deoxyguanosine to urinary creatinine (8‐OHdG/creatinine) have been considered as a good biological indicator of DNA oxidation. Urinary 8‐OHdG/creatinine levels of lung cancer patients were evaluated by enzyme‐linked immunosorbent assay using a monoclonal antibody N45.1 during radiotherapy and chemotherapy. An increase in urinary 8‐OHdG/creatinine was found in non‐small‐cell carcinoma (non‐SCC) patients during the course of radiotherapy. SCC patients showed higher levels of urinary 8‐OHdG/creatinine than the controls. Furthermore, SCC patients with complete or partial response to the chemotherapy showed a significant decrease in urinary 8‐OHdG/creatinine while patients with no change or progressive disease showed an increase.

New biomarker evidence of oxidative DNA damage in patients with non-insulin-dependent diabetes mellitus

Urinary 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) has been reported to serve as a sensitive biomarker of oxidative DNA damage and also of oxidative stress. We have investigated oxidative DNA damage in patients with non‐insulin‐dependent diabetes mellitus (NIDDM) by urinary 8‐OHdG assessments. We determined the total urinary excretion of 8‐OHdG from 24 h urine samples of 81 NIDDM patients 9 years after the initial diagnosis and of 100 non‐diabetic control subjects matched for age and gender. The total 24 h urinary excretion of 8‐OHdG was markedly higher in NIDDM patients than in control subjects (68.2±39.4 μg vs. 49.6±37.7 μg, P=0.001). High glycosylated hemoglobin was associated with a high level of urinary 8‐OHdG. The increased excretion of urinary 8‐OHdG is seen as indicating an increased systemic level of oxidative DNA damage in NIDDM patients.

Generation of Knockout Rats with X-Linked Severe Combined Immunodeficiency (X-SCID) Using Zinc-Finger Nucleases

Background Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. Methodology/Principal Findings We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. Conclusions and Significance The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct

Monoclonal antibodies directed against proteins modified with the major membrane lipid peroxidation product, 4‐hydroxy‐2‐nonenal, have been established and characterized. The monoclonal antibodies specific for HNE‐modified proteins were raised by immunizing mice with a HNE‐keyhole limpet hemocyanin conjugate. The resulting five monoclonal antibodies (mAbs HNEJ‐1–5) recognized HNE‐modified bovine serum albumin (BSA), but not native BSA in Western blot studies. Of the five mAbs, HNEJ‐2 exhibited the highest affinity for HNE‐modified proteins and a much higher affinity for the HNE‐histidine adduct than the HNE‐lysine or HNE‐cysteine adducts. mAb HNEJ‐2 did not cross‐react with proteins that had been treated with other aldehydes, such as 1‐hexenal, 2‐hexenal, 4‐hydroxy‐2‐hexenal, 2‐nonenal, formaldehyde, or glutaraldehyde. These results suggest that the major epitope recognized by mAb HNEJ‐2 is the Michael addition‐type HNE‐histidine adduct.

Increased intratumor Valpha24-positive natural killer T cells: a prognostic factor for primary colorectal carcinomas.

Purpose: Human invariant natural killer T (NKT) cells are novel, distinct lymphocyte populations with a restricted T-cell receptor repertoire (Vα24-Vβ11). They play a pivotal role in immunoregulation and in antitumor activities. This study focused on Vα24+ NKT cells in colorectal carcinomas and their clinicopathologic significance. Experimental Design: Vα24+ NKT-cell infiltration immunohistochemistry was studied in a total of 103 colorectal carcinoma cases. The degree of NKT-cell infiltration in tumors was evaluated as low (<7 NKT cells/5 HPF) or high (≥7 NKT cells/5 HPF). The correlation between the degree of infiltrated Vα24+ NKT cells and clinicopathologic variables was studied statistically. Results: A small number of Vα24+ NKT cells were found in the normal colorectal mucosa (2.6 ± 3.7 cells/5 HPF); however, their number increased remarkably in colorectal carcinomas (15.2 ± 16.3 cells/5 HPF; P = 0.0003) and a majority showed phenotype of activation. Higher NKT-cell infiltration was more frequent in women than in men (P = 0.034) and correlated with fewer lymph node metastases (P = 0.042). Patients with high NKT-cell infiltration showed higher overall (P = 0.018) as well as disease-free (P = 0.0006) survival rates. Intratumor NKT-cell infiltration was an independent prognostic factor for the overall (P = 0.033) and disease-free (P = 0.0064) survival rates. Conclusions: Increased infiltration of Vα24+ NKT cells was observed in colorectal carcinomas. Higher Vα24+ NKT-cell infiltration in colorectal carcinomas was an independent prognostic factor for favorable prognosis.