Joint unloading inhibits articular cartilage degeneration in knee joints of a monosodium iodoacetate-induced rat model of osteoarthritis.

OBJECTIVE: The aim of the study was to examine how mechanical unloading affects articular cartilage degeneration in the patellofemoral (PF) and tibiofemoral (TF) joints of a monosodium iodoacetate (MIA)-induced rat model of osteoarthritis (OA). DESIGN: The study involved 60 male rats. OA was induced by intra-articular injecting MIA into both knee joints. All animals were equally divided into two groups: sedentary (SE) and hindlimb unloading (HU) groups. Histopathological changes in the articular cartilage of the PF and TF joints were evaluated using the Osteoarthritis Research Society International (OARSI) score and modified Mankin score at 2 and 4 weeks after MIA injection. RESULTS: In the SE and HU groups, representative histopathological changes in OA were detected in the PF and TF joints. The OARSI and modified Mankin scores for the PF and TF joints tended to increase over time after the injection of 0.2 mg or 1.0 mg of MIA in the SE and HU groups. Both the scores for the HU group were significantly lower than those for the SE group [OARSI score: P < 0.0001 (1.0-mg injection at 4 weeks); modified Mankin score: P = 0.0116 (0.2-mg injection at 4 weeks); P = 0.0004 and < 0.0001 (1.0-mg injection at 2 and 4 weeks, respectively)]. CONCLUSION: This study revealed new histological evidence that indicates that unloading condition suppresses articular cartilage degeneration and is beneficial in many areas of basal and clinical research involving OA.

Coexisting lateral tibiofemoral osteoarthritis is associated with worse knee pain in patients with mild medial osteoarthritis.

OBJECTIVE: To examine the clinical impact of coexisting lateral osteoarthritis (OA) in knees with mild medial OA. DESIGN: In patients with Kellgren/Lawrence (K/L) grade 2 OA in the medial compartment (n = 100; age: 56-89 years; 80.0% female), anteroposterior knee radiography was used to assess the presence of lateral OA, using grading systems from the Osteoarthritis Research Society International (OARSI) atlas and the K/L classification. The Japanese Knee Osteoarthritis Measure (JKOM), knee range of motion (ROM), and performance-based functional measures (10 m walk, timed up and go and five repetition chair stand maneuvers) were evaluated. The outcomes were compared between patients with and without lateral OA using an analysis of covariance (ANCOVA) or nonparametric rank ANCOVA. Furthermore, ordinal logistic regression analysis was performed, with responses on individual JKOM pain questionnaires as the outcomes and lateral OA as the predictor. RESULTS: Knees with coexisting lateral OA had a significantly worse score of JKOM pain question compared with those without, after adjusting for covariates. The presence of lateral OA was significantly associated with knee pain while ascending/descending stairs and standing. These results were consistent between different definitions of the K/L and OARSI grading systems. The knee ROM and performance-based functional measures were not significantly different between patients with and without lateral OA. CONCLUSION: Knees with concomitant lateral and mild medial OA may be more symptomatic compared to those without lateral OA. These findings might help to define a clinically distinct subgroup based on a simple radiographic finding in mild knee OA.

Physiological exercise loading suppresses post-traumatic osteoarthritis progression via an increase in bone morphogenetic proteins expression in an experimental rat knee model.

OBJECTIVE: To evaluate the dose-response relationship of exercise loading in the cartilage-subchondral bone (SB) unit in surgically-induced post-traumatic osteoarthritis (PTOA) of the knee. DESIGN: Destabilized medial meniscus (DMM) surgery was performed on the right knee of 12-week-old male Wistar rats, and sham surgery was performed on the contralateral knee. Four weeks after the surgery, the animals were subjected to moderate (12 m/min) or intense (21 m/min) treadmill exercises for 30 min/day, 5 days/week for 4 weeks. PTOA development in articular cartilage and SB was examined using histological and immunohistochemical analyses, micro-computed tomography (micro-CT) analysis, and biomechanical testing at 8 weeks after surgery. Gremlin-1 was injected to determine the role of bone morphogenetic protein (BMP) signaling on PTOA development following moderate exercise. RESULTS: Moderate exercise increased BMP-2, BMP-4, BMP-6, BMP receptor 2, pSmad-5, and inhibitor of DNA binding protein-1 expression in the superficial zone chondrocytes and suppressed cartilage degeneration, osteophyte growth, SB damage, and osteoclast-mediated SB resorption. However, intense exercise had little effect on BMP expression and even caused progression of these osteoarthritis (OA) changes. Gremlin-1 injection following moderate exercise caused progression of the PTOA development down to the level of the non-exercise DMM-operated knee. CONCLUSIONS: Exercise regulated cartilage-SB PTOA development in DMM-operated knees in a dose-dependent manner. Our findings shed light on the important role of BMP expression in superficial zone chondrocytes in attenuation of PTOA development following physiological exercise loading. Further studies to support a mechanism by which BMPs would be beneficial in preventing PTOA progression are warranted.

Thinning of articular cartilage after joint unloading or immobilization. An experimental investigation of the pathogenesis in mice.

OBJECTIVE: Moderate mechanical stress generated by normal joint loading and movement is essential for the maintenance of healthy articular cartilage. However, the effects of reduced loading caused by the absence of weight bearing or joint motion on articular cartilage and subchondral bone is still poorly understood. We aimed to characterize morphological and metabolic responses of articular cartilage and subchondral bone to decreased mechanical stress in vivo. METHODS: Mice were subjected to periods of hindlimb unloading by tail suspension or external fixation of the knee joints. The articular surface was observed with digital microscope and the epiphyseal bone was assessed by micro-CT analysis. Articular cartilage and subchondral bone were further evaluated by histomorphometric, histochemical, and immunohistochemical analyses. RESULTS: The joint surface was intact, but thickness of both the total and uncalcified layer of articular cartilage were decreased both after joint unloading and immobilization. Subchondral bone atrophy with concomitant marrow expansion predisposed osteoclast activity at bone surface to invade into cartilaginous layer. Uncalcified cartilage showed decreased aggrecan content and increased aggrecanase expression. Alkaline phosphatase (ALP) activity was increased at uncalcified cartilage, whereas decreased at calcified cartilage. The distributions of hypertrophic chondrocyte markers remained unchanged. CONCLUSION: Thinning of articular cartilage induced by mechanical unloading may be mediated by metabolic changes in chondrocytes, including accelerated aggrecan catabolism and exquisitely modulated matrix mineralization, and cartilage matrix degradation and resorption by subchondral osteoclasts. Cartilage degeneration without chondrocyte hypertrophy under unloading condition indicate the possible existence of mechanism which is different from osteoarthritis pathogenesis.

Exercise intervention increases expression of bone morphogenetic proteins and prevents the progression of cartilage-subchondral bone lesions in a post-traumatic rat knee model.

OBJECTIVE: This study aimed to determine whether treadmill walking (TW) prevents the progression of post-traumatic osteoarthritic changes in cartilage-subchondral bone unit, and whether the exercise timing changes the exercise efficacy in destabilized medial meniscus (DMM) rat knees. DESIGN: Twelve-week-old male Wistar rats underwent DMM surgery on their right knees and sham surgery on their left knees and were assigned to either the sedentary (n = 10) or walking (n = 24) groups. The rats in the walking group were subjected to TW from day 2 through 4 weeks, from 4 through 8 weeks, or from day 2 through 8 weeks (n = 8 per group). Osteoarthritic changes of cartilage and subchondral bone were assessed with micro-computed tomography, histology, and immunohistochemistry 8 weeks after surgery. RESULTS: TW prevented the progression of cartilage and subchondral bone lesions induced by the DMM, and increased bone morphogenetic protein (BMP)-2 and -6 expressions in superficial zone chondrocytes and bone-lining cells including osteoblasts. Furthermore, the TW-induced increase in BMPs varied with the exercise timing. Beginning TW 4 weeks after DMM surgery was the best option for increasing BMPs, coinciding with the most robust prevention of osteoarthritic changes. CONCLUSIONS: TW increased the expression of BMPs and prevented the progression of cartilage-subchondral bone lesions in rat knees with a DMM. Selective exercise timing may be a key factor in the development of an exercise regimen for preventing the progression of post-traumatic osteoarthritis (PTOA). Furthermore, exercise may have favorable effects even after the PTOA has been developed.

Subchondral plate porosity colocalizes with the point of mechanical load during ambulation in a rat knee model of post-traumatic osteoarthritis.

OBJECTIVE: This study investigated the association between spatiotemporal cartilage-subchondral bone plate alterations and mechanical load during ambulation in an experimental rat model of destabilized medial meniscus (DMM). DESIGN: Twelve-week-old Wistar rats (n = 38) underwent DMM surgery on the right knee and sham surgery on the left knee. At 2 and 4 weeks after surgery, subchondral bone changes were evaluated via micro-computed tomography with various knee flexion angles to simulate weight-bearing during rat ambulation under a 3-dimensional motion capture apparatus. Additionally, the biomechanical properties, histology, and ultrastructure of the medial tibia and femoral condyle were evaluated. RESULTS: Focal subchondral bone plate perforations were confirmed in the medial tibia within 2 weeks after surgery and were aggravated rapidly 2 weeks later. This subchondral plate porosity colocalized with articular cartilage lesions as confirmed by histology and scanning electron microscopy, and coincided with the likely point of contact between the posterior femoral condyle and tibial plateau during ambulation. Biomechanical properties were confirmed at the medial tibia, at which stiffness was reduced to approximately half that of the sham-operated knee at 4 weeks after surgery. CONCLUSIONS: Cartilage-subchondral bone plate alterations localized in the region of the point of mechanical load during ambulation in DMM-operated knees, at which the mechanical integrity of cartilage was impaired. These results indicate that DMM-induced increases in mechanical load play an important role in the pathogenesis of early post-traumatic osteoarthritis (OA), and it might accelerate the development of the disease via cartilage-subchondral bone plate crosstalk through increased subchondral plate perforations.

miR-9-3p plays a tumour-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells.

BACKGROUND: The inactivation of the Hippo pathway lead to TAZ (PDZ-binding motif)/YAP (yes-associated protein) overexpression, and is associated with worse prognostic outcomes in various cancers including hepatocellular carcinoma (HCC). Although there are several reports of microRNA (miR) targeting for YAP, miR targeting for TAZ remains unclear. The aim of this study is to identify the miR targeting TAZ expression in HCC. METHODS: MicroRNA expression was analysed using the Human miFinder 384HC miScript miR PCR array, and was compared between low and high TAZ expression cell lines. Then, we extracted miR-9-3p as a tumour-suppressor miR targeting TAZ. We examined the functional role of miR-9-3p using miR-9-3p mimic and inhibitor in HCC cell lines). RESULTS: In HCC cell lines and HCC clinical samples, there was the inverse correlation between miR-9-3p and TAZ expressions. TAZ expression was induced by treatment of miR-9-3p inhibitor and was downregulated by treatment of miR-9-3p mimic. Treatment of miR-9-3p mimic inhibited cell proliferative ability with downregulated phosphorylations of Erk1/2, AKT, and ß-catenin in HLF. Inversely, treatment of miR-9-3p inhibitor accelerated cell growth compared with control in HuH1. CONCLUSIONS: MicroRNA-9-3p was identified as the tumour-suppressor miR targetting TAZ expression in HCC cells.

Effect of LSKL peptide on thrombospondin 1-mediated transforming growth factor ß signal activation and liver regeneration after hepatectomy in an experimental model.

BACKGROUND: A strategy for accelerating liver regeneration after hepatectomy would offer great benefits in preventing postoperative liver failure and improving surgical outcomes. Transforming growth factor (TGF) ß is a potent inhibitor of hepatocyte proliferation. Recently, thrombospondin (TSP) 1 has been identified as a negative regulator of liver regeneration by activation of local TGF-ß signals. This study aimed to clarify whether the LSKL (leucine-serine-lysine-leucine) peptide, which inhibits TSP-1-mediated TGF-ß activation, promotes liver regeneration after hepatectomy in mice. METHODS: Mice were operated on with a 70 per cent hepatectomy or sham procedure. Operated mice received either LSKL peptide or normal saline intraperitoneally at abdominal closure and 6 h after hepatectomy. Perioperative plasma TSP-1 levels were measured by enzyme-linked immunosorbent assay in patients undergoing hepatectomy. RESULTS: Administration of LSKL peptide attenuated Smad2 phosphorylation at 6 h. S-phase entry of hepatocytes was accelerated at 24 and 48 h by LSKL peptide, which resulted in faster recovery of the residual liver and bodyweight. Haematoxylin and eosin tissue staining and blood biochemical examinations revealed no significant adverse effects following the two LSKL peptide administrations. In the clinical setting, plasma TSP-1 levels were lowest on the first day after hepatectomy. However, plasma TSP-1 levels at this stage were significantly higher in patients with subsequent liver dysfunction compared with levels in those without liver dysfunction following hepatectomy. CONCLUSION: Only two doses of LSKL peptide during the early period after hepatectomy can promote liver regeneration. The transient inhibition of TSP-1/TGF-ß signal activation using LSKL peptide soon after hepatectomy may be a promising strategy to promote subsequent liver regeneration. Surgical relevance Although the mechanisms of liver regeneration after hepatectomy have been explored intensively in vivo, no therapeutic tools are thus far available to accelerate liver regeneration after hepatectomy in the clinical setting. Recently, the matricellular protein thrombospondin (TSP) 1, a major activator of latent transforming growth factor (TGF) ß1, has been identified as a negative regulator of liver regeneration after hepatectomy. In this study, the inhibition of TSP-1-mediated TGF-ß signal activation by LSKL (leucine-serine-lysine-leucine) peptide in the early period after hepatectomy accelerated liver regeneration without any adverse effects. In addition, continuous high plasma TSP-1 levels after hepatectomy were associated with liver damage in humans. The transient inhibition of TSP-1/TGF-ß signal activation using LSKL peptide in the early period after hepatectomy could be a novel therapeutic strategy to accelerate liver regeneration after hepatectomy.

Effects of short-term gentle treadmill walking on subchondral bone in a rat model of instability-induced osteoarthritis.

OBJECTIVE: Subchondral bone cyst (SBC) growth, caused by osteoclast activity during early knee osteoarthritis (OA) pathogenesis, should be treated to prevent further progressions of OA. In the present study, we evaluated the effects of gentle treadmill walking on subchondral bone and cartilage changes in an experimental rat model of destabilized medial meniscus (DMM). METHOD: Twelve-week-old Wistar rats underwent DMM surgery in their right knee and sham surgery in their left knee and were assigned to either the sedentary group or walking group (n = 42/group). Animals in the walking group were subjected to treadmill exercise 2 days after surgery, which included walking for 12 m/min, 30 min/day, 5 days/week for 1, 2, and 4 week(s). Subchondral bone and cartilage changes were evaluated by micro-CT analysis, histological analysis, and biomechanical analysis. RESULTS: Treadmill walking had a tendency to suppress SBC growth, which was confirmed by micro-CT (P = 0.06) and positive staining for tartrate-resistant acid phosphatase (TRAP) activity for the osteoclast number per bone surface (P = 0.09) 4 weeks after surgery. These changes coincide with the prevention of cartilage degeneration as evaluated by the Osteoarthritis Research Society International (OARSI) score (P < 0.05) and biomechanically softening (P < 0.05). Furthermore, treadmill walking could suppressed increasing osteocyte deaths (P < 0.01), which was positively correlated with the OARSI score (r = 0.77; P < 0.01). CONCLUSION: These results indicate biomechanical and biological links exist between cartilage and subchondral bone; preventive effects of treadmill walking on subchondral bone deterioration might be partly explained by the chondroprotective effects.

Destabilization of the medial meniscus leads to subchondral bone defects and site-specific cartilage degeneration in an experimental rat model.

OBJECTIVE: This study aimed to investigate subchondral bone changes using micro-computed tomography (micro-CT) and regional differences in articular cartilage degeneration, focusing on changes of cartilage covered by menisci, in the early phase using a destabilization of the medial meniscus (DMM) model. METHOD: The DMM model was created as an experimental rat osteoarthritis (OA) model (12 weeks old; n = 24). At 1, 2, and 4 weeks after surgery, the rats were sacrificed, and knee joints were scanned using a Micro-CT system. Histological sections of the medial tibial plateau, which was divided into inner, middle, and outer regions, were prepared and scored using the modified OARSI scoring system. The cartilage thickness was also calculated, and matrix metalloproteinase 13 (MMP13), Col2-3/4c, and vascular endothelial growth factor (VEGF) expression was assessed immunohistochemically. RESULTS: Subchondral bone defects were observed in the middle region, in which the cartilage thickness decreased over time after surgery, and these defects were filled with MMP13- and VEGF-expressing fibrous tissue. The OARSI score increased over time in the middle region, and the score was significantly higher in the middle region than in the inner and outer regions at 1, 2, and 4 weeks after surgery. Col2-3/4c and MMP13 expression was observed primarily in the meniscus-covered outer region, in which the cartilage thickness increased over time. CONCLUSION: Loss of meniscal function caused cartilage degeneration and subchondral bone defects in the early phase site-specifically in the middle region. Furthermore, our results might indicate cartilage covered by menisci is easily degraded resulting in osmotic swelling of the cartilage in early OA.

Statins inhibit tumor progression via an enhancer of zeste homolog 2-mediated epigenetic alteration in colorectal cancer.

While statin intake has been proven to reduce the risk of colorectal cancer (CRC), the mechanism of antitumor effects and clinical significance in survival benefits remain unclear. Statin-induced antiproliferative effects and its underlying mechanism were examined using six CRC cell lines. Statins except pravastatin showed antiproliferative effects (simvastatin ≥ fluvastatin > atorvastatin) even though both of simvastatin and pravastatin could activate mevalonate pathways, suggesting the statin-mediated antiproliferative effects depended on non-mevalonate pathway. Indeed, statin induced p27(KIP1) expression by downregulation of histone methyltransferase enhancer of zeste homolog 2 (EZH2), which acts as an epigenetic gene silencer. Additionally, the use of simvastatin plus classII histone deacetylase (HDAC) inhibitor (MC1568) induced further overexpression of p27(KIP1) by inhibiting HDAC5 induction originated from downregulated EZH2 in CRC cells and synergistically led to considerable antiproliferative effects. In the clinical setting, Statin intake (except pravastatin) displayed the downregulated EZH2 expression and inversely upregulated p27(KIP1) expression in the resected CRC by immunohistochemical staining and resulted in the significantly better prognoses both in overall survival (p = 0.02) and disease free survival (p < 0.01) compared to patients without statin intake. Statins may inhibit tumor progression via an EZH2-mediated epigenetic alteration, which results in survival benefits after resected CRC. Furthermore, statin plus classII HDAC inhibitor could be a novel anticancer therapy by their synergistic effects in CRC.

CD44s signals the acquisition of the mesenchymal phenotype required for anchorage-independent cell survival in hepatocellular carcinoma.

BACKGROUND: Circulating tumour cells (CTCs) have an important role in metastatic processes, but details of their basic characteristics remain elusive. We hypothesised that CD44-expressing CTCs show a mesenchymal phenotype and high potential for survival in hepatocellular carcinoma (HCC). METHODS: Circulating CD44(+)CD90(+) cells, previously shown to be tumour-initiating cells, were sorted from human blood and their genetic characteristics were compared with those of tumour cells from primary tissues. The mechanism underlying the high survival potential of CD44-expressing cells in the circulatory system was investigated in vitro. RESULTS: CD44(+)CD90(+) cells in the blood acquired epithelial-mesenchymal transition, and CD44 expression remarkably increased from the tissue to the blood. In Li7 and HLE cells, the CD44(high) population showed higher anoikis resistance and sphere-forming ability than did the CD44(low) population. This difference was found to be attributed to the upregulation of Twist1 and Akt signal in the CD44(high) population. Twist1 knockdown showed remarkable reduction in anoikis resistance, sphere formation, and Akt signal in HLE cells. In addition, mesenchymal markers and CD44s expression were downregulated in the Twist1 knockdown. CONCLUSIONS: CD44s symbolises the acquisition of a mesenchymal phenotype regulating anchorage-independent capacity. CD44s-expressing tumour cells in peripheral blood are clinically important therapeutic targets in HCC.

Expression of BAFF and BAFF-R in the synovial tissue of patients with rheumatoid arthritis.

OBJECTIVE: The elevated expression of B-cell-activating factor belonging to the TNF family (BAFF) is associated with systemic autoimmune disease, including rheumatoid arthritis (RA). The present study was undertaken to determine the distribution of BAFF and its receptor BAFF-R in the cells residing in the rheumatoid synovium. METHODS: The expression of BAFF and BAFF-R in synovial tissues obtained from 12 RA patients was examined by immunohistochemistry and flow cytometry. The mRNA expression of these molecules was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Soluble BAFF levels were measured with an enzyme-linked immunosorbent assay (ELISA). Fibroblast-like synoviocytes (FLS) purified from the RA (RA-FLS) were co-cultured with peripheral B cells. The degree of apoptosis in the B cells was measured to assess the effects on the viability of the B cells. RESULTS: The RA synovium showed focal or diffuse infiltration of mononuclear cells (MNCs), and one specimen showed germinal centre (GC)-like structures. Synovial sublining cells, but not lining cells, expressed BAFF. These sublining cells were negative for BAFF-R. BAFF and BAFF-R were expressed in B and T cells extracted from the RA synovium. Notably, RA-FLS spontaneously expressed cytoplasmic BAFF after 4-6 passages; however, they did not express BAFF or BAFF-R on their cell surface. RA-FLS could support the survival of B cells by preventing their apoptosis, but its effect on B cells might not be BAFF dependent. CONCLUSIONS: BAFF and BAFF-R are widely expressed in the RA synovium. The cells residing in the RA synovium might affect each other through BAFF.

Maturation-dependent change and regional variations in acoustic stiffness of rabbit articular cartilage: an examination of the superficial collagen-rich zone of cartilage.

OBJECTIVE: The purpose of the study was to investigate maturation-dependent changes of acoustic (ultrasound) stiffness and other ultrasound features of articular cartilage in healthy rabbit knees. METHODS: Five groups of rabbits of various ages (3 weeks, 8 weeks, 6 months, 1 year, 2.5 years) consisting of five rabbits per group were examined. Signal intensity (index of stiffness), signal duration (index of surface irregularity) and interval between signals (index of thickness) of the ultrasound reflection from articular cartilage were examined at four sites: posterior lateral femoral condyle, posterior medial femoral condyle, lateral tibial plateau, and medial tibial plateau. The sites were observed macroscopically and microscopically with a light microscope and a polarized light microscope. RESULTS: At the lateral and medial condyles and the lateral tibial plateau, signal intensity was least in 3-week-old specimens. The intensity increased until 6 months or 1 year of age. At these sites, the signal durations and intervals between signals were least at the ages of 6 months or 1 year. At the medial tibial plateau, the intensity was the least at 2.5 years of age and the interval between signals was least at 3 weeks of age; there was no effect of age on signal duration. Cartilage surfaces of all specimens were smooth and no degenerative changes were macroscopically or microscopically evident. The surface brightness of cartilage under the polarized light microscope was consistent with signal intensity values. CONCLUSIONS: The response of articular cartilage to ultrasound was maturation-dependent. Acoustic properties differed from mechanical stiffness properties, which were determined using indentation. Ultrasound may detect properties of the surface collagen of the articular cartilage.

[Causes of dementia and bed-ridden state in elderly victims of road traffic accidents in Japan].

BACKGROUND: In Japan, autopsies are seldom performed for the victims of road traffic accidents (RTA). Thus, when an elderly RTA victim dies after long-term hospitalization, the actual cause of death is hard to determine. SUBJECTS: Eight hundred eleven road traffic victims (69 +/- 16 years, M/F = 592/219) for whom insurance companies required us to judge the medical causal relation between the accident and the death from the view point of clinical forensic medicine. METHODS: We discussed and reevaluated the cause of death and decided the cause and effect relation based on medical information. RESULTS: The number of subjects was equivalent to 8.4% of the yearly deaths by RTA in the investigated area, 19.3% of the aged death by RTA. An autopsy was performed only for 5%, ISS was 17.0 +/- 11.5; 94% were hospitalized; 62% continued to be unconscious, involving dementia (32%), persistent vegetative state (17%), coma or semi-coma (13%). Of the cases investigated, 89% died after becoming bedridden, which in 87% of cases was due to the RTA. The odds ratios of a bedridden state for head AIS > = 3, lower limbs AIS > = 2, lower limbs AIS > = 3, spinal AIS > = 3, chest AIS > = 2 were 3.1, 3.2, 7.2, 2.5, 1.8, respectively. The odds ratios of dementia for head AIS > = 3, head AIS > = 4, chest AIS > = 4 were 29.2, 35.9, 1.2, respectively. Patients with head injuries were more likely to be bedridden and senile, and patients with leg fractures tended to be bedridden, particular in the aged. When elderly traffic victims became bedridden or senile, most of them, 399 bedridden victims and 53 non-bedridden victims, died within 242 +/- 283 days, and 356 +/- 284 days from the RTA, respectively. The cause of death could be classified into five categories; pneumonia, other infection, malnutrition and/or dehydration, malnutrition and/or dehydration with pneumonia, malnutrition and/or dehydration with other infection. Traffic deaths were recorded on 31% of death certificates, but were 77% by our reevaluation (p < 0.00001), because the cause-of-death can be difficult to determine in elderly traffic victims. Logistic regression was used to calculate the risk of dementia caused by bone fractures after adjusting for age and gender. The risk of dementia due to bone fractures was influenced by the number of the long fractured bones, a high age group, a lower ADL, and a past history of dementia. Thus, we speculate that traumatic dementia based on bone fractures may occur.

Gene analysis of Vibrio cholerae NAGV14 pilus and its distribution.

Adhesive pilus of Vibrio cholerae 034, strain NAGV14, was genetically analyzed. The deduced amino acid (aa) sequence of the major pilin structural gene (VcfA) was 67% homologous to the MshA pilin in the N-terminal region, but no homology was found in the C-terminal region which contained the antigenic epitopes. Upstream and downstream flanking regions examined were highly homologous to mshB and mshC of the MSHA (mannose-sensitive hemagglutinin) gene locus. A short leader sequence and a pair of cysteines near the C-terminus which are the characteristics of type 4a pilus family were found. The major pilin structural gene of NAGV14 was compared to that of a strain V10 producing non-adhesive pili. The deduced aa sequences showed 60% homology, and the distance between two cysteines in the C-terminal region was different. A total of 177 V. cholerae strains were investigated for the presence of a type 4 pilus gene locus by PCR, and 95% were positive. The major pilin gene of NAGV14 was detected in 4 of 93 V. cholerae non-O1, non-0139 strains tested, but none of the V. cholerae O1 and O139 (72 and 12 strains, respectively). Our result suggested that a type 4 pilus gene locus similar to the MSHA gene locus is widely distributed among V. cholerae strains. We proposed naming this type 4 pilus gene locus the VCF (for V. cholerae flexible pili) gene locus.

[The changes of PCBs and PCDFs as well as symptoms in Yusho patients for 30 years].

Two mass-food poisonings called Yusho and Yucheng occurred in Western Japan and Taiwan in 1968 and 1979, respectively. Blood samples from 5 Yusho patients and 3 Yucheng patients were collected several times from 1982 to 1998 and from 1980 to 1995, respectively, and analyzed for PCB and PCDF congeners. Fat base concentrations of 2,3,4,7,8-penta-CDF in the Yusho patients, which were responsible for about 70% of the dioxin toxicity, are estimated to be decreased from 60 ppb in 1969 to 0.8 ppb in 1997 with the median half-life of 2.9 years in the first 15 years after the onset and 7.7 years in the next stage of 15 years. Fat base concentrations of total PCBs in the blood of Yusho patients are estimated to be decreased from 75 ppm just after the onset to 2.3 ppm after passage of 30 years. Typical Yusho symptoms of acneiform eruption, dermal pigmentation and increased eye discharge were very gradually recovered with lapse of ten years. However, enzyme and/or hormone mediated sign of high serum triglyceride, high serum thyroxin, immunoglobulin disorder and others are persistently maintained for 30 years.

[Assessment of hydroxylated metabolites of polychlorinated biphenyls and polychlorinated dibenzofurans as potential estrogens by yeast two-hybrid system].

The estrogenic activities of several hydroxylated metabolites of PCBs and PCDFs were investigated by yeast two-hybrid assay based on the ligand-dependent interaction of estrogen receptor with coactivator. For the hydroxylated PCBs, the order of estrogenic potency was 4-OH-2',4',6'-triCB > 4-OH-4'-monoCB, 4-OH-biphenyl. These compounds were evaluated as 10(3) to 10(4) less potent than 17 beta-estradiol based on the concentrations of test compounds showing 10% activity of 10(-7) M 17 beta-estradiol. 2-OH-3',4,4'-triCB, 4-OH-2',3,4'-triCB and 3-OH-/4-OH-2,2',5,5'-tetraCB, the metabolites of 2,2',5,5'-tetraCB were inactive as estrogens at the highest concentrations used in this study (10(-5) M). Also 4-OH-3,3',4',5-tetraCB, the metabolite of 3,3',4,4'-tetraCB was inactive as estrogen, indicating that this hydroxylated metabolite did not take part in the estrogenic activity of 3,3',4,4'-tetraCB. OH group at 4-position of biphenyl was necessary for the expression of estrogenicity, but one or two chloro-substitution adjacent to OH group inhibited the activity. For the hydroxylated PCDFs, 8-OH-2-monoCDF, 7-OH-3,4-diCDF, 8-OH-3,4-diCDF, 8-OH-3,4,6-triCDF and 3,8-(OH)2-2-monoCDF exhibited estrogenic activity. The estrogenic activity of 3,8-(OH)2-2-monoCDF was comparable to those of 4-OH-2',4',6'-triCB and 4-nonylphenol (mixture of compounds with branched sidechain). The order of activity was 3,8-(OH)2-monoCDF > 8-OH-3,4-diCDF, 7-OH-3,4-diCDF > 8-OH-2-monoCDF, 8-OH-3,4,6-triCDF. These compounds were evaluated as 2.5 x 10(3) to 3 x 10(4) less potent than 17 beta-estradiol. On the other hand, no estrogenic activity was observed for 2-OH-dibenzofuran, 3-OH-2,8-diCDF, 6-OH-3,4-diCDF and 9-OH-3,4-diCDF at concentrations as high as 10(-4) M. Substitution of OH group at 2(8)- or 3(7)-position of dibenzofuran and no chloro-substitution adjacent to OH group was required for the estrogenic activity.