Disruption of pRb-p16INK4 pathway: a common event in ampullary carcinogenesis

BACKGROUND/AIMS: Aberrations of the pRb (Retinoblastoma gene protein)-p16INK4 pathway play a critical role in carcinogenesis. Our objective is to evaluate its role in tumorigenesis and the development of ampullary cancer. METHODOLOGY: We examined expression status of p16INK4 protein and pRb immunohistochemically and assessed their possible prognostic relevance in 36 ampullary cancers. RESULTS: Thirty-four specimens (94.4%) exhibited alteration of p16INK4 and/or pRb expression, with 63.9% (23/36) of cancers showing p16INK4 negative expression and 94.4% (34/36) pRb abnormal expression. p16INK4 protein negative expression correlated significantly with tumor progression features such as advanced tumor stages (p=0.0291), lymph node metastasis (p=0.005), pancreas invasion (p=0.0002) and duodenum invasion (p=0.0101). Cases with both p16RNK4 protein negative expression and pRb overexpression showed poorer differentiation, more invasive growth (p=0.0425), higher level tumor stages (p=0.0079) and more frequent pancreas invasion (p=0.0024), compared with the others. p16INK4 protein expression showed no relationship with pRb expression (p=0.2199). No association was found in pRb expression status compared with any clinicopathological parameters analyzed. CONCLUSIONS: The disruption of the pRb-p16NK4 pathway plays an important role in ampullary carcinogenesis, the absence of p16INK4 protein expression might be involved in ampullary tumor progression.     

Further chromosome studies on Wilms' tumor cells of patients without aniridia

We studied chromosomes in Wilms' tumor cells of two patients without aniridia who had a normal constitutional karyotype. In both tumors, trisomy for 1q occurred as the result of a t(1;16), although the breakpoints in each chromosome differed in the two tumors. No 11p rearrangements could be detected, whereas in our previous patient an interstitial deletion of 11p13 was present in all tumor cells. Thus, trisomy for 1q may be another pathway leading to the development of Wilms' tumor, although the effect of the deletion of 16q cannot be assessed at present.     

Dominant-Negative Rho, Rac, and Cdc42 Facilitate the Invasion Process of Vibrio parahaemolyticus into Caco-2 Cells

To clarify the invasive process of Vibrio parahaemolyticus, an invasion assay was performed using cells expressing dominant negative small GTPases of the Rho family. This assay showed that the dominant negative host phenotype facilitates bacterial invasion, suggesting that the mechanism of V. parahaemolyticus invasion differs from that reported for other invasive bacteria.     

In vitro and ex vivo blood compatibility study of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer-coated hemodialysis hollow fibers

To identify the advantages of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer-coated polysulfone (PSf) hollow fibers for hemodialyzer and hemofilter minimodules with hollow fibers were made and blood compatibility was evaluated in vitro and ex vivo. Three types of hollow fibers, i.e., pure PSf (no additives), PSf alloyed with poly(1-vinyl-2-pyrrolidone) (PVPy), and PSf coated with the MPC copolymer, were processed in wet conditions. Commercially available hollow fibers (APS) were used as a control sample. The PSf hollow fibers have a condensed structure. A porous structure was observed when the PVPy was alloyed before wet processing, and no effect of the innercoated MPC copolymer on the porous structure was observed. One-tenth-sized minimodules of the conventional hemodialyzer were fabricated with 200 fibers each. The solute permeability of the hollow fibers was evaluated using 10% bovine serum in a buffer solution containing cytochrome C, which is a model protein of ₂-microglobulin. After circulation for 2.5h, the solute permeability of APS and PVPy-alloyed PSf hollow fibers decreased to 50% compared with their initial values. In contrast, the value for the hollow fibers innercoated with the MPC copolymer maintained its initial level. The inner surface of the dialysis membranes was observed with a transmission electron microscope and a layer of adsorbed protein on the PSf, APS, and PVPy-alloyed PSf hollow fibers was observed, but not on the MPC copolymer-coated fibers. Blood cell adhesion was then evaluated by circulation of whole rabbit blood without any anticoagulant ex vivo. Many adherent cells were observed on the PVPy-alloyed PSf hollow fibers; however, blood cells did not adhere or aggregate on the MPC copolymer-coated hollow fibers. From these results, we concluded that the in-situ coating of MPC copolymer on PSf hollow fibers is effective in preventing blood coagulation and maintaining the solute permeability of the fibers.     

Detection of necrotic neural response in super‐acute cerebral ischemia using activity‐induced manganese‐enhanced (AIM) MRI

Immediate and certain determination of the treatable area is important for choosing risky treatments such as thrombolysis for brain ischemia, especially in the super‐acute phase. Although it has been suggested that the mismatch between regions displaying ‘large abnormal perfusion’ and ‘small abnormal diffusion’ indicates a treatable area on an MRI, it has also been reported that the mismatch region is an imperfect approximation of the treatable region named the ‘penumbra’. Manganese accumulation reflecting calcium influx into cells was reported previously in a middle cerebral artery occlusion (MCAO) model using activity‐induced manganese‐enhanced (AIM) MRI. However, in the super‐acute phase, there have been no reports about mismatches between areas showing changes to the apparent diffusion coefficient (ADC) and regions that are enhanced in AIM MRI. It is expected that the AIM signal can be enhanced immediately after cerebral ischemia in the necrotic core region due to calcium influx. In this study, a remote embolic rat model, created using titanium‐oxide macrospheres, was used to observe necrotic neural responses in the super‐acute phase after ischemia. In addition, images were evaluated by comparison between ADC, AIM MRI, and histology. The signal enhancement in AIM MRI was detected at 2 min after the cerebral infarction using a remote embolic method. The enhanced area on the AIM MRI was significantly smaller than that on the ADC map. The tissue degeneration highlighted by histological analysis corresponded more closely to the enhanced area on the AIM MRI than that on the ADC map. Thus, the manganese‐enhanced region in brain ischemia might indicate ‘necrotic’ irreversible tissue that underwent calcium influx. Copyright © 2009 John Wiley & Sons, Ltd.     

Association of VKORC1 and CYP2C9 polymorphisms with warfarin dose requirements in Japanese patients

Warfarin is the most commonly used oral anticoagulant for treatment of thromboembolism, but adjustment of the dose appropriate to each patient is not so easy because of the large inter-individual variation in dose requirement. We analyzed single nucleotide polymorphism (SNP) genotypes of the VKORC1 and CYP2C9 genes using DNA from 828 Japanese patients treated with warfarin, and investigated association between SNP genotype and warfarin-maintenance dose. Five SNPs in VKORC1, 5 flanking–1413A>G, intron 1–136T>C, intron 2+124C>G, intron 2+837T>C and exon 3 343G>A, were in absolute linkage disequilibrium, and showed a significant association with daily warfarin dose of these patients. The median warfarin dose of patients with homozygosity for the minor allele was 4.0 mg/day, which is significantly higher than those heterozygous for the minor allele (3.5 mg/day) or those homozygous for the major allele (2.5 mg/day; P=5.1×10^–11 in the case of intron 1–136T>C SNP). We then genotyped the CYP2C9 gene for the Japanese common genetic variant, CYP2C9*3 and, based on the genotype of these two genes, classified patients into three categories, which we call warfarin-responsive index. The median warfarin daily dose varied significantly in this classification according to the warfarin-responsive index (2.0 mg/day for index 0 group, 2.5 mg/day for index 1 group, and 3.5 mg/day for index 2 group; P=4.4×10^–13). Thus, analysis of the combination of VKORC1 and CYP2C9 genotypes should identify warfarin-sensitive patients who require a lower dose of drug, allowing personalized warfarin treatment.     

Chromosomal numerical abnormalities in early stage lung adenocarcinoma

Chromosomal numerical abnormalities (CNA) are ubiquitous in human cancers. However, the question of when a CNA occurs in the course of tumor generation and progression, is controversial. Recent radiological scrutiny has enabled the identification of small peripheral lesions in the lung. A chromosome-wide investigation encompassing almost all the chromosomal centromeres was performed using modified fluorescence in situ hybridization on the archived pathological samples of 16 atypical adenomatous hyperplasia (AAH) and 30 lung adenocarcioma (AdCa) specimens including those smaller than 1 cm in size. The prevalence of the gain was more extensive in male than in female patients, and in non-smokers than in smokers. It tended to be greater in poorly differentiated AdCa, in moderately differentiated AdCa, and in well-differentiated AdCa cases, in that order. Most AAH had non-specific gains affecting all the examined chromosomes. The prevalence of the gain differed significantly between AAH and bronchioloalveolar carcinoma (BAC) 1 cm. It is proposed that the CNA is a distinct phenomenon occurring in the early or premalignant stage of lung AdCa, and that the CNA itself may not be a sequel in the carcinogenetic process, but a driving factor in carcinogenesis.     

Cartilage regeneration using slow release of bone morphogenetic protein-2 from a gelatin sponge to treat experimental canine tracheomalacia: a preliminary report

We investigated whether saber sheath-type tracheomalacia could be treated by the slow release of bone morphogenetic protein (BMP)-2 from a gelatin sponge. A 1 cm gap was made in the middle portion of each of 10 consecutive tracheal cartilage rings in the canine cervix (control group, n = 3), then a gelatin sponge containing 12 microg of BMP-2 solution was implanted in the gap (12 microg group, n = 3). In another group (120 microg + P group, n = 3), the implanted gelatin sponge contained 120 microg of BMP-2 solution, and the gap was covered with periosteum. All of the control dogs developed saber sheath-type tracheomalacia, whereas tracheomalacia was not observed in the 12 microg and 120 microg + P groups. In the 12 microg group, fibrous cartilage was observed at the ends of the cartilage stumps. In the 120 microg + P group, newly formed bone and cartilage were observed to form a bridge between the cartilage stumps. The regeneration of cartilage or bone induced by the slow release of BMP-2 from a gelatin sponge might be useful for treatment of tracheomalacia.     

Evaluation of anti-parvovirus B19 activity in sera by assay using quantitative polymerase chain reaction

Human parvovirus B19 (B19) infects cells of erythroid lineage. Production of neutralizing antibodies (Abs) is indispensable for recovery from B19-related disease state. In this study, we used a convenient method to measure neutralizing activities in human sera by using a real-time quantitative PCR based assay. Erythroid cell line KU812Ep6 was incubated with test sera before infection with B19 virus. The copy number of B19-DNA in cultures was decreased in the presence of the sera from patients who recovered from acute B19 infection, whereas no decrease in B19-DNA was in cultures incubated with sera from healthy volunteers who had no B19 infection. The decrease in B19-DNA copy number was calculated and the inhibition percentage was expressed as neutralizing activity to B19. A clinical study showed that the levels of neutralizing ability were high in patients who recovered soon after acute B19 infection, but were low in some patients with a prolonged clinical course for recovery from B19 infection. This method is simple and convenient compared with methods described previously, showing its usefulness to evaluate the neutralizing activity to B19.