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We have recently developed an innovative bone marrow transplantation (BMT) method, intra-bone marrow (IBM)-BMT, in which donor bone marrow cells (BMCs) are injected directly into the recipient bone marrow (BM), resulting in the rapid recovery of donor hemopoiesis and permitting a reduction in radiation doses as a pretreatment for BMT. However, even with this IBM injection, some of the injected BMCs were found to enter into circulation. Therefore, we attempted to modify the method to allow the efficient retention of injected BMCs in the donor BM. The BMCs of enhanced green fluorescent protein transgenic mice (C57BL/6 background) were suspended in collagen gel (CG) or phosphate-buffered saline (PBS), and these cells were then injected into the BM of irradiated C57BL/6 mice. The numbers of retained donor cells in the injected BM, the day 12 colony-forming units of spleen (CFU-S) counts, and the reconstitution of donor cells after IBM-BMT were compared between the CG and PBS groups. The number of transplanted cells detected in the injected BM in the CG group was significantly higher than that in the PBS group. We next carried out CFU-S assays. The spleens of mice in the CG group showed heavier spleen weight and considerably higher CFU-S counts than in the PBS group. Excellent reconstitution of donor hemopoietic cells in the CG group was observed in the long term (>100 days). These results suggest that the IBM injection of BMCs suspended in CG is superior to the injection of BMCs suspended in PBS. Disclosure of potential conflicts of interest is found at the end of this article.

Neural cell adhesion molecules (CD56) are important adhesion molecules that are mainly expressed on neural cells and natural killer cells. Although freshly isolated cynomolgus monkey bone marrow cells (BMCs) contained only a few CD56-positive cells, almost all the BM adherent cells (obtained after a 2- to 3-week culture of the BMCs) were stained positively with anti-CD56 monoclonal antibody (mAb). The BM adherent cells showed uniformly fibroblastic morphology and were negative for hematolymphoid markers (CD4, CD8, CD11b, CD14, CD34, and CD45). Adipogenesis and osteogenesis were observed under inductive culture conditions. The BM adherent cells had the ability to support hemopoiesis of hemopoietic stem cells (HSCs) in vitro, and the proliferation of HSCs was significantly inhibited by the addition of anti-CD56 mAb to the coculture system. CD56 molecules were also expressed on HSCs because about 20% of an HSC-enriched population (lineage-negative and blast-gated cells) was positive for CD56. In addition, the immunostaining of monkey BM sections revealed that many stromal cells were CD56-positive, and some CD56-positive stromal cells came into direct contact with CD56-positive hemopoietic cells. These results indicate that the CD56 molecule is expressed on both HSCs and BM stromal cells (containing MSCs) in monkeys, and therefore it can be speculated that CD56 also contributes to the human hematopoietic system.

Cytoglobin (Cygb) is a recently discovered member of the vertebrate globin family, which includes probably most extensively studied proteins, hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb). It has been reported that Cygb is expressed ubiquitously at the mRNA or protein level. However, details of the distribution of Cygb in the various tissues have hitherto been unclear. In this experiment, we clarified the distribution of Cygb in various human tissues by immunohistochemical staining. First, we prepared a rabbit anti human Cygb polyclonal antibody. Using the antibody, we stained a tissue array slide containing 60 normal tissues from 40 human organs. We confirmed the staining patterns of the antibodies in these various tissues using autopsy samples from our university. In general, Cygb is positive in the epithelial cells, hepatocytes, pancreatic acinar cells, cardiomyocytes and skeletal muscle but rarely so in cells in the interstitial tissues. Cytoglobin is usually positive in the cytoplasm, but is also positive in the nucleus in some hepatocytes. In contrast, Cygb is negative in the smooth muscle. The distribution of Cygb could suggest its roles.

Female moths of Lyclene dharma dharma (Arctiidae, Lithosiinae) produced three pheromone components (I-III), which strongly stimulated male antennae. Using GC-MS analysis and chemical derivatizations, the following structures were estimated: 6-methyl-2-octadecanone (I), 14-methyl-2-octadecanone (II), and 6,14-dimethyl-2-octadecanone (III). While the stereochemistry of the chiral centers could not be determined because it was difficult to collect a sufficient amount of the natural pheromone, the plain structures of I and II were confirmed by synthesis of the racemic mixtures starting from diols. These methyl-branched ketones have not been identified as a natural product, indicating that they constitute a new chemical group of lepidopteran female sex pheromones.

Background— The involvement of Ca 2+ -dependent tyrosine kinase PYK2 in the Akt/endothelial NO synthase pathway remains to be determined. Methods and Results— Blood flow recovery and neovessel formation after hind-limb ischemia were impaired in PYK2 −/− mice with reduced mobilization of endothelial progenitors. Vascular endothelial growth factor (VEGF)–mediated cytoplasmic Ca 2+ mobilization and Ca 2+ -independent Akt activation were markedly decreased in the PYK2-deficient aortic endothelial cells, whereas the Ca 2+ -independent AMP-activated protein kinase/protein kinase-A pathway that phosphorylates endothelial NO synthase was not impaired. Acetylcholine-mediated aortic vasorelaxation and cGMP production were significantly decreased. Vascular endothelial growth factor–dependent migration, tube formation, and actin cytoskeletal reorganization associated with Rac1 activation were inhibited in PYK2-deficient endothelial cells. PI3-kinase is associated with vascular endothelial growth factor–induced PYK2/Src complex, and inhibition of Src blocked Akt activation. The vascular endothelial growth factor–mediated Src association with PLCγ1 and phosphorylation of 783 Tyr-PLCγ1 also were abolished by PYK2 deficiency. Conclusion— These findings demonstrate that PYK2 is closely involved in receptor- or ischemia-activated signaling events via Src/PLCγ1 and Src/PI3-kinase/Akt pathways, leading to endothelial NO synthase phosphorylation, and thus modulates endothelial NO synthase–mediated vasoactive function and angiogenic response.

Hedgehog-interacting protein (HHIP) was identified as a putative antagonist of the Hh pathway and as a target of Hh signalling. Our aim was to clarify the expression profiles and epigenetic alterations of the HHIP gene in gastrointestinal cancer. The expression and promoter epigenetic status of HHIP in cancer cell lines and freshly resected gastrointestinal cancer tissues were examined using RT-PCR, tissue microarray analysis, methylation-specific PCR, and chromatin immunoprecipitation assay. Cells were treated with the demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. WST-8 assays and in vitro invasion assays after treatment with HHIP-specific siRNA were performed. HHIP expression levels were reduced in most of the gastrointestinal cancer cell lines and in a certain subset of cancer tissues, and these were correlated with promoter hypermethylation. A heterochromatic structure characterized by neither acetylated H3 nor acetylated H4, and histone H3 lysine 9 hypermethylation and histone H3 lysine 4 hypomethylation was observed in cancer cells in which the HHIP gene was aberrantly silenced. On the other hand, overexpression of the HHIP gene was also found in some cancer tissues and there were significant correlations between protein expression levels of HHIP and those of Sonic hedgehog (Shh), Indian hedgehog, Patched, and glioma-associated oncogene homologue-1. An association was found between lymph node metastasis and HHIP silencing in colorectal cancer tissues with strong Shh expression and between advanced TNM stage and HHIP silencing in diffuse-type gastric cancer tissues with strong Shh expression. Down-regulation of HHIP expression by siRNA resulted in a significant increase in colon cancer cell growth and invasion in vitro. Silencing of the HHIP gene due to hypermethylation and chromatin remodelling appears to be frequently involved in gastrointestinal tumourigenesis.

Neural cell transplantation, a new therapeutic strategy for replacing injured neural components and obtaining functional recovery, has shown beneficial effects in animal models. Use of this strategy in human patients, however, requires that a number of serious issues be addressed, including ethics, immunorejection, and the therapeutic time window within which the procedure will be effective. Bone marrow-derived mononuclear cells (BM-MNC) are attractive for transplantation because they can be used as an autograft, can be easily collected within a short time period, and do not have to be cultured. In a rat model of spinal cord injury (SCI), we transplanted BM-MNC at 1 h after SCI at Th 8-9 by injecting them into the cerebrospinal fluid (CSF), and investigated the effect of this on neurologic function. In the acute stage of injury, we found a neuroprotective antiapoptotic effect, with an elevated concentration of hepatocyte growth factor in CSF. At 1 week after transplantation, the Basso-Beattie-Bresnahan locomotor score had increased significantly over its base-line value. In the chronic stage of injury, we observed suppressed cavity formation and functional improvement. We conclude that transplantation of BM-MNC after SCI has a remarkable neuroprotective effect in the acute stage of injury, suppressing cavity formation, and contributing to functional recovery. Our results suggest that transplantation of BM-MNC via the CSF is a potentially effective means of enhancing functional recovery after SCI in humans.

The collection of bone marrow cells (BMCs) using a perfusion method has been advantageous not only because of the low contamination of BMCs with T cells from the peripheral blood but also the enrichment of stromal cells, which support hemopoiesis. Before the application of this new method to humans, its safety needed to be confirmed using cynomolgus monkeys. We therefore performed the perfusion method on more than 100 cynomolgus monkeys using the long bones (such as the humerus and femur) and also the iliac bones (for human application); in the more than 150 trials to date, there have been no accidental deaths. Furthermore, the technical safety of a new method for the intra-bone marrow (IBM) injection of BMCs (termed IBM-bone marrow transplantation) has also been confirmed using 30 monkeys. Disclosure of potential conflicts of interest is found at the end of this article.

Summary RNA interference is a phenomenon whereby small double-stranded RNA knocks down the expression of a sequence-specific gene. Double-stranded siRNA transfection, as currently used, is considered to have transient and low transfection efficiency. We constructed an adenoviral vector-based short hair-pin(sh)RNA system to overcome the limitations of the genetic blockade of IGF-1R, one of most important cancer therapy targets. We constructed three different IGF-1R specific shRNAs (612, 801, and 3425) and generated three ad-shIGF-1Rs using BD Adeno-X™ expression system. We assessed the effect of ad-shIGF-1R on signal transduction, induction of apoptosis, and in vitro tumorigenicity of lung cancer cell lines. Western blot and FACS assays demonstrated that endogenous IGF-1R expression was efficiently suppressed after transduction of lung cancer cell lines with the three different ad-shIGF-1Rs. IGF-1R blockade by ad-shIGF-1R inhibited ligand induced phosphorylation of pAkt and pErk, and ad-shIGF-1R effectively blocked the in vitro tumorigenicity of lung cancer cell lines. Moreover, the transduction of a human lung cancer cell line with ad-IGF-1R(3425) enhanced chemosensitivity to anticancer drugs. We conclude that the adenoviral vector-based approach to the RNA interference of IGF-1R induced effective IGF-1R silencing in lung cancer cell lines as manifested by effective blocking of the downstream pathway of IGF-1R and by an antitumor effect. We believe that this system can be usefully applied to other cancer targets.

Gastric cancers with and without high-frequency microsatellite instability (MSI-H) represent distinctive pathways of carcinogenesis. The aim of this study was to clarify if human leukocyte antigen (HLA) class I antigen subunits and antigen processing machinery (APM) components are differentially downregulated in these two groups of tumours. Using reverse transcription PCR (RT-PCR), loss of heterozygosity (LOH) analysis, methylation-specific PCR (MSP), DNA sequencing, immunohistochemistry, and flow cytometry, we analysed expression and/or alteration of HLA class I antigen subunits and APM components, including low molecular weight polypeptide proteasome subunit (LMP)2, LMP7, LMP10, transporter associated with antigen processing (TAP)1, TAP2, tapasin, proteasome activator (PA) 28alpha, and PA28beta in two stage-matched panels of 30 MSI-H and 30 microsatellite stable (MSS) gastric cancers. Mutations at coding microsatellites (cMS) located within beta2-microglobulin (beta2m) and genes encoding APM components, including endoplasmic reticulum (ER) chaperone protein genes, such as calnexin, SEC63, SEC31, and P4HB (p55), were also analysed. HLA class Ia transcripts were totally downregulated in 18.3% of cancer cases. Locus-specific downexpression of HLA-A, -B, and -C was detected in 41.7%, 45.0%, and 31.7% of cases. Loss of HLA-A was significantly more frequent in MSI-H cancers. The LOH ratios of the HLA-A, -B, and -C loci microsatellite markers were relatively low: 5/32 (15.6%) for D6S306, 4/32 (12.5%) for D6S258, 4/33 (12.1%) for D6S273, and 4/30 (13.3%) for D6S1666. Methylation of HLA-A, -B, and -C was detected in 38.3%, 40.0%, and 28.3% of cases. A significant association between methylation and reduction in expression was observed in gastric cancer tissues. Mutations at cMS of beta2m and APM components were detected in 3.3-46.7% of MSI-H cancers but in none of MSS cancers. These data show that gastric cancers have various defects in HLA class I antigen subunits and APM components and that the MSI phenotype is associated with frequent HLA-A inactivation and frameshift mutations of the beta2m and APM genes.

We have recently found that allogeneic intrabone marrow-bone marrow transplantation (IBM-BMT) + donor lymphocyte infusion (DLI) using CD4+ cell-depleted spleen cells (CD4− cells) can prevent graft-versus-host disease (GvHD) but suppress tumor growth (Meth A: fibrosarcoma) in mice. In the present study, we show that allogeneic IBM-BMT + DLI using CD4− cells also has suppressive effects on the growth of colon cancer cells implanted not only in the skin but also in the liver of rats. First, we examined the effects of allogeneic IBM-BMT + DLI on the subcutaneously inoculated ACL-15 (rat colon cancer cell line). Lethally irradiated Fischer rats (F344 rats) were transplanted with T-cell-depleted bone marrow cells (BMCs) from Brown Norway (BN) rats. Simultaneously, DLI was performed using whole spleen cells (whole cells), CD4+ cell-depleted spleen cells (CD4− cells) or CD8+ cell-depleted spleen cells (CD8− cells) of BN rats. Although allogeneic IBM-BMT + DLI suppressed tumor growth, a considerable number of rats treated with allogeneic IBM-BMT + DLI using whole cells or CD8− cells died due to GvHD. In contrast, allogeneic IBM-BMT + DLI using CD4− cells also suppressed tumor growth, but there was no GvHD. Based on these findings, we next examined the effects of allogeneic IBM-BMT + DLI using CD4− cells on the cancer cells implanted in the liver. Allogeneic IBM-BMT + DLI using CD4− cells via the portal vein significantly prolonged the survival. These results suggest that allogeneic IBM-BMT + DLI using CD4− cells could become a new strategy for the treatment of solid tumors.

Morphologically, early colorectal tumors are divided into two groups, protruded-type tumors and flat-type tumors. Although some studies have shown genetic alterations in protruded-type tumors, little is known about genetic and epigenetic alterations in flat-type tumors, as well as pT1 (early invasive) colorectal cancers (CRCs). In the current study, we compared the frequencies of genetic and epigenetic alterations of the RAS-RAF and Wnt signaling pathways in flat-type and protruded-type tumors. In addition, we investigated the relationship between those alterations and invasive potential of pT1 CRCs. Methylations of RASSF2, O-6-methylguanine-DNA methyltransferase (MGMT), Wnt inhibitory factor-1 (WIF-1), EPHB2, CDKN2A and MLH1 were detected in 44.3, 30.3, 81.4, 7.5, 43.6 and 13.4% of the 307 early colorectal tumors, respectively. Mutations of KRAS, BRAF, catalytic subunit alpha of phosphatidylinositol 3'-kinase (PIK3CA) and beta-catenin were detected in 25.4, 4.6, 1.6 and 9.4% of those tumors, respectively. Methylations of MGMT, WIF-1 and CDKN2A were detected in significantly higher percentages of protruded-type tumors than in flat-type tumors. Mutation of at least one gene was detected in a significantly higher percentage of flat-type tumors than in protruded-type tumors. RASSF2 methylation was correlated significantly with KRAS, BRAF or PIK3CA mutation. Multiple logistic analysis showed that lymphatic invasion and RASSF2 methylation with KRAS, BRAF or PIK3CA mutation were independent risk factors for venous invasion in pT1 CRCs. In conclusion, since genetic alterations of these pathways have frequently occurred in flat-type tumors, flat-type tumors seem to have a distinct genetic profile different from that of protruded-type tumors. RASSF2 methylation with oncogenic activation is a promising biomarker for predicting invasive potential of pT1 CRCs.

Morphologically, colorectal adenomas can be divided into two groups, protruded-type and flat-type. However, the accurate frequencies of genetic and epigenetic alterations in flat-type colorectal advanced adenomas (laterally spreading tumors) have remained largely unknown. In the current study, we investigated genetic and epigenetic alterations in 101 flat-type colorectal advanced adenomas and 68 protruded-type colorectal advanced adenomas by using direct DNA sequencing and quantitative real-time PCR (MethyLight), respectively. KRAS mutation was detected in a significantly higher percentage of flat-type adenomas (35%) than in protruded-type adenomas (13%). When the samples were limited to the tumors in the distal colon, the difference of KRAS mutation was still significant. KRAS mutation in G-to-A transitions at codons 12 and 13 was detected in a significantly higher percentage of flat-type adenomas (26%) than in protruded-type adenomas (9%). BRAF and beta-catenin mutations were detected in 3 and 8% of the 101 flat-type adenomas, respectively. No significant difference was found between frequencies of those mutations in flat-type adenomas and protruded-type adenomas. Methylations of MGMT, CDKN2A (p16) and MLH1 were detected in 28, 33 and 9% of the 101 flat-type adenomas, respectively. CDKN2A methylation was detected in a significantly lower percentage of flat-type adenomas than in protruded-type adenomas (63%). Methylation of at least one gene was detected in a significantly lower percentage of flat-type adenomas (54%) than in protruded-type adenomas (78%). In conclusion, KRAS mutation was frequently detected in flat-type advanced adenomas and the mutational patterns in most of them with KRAS mutations were a transition from G-to-A. Therefore, these genetic alterations seem to play an important role in the development of flat-type advanced adenomas, especially in the distal colon. Epigenetic alterations infrequently occurred in flat-type advanced adenomas, suggesting that they have different genetic and epigenetic alterations from those of protruded-type advanced adenomas.

An intra-axial tumor measuring about 4 cm was excised from the right temporal lobe of a 35-year-old woman, who had a past history of resection of craniopharyngioma and postoperative radiation 21 years earlier. The tumor involved both the cortex and white matter, but was not attached to the dura mater. It consisted of a dense, interlacing, fascicular proliferation of atypical fibroblastic cells and was associated with an extensive, lace-like, desmoplastic change mainly involving the perivascular region around the tumor and overlying the subarachnoid space. The histopathological features of the desmoplastic change resembled meningioangiomatosis, but no proliferation of meningothelial cells was noted. The patient has been free from recurrence for 12 months since the operation. The association of primary cerebral fibrosarcoma with an extensive, lace-like, perivascular, desmoplastic change has not been documented in the literature. The radiation administered 21 years previously may have played some pathogenetic role in the perivascular desmoplastic change, and a malignant transformation of fibroblasts within the perivascular collagenous tissue is considered the most likely origin of the fibrosarcoma.

The human intestinal microbiome plays a major role in human health and diseases, including colorectal cancer. Colorectal carcinogenesis represents a heterogeneous process with a differing set of somatic molecular alterations, influenced by diet, environmental and microbial exposures, and host immunity. Fusobacterium species are part of the human oral and intestinal microbiota. Metagenomic analyses have shown an enrichment of Fusobacterium nucleatum (F. nucleatum) in colorectal carcinoma tissue. Using 511 colorectal carcinomas from Japanese patients, we assessed the presence of F. nucleatum. Our results showed that the frequency of F. nucleatum positivity in the Japanese colorectal cancer was 8.6% (44/511), which was lower than that in United States cohort studies (13%). Similar to the United States studies, F. nucleatum positivity in Japanese colorectal cancers was significantly associated with microsatellite instability (MSI)-high status. Regarding the immune response in colorectal cancer, high levels of infiltrating T-cell subsets (i.e., CD3+, CD8+, CD45RO+, and FOXP3+ cells) have been associated with better patient prognosis. There is also evidence to indicate that molecular features of colorectal cancer, especially MSI, influence T-cell-mediated adaptive immunity. Concerning the association between the gut microbiome and immunity, F. nucleatum has been shown to expand myeloid-derived immune cells, which inhibit T-cell proliferation and induce T-cell apoptosis in colorectal cancer. This finding indicates that F. nucleatum possesses immunosuppressive activities by inhibiting human T-cell responses. Certain microRNAs are induced during the macrophage inflammatory response and have the ability to regulate host-cell responses to pathogens. MicroRNA-21 increases the levels of IL-10 and prostaglandin E2, which suppress antitumor T-cell-mediated adaptive immunity through the inhibition of the antigen-presenting capacities of dendritic cells and T-cell proliferation in colorectal cancer cells. Thus, emerging evidence may provide insights for strategies to target microbiota, immune cells and tumor molecular alterations for colorectal cancer prevention and treatment. Further investigation is needed to clarify the association of Fusobacterium with T-cells and microRNA expressions in colorectal cancer.

We report a rare case of mucosa-associated lymphoid tissue (MALT) lymphoma of the common bile duct and the papilla of Vater. A 66-year-old man was admitted to our hospital with right upper quadrant pain and jaundice. Endoscopic retrograde cholangiopancreatography and magnetic resonance cholangiopancreatography showed common bile duct stenosis. Further endoscopy revealed swelling of the papilla of Vater, with erosion at the ileal end. The patient was pathologically diagnosed with MALT lymphoma and treated for Helicobacter pylori eradication, after which the lesions improved. However, 1 year later, the lesions began to enlarge again;thus, he was treated with 6 courses of combination chemotherapy (R-CHOP). After treatment, the tumor and erosion of the papilla of Vater disappeared and the stenosis of the common bile duct improved.

Background: The population in Japan is aging more rapidly than in any other country. However, no studies have determined the characteristics of the large population of elderly patients with colorectal tumors. Therefore, we examined the clinicopathological and molecular features of these tumors in elderly patients. Methods: In total, 1,627 colorectal tumors (393 serrated lesions, 277 non-serrated adenomas and 957 colorectal cancers) were acquired from patients. Tumor specimens were analyzed for BRAF and KRAS mutations, CpG island methylator phenotype-specific promoters (CACNA1G, CDKN2A, IGF2 and RUNX3), IGFBP7, MGMT, MLH1 and RASSF2 methylation, microsatellite instability (MSI) and microRNA- 31 (miR-31). Results: The frequency of elderly patients (aged ≥75 years) with sessile serrated adenomas (SSAs) with cytological dysplasia was higher than that of those with other serrated lesions and non-serrated adenomas (p < 0.0001). In elderly patients, all SSAs were located in the proximal colon (particularly the cecum to ascending colon). High miR-31 expression, MLH1 methylation and MSI-high status were more frequently detected in SSAs from elderly patients than in those from non-elderly patients. In contrast, no significant differences were found between older age of onset and high-grade dysplasia for traditional serrated adenomas or non-serrated adenomas in any of these molecular alterations. Conclusion: In elderly patients, all SSAs were located in the proximal colon. Furthermore, cytological dysplasia and molecular alterations were more frequently detected in elderly patients with SSAs than in non-elderly patients. Thus, careful colonoscopic examinations of the proximal colon are necessary for elderly patients because SSAs in those patients may exhibit malignant potential.

Fetal liver (FL) contains hepatic stem cells, hematopietic stem cells, and mesenchymal stem cells, as well as pluripotent stem cells and very small embryonic-like stem cells. FL has thus been available as a source of stem cells for regenerative medicine. Our previous report suggested that combining FL cell transplantation with the transplantation of fetal thymus prevented tumor growth in tumor-bearing mice. Moreover, in animal models, intra bone marrow-bone marrow transplantation (IBM-BMT) has proven to be the best approach for allogenic BMT. We here propose that transplanting FL cells by IBM-BMT can improve adipocyte functions in obese mice. FL cells were collected from FLs of C57BL/6 mice at 16 days post coitum, and then transplanted by IBM-BMT to db/db mice, an animal model of type 2 diabetes with obesity. Our results showed that the body weight was significantly lower in the treated db/db mice than in the sham-treated db/db mice. The plasma IL-6 level significantly decreased and adiponectin level significantly increased after the transplantation of FL cells. Blood glucose levels also significantly decreased although not to within the normal range. This is the first report that the transplantation of FL cells may improve adipocyte functions, resulting in decreased body weight in obese mice.

GVHD is a crucial mortality factor in allogeneic bone marrow transplantation (ABMT). In this paper, we show that dehydroxymethylepoxyquinomicin (DHMEQ), a novel inhibitor of nuclear factor-κB, suppresses GVHD, resulting in an improved mortality rate in a mouse ABMT model. Bone marrow cells from C57BL/6 mice (B6 mice) were transplanted into lethally irradiated BALB/c mice. Two weeks later, spleen cells from B6 mice were transplanted into the irradiated BALB/c mice. From one week after the injection of spleen cells, when the mice started to show GVHD, the mice were also injected intraperitoneally daily with DHMEQ or vehicle only (DMSO) for 4 weeks. By 80 days after the ABMT, 6/14 of the vehicle-injected mice (43%) had died because of GVHD, whereas all DHMEQ-injected mice survived this observation period and developed milder GVHD than the vehicle-injected mice. When regulatory T cells were reduced by the injection of anti-folate receptor 4 (FR4) antibody, the effects of DHMEQ were reduced. These findings suggest that administration of DHMEQ could become a new strategy for preventing fatalities from GVHD.

Most renal cell carcinomas (RCCs) are clear cell RCCs, the second most common being papillary RCCs (PRCCs). PRCCs are sub classified as type 1, type 2A, type 2B and mixed type. Pure type 2A PRCCs are estimated to account for only approximately 1-2% of malignant tumors of the kidney. In this paper, we show a case of an exophytic type 2A PRCC with massive necrosis arising in an 80-year-old male. The tumor, which measured 45mm in diameter and showed exophytic growth, was in the left kidney. The tumor was resected by a partial resection of the kidney and was found to be mainly necrotic, while the viable part showed papillary growth. There were small cuboidal cells with eosinophilic cytoplasm covering thin papillae with a single line of uniform small-to-medium sized nuclei and small nucleoli. The tumor was also positive for cytokeratin 7. Following Yang et al, we diagnosed the tumor as a type 2A PRCC. Conclusion: Type 2 PRCCs should be clearly differentially diagnosed into type 2A or 2B, since the prognosis is different between these two types.

Morphologically, early colorectal tumours can be divided into two groups, protruded-type and flat-type. However, little is known about genetic mechanisms of the latter. We investigated mutations of beta-catenin, KRAS, BRAF, and PIK3CA in 310 early colorectal tumours. beta-catenin mutation was detected in 7.1% of 310 tumours. beta-catenin mutation was detected in a significantly higher percentage of flat-type tumours with depressed areas (4/17, 23.5%) than in other tumours (18/293, 6.1%; p=0.0246). KRAS, BRAF, and PIK3CA mutations were detected in 21.6%, 5.4%, and 1.0% of 310 tumours, respectively. Concomitant mutations of beta-catenin and KRAS or BRAF were detected in seven tumours. Mutation of at least one gene was detected in a significantly higher percentage of flat-type tumour tissues (75/193, 38.9%) than in protruded-type tumour tissues (25/117, 21.4%; p=0.0014), and it was correlated significantly with size (p=0.0001). In conclusion, beta-catenin mutation seemed to play an important role in flat-type tumours, especially in those with depressed areas. The genetic abnormalities could arise and accumulate in the early stage of colorectal tumourigenesis, and seem to contribute to the development of flat-type tumour.

We have established a new method for allogeneic pancreatic islet (PI) transplantation: relatively low doses of irradiation followed by simultaneous transplantation of PIs and bone marrow cells (BMCs) via the portal vein (PV). In the present study, we have compared this method with intra-bone marrow (IBM)-bone marrow transplantation (BMT), and with a combination of both methods. Streptozotocin (STZ)-induced diabetic-recipient rats, Fischer 344 (F344, RT1A(l), RT1B(l)), were irradiated 1 day before transplantation. PIs of Brown Norway rats (BN, RT1A(n), RT1B(n)) were transplanted into the liver of the diabetic F344 rats via the PV. BMCs from BN rats were injected into the recipients' bone marrow (IBM), PV or intravenously (IV) or by a simultaneous combination of PV plus IBM (PV+IBM). We compared graft survival among the groups of '9 Gy+IBM'(10/10 accepted), '9 Gy+PV'(7/10 accepted), '9 Gy+IV'(0/7 accepted), '9 Gy+PV+IBM'(8/8 accepted), '8.5 Gy+IBM'(4/9 accepted), '8.5 Gy+PV'(0/7 accepted), '8.5 Gy+IV'(0/7 accepted), '8.5 Gy+PV+IBM'(9/12 accepted), '8 Gy+IBM'(2/10 accepted) and '8 Gy+PV+IBM'(2/8 accepted). As we reported previously, PV-BMT is more effective in inducing the acceptance of allogeneic PIs than IV-BMT. However, IBM-BMT requires less pretreatment than PV-BMT. (PV+IBM)-BMT was found to be the most effective in inducing the acceptance of allogeneic PIs. These results suggest that allogeneic PI-transplantation in conjunction with (PV+IBM)-BMT could become a viable strategy.

Abnormal activation of growth factor receptors and their signal pathways are required for neoplastic transformation and tumor progression. The concept of targeting specific tumorigenic receptors has been validated by successful clinical application of multiple new drugs, such as those acting against HER2/neu, epidermal growth factor receptor 1, and c-Kit. In this review, we focus on the next promising therapeutic molecular target of insulin-like growth factor (IGF)-I receptor (IGF-Ir). The IGF/IGF-Ir system is an important modifier of cancer cell proliferation, survival, growth, and treatment sensitivity in a number of neoplastic diseases, including human gastrointestinal carcinomas. Preclinical studies demonstrated that downregulation of IGF-Ir signals reversed the neoplastic phenotype and sensitized cells to antitumor treatments. We summarize a variety of ways to disrupt IGF-Ir function. Then, we introduce our strategy of adenoviruses expressing dominant negative of IGF-Ir (IGF-Ir/dn) against gastrointestinal cancers, including stomach, colon, and pancreas. IGF-Ir/dn suppresses tumorigenicity both in vitro and in vivo and increases stressor-induced apoptosis. IGF-Ir/dn expression upregulates chemotherapy-induced apoptosis and these combination therapies with chemotherapy are very effective against tumors in mice. Some drugs blocking IGF-Ir function are now entering clinical trial, thus IGF-Ir might be a candidate for a therapeutic target in several gastrointestinal malignancies.

Heme oxygenase-1 (HO-1) degrades heme into biliverdin, iron and CO. The enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli, and is induced in response to reactive oxygen species (ROS). 2',7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) is a common reagent used to detect ROS by the oxidation of 2',7'-dichlorodihydrofluorescin (DCFH) to fluorescent dichlorodihydrofluorescein. We previously found that rapid oxidation of DCFH occurred with heme-compounds as well as ROS [Ohashi, T. et al. (2002) FEBS Lett. 511, 21-27], and then examined the effect of DCFH-DA on the induction of HO-1 expression by arsenite, cadmium and hemin, which induce oxidative stress and cytotoxicity. We found suppression of the arsenite-, cadmium- and hemin-dependent induction of HO-1 with DCFH-DA. The suppression occurred at the transcriptional level since the promoter activity of the Maf-recognition site of the HO-1 gene decreased with the DCFH-DA treatment. DCFH abolished the phosphorylation of extracellular signal-regulated kinase, the nuclear translocation of a transcriptional activator Nrf2, and cell death. An antioxidant, N-acetylcysteine (NAC), also suppressed the induction by arsenite and cadmium, but not that by hemin, indicating that DCFH blocked a different site in the stress signal pathway from NAC. Considering that the oxidation of DCFH diminishes ROS generated by various stressors, our findings provide a potential strategy for protection of cells from toxic insults using DCFH-like molecules.

Matrix metalloproteinase (MMP)-7, also known as matrilysin, is a “minimal domain MMP” that exhibits proteolytic activity against components of the extracellular matrix (ECM). Matrilysin is frequently overexpressed in human cancer tissues and is associated with cancer progression. Tumorigenesis is a multi-step process involving cell growth, invasion, metastasis, and angiogenesis. Matrilysin has been shown to play important roles not only in degradation of ECM proteins, but also in the regulation of several biochemical processes such as activation, degradation, and shedding of non-ECM proteins. This minireview provides a summary of the current literature on the roles of matrilysin in tumorigenesis with a focus on the roles of modifications of non-ECM proteins by matrilysin and other related MMPs in tumorigenesis. Proteolysis of insulin-like growth factor binding protein by matrilysin results in increased bioavailability of insulin-like growth factors and enhanced cellular proliferation. Matrilysin has also been implicated in the ectodomain shedding of several cell surface molecules. Heparin-binding epidermal growth factor precursor (proHB-EGF) is cleaved by matrilysin into mature HB-EGF, which promotes cellular proliferation. Membrane-bound Fas ligand (FasL) is cleaved into soluble FasL, which increases apoptosis of cells adjacent to tumor cells. E-cadherin is converted to soluble E-cadherin to promote invasion. Tumor necrosis factor (TNF)-alpha precursor is cleaved to release soluble TNF-alpha to increase apoptosis. We propose that these matrilysin-mediated pathways provide the necessary and logical mechanisms to promote cancer progression.

Background Although lung transplantation is now an established treatment for end-stage lung diseases, allogeneic transplantation of parenchymal organs requires immunosuppressive therapy to prevent rejection. It has been reported that bone marrow transplantation (BMT) induces specific tolerance to donor organs. We have recently discovered a new method for BMT, which is called intra–bone marrow (IBM) BMT, in which bone marrow cells (BMCs) are injected directly into the bone marrow cavity. We demonstrate that IBM-BMT can be used to induce tolerance even in simultaneous lung transplantations in rats without administering any immunosuppressants. Methods Allogeneic lung transplantation was carried out from Brown Norway to Lewis rats. Simultaneously, IBM-BMT was carried out. Results Transplantation of nonirradiated lung or nontreated BMCs (T cell–containing BMCs) induced graft vs host disease. Therefore, the donor lung was irradiated, and T cells in the BMCs were depleted by anti-CD4, anti-CD5, and anti-CD8 antibodies plus anti-mouse antibody–coated magnetic beads. Lung allografts with conventional (intravenous) BMT failed to induce tolerance. However, the recipients treated with lung allografts plus IBM-BMT, which showed either mixed chimerism or full chimerism of hematopoietic cells, did not show symptoms of graft rejection or graft vs host disease, even without the use of immunosuppressants. Conclusions These results suggest that simultaneous lung transplantation and IBM-BMT (but not conventional BMT) is effective in inducing persistent tolerance without the use of immunosuppressants.

Background and aims: Insulin-like growth factor (IGF) I receptor (IGF-Ir) signalling is required for carcinogenicity and proliferation of many tumours but this pathway has not been studied in detail in gastric cancer. We have previously shown successful therapy for colorectal, pancreatic, and lung cancer using recombinant adenoviruses expressing dominant negative (dn) IGF-Ir. In this study, we sought to better dissect the role of IGF-Ir on progression of gastric cancer and determine whether IGF-Ir targeted adenoviruses represent potentially effective therapeutics for human gastric cancer. Methods: We assessed the effect of IGF-Ir ligands on proliferation and survival in gastric cancer cells in culture. Then, recombinant adenoviruses expressing truncated IGF-Ir (482 and 950 amino acids long, IGF-Ir/dn) that function as dn inhibitors were studied in the treatment of human gastric cancer xenografts. We characterised the effects of IGF-Ir/dn on signalling blockade, growth, apoptosis induction, and in vivo therapeutic efficacy. Results: IGF-Ir signalling promoted tumour growth and survival in gastric cancer. IGF-Ir/dn expression suppressed tumorigenicity both in vitro and in vivo and upregulated stressor induced apoptosis. IGF-Ir/dn blocked Akt-1 activation induced by IGF-I, IGF-II, and des(1-3)IGF-I, but not by insulin. IGF-Ir/dn expression increased radiation and chemotherapy induced apoptosis and the combination of IGF-Ir/dn and chemotherapy was very effective against tumours in mice. In an intraperitoneal model, IGF-Ir/dn therapy also suppressed peritoneal dissemination. Conclusions: IGF-Ir is involved in the regulation of survival and cell growth in human gastric cancer and may be a good molecular therapeutic target. Adenovirus-IGF-Ir/dn may thus have therapeutic use in gastric cancer.

Choriocarcinomas usually develop in the uterus and ovaries in the female, being extremely rare in the extragenital organs in the male. Extragenital choriocarcinomas in the male usually develop in the mediastinum or retroperitoneum. The frequency of choriocarcinoma in the urinary bladder is extremely low. The purpose of the present paper was to report an autopsy case of choriocarcinoma in the urinary bladder in the male. An 81-year-old male patient with macrohematuria was first diagnosed with transitional cell carcinoma (TCC). At autopsy a hemorrhagic necrotic tumor, which was found in the urinary bladder with metastatic lesions in the lungs, was diagnosed as choriocarcinoma microscopically. There was no evidence for choriocarcinoma derived from any other organs than the urinary bladder, although there were metastatic lesions in both lungs and the direct invasion into the prostate. From these findings it is concluded that the tumor was a primary choriocarcinoma in the urinary bladder in a male patient. Choriocarcinoma of the urinary bladder is very rare, but the prognosis is extremely poor in comparison with TCC even in the urinary bladder. Therefore, it is essential to clearly discriminate between choriocarcinomas and TCC.

It is generally accepted that most colorectal carcinomas arise in pre-existing adenomas. Morphologically, colorectal adenomas can be divided into two groups, protruded type and flat type. The aim of this study was to clarify relevant alterations of gene expression associated with the early stage of colorectal carcinogenesis. Using cDNA array, we analysed the expression profiles of 550 cancer-related genes in 36 colorectal adenomas (18 flat-type and 18 protruded-type adenomas) and 14 early invasive carcinomas. Among the 550 genes, we chose 32 genes the average expression levels of which were at least three-fold up- or downregulated in tumour tissues compared with levels in matched normal tissues. A total of 13 and 19 genes were identified as up- and downregulated genes in tumour tissues, respectively. Among the upregulated genes, the average expression levels of E1AF, bone morphogenic protein (BMP)-4, insulin-like growth factor (IGF)-2, inducible nitric oxide synthase (iNOS), tissue inhibitors of metalloproteinase (TIMP)-1, Smad4, and nm23 in tumour tissues were over five times higher than those in matched normal tissues. Colorectal adenomas and early invasive carcinomas were divided into two major clusters by clustering analysis. Moreover, flat- and protruded-type adenomas were divided into two major clusters by clustering analysis. The expression profiles obtained by the cDNA array clearly indicate that colorectal adenomas and early invasive carcinomas have specific expression profiles. Likewise, the gene expression profiles of flat- and protruded-type adenomas are different. These results indicate that molecular classification of early colorectal tumours by a cDNA array is feasible.

Donor lymphocyte infusion (DLI) is clinically used for the treatment of malignant tumors. We have found recently that intra-bone marrow–bone marrow transplantation (IBM-BMT) can be used to treat various autoimmune diseases, even when radiation doses are reduced. In addition, recently we have found that IBM-BMT can prevent not only graft failure but also graft-versus-host disease (GvHD). Based on these findings, we attempted to prevent and treat the progression of a tumor (Meth-A cell line: BALB/c-derived fibrosarcoma) by DLI plus IBM-BMT. When the tumors had grown to approximately 10 × 10 mm, the tumor-bearing BALB/c (H-2d) mice were irradiated with 5 Gy, and whole spleen cells from C57BL/6J (B6) (H-2b) mice (as DLI) were then intravenously injected into the BALB/c mice. Simultaneously, bone marrow cells (BMCs) from B6 mice were injected directly into the bone marrow cavity of the BALB/c mice (IBM-BMT). The tumors decreased in size, but the mice died of GvHD. However, when CD4+ T-cell–depleted spleen cells were used for DLI, the recipients showed only mild GvHD and survived longer, due to the slow growth of the tumor. In contrast, when CD8+ T-cell–depleted spleen cells were used for DLI, the recipients showed more severe GvHD than those injected with whole spleen cells. These results suggest that IBM-BMT plus DLI (the depletion or reduction of a certain cell population like CD4+ T cells) could be helpful to suppress both GvHD and tumor growth.

Cisplatin, which is a broadly used anticancer drug, is widely known to induce acute renal failure as a result of renal tubular injury. This article examines whether G-CSF and/or M-CSF rescues mice from renal failure induced by cisplatin. BALB/c mice received intraperitoneal injections with or without G-CSF and/or M-CSF for 5 d (from day -5 to day -1). The day after the last injection of G-CSF and/or M-CSF (day 0), the mice received an intraperitoneal injection of cisplatin. When pretreated with G-CSF or G-CSF + M-CSF, the mice showed longer survival and lower serum creatinine and blood urea nitrogen levels than mice that had been received injections of M-CSF or saline. Histologically, pretreatment with G-CSF or G-CSF + M-CSF attenuated the damage to renal tubules induced by cisplatin. BALB/c mice that had received a transplant of bone marrow cells of enhanced green fluorescent protein (EGFP)-transgenic mice ([EGFP-->BALB/c] mice) were treated with or without G-CSF and/or M-CSF, followed by injection of cisplatin as well as above. [EGFP-->BALB/c] mice that were treated with G-CSF or G-CSF + M-CSF showed a significantly higher number of EGFP(+) tubular epithelial cells in the kidney than mice that were treated with only M-CSF or saline. These results suggest that bone marrow cells mobilized by G-CSF accelerate the improvement in renal functions and prevent the renal tubular injury induced by cisplatin and that M-CSF enhances the effects of G-CSF.

We report an autopsy case of malignant lymphoma of the cranial vault. The patient was an 85-year-old woman who exhibited a painless subcutaneous scalp lump associated with no neurological abnormalities. CT scan and magnetic resonance imaging of the head showed an extra-intra cranial isodensity lesion of the cranial vault. Autopsy revealed that the tumor was composed of medium-sized cells which were immunoreactive with CD45, CD20, CD79a, and CD10, and a diagnosis of peripheral B cell lymphoma, Burkitt-like, was made. Non-Hodgkin lymphoma originating from the cranial vault is extremely rare; a search of the English literature revealed only 16 previously reported cases. Herein, we review this singular calvarial lymphoma.

Background Hepatic portal venous gas (HPVG) is a rare entity commonly associated with intestinal necrosis and fatal outcome, and various underlying diseases have been reported. Pancreatic solitary metastasis without local extension is also rare in esophageal squamous cell carcinoma. Methods This report describes an interesting and unusual case of HPVG arising from pancreatic tumor. Autopsy revealed pathogenesis of HPVG and synchronous tumors of the esophagus and pancreas. Results A 73-year-old man developed synchronous double tumor in the esophagus and pancreas several months before acute abdomen and his death, which were generated by HPVG. Autopsy revealed that HPVG was caused by gastric wall infarction owing to expansion of an isolated pancreatic metastasis from esophageal squamous cell carcinoma. Conclusions This is the first case of HPVG that was derived from pancreatic tumor infiltration. If he had been diagnosed with solitary pancreatic metastasis from esophageal squamous cell carcinoma in the first time, he might have an option for chemotherapy, which could let him live longer.

Purpose: The aim of this study was to characterize the relationship of insulin-like growth factor (IGF)-II expression with IGF-I, IGF-I receptor (IGF-IR), cyclooxygenase-2 (COX-2), and matrix metalloproteinase (MMP)-7 in early colorectal carcinogenesis. Experimental Design: With the semiquantitative reverse transcriptase-PCR, 90 human colorectal tumor tissues (63 adenomas and 27 submucosal pT1 cancers) were analyzed for IGF-II, IGF-IR, IGF-I, COX-2, and MMP-7 expression. Ninety-nine adenoma tissues and 60 pT1 cancer tissues were also analyzed immunohistochemically for IGF-II expression. Loss of imprinting of the IGF-II gene was analyzed. Paired carcinoma and adenoma tissues obtained from a carcinoma in adenoma lesion was analyzed by a cDNA array. Results: IGF-II mRNA expression was detected in 37.8% of the 90 colorectal tumor tissues. The frequency of IGF-II mRNA expression was significantly higher in pT1 cancer (70.4%) than in adenoma (23.8%). Immunohistochemical IGF-II expression was also more frequently detected in pT1 cancer (58.3%) than in adenoma (25.3%). Loss of imprinting of the IGF-II gene was observed in 15 (44.1%) of the 34 colorectal tumors in which IGF-II was overexpressed. IGF-II expression was positively correlated with the expression of IGF-IR and IGF-I. COX-2 and MMP-7 mRNA expression was detected in 42.2% and 77.8% of the tumor tissues, respectively, and both were positively correlated with IGF-I, IGF-II, and IGF-IR expression. IGF-II was the most differentially expressed gene between carcinoma and adenoma lesions. Conclusions: IGF-II, in conjunction with IGF-IR, IGF-I, COX-2, and MMP-7, seems to play a key role in the early stage of colorectal carcinogenesis.

Pulmonary hypertension associated with increased pulmonary vascular resistance occurring in the setting of portal hypertension, referred to as "portopulmonary hypertension", is a complication of chronic liver disease, and occurs in 2% to 3% of patients with portal hypertension. Portal hypertension is a relatively common finding in patients with chronic myeloproliferative disorder (CMPD). Pulmonary hypertension is also an occasional finding in CMPD patients. Latent myeloproliferative disorder, on the other hand does not fulfill the diagnostic criteria of classical CMPD and is characterized by younger age of onset, slow disease progression, a high risk of thrombosis, platelet dysfunction, and normal or increased platelet count in spite of the presence of splenomegaly. We report findings in a 50-year-old woman with portal hypertension for which there were three major etiological findings-increased splenic blood flow, infiltration of hematopoietic cells in the liver, and thrombosis in the portal or hepatic vein-over a 25-year clinical course, during which there was also reversible stenosis of the portal vein. Twenty-three years after her first admission, her condition was diagnosed as latent myeloproliferative disorder, and she developed pulmonary hypertension. Her clinical history and data indicated that the portopulmonary hypertension was due to the latent myeloproliferative disorder.

Any alteration in the synthesis and breakdown of the extracellular matrix is important in tissue remodeling during inflammation and wound healing. The degradation of the extracellular matrix components is regulated by a cascade of matrix metalloproteinases (MMPs). The present study attempted to assess the relationship between MMPs and the degree of inflammation in ulcerative colitis. Methods: The expression of MMPs, including MMP-1, -2, -3, -7 (matrilysin), and -9, and that of their inhibitors (tissue inhibitor of metalloproteinases [TIMP]-1 and -2) were analyzed immunohistochemically by using 52 formalin-fixed and paraffin-embedded specimens from patients with ulcerative colitis who had undergone a biopsy or surgery. Results: It was observed that MMP-1, -2, and -9, and the TIMPs were expressed in stromal cells, MMP-3 was expressed in both the epithelial cells and stromal components, and matrilysin was expressed only in the epithelial cells on the edge of ulcers. The expression of the MMPs was increased compared with that of the TIMPs. The frequency of matrilysin expression was increased corresponding to the severity of the inflammation. Matrilysin was also expressed in epithelial cells with dysplasia and cancer. Conclusions: Matrilysin expression could be an important marker of activity and could be used for the prediction of subsequent transformation in patients with ulcerative colitis.

Lesions of the pancreas induced by viral infection have drawn relatively little attention because of their low incidence, and the histopathologic features of viral pancreatitis have not been fully elucidated. We report the autopsy findings of 2 patients, a 59-year-old woman with allergic granulomatous angiitis and a 73-year-old man with invasive pulmonary aspergillosis who had a disseminated visceral herpes simplex virus (HSV) infection. In both cases, the liver was the organ most severely affected by the viral infection. The pancreas showed multiple small foci of hemorrhagic necrosis, which were not accompanied by fat necrosis of the surrounding adipose tissue. Histopathologically, Cowdry type A intranuclear inclusions and a ground-glass appearance of the nuclei were found in many degenerated acinar cells around the necrotic foci. The gross appearance and histopathologic features of HSV pancreatitis were characteristic and, in particular, distinct from those of the more common acute hemorrhagic pancreatitis. Immunohistochemistry using an anti-HSV antibody revealed immunoreactivity in the intranuclear inclusions and ground-glass nuclei, and polymerase chain reaction analysis disclosed that the causative virus in these 2 cases was HSV-1. Herpes simplex virus pancreatitis constitutes a rare, but distinct pathologic entity among a group of acute pancreatitis diseases with diverse etiopathogenesis.

The objective of this report was to review 365 cases of Brånemark Implant Bridge including 1,444 fixtures in patients of Tokyo Dental College Chiba Hospital. The term of implantation was divided into several phases; less than 1 year, from 1 year to 3 years, from 3 years to 5 years, from 5 years to 7 years, from 7 years to 10 years, more than 10 years, and the survival rate was calculated for each phase. The removal rate of fixture after connecting the superstructure was 13% in maxillary cases and 2% in mandibular cases. The functioning survival rate in maxillary cases slightly decreased from 91% in less than 1 year to 87% after more than 10 years; however, the functioning survival rate in mandibular cases was about 99% in all periods. The removal rate of fixtures per patient was 23% in maxillary cases and 6% in mandibular ones. The average removal number of fixtures was 1.8 in maxillary cases and 1.2 in mandibular ones. The removal of the fixture occurred most frequently at less than 1 year in maxillary cases, but there was no tendency for a pattern of removal of fixture in mandibular cases.

Overexpression of the matrix serine protease (MSP) trypsin has been implicated in tumour growth, invasion, and metastasis. The objective of this study was to clarify the clinicopathological and prognostic significance of trypsin expression in colorectal cancer. This study analysed the association between immunohistochemically detected trypsin expression in colorectal cancer and clinicopathological characteristics, and investigated whether trypsin is a predictor of recurrence and/or survival. Trypsin immunoreactivity was more intense at the invasive front than in the superficial part of the tumour. Sections with immunostaining signals in more than 30% of carcinoma cells at the invasive front, which were observed in 48 cases (48%), were judged to be positive for trypsin. Trypsin positivity was significantly correlated with depth of invasion, lymphatic and venous invasion, lymph node and distant metastasis, advanced pathological tumour-node-metastasis (TNM) stage, and recurrence. Patients with trypsin-positive carcinoma had significantly shorter overall and disease-free survival periods than did those with trypsin-negative carcinoma. Trypsin retained its significant predictive value for overall and disease-free survival in multivariate analysis that included conventional clinicopathological factors. It is well known that trypsin activates matrilysin (matrix metalloproteinase-7), which plays an important role in colorectal cancer progression. Patients with concordant overexpression of trypsin and matrilysin at the invasive front, in which they were often co-localized, had the worst prognosis. Trypsinogen-1-transfected HCT116 colon cancer cells showed not only trypsin activity, but also active matrilysin activity and were more invasive in vitro than mock-transfected HCT116 cells. These results suggest that trypsin plays a key role in the progression of colorectal cancer. Detection of trypsin expression as well as matrilysin is useful for the prediction of recurrence in and poor prognosis of colorectal cancer patients.

The continuous growth of tumors depends on the altered regulation of the cell cycle, which is in turn modulated by signals from growth factors and their receptors. Blockade of insulin-like growth factor (IGF)-I and IGF-IR by antisense or dominant negative plasmid transfection can suppress tumorigenicity and induce regression of established tumors. We have constructed two recombinant adenoviruses: an adenovirus expressing truncated IGF-IR (ad-IGF-IR/950) with an engineered stop codon at amino acid residue 950, and an adenovirus expressing the soluble extracellular domain of IGF-IR (ad-IGF-IR/482) with an engineered stop codon at amino acid residue 482. Ad-IGF-IR/950 produces a defective receptor with an intact alpha subunit and a defective beta subunit lacking the tyrosine kinase domain. Dominant negative inhibition results from competition of the defective receptor with normal IGF-IR subunits, or the competition with normal IGF-IR for ligand by the soluble receptor. We were able to show here that ad-IGF-IR/950 induced the increased expression of IGF-IR on the cell surface and ad-IGF-IR/482 induced the secretion of the soluble fragment of IGF-IR. The transduction of both ad-IGF-IR/950 and ad-IGF-IR/482 could blunt the growth-stimulatory effect of IGF-I on human lung cancer cell lines. Both ad-IGF-IR/950 and ad-IGF-IR/482 effectively blocked IGF-I-induced Akt kinase activation. Intratumoral injection of ad-IGF-IR/482 virus showed significant growth suppression in established lung cancer xenografts. These findings suggest that these ad-IGF-IR/dn (950, 482) have the potential to be effective and practical cancer gene therapy strategies.

Process extension is a most marked and characteristic neuronal feature that is observed during the development, regeneration and plasticity of nervous system tissues. Neuro-p24, a novel membranous protein with a molecular weight of 24 kDa, is specifically localized in neurons, particularly in the neurites. Based on its molecular structure and distribution pattern in the brain we proposed that Neuro-p24 plays a role in neurite extension. In the present study we have made several findings that support this hypothesis; first, Neuro-p24 was abundant in motor axonal fibers, neurites of dorsal root ganglia neurons and apical dendrites of cerebral cortex neurons when their extension or arborization was proceeding very actively. Secondly, when COS-7 epithelial cells were transfected with either wild-type or deletion-mutated Neuro-p24 cDNAs, ectopic expression of wild-type cDNA caused morphological alterations resulting in a neuron-like appearance. These observations firmly support our proposal and indicate that Neuro-p24 plays an important role in the nervous tissue.

An 88-year-old man developed pneumothorax and pleural effusion. After the finding of ingesta in the pleural effusion, a gastropleural fistula was diagnosed. A biopsy specimen of the stomach revealed non-Hodgkin lymphoma, diffuse large-cell, B-cell type. Autopsy examination demonstrated that the malignant lymphoma had originated from high-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Gastropleural fistula is an uncommon condition, as the diaphragm forms a thick barrier between the stomach and the thoracic cavity. We review 25 reported cases of gastropleural fistula found in a MEDLINE search from 1966 to 2000. In only 3 of the 25 patients was malignant lymphoma reported to have caused the gastropleural fistula.

It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. We therefore examined whether bone marrow stem cells can differentiate into retinal neural cells in adult rats. PKH-67-labeled stem cell-enriched bone marrow cells (BMCs) were injected into the vitreous space of eyes in which the retinas had been mechanically injured using a hooked needle. Two weeks after the injection of these cells, immunohistochemical examinations were carried out. The stem cell-enriched BMCs had been incorporated and had differentiated into retinal neural cells in the injured retina. The stem cell-enriched BMCs had accumulated mainly in the outer nuclear layer around the injured sites. The incorporated cells expressed glial fibrillary acidic protein, calbindin, rhodopsin, and vimentin. These results raise the possibility that stem cell-enriched BMCs have the ability to differentiate into retinal neural cells, and that the injection of stem cell-enriched BMCs into the retina would help repair damaged retinal cells.

Pleomorphic adenomas gene-like 2 (PLAGL2) protein containing seven C(2)H(2) zinc finger motifs exhibits DNA binding and transcriptional activation activity and is expressed in response to hypoxia or iron deficiency. To identify the target genes of PLAGL2, we transfected mouse PLAGL2 cDNA into Balb/c3T3 fibroblasts and neuroblastoma Neuro2a cells. Both cells were induced to undergo apoptosis by the expression of PLAGL2 as judged by assays of TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling), DNA fragmentation, propidium iodide staining, and the binding of annexin V to the cell surface. The treatment of the cells with an iron chelator, desferrioxamine, resulted in the induction of apoptosis with a concomitant accumulation of PLAGL2 in the nucleus. The expression of PLAGL2 in Balb/c3T3 cells led to the mRNA expression of a proapoptotic factor, Nip3, which can dimerize with Bcl-2. Nip3 mRNA was also induced in desferrioxamine-treated cells. Furthermore, the Nip3 promoter containing a hypoxia-responsive element was activated by PLAGL2, independent of hypoxia-inducible factor-1 (HIF-1). The transfection of antisense oligonucleotide to mouse Nip3 mRNA into PLAGL2-expressing cells led to a decrease in apoptotic cells compared with sense oligonucleotide-transfected cells. Despite the activation of DNA-HIF-1 binding activity under hypoxic conditions, neither an accumulation of HIF-1 alpha nor the activation of HIF-1 was observed following the expression of PLAGL2. These results indicate that PLAGL2 is located downstream of HIF-1 and suggest that PLAGL2 functions as a tumor suppressor in association with HIF-1.

Background. We have established a new method for the transplantation of allogeneic pancreatic islets (PIs) using sublethal irradiation (9 Gy) plus simultaneous transplantation of PIs and bone marrow cells (BMCs) via the portal vein (PV) followed by intravenous (i.v.) injection of donor BMCs (9 Gy + PV + i.v.). Methods. Approximately 600 PIs of Brown Norway (BN: RT1A n , RT1B n ) rats were transplanted into the liver of streptozotocin-induced diabetic Fischer 344 (F344: RT1A 1 , RT1B 1 ) rats via the PV. BMCs (3×10 8 ) of BN rats were injected via the PV or i.v. into the recipients simultaneously. In some groups, additional i.v. injections of BMCs from BN rats were given 5 days after the PI transplantation. Results. All the recipients (10 of 10) in the 9 Gy + PV + i.v. group showed normoglycemia for more than 1 year, whereas PIs were rejected within 30 days after transplantation in the group of 9 Gy + i.v. + i.v. Conclusions. These results suggest that simultaneous transplantation of PIs and BMCs via the PV is effective in inducing persistent tolerance.

It is well known that dendritic cells (DCs) are developed from the peripheral blood of mice when peripheral blood mononuclear cells (PBMCs) are cultured with GM-CSF. We have previously found that immature DCs are present in the blood even in humans. In the present study, we show that CD11c+ CD3- B220- cells in the mouse peripheral blood are immature DCs. The percentage of CD11c+ CD3- B220- cells in the (PBMCs) of normal mice ranges from 0.5 to 2.5%. The CD11c+ CD3- B220- cells in the PBMCs show dendrites, similar in shape to the CD11c+ CD3- B220- cells in the spleen, which are thought to be DCs definitely. However, they have practically no capacity to stimulate the proliferation of allogeneic T cells, and show a lower expression of MHC class II, B7-1 and B7-2 than CD11c+ CD3- B220- cells in the spleen. When the CD11c+ CD3- B220- cells in the PBMCs are cultured with GM-CSF, they show not only the potent ability to stimulate the proliferation of allogeneic T cells but also a higher expression of MHC class II, B7-1 and B7-2. Moreover, they migrate into the spleen when they are injected intravenously. These results suggest that CD11c+ CD3- B220- cells in the PBMCs are immature DCs, and that they migrate into the spleen, where they mature.