Usefulness of endoscopic biopsy using immunostaining of p53 and Ki-67 in tumors of the ampulla of Vater

We investigated the diagnostic and prognostic value of p53 expression and proliferative activity, as indicated by the Ki-67, in endoscopic biopsy specimens. Specimens were immunologically stained with p53 and MIB-1 (Ki-67), and the MIB-1/Ki-67 labeling index (LI) was calculated. Classification of adenomas was based on findings of H&E-stained preparations into those with low- or high-grade atypia. Well-differentiated tubular and papillary adenocarcinomas were classified as carcinomas with low- or high-grade atypia. There were significant differences among the control and adenoma patients in MIB-1/Ki-67 LI (P < 0.05). No significant difference was identified between adenomas with high grade atypia and carcinomas with low grade atypia. The p53 expression was negative in all adenomas, but it was positive in 68.2% of carcinomas. The current study demonstrated that p53 protein expression in endoscopic biopsy specimens was of preoperative diagnostic value for carcinoma of the ampulla of Vater. The p53 protein positive tumors had a relatively higher malignant potential than p53 protein negative ones. The MIB-1/Ki-67 LI was useful in differentiating non-tumorous lesions from adenomas and adenomas with low- or high-grade atypia. The MIB-1/Ki-67 LI had a prognostic value because clinicopathological factors of carcinoma of ampulla of Vater correlated with MIB-1/Ki-67 LI.     

Mechanisms of inactivation of the p16INK4a gene in leiomyosarcoma of soft tissue: decreased p16 expression correlates with promoter methylation and poor prognosis

The p16INK4a tumour suppressor gene, encoding p16 protein, plays a crucial role in regulation of the G1 cell-cycle phase. To investigate the potential role of p16 in soft tissue leiomyosarcoma (LMS), an immunohistochemical analysis was performed of 77 LMSs for p16 expression. Decreased expression of the p16 protein was identified in 25 of 77 LMSs (32%). Decreased expression of p16 correlated significantly with large tumour size (p=0.0038). In a univariate analysis, large tumour size and decreased expression of p16 were statistically significant adverse prognostic factors (p=0.025 and p=0.0021, respectively). In a multivariate analysis including conventional clinicopathological parameters, decreased expression of p16 protein was revealed as the only independent unfavourable prognostic factor (p=0.012). To elucidate the mechanisms of inactivation of the p16INK4a gene, 49 LMSs for which genomic DNA was available were examined; analysis for homozygous deletion, mutation, and promoter hypermethylation was conducted using differential PCR, PCR-SSCP, and methylation-specific PCR, respectively. Promoter hypermethylation was detected in 11 of 49 LMS cases (22%); homozygous deletion was detected in 3 of 49 cases (6%); and mutation was not recognized in any of the cases studied. Eight of 15 cases (53%) with decreased expression of p16 protein revealed methylation of the p16INK4a gene promoter. Promoter hypermethylation correlated closely with decreased expression and poor prognosis (p=0.0014 and p=0.0088, respectively). These results suggest that decreased expression of p16 protein can be considered as an independent reliable prognostic parameter in patients with soft tissue LMS. Furthermore, promoter methylation was more frequent than either homozygous deletion or mutation in this tumour, and promoter methylation was also shown to have a strong association with inactivation of the p16INK4a gene.     

Protective effect of S-allyl-L-cysteine,a garlic compound,on amyloid beta-protein-induced cell death in nerve growth factor-differentiated PC12 cells

Aged garlic extract (AGE) contains several neuroactive compounds, including S-allyl-L-cysteine (SAC) and allixin. We characterized cell death induced by amyloid beta-protein (Abeta), 4-hydroxynonenal (HNE), tunicamycin, an endoplasmic reticulum (ER) stressor, or trophic factor deprivation, and investigated whether and how SAC could prevent this in nerve growth factor (NGF)-differentiated PC12 cells, a model of neuronal cells. Exposure of the cells to amyloid beta-protein(1-40) (Abeta(1-40)) decreased the extent of [3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) reduction activity and loss of neuronal integrity, but these effects were not prevented by Ac-DEVD-CHO, a caspase-3 inhibitor. Simultaneously applied SAC protected the cells against Abeta-induced cell death in a concentration-dependent manner. It also protected them against tunicamycin-induced neuronal death. In contrast, it afforded no protection against cell death induced by HNE and trophic factor deprivation, which is mediated by a caspase-3-dependent pathway. These results suggest that SAC may selectively protect cell death induced by Abeta and tunicamycin, which may be triggered by ER dysfunction in NGF-differentiated PC12 cells.     

Provoked anterior knee pain in medial osteoarthritis of the knee

The incidence and the causes of provoked anterior knee pain in medial osteoarthritis (OA) of the knee were investigated clinically and radiographically. A retrospective study was performed in 179 primary osteoarthritic knees of 129 patients. Provocative tests were conducted on the patellofemoral (PF) joint to induce retropatellar crepitation, grating pain, tenderness around the patella, and pain on deviating the patella. The femorotibial angle (FTA) was measured on standing anteroposterior radiographs as a parameter of limb alignment. The widths of the medial and lateral joint space of the PF joint were measured on skyline views of standing or 30, 60 or 90° knee flexion. The angle of flexion contracture was measured on lateral radiographs of knees with maximum extension. The lateral shift and tilt of the patella were measured on standing skyline views. Retropatellar crepitation was found in 70% of knees, while provoked anterior knee pain was observed in 35–45% of knees with medial OA of the knee. Standing FTA was significantly greater in knees with tenderness around the patella and pain on deviating the patella than in those without these symptoms (P<0.05). The angle of flexion contracture was significantly greater in knees with provoked symptoms in the PF joint than in those without symptoms (P<0.05). The degree of lateral shift was greater in knees with provoked symptoms (P<0.05). Flexion contracture and varus deformity of the knee with lateralization of the patella may be factors aggravating provoked PF symptoms in medial OA of the knee. The radiographic assessment in this series failed to show a significant relationship between the width of the PF joint space and the incidence of provoked PF symptoms.     

Correlation between ventricular flow field and valve closing sound of mechanical mitral prostheses

The Jyros (JR) valve and the newer On-X and MIRA valves, all installed antianatomically, were compared with the St. Jude Medical (SJM) valve in the mitral position to study the effects of valve design differences on the down-stream flow field and the associated valve closing sound. The dynamic particle image velocimetry method utilizing a high-speed video flow visualization technique was used to map the velocity field, and wavelet analysis of the sound was used to find the correlation between the ventricular flow field and the valve closing sound. Based on the experimental data, the following general conclusions can be made. In the velocity field directly below the mitral valve, where the distinct characteristic differences of the valve designs will be evident, twin symmetrical circulations were observed due to the divergent nature of the flow generated by the two inclined half-disks with the valve installed in the anti-anatomical orientation; the SJM, the On-X, and the MIRA valves generated a centrally downward circulation that opposed the valve leaflet closing movement, and resulted in relatively loud valve closing sounds.     

Possible molecular mechanism of the relationship between NS5B polymorphisms and early clearance of hepatitis C virus during interferon plus ribavirin treatment

We previously reported the relationship between viral polymerase polymorphisms and the initial decline in viral load induced by interferon-alpha and ribavirin therapy in genotype 1b-related chronic hepatitis C patients. The presence of E124K and I85V of NS5B was closely associated with viral clearance at 8 weeks of treatment. The aim of this study was to investigate the mechanisms by which this polymorphism of NS5B protein affects early viral clearance. We used a replicon system derived from strain O, genotype 1b virus. Three mutants (V85I), (K124E), and (V85I/K124E) were introduced to the replicon. OR6c, a derivative of HuH7 cells, was transfected with the replicon including a luciferase reporter gene. Luciferase activities were measured 72 hr post-transfection. All three mutants showed higher luciferase activity than that of the wild type, and the V85I mutant showed the highest activity. This result was also confirmed by neomycin gene-containing replicons with same mutations. All replicons were down-regulated by ribavirin, but the level of reduction in the V85I mutant was the lowest. Our results suggested that this mutation at least partly contributes to resistance to early viral clearance during interferon and ribavirin combination therapy.     

Advance in animal experiments with the undulation pump total artificial heart: 50 and 54 day survival periods with 1/R control

The undulation pump total artificial heart (UPTAH) is a unique, implantable, total artificial heart (TAH) that uses undulation pumps. To achieve long-term survival in animals with physiologic hemodynamic conditions, a control method based on conductance and arterial pressure was applied to UPTAH. With this control method, called 1/R control, survival periods of 50 days (No. 0016, 49.6 kg) and 54 days (No. 0030, 42.5 kg) were obtained in adult female goats. In No. 0016, 1/R control was applied to the left pump, whereas in No. 0030, it was applied to the right pump. Another pump was used for left-right balance control. The control stability was better in No. 0030 than in No. 0016. The sucking effect of the left atrium was remarkable in No. 0016, possibly because of a time delay when left-right balance control was performed with the right pump. In No. 0016, the cause of death was probably a thrombus flown from a panus in the left atrium. It is possible that the left atrial suction effect influenced the thrombus and panus formation in the left atrium. In No. 0030, the cause of death was a small rupture of the membrane in the right pump. The rupture may have been caused by excessive negative pressure inside the pump. This pressure resulted from suction of the right atrium because of an unexpected control excursion, which was probably caused by a software bug. It will be necessary to redesign the undulation pump and improve the software to achieve longer survival periods for animals with physiologic hemodynamic conditions.     

Mechanism of acidic pH‐induced contraction in spontaneously hypertensive rat aorta: role of Ca2+ release from the sarcoplasmic reticulum

Aim: This study was conducted to investigate the mechanism of acidic pH‐induced contraction (APIC) with regard to Ca²⁺ handling using isometric tension recording experiments. Results: Decreasing extracellular pH from 7.4 to 6.5 produced a marked and sustained contraction of spontaneously hypertensive rat (SHR) aorta, that was 128.7 ± 2.0% of the 64.8 mm KCl‐induced contraction. Verapamil, an inhibitor of voltage‐dependent Ca²⁺ channels (VDCC) significantly inhibited the APIC. In Ca²⁺‐deficient solution, sustained contraction induced by acidic pH was abolished completely, while a transient contraction was still observed suggesting the release of Ca²⁺ from intracellular site. Ryanodine (1 μm ), a ryanodine receptor blocker, and 10 μm cyclopiazonic acid (CPA; a sarco/endoplasmic reticulum Ca²⁺ ATPase inhibitor) abolished the transient contraction induced by acidosis. In normal Ca²⁺‐containing solution, ryanodine significantly decreased the rate of rise as well as maximum level of APIC. Interestingly, ryanodine and CPA showed an additive inhibitory effect with verapamil and the combined treatment of ryanodine or CPA with verapamil nearly abolished the APIC. Conclusions: It is concluded that acidic pH induces Ca²⁺ release from ryanodine/CPA‐sensitive store of sarcoplasmic reticulum in SHR aorta. This Ca²⁺ plays an important role in the facilitation of the rate of rise of APIC, as well as contributing to the sustained contraction via a mechanism which is independent of Ca²⁺ influx through VDCC.     

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.