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.

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.

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.

Influence of androgen on tyrosine hydroxylase mRNA in adrenal medulla of spontaneously hypertensive rats.

We investigated the effects of castration and testosterone propionate on tyrosine hydroxylase mRNA, its activity, and catecholamine synthesis in the adrenal medulla of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Four-week-old male rats were castrated. Testosterone propionate (500 micrograms per rat) was administered subcutaneously twice a week to castrated rats (between 14 and 25 weeks of age). Systolic pressure was measured at the age of 25 weeks, and rats were decapitated. The systolic pressure of castrated SHR was significantly lower than that of control and testosterone-replaced SHR. Epinephrine and norepinephrine levels, tyrosine hydroxylase activity, and tyrosine hydroxylase mRNA in the adrenal medulla of castrated SHR were significantly lower than in control and testosterone-replaced SHR. Systolic pressure and epinephrine and norepinephrine levels, tyrosine hydroxylase activity, and tyrosine hydroxylase mRNA levels in the adrenal medulla of WKY showed no significant differences among the control, castrated, and testosterone-replaced groups. These results suggest that androgens contribute to the development and maintenance of hypertension in SHR via sustained enhancement of tyrosine hydroxylase synthesis in the adrenal medulla, leading to increased epinephrine and norepinephrine levels.

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.

The activity of dihydropyrimidine dehydrogenase from rat liver was not affected by any of five 5-hydroxytryptamine antagonists.

We estimate the influence of five 5-hydroxytryptamine receptor (5-HT3) antagonists on the activity of dihydropyrimidine dehydrogenase (DPDase), the rate-limiting enzyme in 5-fluorouracil (5FU) metabolism. The activity of DPDase from the rat liver was compared in the cytosol mixture of 5FU incubated with or without each of five 5-HT3 antagonists. DPDase activity was not altered in the presence of any 5-HT3 antagonist studied here. It may be inferred from these results that the any 5-HT3 receptor antagonist examined in this study has little or no effect on fluorouracil catabolism.

Multiple cytochrome P-450 subfamilies are co-induced with P-glycoprotein by both phenothiazine and 2-acetylaminofluorene in rats.

We studied the effects of two P-glycoprotein (P-gp) inducers, 2-acetylaminofluorene (2-AAF) and phenothiazine (PTZ), administered intraperitoneally, on the activities and content of hepatic cytochrome P-450 (CYP) subfamilies in hepatic microsomes of Sprague-Dawley rats. After 4-day administration of 2-AAF or PTZ, the P-gp content was increased. The total CYP content after PTZ treatment was significantly increased compared with that of controls. The CYP1A, CYP2B and CYP3A2 contents were induced, while the CYP2C6, CYP2C11 and CYP2E1 contents remained unaffected. A marked increase in CYP1A1 was found after administration of each compound. Ethoxyresorufin O-deethylase, pentoxyresorufin O-deethylase, and testosterone 6beta hydroxylation activities showed a significant increase after both 2-AAF and PTZ treatments. In particular, ethoxyresorufin O-deethylase exhibited more than ten times greater activity than that of the controls after the treatments. These results suggest that P-gp inducers affect several CYP subfamilies in addition to CYP3A, which is reported to be up-regulated coordinately with P-gp by a CYP3A inducer.

Elevated tyrosine hydroxylase mRNA levels in medulla oblongata of spontaneously hypertensive rats.

The present study has investigated the expression of tyrosine hydroxylase (TH) mRNA and its activity in medulla oblongata of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). The TH mRNA levels were determined by Northern blot and dot blot analyses. The TH activity and the expression of TH mRNA in medulla oblongata of SHR were significantly higher than those of WKY. These results suggest that the hypertension of SHR may be related to the high activity of TH due to the high level of TH mRNA which increases norepinephrine levels in the medulla oblongata.

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.

Reflex discharge evoked by water stimulation on the frog tongue.

The frog tongue contains the chemoreceptor sensitive to water, the role of which has not been clarified. If the water on the tongue reflexly keeps the mouth closed as suggested by Zotterman, reflex discharges should be evoked in the nerve innervating the mandible elevator muscles. So far as we have studied, however, pronounced reflex discharges were evoked in the nerve innervating the submental and submaxillary muscles, but not in the nerves innervating the elevator muscles. This result suggests that the chemoreceptor sensitive to water in the frog tongue plays a role in the nostril closing mechanism.

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.

Properties of the lingual and LVP branches of the glossopharyngeal nerve.

We investigated the effects of various stimuli on the afferent and efferent branches of the glossopharyngeal nerve in the rat soft palate. One of the sensory components, the lingual branch, responded to tactile stimulation, while the LVP branch responded to stretching of the levator veli palatini muscle. We also obtained physiological and morphological evidence of the existence of muscle spindles in the levator veli palatini muscle and showed that tactile stimulation of the contralateral soft palate and stretching of the contralateral LVP modulated discharges from the motor component of the ipsilateral levator veli palatini muscle. Our results suggest that these receptor units with both sensory and motor efferents may be the main determinants of modulation of respiratory movements in the upper airway by the IXth nerve.

Role of proprioceptors in the mylohyoid muscle.

Afferent discharges of the mylohyoid muscle branch during respiration were studied electrophysiologically in the rat. Afferent discharges from the mylohyoid muscle branch of the mylohyoid nerve were found to be synchronized with respiration. Stretching of the mylohyoid muscle elicited afferent discharges of the mylohyoid muscle branch, suggesting that lengthening of the mylohyoid muscle caused electrical activity in the proprioceptors. When the central cut end of the mylohyoid muscle branch was stimulated electrically, reflex discharges were recorded from the EMG lead at the sternohyoid muscle where it is innervated by the cervical nerve. The latency between the electrical stimulation and the action potential in the sternohyoid muscle was 3-4 ms. Therefore, the mylohyoid muscle branch may transmit information to the sternohyoid muscle regarding the stretching actions of the mylohyoid muscle resulting from movements of the hyoid bone.

Airflow receptors in the lip and buccal mucosa.

Airflow receptor afferents in the oral mucosa responding to changes in intraoral air pressure during blowing were found to be innervated by the infraorbital nerve. They provided one response corresponding to the onset of blowing, a second related to an increase in air pressure, a third corresponding to the cessation of blowing, and a fourth that exhibited little change throughout sustained blowing. Intraoral air pressure in the cavity between the lips and the velopharyngeal portal may be monitored by these receptors, and the data they provide may contribute to the control of phonation.

Effects of anti-androgen treatment on the catecholamine synthetic pathway in the adrenal medulla of spontaneously hypertensive rats.

We investigated the effects of the antiandrogen flutamide on the activity of tyrosine hydroxylase, the levels of its encoding mRNA, and catecholamine levels in the adrenal medulla of male spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Flutamide (30 mg/kg) was administered subcutaneously daily (between 9 and 15 weeks of age). The systolic blood pressure of flutamide-treated SHR rats was lower than that of control SHR. Epinephrine and norepinephrine levels, tyrosine hydroxylase activity, and the levels of encoding mRNA in the adrenal medulla were significantly lower in flutamide-treated SHR rats than in paired controls. Systolic blood pressure, epinephrine and norepinephrine levels, tyrosine hydroxylase activity, and encoding mRNA in the adrenal medulla of WKY rats showed no significant differences between flutamide-treated and control groups. These findings suggested that flutamide may have cardiovascular effects through alteration of the catecholamine synthetic pathway caused by removal of androgen receptor stimulation on the expression of tyrosine hydroxylase in the adrenal medulla of male SHR rats.

Preliminary examination of the influence of incubation time or cytosolic protein concentration on dihydropyrimidine dehydrogenase activity.

We examined the influence of incubation time or cytosolic protein concentration on the metabolite production of 5-fluorouracil (5FU). Although the activity of dihydropyrimidine dehydrogenase (DPDase) from rat liver is considered to be retained for up to 60 min, the production rate of 5FU metabolites was reduced, probably due to depletion of the substrate in 40 mumol/l and lower concentration of 5FU. Since the ratio of the metabolite production rate to the cytosolic protein became smaller in higher concentrations of cytosolic protein, the DPDase activity should be compared in the same concentration of cytosolic protein. The production rate of 5FU metabolites was considered to be linear up to 40 mumol/l 5FU incubated with 500 micrograms cytosolic protein. The rate of the metabolite production calculated by one-point sampling significantly correlated with the enzyme activity by the multi-point sampling method. Minimizing sampling points to determine the DPDase activity would save time and expense.

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.

CYP3A is responsible for N-dealkylation of haloperidol and bromperidol and oxidation of their reduced forms by human liver microsomes.

We studied the biotransformation of haloperidol, bromperidol and their reduced forms by human liver microsomes. Nifedipine oxidation (CYP3A) activity correlated significantly with N-dealkylation rates of haloperidol and bromperidol and oxidation rates of their reduced forms, while neither ethoxyresorufin O-deethylation (CYP1A2) activity nor dextromethorphan O-deethylation (CYP2D6) activity did. In chemical and immunoinhibition studies, only troleandomycin and anti-CYP3A4 serum inhibited both formation rates of 4-fluorobenzoylpropionic acid, a metabolite of haloperidol and bromperidol, and back oxidation rates. Among 10 recombinant isoforms examined, only CYP3A4 showed catalytic activity. The Vmax and Km values of N-dealkylation of bromperidol and reoxidation of reduced bromperidol were similar to those of haloperidol and reduced haloperidol, respectively. The present study indicates that CYP3A plays a major role in N-dealkylation of and oxidation back to bromperidol as well as haloperidol and suggests that modification of in vivo CYP3A activity by inhibition or induction may affect the pharmacokinetics and therapeutic effects of haloperidol and bromperidol.