Histological and molecular characterization of the femoral attachment of the human ligamentum capitis femoris.

The ligamentum capitis femoris (LCF) has increased in clinical significance through the development of hip arthroscopy. The histological pathologies and molecular composition of the femoral attachment of the LCF and the degeneration caused by LCF disruption were investigated in the human hip joint. Twenty-four LCFs were retrieved at surgery for femoral neck fracture (age range: 63-87 years). In the "intact" (i.e., intact throughout its length, n = 12) group, the attachment consisted of rich fibrocartilage. Fibrocartilage cells were present in the midsubstance. In contrast, the construction of the attachment in the "disrupted" (i.e., ligament no longer attached to the femoral head, n = 12) group had disappeared. The attachment in the disrupted group was not labeled for type II collagen or aggrecan, while that in the intact group was labeled for types I, II and III collagen, chondroitin 4-sulfate, chondroitin 6-sulfate, aggrecan, and versican. The percentage of single-stranded DNA-positive chondrocytes was significantly higher in the disrupted group than in the intact group. We conclude that the femoral attachment of the LCF has a characteristic fibrocartilaginous structure that is likely to adjust to the mechanical load, and suggest that its degeneration is advanced by disruption and should be regarded as a clinical pathology.

EGFR inhibitors augment antitumour helper T-cell responses of HER family-specific immunotherapy.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer-related morbidity and mortality worldwide. Epidermal growth factor receptor (EGFR)-targeted therapy is an attractive strategy alternative to conventional cancer treatments for HNSCC, but its efficacy remains controversial. T-cell-based immunotherapy has been proposed as a novel therapeutic approach to improve the clinical outcome for HNSCC. In this study, we report human epidermal receptor (HER) family epitopes that induced CD4 T-cell responses to HNSCC. The results provide support for a novel strategy to treat HNSCC by combining EGFR-targeted therapy with T-cell-based immunotherapy. METHODS: We evaluated the capacity of predicted CD4 T-cell peptide epitopes from EGFR to induce antitumour immune responses in vitro. In addition, EGFR inhibitors were evaluated for their ability to augment tumour MHC class II expression in HNSCC cell lines and subsequently increase T-cell recognition. RESULTS: Among several predicted peptide epitopes, EGFR875-889 elicited CD4 T-cell responses that were restricted by HLA-DR4, DR15, or DR53 molecules, indicating that the peptide functions as a promiscuous T-cell epitope. The peptide-reactive T cells responded to autologous dendritic cells loaded with EGFR-expressing tumour cell lysates, indicating that these epitopes are naturally processed. In addition, the CD4 T cells were capable of directly recognising and killing HNSCC cells expressing EGFR and the appropriate HLA class II molecule. T cells reactive with the EGFR875-889 epitope could be detected in the blood of HNSCC patients. EGFR875-889-reactive CD4 T cells were also able to recognise several peptide analogues derived from homologous regions of EGFR family members, HER-2, HER-3 and c-MET. Finally, we examined the effects of EGFR tyrosine kinase inhibition or EGFR-blocking antibodies on CD4 T-cell tumour reactivity. Treatment of tumour cells with the EGFR inhibitors enhanced tumour recognition by EGFR875-889-reactive T cells presumably due to the upregulation of HLA-DR expression in the HNSCC cells. CONCLUSION: We identified novel CD4 T-cell EGFR epitopes and amongst these, EGFR875-889 functions as a promiscuous helper T-cell epitope that can elicit effective antitumour T-cell responses against tumours expressing HER family members and c-MET. These observations should facilitate the translation of T-cell-based immunotherapy into the clinic for the treatment of HNSCC and provide a rational basis for EGFR inhibition, immune-targeted combination therapy.

The use of a ceramic talar body prosthesis in patients with aseptic necrosis of the talus.

The purpose of this study was to evaluate the clinical results of a newly designed prosthesis to replace the body of the talus in patients with aseptic necrosis. Between 1999 and 2006, 22 tali in 22 patients were replaced with a ceramic prosthesis. A total of eight patients were treated with the first-generation prosthesis, incorporating a peg to fix into the retained neck and head of the talus, and the remaining 14 were treated with the second-generation prosthesis, which does not have the peg. The clinical results were assessed by the American Orthopaedic Foot and Ankle Society ankle/hindfoot scale. The mean follow-up was 98 months (18 to 174). The clinical results of the first-generation prostheses were excellent in three patients, good in one, fair in three and poor in one. There were, however, radiological signs of loosening, prompting a change in design. The clinical results of the second-generation prostheses were excellent in three patients, good in five, fair in four and poor in two, with more favourable radiological appearances. Revision was required using a total talar implant in four patients, two in each group. Although the second-generation prosthesis produced better results, we cannot recommend the use of a talar body prosthesis. We now recommend the use of a total talar implant in these patients.

Low tibial osteotomy for varus-type osteoarthritis of the ankle.

In this retrospective study we have assessed the results of low tibial valgus osteotomy for varus-type osteoarthritis of the ankle and its indications. We performed an opening wedge osteotomy in 25 women (26 ankles). The mean follow-up was for eight years and three months (2 years 3 months to 17 years 11 months). Of the 26 ankles, 19 showed excellent or good clinical results. Their mean scores for pain, walking, and activities of daily living were significantly improved but there was no change in the range of movement. In the ankles which were classified radiologically as stage 2 according to our own grading system, with narrowing of the medial joint space, and in 11 as stage 3a, with obliteration of the joint space at the medial malleolus only, the joint space recovered. In contrast, such recovery was seen in only two of 12 ankles classified as stage 3b, with obliteration of the joint space advancing to the upper surface of the dome of the talus. Low tibial osteotomy is indicated for varus-type osteoarthritis of stage 2 or stage 3a.

Regional variations in human patellar trabecular architecture and the structure of the proximal patellar tendon enthesis.

Proximal patellar tendinopathy occurs as an overuse injury in sport and is also characteristic of ankylosing spondylitis patients. It particularly affects the posteromedial part of the patellar tendon enthesis, although the reason for this is unclear. We investigated whether there are regional differences in the trabecular architecture of the patella or in the histology of the patellar tendon enthesis that could suggest unequal force transmission from bone to tendon. Trabecular architecture was analysed from X-rays taken with a Faxitron radiography system of the patellae of dissecting room cadavers and in magnetic resonance images of the knees of living volunteers. Structural and fractal analyses were performed on the Faxitron digital images using MatLab software. Regional differences at the enthesis in the thickness of the uncalcified fibrocartilage and the subchondral plate were evaluated histologically in cadaveric material. The radiological studies showed that the quantity of bone and the apparent trabecular thickness in the patella were greatest medially, and that in the lateral part of the patella there were fewer trabeculae which were orientated either antero-posteriorly or superiorly inferiorly. The histological study showed that the uncalcified fibrocartilage was most prominent medially and that the subchondral plate was thinner laterally. Overall, the results indicate that mechanical stress at the proximal patellar tendon enthesis is asymmetrically distributed and greater on the medial than on the lateral side. Thus, we suggest that the functional anatomy of the knee is closely related to regional variations in force transmission, which in turn relates to the posteromedial site of pathology in proximal patellar tendinopathy.

Pleiotropic effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors: candidate mechanisms for anti-lipid deposition in blood vessels.

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) are considered first-line therapeutic agents for the prevention of coronary heart disease and atherosclerotic disorders related to hypercholesterolemia. Statins inhibit lipid deposition in the aortic endothelium. Although it has been accepted that the statins are potent inhibitors of cholesterol biosynthesis in the liver and that they lower circulating cholesterol levels, several cholesterol-independent (pleiotropic) effects have been reported. The cholesterol-independent effects of statins involve normalization of the nitric oxide (NO)-NO synthase system, anti-inflammatory effects through the inhibition of cytokine/chemokine production, inhibition of vascular smooth muscle cell proliferation and migration, and inhibition of platelet thrombus formation/reduction of the thrombotic response. Some pleiotropic effects of statins may depend on the inhibition of the biosynthesis of farnesyl- and geranylgeranyl-nonsterol compounds from mevalonate in the cells. The Rho/Rho kinase pathway and the phospatidylinositol-3 kinase/Akt pathway mediate the pleiotropic effects of statins. As variations occur in absorption, metabolism, and excretion mechanisms due to the characteristics of specific statins including their hydrophilicity and lipophilicity, there are differences in the transfer mechanisms of statins into tissues. However, the pleiotropic effects occur regardless of statin hydrophilicity and lipophilicity. This review summarizes the pleiotropic effects of statins on lipid deposition in blood vessels.

Cortical neuromagnetic fields preceding voluntary jaw movements.

Slow cortical potentials (readiness potentials, RPs) reflecting the central programming of voluntary jaw movements were reported to appear preceding the movements. However, the current source producing the RP has not yet been localized. This study aimed to determine the cortical regions involved in the central programming of bilaterally symmetrical voluntary jaw movements, by locating the current source of the neuromagnetic counterpart of the RP (readiness field, RF). The RFs were found in the fronto-lateral region bilaterally, starting around 860 and 600 ms prior to the onset of masseter and digastric electromyograms (EMGs), respectively, and gradually increasing in magnitude to the peak within 100 ms before the EMG onset. Thus, the RFs appeared long before the reported onset of the excitability increase of pyramidal tract neurons. The current sources producing the RFs were located in the precentral gyrus bilaterally, with no bilateral differences in strength. We conclude that the primary motor cortex is involved bilaterally in central programming as well as in execution of bilaterally symmetrical voluntary jaw movements.

The histological structure of the malleolar groove of the fibula in man: its direct bearing on the displacement of peroneal tendons and their surgical repair.

The peroneal (fibularis) tendons are held in place within the malleolar groove by the superior peroneal retinaculum. If this is torn, the tendons can subluxate or dislocate. Understanding the anatomy of the region is important for treating these injuries when it becomes necessary to reconstruct the malleolar groove surgically. Serial transverse sections of the groove were cut from 10 dissecting room cadavers after routine histology processing. The structure of the malleolar groove differed significantly in its proximal and distal parts. Distally, the bone is convex and the shape of the groove is determined by a thick periosteal cushion of fibrocartilage that covers the bone surface. Proximally, the groove shape is determined by the bone itself, and the periosteum is thin and fibrous. The restriction of a periosteal fibrocartilage to the distal end suggests that it serves to adapt the shape of the malleolar groove to that of the tendons within it and thus promotes stress dissipation. Paradoxically, however, it increases the risk of damage to subluxated tendons, because these can be sliced longitudinally by a sharp ridge created from periosteal fibrocartilage when the retinaculum is torn. Our results suggest that if bone-block surgical procedures are used to reconstruct the malleolar groove, they are best restricted to its proximal part.

The skeletal attachment of tendons--tendon "entheses".

Tendon entheses can be classed as fibrous or fibrocartilaginous according to the tissue present at the skeletal attachment site. The former can be "bony" or "periosteal", depending on whether the tendon is directly attached to bone or indirectly to it via the periosteum. At fibrocartilaginous entheses, the uncalcified fibrocartilage dissipates collagen fibre bending and tendon narrowing away from the tidemark; calcified fibrocartilage anchors the tendon to the bone and creates a diffusion barrier between the two. Where there are additional fibrocartilaginous specialisations in the tendon and/or bone next to the enthesis, an "enthesis organ" is created that reduces wear and tear. Little attention has been paid to bone at entheses, despite the obvious bearing this has on the mechanical properties of the interface and the clinical importance of avulsion fractures. Disorders at entheses (enthesopathies) are common and occur in conditions such as diffuse idiopathic skeletal hyperostosis and the seronegative spondyloarthropathies. They are also commonly seen as sporting injuries such as tennis elbow and jumper's knee.

The functional anatomy of the human anterior talofibular ligament in relation to ankle sprains.

The anterior talofibular ligament is the most commonly injured ligament in the ankle. Despite considerable interest in the clinical outcome of treatment protocols, we do not know whether the distinctive pattern of localization of the injuries relates to regional differences in the structure and molecular composition of the ligament. To address this issue, ligaments were examined by histology and immunohistochemistry. Differences in the structure of its two attachments (i.e. entheses) were evaluated with quantitative, morphometric techniques, and regional differences in the distribution of collagens, glycosaminoglycans and proteoglycans were determined qualitatively by immunolabelling. Morphometric analyses showed that bone density was less at the fibular attachment, but that enthesis fibrocartilage was more prominent. Immunohistochemistry revealed the presence of a fibrocartilage (containing type II collagen and aggrecan) at the site where the ligament wraps around the lateral talar articular cartilage in a plantarflexed and inverted foot: the fibrocartilage is regarded as an adaptation to resisting compression. We propose that avulsion fractures are less common at the talar end of the ligament because (1) bone density is greater here than at the fibular enthesis, and (2) stress is dissipated away from the talar enthesis by the 'wrap-around' fibrocartilaginous character of the ligament near the talar articular facet.

Fixation of osteochondral lesions of the talus using cortical bone pegs.

We have treated osteochondral lesions of the talus using cortical bone pegs. We examined 27 ankles (27 patients) after a mean follow-up of 7.0 years (2 to 18.8). The mean age of the patients was 27.8 years (12 to 62). An unstable osteochondral fragment or osteosclerotic changes in the bed of the talus were regarded as indications for the procedure. The clinical results were good in 24 ankles (89%) and fair in three (11%); none had a poor result. There was also radiological improvement in 24 ankles. Repair of the articular surface and stability of the lesion can be achieved even in unstable chronic lesions.

Tyrosine hydroxylase antisense gene therapy causes hypotensive effects in the spontaneously hypertensive rats.

We investigated the effect of antisense oligodeoxynucleotides (AS ODN) against tyrosine hydroxylase (TH) on hypertension and sympathetic nervous system activity in spontaneously hypertensive rats (SHR). Systolic blood pressure (SBP) in SHR treated with TH AS ODN (50, 200 microg/rat, i.v.) was significantly lower than that of control SHR. Epinephrine and norepinephrine levels, TH activity, and TH protein levels in the adrenal medulla of SHR were reduced concomitant with TH AS ODN treatment-induced changes in SBP. In contrast, TH AS ODN (200 microg/rat) had no effect on SBP in Wistar-Kyoto rats (WKY), despite significantly decreased catecholamine levels, TH activity, and TH protein levels. These findings suggest that peripheral systemic injection of TH AS ODN may be effective as hypotensive therapy in SHR.

Impaired testicular function in rats with diet-induced hypercholesterolemia and/or streptozotocin-induced diabetes mellitus.

Hypercholesterolemia and diabetes mellitus are known to be accompanied by reproductive dysfunction. In this study, we investigated the effects of hypercholesterolemia, hyperglycemia, and these conditions combined, on testosterone (T) and testicular luteinizing hormone/human chorionic gonadotropin (LH/hCG) binding. Sprague-Dawley rats (8 weeks old) were divided into four groups: Group 1 was the control, group 2 was fed standard chow containing 2% cholesterol (C-diet), group 3 was administered streptozotocin (STZ, 65 mg/kg, i.p.), group 4 was treated with both the C-diet and STZ. After 4 weeks, rats were sacrificed. Serum glucose was significantly higher in the STZ group (304% that of controls) and the C-diet plus STZ group (345%), but there was no difference between the C-diet group (89%) and the control group. Serum cholesterol was significantly higher in the C-diet group (206% that of controls), the STZ group (452%) and the C-diet plus STZ group (2042%). Serum T, testicular T, and LH/hCG binding were significantly lower in the C-diet group (49%, 52%, and 81% that of controls, respectively), the STZ group (15%, 32%, and 72%) and the C-diet plus STZ group (8%, 21%, and 57%). These results suggest that hypercholesterolemia is an independent risk factor for testicular dysfunction and that the reduction of serum and testicular T levels is due at least in part to a reduction in testicular LH/hCG binding in rats with hypercholesterolemia, hyperglycemia, and these conditions combined. It is further suggested that the reduction in LH/hCG binding is mainly related to a rise in serum cholesterol levels.

Effects of PR-350, a newly developed radiosensitizer, on dihydropyrimidine dehydrogenase activity and 5-fluorouracil pharmacokinetics.

This study was designed to investigate the effects of PR-350, a newly developed radiosensitizer, on dihydropyrimidine dehydrogenase (DPD) activity and 5-fluorouracil (5-FU) pharmacokinetics in 8-week-old male Sprague-Dawley rats. In an in vitro study with hepatic cytosol, DPD activity was dose-dependently reduced by PR-350 at 0.5, 1.0, and 2.0 mmol/l to 75.5%, 64.9%, and 61.5%, respectively, of the control values. In an ex vivo study, DPD activities in hepatic cytosols obtained from animals which had received PR-350 over 4 days (200 mg/kg per day) were not significantly different from those in animals which had not. In an in vivo study, none of the pharmacokinetic parameters obtained from the plasma concentration-time profile of 5-FU were significantly altered by single i.v. injections of PR-350 (50, 100, or 200 mg/kg). However, (E)-5-(2)-(bromovinyl)uracil (BVU), a DPD inhibitor, significantly increased the half-life and area under the curve of 5-FU to 238.1% and 323.2%, respectively, of the control values. Administration of PR-350 over 4 days (200 mg/kg per day) did not affect either of these parameters. The administration of PR-350 significantly reduced the clearance (73.5% of control) and volume of distribution (71.0% of control) of 5-FU, but the alterations were much less than those caused by BVU. These results suggest that the effect of PR-350 on 5-FU pharmacokinetics is much less than that of BVU and that the enhancement of 5-FU toxicity by PR-350 is less than we initially anticipated.

Involvement of tyrosine hydroxylase upregulation in cyclosporine-induced hypertension.

To identify the mechanism of cyclosporine-induced hypertension, we studied the effect of cyclosporine on the catecholamine synthetic pathway in rats. We administered cyclosporine (10 mg/kg per day, s.c.) for 3 days to 10-week-old male Wistar rats. Systolic blood pressure increased significantly in the cyclosporine-treated group in comparison to that in the control group. Norepinephrine and epinephrine levels in the adrenal medulla and plasma of cyclosporine-treated rats were also significantly higher than levels in the control rats. Moreover, tyrosine hydroxylase (TH) activity and TH mRNA expression in the adrenal medulla of cyclosporine-treated rats were significantly elevated. Administration of the TH inhibitor alphamethyl-p-tyrosine (200 mg/kg, b.i.d., s.c.) for 3 days significantly suppressed cyclosporine-induced increases in systolic blood pressure. Phosphorylation of cyclic AMP responsive element-binding protein (CREB) and its binding activity to DNA in the nuclear fraction from the adrenal medulla of cyclosporine-treated rats were much higher than that of the control rats. Calcineurin protein expression of cyclosporine-treated rats was less than that of the control rats. These results suggest that cyclosporine increased blood pressure via activation of the catecholamine synthetic pathway due to the activation of transcription factor CREB.

Effects of estrogen on serum leptin levels and leptin mRNA expression in adipose tissue in rats.

OBJECTIVE: In this study, we examined changes in serum leptin levels during the estrus cycle and the role of estrogen in these changes. METHODS: We measured serum leptin levels during normal estrus cycles in intact rats and estradiol-17beta (E2)-induced artificial estrus cycles in ovariectomized rats. RESULTS: Serum leptin levels increased 1.6-fold from 4.2 +/- 0.2 ng/ml during diestrus stage 2 to 6.7 +/- 0.9 ng/ml during proestrus stage during the 4-day estrus cycle. During the E2-induced estrus cycle, serum leptin levels increased 2.3-fold from 2.3 +/- 0.1 ng/ml at estrus to 5.4 +/- 1.2 ng/ml at proestrus. E2 also increased serum leptin concentrations and leptin mRNA expression in adipose tissue of immature rats. DISCUSSION: These findings suggest that increased serum leptin induced by estrogen during proestrus may trigger the preovulatory release of luteinizing hormone. Furthermore, our findings indicate that estrogen has a positive effect on leptin production in adipose tissue.

Involvement of tyrosine hydroxylase up regulation in dexamethasone-induced hypertension of rats.

We investigated the effect of dexamethasone (DEX) on tyrosine hydroxylase (TH) mRNA level, and TH activity and catecholamine levels in the adrenal medulla of the rat. DEX (1 mg/kg/day, s.c.) was administered for 2 days, and a control group was given corn oil. DEX significantly increased systolic blood pressure. TH mRNA level, TH activity, epinephrine level, and norepinephrine level in the adrenal medulla of DEX-treated rats were significantly higher than those of control rats. Also, epinephrine and norepinephrine levels in plasma were significantly higher in DEX-treated rats than in controls. alpha-Methyl-p-tyrosine prevented the DEX-induced blood pressure increase. These results suggest that the catecholamine synthetic pathway may be involved in DEX-induced hypertension.

Precise anatomic configuration changes in the first ray of the hallux valgus foot.

To detect precise anatomical configuration of the first ray in feet with hallux valgus, a two-dimensional coordinate system was devised for evaluation of a weight-bearing dorsoplantar radiograph. The radiographs, taken from 229 feet of 114 patients with symptomatic hallux valgus and 94 normal feet, were investigated. A comparative study showed the first metatarsal head of a foot with hallux valgus was located on the medial side of that of the normal foot and the base of the proximal phalanx of the hallux valgus foot was located on the same point of that of the normal foot. Lateral translation of the base of the proximal phalanx occurred only in cases with overlap toes. Our study shows that subluxation of the metatarsophalangeal joint in hallux valgus is primarily caused by metatarsus primus varus. This study indicated that first metatarsal osteotomies should be given the first priority in consideration for bunion surgery, because these procedures could move the first metatarsal heads laterally in a more normal position.

Effect of antenatal dexamethasone treatment on Ca2+-dependent nitric oxide synthase activity in rat lung.

We investigated the effects of dexamethasone on nitric oxide synthase activity, nitrate/nitrite concentration, and cGMP concentration in the lungs of premature and full-term neonate rats. Dexamethasone or vehicle alone was administered to the mother (1 mg/kg/d, s.c., 2 d), and the neonate was killed 24 h after birth. Ca2+-dependent nitric oxide synthase activity and nitrate/nitrite and cGMP concentrations in lungs of dexamethasone-treated neonates, both premature and full-term, were significantly higher than those in the lungs of the control rats. Ca2+-dependent nitric oxide synthase activity, nitrate/nitrite concentration, and cGMP concentration in the lungs of control rats showed developmentally associated increases during late gestation and in the early postnatal period. The activation of the nitric oxide synthasenitric oxide-cGMP system by antenatal dexamethasone treatment may be related to the improvement of pulmonary function by antenatal glucocorticoid therapy to minimize respiratory distress syndrome.