Angiogenic activity of subchondral bone during the progression of osteoarthritis in a rabbit anterior cruciate ligament transection model.

OBJECTIVE: To investigate the longitudinal angiogenic activity of subchondral bone and cartilage during the progression of osteoarthritis (OA) using a rabbit model of OA. MATERIALS AND METHODS: OA was surgically induced by anterior cruciate ligament transaction (ACLT) in left knee of 12 months old female New Zealand white rabbits (n = 33). Histological examination, immunohistochemistry, and angiogenic activity assay was done at 0, 2, 4, 6, 8, 12 weeks after ACLT. Histologic evaluation was performed with haematoxylin and eosin, safranin-O staining to assess the OA change of medial femoral condyle (MFC) and lateral femoral condyle (LFC). CD31 immunohistochemistry was performed to confirm the vascular invasion at osteochondral junction. A co-cultured tubule formation assay was conducted to evaluate angiogenic activity of the subchondral bone and cartilage of MFC and LFC as well as synovium. Association between histological changes, angiogenic activity, and vascular invasion were evaluated. RESULTS: OA changes increased in a time-dependent manner both in MFC and LFC. Angiogenic activity of subchondral bone showed a monomodal change during the OA progression, achieved a peak in the early to progressive stage and decreased to normal level in the late stage of OA. Surge of vascular invasion was observed following the increase of angiogenic activity in the progressive stage of OA. Angiogenic activity of cartilage did not change during the course of OA progression. CONCLUSION: Angiogenic activity of subchondral bone was elevated in the early to progressive stage of OA and vascular invasion into the osteochondral junction followed.

IL-6 SNP diversity among four ethnic groups as revealed by bead-based liquid array profiling.

In the present work, we established a rapid, cost-effective and high-throughput method for genotyping using a multiplexed microsphere-based suspension array platform - Luminex(®) ×MAP™, which enabled us to analyse two SNPs in the promoter of IL-6 gene, determining haplotypes associated with different levels of expression. Using this system, IL-6 diversity in four different ethnic groups - East Asians, Caucasians, Hispanic and African Americans - was assessed. Results showed a significant variability in terms of allele, genotype and haplotype distribution. Considering the important immunoregulatory role of this cytokine and as a clinically relevant marker, this genotyping approach will provide a powerful tool for disease association or transplant outcome studies.

Joint distraction and movement for repair of articular cartilage in a rabbit model with subsequent weight-bearing.

We have previously shown that joint distraction and movement with a hinged external fixation device for 12 weeks was useful for repairing a large articular cartilage defect in a rabbit model. We have now investigated the results after six months and one year. The device was applied to 16 rabbits who underwent resection of the articular cartilage and subchondral bone from the entire tibial plateau. In group A (nine rabbits) the device was applied for six months. In group B (seven rabbits) it was in place for six months, after which it was removed and the animals were allowed to move freely for an additional six months. The cartilage remained sound in all rabbits. The areas of type II collagen-positive staining and repaired soft tissue were larger in group B than in group A. These findings provide evidence of long-term persistence of repaired cartilage with this technique and that weight-bearing has a positive effect on the quality of the cartilage.

Modified ELISPOT assay may predict T-cell hyporesponsiveness to non-inherited maternal antigens.

Clinical reports have suggested the existence of immunological tolerance to noninherited maternal antigens (NIMA) in human leukocyte antigen (HLA) mismatched allogeneic stem cell transplantation (allo-SCT). We studied the T-cell reactivity using IFN-gamma enzyme-linked immunospot (ELISPOT) assay in three HLA fully matched allo-SCT cases and one healthy volunteer family case. In HLA fully matched allo-SCT cases, ELISPOT assay could detect the hyporesponsiveness of T cells from donors to the B cells from recipients. Moreover, ELISPOT assay showed that the T cells from an individual responded to B cell from his mother significantly weakly than those from an unrelated HLA-haploidentical individual. These observations suggest that our IFN-gamma ELISPOT assay-based method may predict the presence of immunological tolerance to NIMA.

Restoration of elbow function by intercostal nerve transfer for obstetrical paralysis with co-contraction of the biceps and the triceps.

Among the conservatively treated patients with obstetrical paralysis after delivery in the vertex presentation, cross reinnervation sometimes occurs in the course of their recovery and co-contraction of multiple muscles impairs smooth upper limb motion. Such co-contraction of the biceps and the triceps inhibits normal elbow motion, making it impossible to use the elbow effectively in daily activities, despite adequate strength in these muscles. To overcome biceps/triceps co-contraction, we performed intercostal nerve transfer to the musculocutaneous nerve for three patients of age 11 months, 6 years and 9 years, respectively, and to the motor branches of the triceps for two patients of age 4 and 14 year-old, respectively. During the average follow-up period of 5.6 (range 1-11.5) years, the power of the reinnervated muscle improved to more than M3, and smooth motion of the elbow independently of shoulder motion was restored.

Effect of progesterone on recovery from nerve injury during leg lengthening in rats.

We investigated the effect of progesterone on the nerve during lengthening of the limb in rats. The sciatic nerves of rats were elongated by leg lengthening for ten days at 3 mm per day. On alternate days between the day after the operation and nerve dissection, the progesterone-treated group received subcutaneous injections of 1 mg progesterone in sesame oil and the control group received oil only. On the fifth, tenth and 17th day, the sciatic nerves were excised at the midpoint of the femur and the mRNA expression level of myelin protein P0 was analysed by quantitative real time polymerase chain reaction. On day 52 nodal length was examined by electron microscopy, followed by an examination of the compound muscle action potential (C-MAP) amplitude and the motor conduction velocity (MCV) of the tibial nerve on days 17 and 52. The P0 (a major myelin glycoprotein) mRNA expression level in the progesterone-treated group increased by 46.6% and 38.7% on days five and ten, respectively. On day 52, the nodal length in the progesterone-treated group was smaller than that in the control group, and the MCV of the progesterone-treated group had been restored to normal. Progesterone might accelerate the restoration of demyelination caused by nerve elongation by activating myelin synthesis.

The effects of radial shock waves on gene transfer in rabbit chondrocytes in vitro.

OBJECTIVE: The purpose of this study was to develop a new technique of gene transfer utilizing radial shock waves. The effects of radial shock waves on gene transfer in rabbit chondrocytes were examined by varying the parameters of exposure conditions in vitro. METHODS: Chondrocytes were obtained from New Zealand white rabbits and cultured in a monolayer. A luciferase-encoding gene expression vector, or vector alone, was added to chondrocyte cell suspensions, and the cells were then exposed to radial shock waves. Parameters such as pressure amplitude, number of pulses, frequency, and DNA concentration were varied, and luciferase activity was measured 48h after transfection. Transfection efficiency of radial shock waves was compared with the FuGENE6 transfection method using a green fluorescence protein (GFP)-encoding gene vector by fluorescent-activated cell sorter (FACS) analysis. RESULTS: Radial shock wave exposure significantly increased luciferase activity over 140-fold as compared to the control under the optimal exposure conditions. Both pressure amplitude and number of pulses were relevant to transfection efficiency and cell viability, but frequency was not. Transfection efficiency increased in a dose-dependent manner with DNA concentration. FACS analysis showed 4.74% of GFP-encoding gene using radial shock waves. FuGENE6 transfection was almost similar in transfection efficiency to radial shock wave. CONCLUSION: In spite of certain degree of cell disruption, radial shock waves significantly augmented reporter gene transfection in rabbit chondrocytes in vitro. Radial shock waves may potentially contribute to the treatment of the cartilage morbidities by enhancing the potency of tissue healing and gene transfection of growth factors.

Extracorporeal shock wave therapy improves motor dysfunction and pain originating from knee osteoarthritis in rats.

OBJECTIVE: Although there have been several reports on the use of extracorporeal shock wave therapy (ESWT), the efficacy of ESWT for knee osteoarthritis (OA) has not been clarified. The aim of this study is to investigate the effect of ESWT on OA in a rat knee model. METHODS: The rats were divided into three groups: (1) control, (2) OA, and (3) ESWT (knee OA+shock wave therapy). Behavioral analysis consisted of measuring the duration of walking on a treadmill. The expression of calcitonin gene-related peptide (CGRP) in dorsal root ganglion (DRG) neurons innervating the knee using immunohistochemistry was examined in the three groups at their peak time point on the treadmill. RESULTS: Walking duration was significantly extended 4, 7 and 14 days after ESWT in rats with knee OA (peak time point: 4 days), again decreasing by days 21 and 28. Immunohistochemical studies revealed that the OA group had significantly higher percentages of CGRP positive neurons in the DRG than were found in the control group. In addition, ESWT reduced the ratio of CGRP positive DRG neurons in the OA model. CONCLUSION: The improvement in walking ability and the reduction of CGRP positive neurons in DRG indicates that ESWT is a useful treatment for knee OA.

Unchanged high prevalence of antibodies to hepatitis E virus (HEV) and HEV RNA among blood donors with an elevated alanine aminotransferase level in Japan during 1991-2006.

Hepatitis E is rare in Japan but is occurring more frequently than previously thought. To investigate whether de novo subclinical infection of hepatitis E virus (HEV) has recently increased in Japan, HEV RNA was assayed in serum samples obtained from 4019 Japanese voluntary blood donors with alanine aminotransferase (ALT) of > or =61 IU/l, who are likely to have ongoing HEV infection, during 1991-2006. The overall rates of IgG-class antibody to HEV (anti-HEV IgG), anti-HEV IgM/IgA and HEV RNA among 3185 donors in 2004-2006 were comparable with those among 594 donors in 1998 (5.3 vs. 5.2%, 0.2 vs. 0.5%, and 0.2 vs. 0.3%, respectively). Among blood donors with ALT > or = 201 IU/l in three groups according to the year of blood collection (1991-1995 [n = 156], 1996-1999 [n = 116] and 2004-2006 [n = 61]), there were no appreciable differences in the prevalence of anti-HEV IgG (5.8, 4.3, and 6.6%, respectively), anti-HEV IgM/IgA (1.9, 3.4, and 3.3%, respectively) and HEV RNA (1.3, 3.4, and 3.3%, respectively). The eleven HEV isolates obtained in the present study differed from each other by 1.7-22.8% in the ORF2 sequence and segregated into genotype 3 or 4. The occurrence rate of subclinical infection with divergent HEV strains has essentially remained unchanged during 1991-2006 in Japan.

Effects of Green Manure Type and Amendment Rate on Verticillium Wilt Severity and Yield of Russet Burbank Potato.

Increasing restriction of agrochemicals is motivating development of ecology-based cropping systems, including green manures, to manage soilborne diseases. Green manures have shown promise in suppressing Verticillium dahliae, but information about effect of different green manures and optimal application rates remains limited. Therefore, we conducted two single-year field experiments comparing effects of Austrian winter pea (Pisum sativum 'Melrose'), broccoli (Brassica oleracea var. botrytis 'Excelsior'), and Sudan grass (Sorghum vulgare var. sudanense 'Monarch'), amended at 6, 12, or 24 Mg ha-1, on soil populations and root infection by V. dahliae, wilt severity, and yield of Russet Burbank potato. Inoculum density was reduced relative to the nonamended, infested control by all broccoli treatments, Austrian winter pea applied at 12 and 24 Mg ha-1, and Sudan grass applied at 12 Mg ha-1. Root infection was not reduced by any green manure treatment. Median wilt severity was reduced approximately 70% by all green manures applied at 24 Mg ha-1 and 74% by Austrian winter pea applied at 12 Mg ha-1. Tuber yield was reduced approximately 20% in nonamended, V. dahliae-infested controls relative to the noninfested controls. No green manure treatment improved yield relative to the nonamended, infested control.

Repair of large full-thickness articular cartilage defects in the rabbit: the effects of joint distraction and autologous bone-marrow-derived mesenchymal cell transplantation.

We produced large full-thickness articular cartilage defects in 33 rabbits in order to evaluate the effect of joint distraction and autologous culture-expanded bone-marrow-derived mesenchymal cell transplantation (ACBMT) at 12 weeks. After fixing the knee on a hinged external fixator, we resected the entire surface of the tibial plateau. We studied three groups: 1) with and without joint distraction; 2) with joint distraction and collagen gel, and 3) with joint distraction and ACBMT and collagen gel. The histological scores were significantly higher in the groups with ACBMT collagen gel (p < 0.05). The area of regenerated soft tissue was smaller in the group allowed to bear weight (p < 0.05). These findings suggest that the repair of large defects of cartilage can be enhanced by joint distraction, collagen gel and ACBMT.

A paired study of Perthes' disease comparing conservative and surgical treatment.

We performed a paired study of mature patients with Perthes' disease to compare the radiological results after treatment between conservatively- and surgically-treated groups. One patient was selected from each group to create the pairs for this study. Each pair was strictly matched for gender, body mass index, age at onset, stage at the first visit, necrotic area and radiological at-risk signs and each was assessed by comparing the values of six radiological measurements. Eighteen pairs (36 hips) fitted the criteria. The radiological measurements which showed a statistically better result in the surgical groups were Mose's method, the acetabular-head index and leg-length discrepancy. There were no statistical differences in the slope of the acetabular roof and the articulotrochanteric distance. Four hips in the conservative group were in Stulberg class II, five in class III and nine in class IV. In the surgical group, 13 were in Stulberg class II, four in class III and one in class IV. We conclude that surgical treatment improved the sphericity of the femoral head and provided greater acetabular cover, but did not reform the acetabular roof. It was noteworthy that a greater leg-length discrepancy and a smaller articulotrochanteric distance were not seen in the surgical group. Our study suggests that surgical treatment is preferable in patients with severe Perthes' disease.

Long-term implantation of zinc-releasing calcium phosphate ceramics in rabbit femora.

Zinc is an essential trace element that has stimulatory effects on bone formation. Recently, we developed zinc-releasing calcium phosphate ceramics in order to add the pharmacologic effect of zinc to calcium phosphate ceramics. In our previous study, we showed that the optimum zinc content for promoting bone formation was 0.316 wt %. Therefore a zinc composite ceramic of zinc-containing beta-tricalcium phosphate and hydroxyapatite, with a zinc content of 0.316 wt %, was chosen for long-term implantation. Cylindrical rods of the zinc composite ceramic were implanted in rabbit femora for 2 to 60 weeks. Using computer-aided image analysis, a histomorphometric study was carried out to investigate bone formation and resorption around the implants. The control was a composite ceramic of beta-tricalcium phosphate and hydroxyapatite without zinc. The addition of zinc to the implant demonstrated both favorable and unfavorable effects on bone remodeling. The favorable effect was enhanced bone apposition to the implant surface, demonstrated by a significant increase in intramedullary bone apposition rate at 6 weeks and in cortical bone apposition rate at 24 and 60 weeks (p < 0.05). The unfavorable effect was increased bone resorption, demonstrated by a significant increase in medullary cavity area at 60 weeks (p < 0.05). In order to utilize the favorable effect and avoid the unfavorable effect of zinc, either a reduction in zinc content in the zinc composite ceramic or the selection of implantation sites that do not have excessive exposure to bone marrow are required.

Determination of stale-flavor carbonyl compounds in beer by stir bar sorptive extraction with in-situ derivatization and thermal desorption-gas chromatography-mass spectrometry.

A method for the determination of stale-flavor carbonyl compounds including E-2-octenal, E-2-nonenal, E,Z-2,6-nonadienal and E,E-2,4-decadienal in beer was developed using stir bar sorptive extraction (SBSE) with in-situ derivatization followed by thermal desorption-GC-MS analysis. The derivatization conditions with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine and the SBSE conditions--sampling mode, salt addition, sample volume, polydimethylsiloxane volume (sample/polydimethylsiloxane phase ratio) and extraction time--were examined. The method showed good linearity over the concentration range from 0.1 to 10 ng ml(-1) for all analytes and the correlation coefficients were higher than 0.9993. The limits of detection ranged from 0.021 to 0.032 ng ml(-1) for all analytes. The recoveries (98-101%) and precision (RSD 2.4-7.3%) of the method were examined by analyzing beer samples fortified at the 0.5-ng ml(-1) level. The method was successfully applied to low-level concentration samples.

Successful non-T cell-depleted HLA haplo-identical three-loci mismatched hematopoietic stem cell transplantation from mother to son based on the feto-maternal microchimerism in chronic myelogenous leukemia.

A 17-year-old male with chronic myelogenous leukemia in blast crisis received a non-T cell-depleted (TCD) HLA haplo-identical three-loci mismatched hematopoietic stem cell transplant (HSCT) from his mother. Long-term feto-maternal microchimerism was detected by nested polymerase chain reaction with sequence-specific primer typing. The post-transplantation prophylaxis against graft-versus-host disease (GVHD) was tacrolimus with minidose methotrexate. Sustained engraftment was obtained. Acute GVHD (grade 2) developed, but improved rapidly. Bone marrow aspiration on day 120 showed complete remission. Non-TCD HLA haplo-identical HSCT based on feto-maternal microchimerism might be feasible and has important implications in the selection of alternative family donors in HSCT.

Determination of trace amounts of off-flavor compounds in drinking water by stir bar sorptive extraction and thermal desorption GC-MS.

A method for the determination of trace amounts of off-flavor compounds including 2-methylisoborneol, geosmin and 2,4,6-trichloroanisole in drinking water was developed using the stir bar sorptive extraction technique followed by thermal desorption-GC-MS analysis. The extraction conditions such as extraction mode, salt addition, extraction temperature, sample volume and extraction time were examined. Water samples (20, 40 and 60 ml) were extracted for 60-240 min at room temperature (25 degrees C) using stir bars with a length of 10 mm and coated with a 500 microm layer of polydimethylsiloxane. The extract was analyzed by thermal desorption-GC-MS in the selected ion monitoring mode. The method showed good linearity over the concentration range from 0.1 or 0.2 or 0.5 to 100 ng l(-1) for all the target analytes, and the correlation coefficients were greater than 0.9987. The detection limits ranged from 0.022 to 0.16 ng l(-1). The recoveries (89-109%) and precision (RSD: 0.80-3.7%) of the method were examined by analyzing raw water and tap water samples fortified at the 1 ng l(-1) level. The method was successfully applied to low-level samples (raw water and tap water).