A high incidence of WT1 abnormality in bilateral Wilms tumours in Japan, and the penetrance rates in children with WT1 germline mutation.

BACKGROUND: Bilateral Wilms tumours (BWTs) occur by germline mutation of various predisposing genes; one of which is WT1 whose abnormality was reported in 17-38% of BWTs in Caucasians, whereas no such studies have been conducted in East-Asians. Carriers with WT1 mutations are increasing because of improved survival. METHODS: Statuses of WT1 and IGF2 were examined in 45 BWTs from 31 patients with WT1 sequencing and SNP array-based genomic analyses. The penetrance rates were estimated in WT1-mutant familial Wilms tumours collected from the present and previous studies. RESULTS: We detected WT1 abnormalities in 25 (81%) of 31 patients and two families, which were included in the penetrance rate analysis of familial Wilms tumour. Of 35 BWTs from the 25 patients, 31 had small homozygous WT1 mutations and uniparental disomy of IGF2, while 4 had large 11p13 deletions with the retention of 11p heterozygosity. The penetrance rate was 100% if children inherited small WT1 mutations from their fathers, and 67% if inherited the mutations from their mothers, or inherited or had de novo 11p13 deletions irrespective of parental origin (P=0.057). CONCLUSIONS: The high incidence of WT1 abnormalities in Japanese BWTs sharply contrasts with the lower incidence in Caucasian counterparts, and the penetrance rates should be clarified for genetic counselling of survivors with WT1 mutations.

The role of mitochondria-derived reactive oxygen species in the pathogenesis of non-steroidal anti-inflammatory drug-induced small intestinal injury.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been implemented in clinical settings for a long time for their anti-inflammatory effects. With the number of NSAID users increasing, gastroenterological physicians and researchers have worked hard to prevent and treat NSAID-induced gastric mucosal injury, an effort that has for the large part being successful. However, the struggle against NSAID-induced mucosal damage has taken on a new urgency due to the discovery of NSAID-induced small intestinal mucosal injury. Although the main mechanism by which NSAIDs induce small intestinal mucosal injury has been thought to depend on the inhibitory effect of NSAIDs on cyclooxygenase (COX) activity, recent studies have revealed the importance of mitochondria-derived reactive oxygen species (ROS) production, which occurs independently of COX-inhibition. ROS production is an especially important factor in the increase of small intestinal epithelial cell permeability, an early stage in the process of small intestinal mucosal injury. By clarifying the precise mechanism, together with its clinical features using novel endoscopy, effective strategies for preventing NSAID-induced small intestinal damage, especially targeting mitochondria-derived ROS production, may be developed.

Reversed intestinal malrotation with concurrent cecal carcinoma.

A 57-year-old man was admitted with a type 2 (ulcerated with clear margin) cancer in the cecum. Contrast-enhanced CT showed that the superior mesenteric vein was anterior to the superior mesenteric artery, and the patient was suspected of having intestinal malrotation. A laparoscopic-assisted ileocecal resection was performed. At operation, the cecum and the transverse colon passed through the root of the mesentery behind the superior mesenteric artery with the duodenum. Therefore, this was thought to be a reversed-type intestinal malrotation. After the operation, 3D-CT colonography with duodenography images were reconstructed to retrospectively confirm the diagnosis of a reversed malrotation. These images clearly demonstrated the abnormal anatomy and overall orientation of the intestine. Patients with a reversed intestinal malrotation and concurrent cecal cancer are extremely rare. Herein, we present a patient who underwent a laparoscopic-assisted ileocecal resection for cecal cancer that presented concurrently with a reversed intestinal malrotation.

Gene expression signature and response to the use of leucovorin, fluorouracil and oxaliplatin in colorectal cancer patients

PURPOSE: FOLFOX (a combination of leucovorin, fluorouracil and oxaliplatin) has achieved substantial success in the treatment of colorectal cancer (CRC) patients. However, about half of all patients show resistance to this regimen and some develop adverse symptoms such as neurotoxicity. In order to select patients who would benefit most from this therapy, we aimed to build a predictor for the response to FOLFOX using microarray gene expression profiles of primary CRC samples. PATIENTS AND METHODS: Forty patients who underwent surgery for primary lesions were examined. All patients had metastatic or recurrent CRC and received modified FOLFOX6. Responders and nonresponders were determined according to the best observed response at the end of the first-line treatment. Gene-expression profiles of primary CRC were determined using Human Genome GeneChip arrays U133. We identified discriminating genes whose expression differed significantly between responders and nonresponders and then carried out supervised class prediction using the k-nearest-neighbour method. RESULTS: We identified 27 probes that were differentially expressed between responders and nonresponders at significant levels. Based on the expression of these genes, we constructed a FOLFOX response predictor with an overall accuracy of 92.5%. The sensitivity, specificity, positive and negative predictive values were 78.6%, 100%, 100% and 89.7%, respectively. CONCLUSION: The present model suggests the possibility of selecting patients who would benefit from FOLFOX therapy both in the metastatic and the adjuvant setting. To our knowledge, this is the first study to establish a prediction model for the response to FOLFOX chemotherapy based on gene expression by microarray analysis (AU)

Galectin-1 promotes basal and kainate-induced proliferation of neural progenitors in the dentate gyrus of adult mouse hippocampus.

We examined the expression of galectin-1, an endogenous lectin with one carbohydrate-binding domain, in the adult mouse hippocampus after systemic kainate administration. We found that the expression of galectin-1 was remarkably increased in activated astrocytes of the CA3 subregion and dentate gyrus of the hippocampus, and in nestin-positive neural progenitors in the dentate gyrus. Quantitative reverse transcription PCR (RT-PCR) analysis revealed that the galectin-1 mRNA level in hippocampus began to increase 1 day after kainate administration and that a 13-fold increase was attained within 3 days. Western blotting analysis confirmed that the level of galectin-1 protein increased to more than three-fold a week after the exposure. We showed that isolated astrocytes express and secrete galectin-1. To clarify the significance of the increased expression of galectin-1 in hippocampus, we compared the levels of hippocampal cell proliferation in galectin-1 knockout and wild-type mice after saline or kainate administration. The number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells detected in the subgranular zone (SGZ) of galectin-1 knockout mice decreased to 62% with saline, and to 52% with kainate, as compared with the number seen in the wild-type mice. Most of the BrdU-positive cells in SGZ expressed doublecortin and neuron-specific nuclear protein, indicating that they are immature neurons. We therefore concluded that galectin-1 promotes basal and kainate-induced proliferation of neural progenitors in the hippocampus.

Loss of imprinting of IGF2 correlates with hypermethylation of the H19 differentially methylated region in hepatoblastoma.

IGF2, a maternally imprinted foetal growth factor gene, is implicated in many childhood tumours including hepatoblastoma (HB); however, the genetic and epigenetic alterations have not comprehensively been studied. We analysed the methylation status of the H19 differentially methylated region (DMR), loss of heterozygosity (LOH) and allelic expression of IGF2 in 54 HB tumours, and found that 12 tumours (22%) with LOH, 9 (17%) with loss of imprinting (LOI) and 33 (61%) with retention of imprinting (ROI). Biallelic and monoallelic IGF2 expressions correlated with hypermethylation and normal methylation of H19 DMR, respectively, in two tumours with LOI and seven tumours with ROI. Quantitative RT-PCR analysis showed minimal expression of H19 mRNA and substantial expression of IGF2 mRNA in tumours with LOH or LOI, and substantial expression of both H19 and IGF2 mRNAs in tumours with ROI. Increased IGF2 expression with predominant embryonic P3 transcript was found in the majority of HBs with ROI and foetal livers. In contrast to the earlier reports, our findings suggest that the disruption of the enhancer competition model reported in Wilms' tumour may also occur in HB. Both frequencies of LOH and LOI seem to be lower in HB than in Wilms' tumour, reflecting the different tissue origins.

Metformin, but not pioglitazone, decreases postchallenge plasma ghrelin levels in type 2 diabetic patients: a possible role in weight stability?

AIM: Effects of metformin and pioglitazone on body weight are clearly different. Recently, the role of ghrelin, an orexigenic peptide derived from stomach, has been appreciated. Plasma ghrelin levels display a preprandial peak and postprandial suppression, suggesting its physiological role. We hypothesized that metformin or pioglitazone may modulate circulating ghrelin levels and this modulation may be related to differential effects on body weight with these agents. METHODS: Thirty-five Japanese type 2 diabetic patients [21 men and 14 women, age 62 +/- 2 years, body mass index (BMI) 26.6 +/- 0.5 kg/m(2) and haemoglobin A1c (HbA1c) 8.2 +/- 0.1%, mean +/- s.e.] were randomly assigned to groups for the addition of metformin or pioglitazone. At baseline and 4 months later, a 75-g oral glucose tolerance test (OGTT) was performed to measure plasma ghrelin levels. RESULTS: In 33 subjects who completed the study, the overall decrease in HbA1c ( approximately 1%) was comparable between the two groups. As expected, BMI increased in the pioglitazone group but not in the metformin group. After the treatment, plasma ghrelin levels at each point of OGTT remained unchanged in the pioglitazone group. In the metformin group, fasting ghrelin levels were unaltered, whereas the absolute levels at 30, 60 and 120 min decreased significantly. The area under the curve for the 2-h ghrelin profile also decreased significantly. CONCLUSIONS: Metformin, but not pioglitazone, decreased plasma ghrelin levels after the glucose load. This decrease may in part account for weight stability in type 2 diabetic patients treated with metformin.

Assessment of efficacy of a live oral poliovirus vaccine for virulent Sabin-like poliovirus 1 strains in Japan.

Virulent Sabin-like poliovirus (VSLP) was isolated from river and sewage waters between October 1993 and September 1995 in Toyama Prefecture, Japan (Yoshida et al., Lancet 356, 1461-1463, 2000). In this study, to assess the possibility of an epidemic of poliomyelitis caused by a VSLP in Japan under the current vaccination policy of administration of live attenuated oral poliovirus vaccine (OPV), we determined titers of serum neutralizing antibodies to poliovirus 1 (PV-1) strains Sabin (vaccine strain), Mahoney (wild-type strain) and G4-12 (VSLP) in various groups of residents of Toyama Prefecture, Japan. The seropositivity and geometric mean neutralizing antibody titers against these strains in the individuals who obtained two doses of OPV were 99.1%, 94.5% and 95.5%, respectively, and 564, 186 and 194, respectively. Although the antibody titers to G4-12 were lower compared with those to Sabin, these results indicate that the OPV vaccination policy in Japan has been effective in preventing poliomyelitis caused by VSLPs. These results also suggest that (i) an epidemic of poliomyelitis caused by a VSLP has not occurred in Japan due to herd immunity, and (ii) the possibility of reemergence of VSLPs will be prevented if sufficient herd immunity is acquired immediately after completion of the OPV vaccination in accordance with the poliomyelitis eradication program.

Development of inactivated poliovirus vaccine derived from Sabin strains.

In the course of Sabin-inactivated poliovirus vaccine (S-IPV) development, we have established high-yield virus production techniques based on Vero cell micro-carrier cultures. Development of specific ELISA tests to quantify the antigen content of S-IPV has been achieved. To adjust the immunogenicity of S-IPV so as to be comparable with the conventional-IPV, a new formulation was determined using a potency test using rats. The reformulated S-IPV was shown to be efficacious for the immunization of monkeys.

Regulation of neuronal and glial galectin-1 expression by peripheral and central axotomy of rat primary afferent neurons.

Galectin-1 (Gal1) is an endogenously-expressed protein important for the embryonic development of the full complement of primary sensory neurons and their synaptic connections in the spinal cord. Gal1 also promotes axonal regeneration following peripheral nerve injury, but the regulation of Gal1 by axotomy in primary afferent neurons has not yet been examined. Here, we show by immunohistochemistry and in situ hybridization that Gal1 expression is differentially regulated by peripheral nerve injury and by dorsal rhizotomy. Following peripheral nerve injury, the proportion of Gal1-positive DRG neurons was increased. An increase in the proportion of large-diameter DRG neurons immunopositive for Gal1 was paralleled by an increase in the depth of immunoreactivity in the dorsal horn, where Gal1-positive terminals are normally restricted to laminae I and II. Dorsal rhizotomy did not affect the proportions of neurons containing Gal1 mRNA or protein, but did deplete the ipsilateral dorsal horn of Gal1 immunoreactivity, indicating that it is transported centrally by dorsal root axons. Dorsal rhizotomy also resulted in an increase in Gal1 mRNA the nerve peripheral to the PNS-CNS interface (likely within Schwann cells and/or macrophages), and to a lesser extent within deafferented spinal cord regions undergoing Wallerian degeneration. This latter increase was notable in the dorsal columns and along the prior trajectories of myelinated afferents into the deeper dorsal horn. These results show that neuronal and glial expressions of Gal1 are tightly correlated with regenerative success. Thus, the differential expression pattern of Gal1 following peripheral axotomy and dorsal rhizotomy suggests that endogenous Gal1 may be a factor important to the regenerative response of injured axons.

Selective induction of DeltaFosB in the brain after transient forebrain ischemia accompanied by an increased expression of galectin-1, and the implication of DeltaFosB and galectin-1 in neuroprotection and neurogenesis.

Transient forebrain ischemia causes selective induction of DeltaFosB, an AP-1 (activator protein-1) subunit, in cells within the ventricle wall or those in the dentate gyrus in the rat brain prior to neurogenesis, followed by induction of nestin, a marker for neuronal precursor cells, or galectin-1, a beta-galactoside sugar-binding lectin. The adenovirus-mediated expression of FosB or DeltaFosB induced expression of nestin, glial fibrillary acidic protein and galectin-1 in rat embryonic cortical cells. DeltaFosB-expressing cells exhibited a significantly higher survival and proliferation after the withdrawal of B27 supplement than the control or FosB-expressing cells. The decline in the DeltaFosB expression in the survivors enhanced the MAP2 expression. The expression of DeltaFosB in cells within the ventricle wall of the rat brain also resulted in an elevated expression of nestin. We therefore conclude that DeltaFosB can promote the proliferation of quiescent neuronal precursor cells, thus enhancing neurogenesis after transient forebrain ischemia.

Galectin-1 in regenerating motoneurons.

The exogenous application of recombinant galectin-1 has recently been shown to promote the rate of peripheral nerve regeneration. Endogenous neuronal galectin-1 expression has recently been demonstrated to increase after axotomy. Here we demonstrate a significant increase in the endogenous neuronal expression of galectin-1 mRNA in facial motoneurons after either a nerve resection or crush injury in mice. This increase in galectin-1 expression was due in part to the loss of target-derived factor(s) as indicated by both the return of galectin-1 expression to control levels following target re-innervation and the increase in galectin-1 expression after blockade of axonal transport by an interneuronal colchicine injection. Furthermore, interneuronal injections of glial-derived neurotrophic factor into the uninjured nerve also increased galectin-1 mRNA expression within facial motoneurons suggesting that positive signals may also be involved in the regulation of galectin-1 expression. Galectin-1 null mutant mice showed an attenuated rate of functional recovery of whisking movement after a facial nerve crush.

Galectin-1 expression correlates with the regenerative potential of rubrospinal and spinal motoneurons.

Axotomized spinal motoneurons are able to regenerate to their peripheral targets, whereas injured rubrospinal neurons that lie completely within the CNS fail to regenerate. The differing cell body reactions to axotomy of these two neuronal populations have been implicated in their disparate regenerative ability. Recently, the lectin galectin-1 has been shown to be involved in both spinal motoneurons and primary afferent regeneration. Using in situ hybridization, we compared the endogenous galectin-1 mRNA expression in spinal motoneurons and rubrospinal neurons after axotomy. We found that 7 and 14 days after axotomy, galectin-1 mRNA increased in spinal motoneurons but decreased in rubrospinal neurons. Infusion of the brain-derived neurotrophic factor into the vicinity of the injured rubrospinal nucleus, which we have previously shown to increase the regenerative capacity of rubrospinal neurons, significantly increased galectin-1 mRNA compared with uninjured control levels. Thus, the expression of galectin-1 in neurons correlates with the regenerative propensity.

High expression of telomerase is an independent prognostic indicator of poor outcome in hepatoblastoma.

Telomerase, an enzyme related with cellular immortality, has been extensively studied in many kinds of malignant tumours for clinical diagnostic or prognostic utilities. Telomerase activity is mainly regulated by the expression of hTERT (human telomerase reverse transcriptase), which is a catalytic component of human telomerase. To evaluate whether the levels of hTERT mRNA provides a molecular marker of hepatoblastoma malignancy, we examined hTERT mRNA expression levels in the primary hepatoblastoma tissues by fluorescent RT-PCR using LightCycler technology and followed up the clinical outcomes in 63 patients listed in the Japanese Study Group of Pediatric Liver Tumor between 1991 and 2002. The hTERT mRNA expression was detected in 61 (96.8%) specimens and their expression levels ranged between 0.1/1000 and 745.1/1000 copies of PBGD gene that was used as an internal control. Among these cases, frozen 39 tumour samples and 14 adjacent noncancerous liver tissues were analysed for semiquantitative telomerase assay. In the 39 tumour samples, the levels of telomerase activity ranged between 0.11 and 2709 TPG and 12 (30.7%) had high telomerase activity (>100 TPG), whereas only nine of 14 noncancerous liver tissue samples showed telomerase activity which was less than 1.0 TPG. The levels of telomerase activity were significantly correlated with the levels of hTERT mRNA expression (P<0.001). The frequency of high hTERT mRNA expression and/or high telomerase activity did not significantly associate with the clinicopathological factors except for stage of disease. The prognosis of the patients with high hTERT mRNA expression was significantly worse than that of others (P<0.01), as was the patients with high telomerase activity (P<0.01). Multivariate analysis indicated that high levels of hTERT mRNA expression as well as telomerase activity are independent prognosis-predicting factors in patients with hepatoblastoma.

Galectin-1beta, a natural monomeric form of galectin-1 lacking its six amino-terminal residues promotes axonal regeneration but not cell death.

We previously identified a novel N-terminally processed form of galectin-1, galectin-1beta (Gal-1beta) whose expression was induced by DeltaFosB. In the present study, the biochemical properties and biological functions of Gal-1beta were compared with the full-length form of galectin-1 (Gal-1alpha). We first purified recombinant mouse Gal-1alpha and beta (rmGal-1alpha, beta) to near homogeneity. The rmGal-1alpha exists as a monomer under oxidized conditions and forms a dimer under reduced conditions, while the rmGal-1beta exists as a monomer regardless of redox conditions. The affinity of rmGal-1beta to beta-lactose was approximately two-fold lower than that of rmGal-1alpha under reduced conditions. The viability of Jurkat cells efficiently decreased when they were exposed to rmGal-1alpha, however, rmGal-1beta barely induced such a reduction. In contrast, both rmGal-1alpha and rmGal-1beta exhibited an equivalent capacity to promote axonal regeneration from the dorsal root ganglion explants. Our results suggest that the biochemical properties of rmGal-1beta determine its biological functions.

Distribution of the galectin-1 mRNA in the rat nervous system: its transient upregulation in rat facial motor neurons after facial nerve axotomy.

Galectin-1 is a member of the animal lectin family that displays conserved consensus sequences and similar carbohydrate binding specificities. Recent analyses revealed that galectin-1 plays an important role in the process of nerve regeneration. We analyzed the topological expression of galectin-1 mRNA in adult rat nervous system. Galectin-1 mRNA was predominantly observed in the cell bodies of neurons such as oculomotor nucleus (III), trochlear nucleus (IV), trigeminal motor nucleus (V), abducens nucleus (VI), facial nucleus (VII), hypoglossal nucleus (XII), red nucleus, and locus ceruleus. Neurons in pineal gland and dorsal root ganglia expressed galectin-1 mRNA. We next tested whether the axotomy of facial nerve altered the expression of galectin-1 mRNA in motor neurons. In the adult rats, the axotomy of facial nerve induced transient upregulation of galectin-1 mRNA around 6 h after axotomy. These results indicate that galectin-1 may play roles in the early event of the nerve injury and regeneration through the transient change of its expression level.

DeltaFosB, but not FosB, induces delayed apoptosis independent of cell proliferation in the Rat1a embryo cell line.

The fates of Rat1a cells expressing FosB and DeltaFosB as fusion proteins (ER-FosB, ER-DeltaFosB) with the ligand binding domain of human estrogen receptor were examined. The binding of estrogen to the fusion proteins resulted in their nuclear translocation and triggered cell proliferation, and thereafter delayed cell death was observed only in cells expressing ER-DeltaFosB. The proliferation of Rat1a cells, but not cell death triggered by ER-DeltaFosB, was completely abolished by butyrolactone I, an inhibitor of cycline-dependent kinases, and was partly suppressed by antisense oligonucleotides against galectin-1, whose expression is induced after estrogen administration. The cell death was accompanied by the activation of caspase-3 and -9, the fragmentation of the nuclear genome and cytochrome c release from the mitochondria, and was suppressed by zDEVD-fmk and zLEHD-fmk but not zIETD-fmk. The cell death was not suppressed by exogenous His-PTD-Bcl-x(L) at all, suggesting involvement of a Bcl-x(L)-resistant pathway for cytochrome c release.

Probiotic mixture decreases DNA adduct formation in colonic epithelium induced by the food mutagen 2-amino-9H-pyrido[2,3-b]indole in a human-flora associated mouse model.

Consumption of probiotic bacteria such as bifidobacteria has been shown to reduce the risk of colon cancer in animal models. However, the composition and metabolic activities of the intestinal flora of experimental animals are significantly different from those of humans. The aim of the study was to examine whether the probiotic mixture, which consisted of Streptococcus faecalis, Clostridium butyricum and Bacillus mesentericus, could decrease DNA adduct formation induced by 2-amino-9H-pyrido[2,3-b]indole (2-amino-alpha-carboline; AAC) in the colonic epithelium of a human-flora-associated (HFA) mouse model. Ten HFA mice were divided into a control group (n=4) and a probiotic group (n=6). The control group was administered AAC for 3 days and sacrificed 24 h after the last dose. The probiotic group was administered the probiotic mixture for 2 weeks prior to the administration of AAC. Analysis of DNA adducts with the 32P-high-performance liquid chromatography method was performed on stomach, jejunum and colonic epithelium, representing direct exposure sites of AAC, and colon wall, liver and kidney, representing indirect exposure sites. The mean level of the DNA adducts in the colonic epithelium of the probiotic group was significantly lower than that of control group, while the mean levels at the other sites did not differ significantly between the groups. The results indicated that the probiotic mixture could decrease the DNA adduct formation in the colonic epithelium induced by AAC.

Fatal sepsis associated with acute pancreatitis caused by Moraxella catarrhalis in a child.

We describe a 4-year-old boy with Cornelia de Lange syndrome who died of septic shock caused by Moraxella catarrhalis bacteremia. At autopsy there was evidence of acute hemorrhagic pancreatitis with abscesses. Gram-negative diplococci were seen histologically in the abscesses and pancreatic ducts.