The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo.

OBJECTIVES: The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy. METHODS: Using tenocytes from normal Sprague-Dawley rats, cultured both in control and high glucose conditions, reactive oxygen species (ROS) production, cell proliferation, messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, interleukin-6 (IL-6), matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 and type I and III collagens were determined after 48 and 72 hours in vitro. In an in vivo study, using diabetic rats and controls, NOX1 and 4 expressions in Achilles tendon were also determined. RESULTS: In tenocyte cultures grown under high glucose conditions, gene expressions of NOX1, MMP-2, TIMP-1 and -2 after 48 and 72 hours, NOX4 after 48 hours and IL-6, type III collagen and TIMP-2 after 72 hours were significantly higher than those in control cultures grown under control glucose conditions. Type I collagen expression was significantly lower after 72 hours. ROS accumulation was significantly higher after 48 hours, and cell proliferation after 48 and 72 hours was significantly lower in high glucose than in control glucose conditions. In the diabetic rat model, NOX1 expression within the Achilles tendon was also significantly increased. CONCLUSION: This study suggests that high glucose conditions upregulate the expression of mRNA for NOX1 and IL-6 and the production of ROS. Moreover, high glucose conditions induce an abnormal tendon matrix expression pattern of type I collagen and a decrease in the proliferation of rat tenocytes.Cite this article: Y. Ueda, A. Inui, Y. Mifune, R. Sakata, T. Muto, Y. Harada, F. Takase, T. Kataoka, T. Kokubu, R. Kuroda. The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo. Bone Joint Res 2018;7:362-372. DOI: 10.1302/2046-3758.75.BJR-2017-0126.R2.

Effects of platelet-rich plasma and triamcinolone acetonide on interleukin-1ß-stimulated human rotator cuff-derived cells.

OBJECTIVES: Triamcinolone acetonide (TA) is widely used for the treatment of rotator cuff injury because of its anti-inflammatory properties. However, TA can also produce deleterious effects such as tendon degeneration or rupture. These harmful effects could be prevented by the addition of platelet-rich plasma (PRP), however, the anti-inflammatory and anti-degenerative effects of the combined use of TA and PRP have not yet been made clear. The objective of this study was to determine how the combination of TA and PRP might influence the inflammation and degeneration of the rotator cuff by examining rotator cuff-derived cells induced by interleukin (IL)-1ß. METHODS: Rotator cuff-derived cells were seeded under inflammatory stimulation conditions (with serum-free medium with 1 ng/ml IL-1ß for three hours), and then cultured in different media: serum-free (control group), serum-free + TA (0.1mg/ml) (TA group), serum-free + 10% PRP (PRP group), and serum-free + TA (0.1mg/ml) + 10% PRP (TA+PRP group). Cell morphology, cell viability, and expression of inflammatory and degenerative mediators were assessed. RESULTS: Exposure to TA significantly decreased cell viability and changed the cell morphology; these effects were prevented by the simultaneous administration of PRP. Compared with the control group, expression levels of inflammatory genes and reactive oxygen species production were reduced in the TA, PRP, and TA+PRP groups. PRP significantly decreased the expression levels of degenerative marker genes. CONCLUSIONS: The combination of TA plus PRP exerts anti-inflammatory and anti-degenerative effects on rotator cuff-derived cells stimulated by IL-1ß. This combination has the potential to relieve the symptoms of rotator cuff injury.Cite this article: T. Muto, T. Kokubu, Y. Mifune, A. Inui, R. Sakata, Y. Harada, F. Takase, M. Kurosaka. Effects of platelet-rich plasma and triamcinolone acetonide on interleukin-1ß-stimulated human rotator cuff-derived cells. Bone Joint Res 2016;5:602-609. DOI: 10.1302/2046-3758.512.2000582.

Dose- and time-dependent effects of triamcinolone acetonide on human rotator cuff-derived cells.

OBJECTIVES: To investigate the appropriate dose and interval for the administration of triamcinolone acetonide (TA) in treating tendinopathy to avoid adverse effects such as tendon degeneration and rupture. METHODS: Human rotator cuff-derived cells were cultured using three media: regular medium (control), regular medium with 0.1 mg/mL of TA (low TA group), and with 1.0 mg/mL of TA (high TA group). The cell morphology, apoptosis, and viability were assessed at designated time points. RESULTS: In the low TA group, the cells became flattened and polygonal at seven days then returned to normal at 21 days. The cell apoptosis ratio and messenger ribonucleic acid expression of caspase-3, 7, 8, and 9 increased, and viability was reduced in the low and high groups at seven days. In the low TA group, apoptosis and viability returned to normal at 21 days, however, in the high TA group, the cell morphology, apoptosis ratio, caspase-3, 7, 8, and 9 and viability did not return by day 21. Re-administration was performed in the low TA group at 7-, 14-, and 21-day intervals, and cell viability did not return to the control level at the 7- and 14-day intervals. CONCLUSION: A 0.1 mg/mL dose of TA temporarily decreased cell viability and increased cell apoptosis, which was recovered at 21 days, however, 1 mg/mL of TA caused irreversible damage to cell morphology and viability. An interval > three weeks was needed to safely re-administer TA. These findings may help determine the appropriate dose and interval for TA injection therapy. Cite this article: Bone Joint Res 2014;3:328-34.

Repair of osteochondral defects with a new porous synthetic polymer scaffold.

We developed a new porous scaffold made from a synthetic polymer, poly(DL-lactide-co-glycolide) (PLG), and evaluated its use in the repair of cartilage. Osteochondral defects made on the femoral trochlear of rabbits were treated by transplantation of the PLG scaffold, examined histologically and compared with an untreated control group. Fibrous tissue was initially organised in an arcade array with poor cellularity at the articular surface of the scaffold. The tissue regenerated to cartilage at the articular surface. In the subchondral area, new bone formed and the scaffold was absorbed. The histological scores were significantly higher in the defects treated by the scaffold than in the control group (p<0.05). Our findings suggest that in an animal model the new porous PLG scaffold is effective for repairing full-thickness osteochondral defects without cultured cells and growth factors.

Isolation of chlamydia psittaci from domestic cats with oculonasal discharge in Japan.

Eight strains of Chlamydia psittaci were isolated in Japan from the nasal and conjunctival swabs of six household cats using the L929 cell line of mouse fibroblast origin. The isolates were identified as C. psittaci on the basis of the formation of characteristic inclusion bodies in the cell culture detected by Giemsa stain and immunofluorescence. Comparison of nucleotide sequences of the ompA gene amplified from the three isolates with the published sequence of feline FEPN strain of C. psittaci showed almost 100% homology.

Low-intensity pulsed ultrasound initiates bone healing in rat nonunion fracture model.

Low-intensity pulsed ultrasound exposure has been shown clinically to shorten the fracture repair process and to induce healing of nonunions in humans, but its mechanism of action remains unclear. In this study we investigated the effect and mechanism of low-intensity pulsed ultrasound on nonunion fracture healing in rat tibias. A consistently reproducible nonunion was produced in rat tibias by muscle interposition without osteotomy. This model was produced by creating a closed tibial fracture with only the distal end of the tibialis anterior muscle interposed into the fracture site. One limb was noninvasively exposed to low-intensity pulsed ultrasound (a 200-millisecond burst of sine waves of 1.5 MHz, repeating at 1.0 kHz) for 20 minutes daily. The incident intensity was approximately 30 mW/cm2. Rats were killed at intervals between 2 and 6 weeks. The events were assessed by radiographs, microfocus X-ray computed tomograms, and histologic examination. After 6 weeks of exposure, 7 of 14 nonunion fractures showed healing on radiologic assessment. The results of three-dimensional microfocus X-ray computed tomographic reconstruction and histologic examination also supported this finding. On the other hand, all control tibias remained in a state of nonunion during the same period. These results indicate that low-intensity pulsed ultrasound promotes healing in the rat nonunion fracture model.

Low intensity pulsed ultrasound exposure increases prostaglandin E2 production via the induction of cyclooxygenase-2 mRNA in mouse osteoblasts.

Both prostaglandin E2 (PGE2) and low intensity pulsed ultrasound (U/S) exposure have been reported to accelerate fracture repair. We hypothesized that these two pathways are interactive. To verify this hypothesis, we examined the regulation of PGE2 production by U/S exposure (sine wave of 1.5MHz repeating at 1kHz, 30mW/cm2, 20 minutes) in mouse osteoblastic cell line, MC3T3-E1. The production of PGE2 in osteoblasts was augmented by U/S, which was threefold at 60 min. in comparison with unexposed samples. Then we evaluated the expression of cyclooxygenase-2 (COX-2) mRNA, which is a critical enzyme for PGE2 production. U/S rapidly up-regulated the expression of COX-2 mRNA in a time dependent manner. In addition, PGE2 production by U/S was drastically suppressed by a selective inhibitor of COX-2. These results provide strong evidence that PGE2 production in osteoblasts is dependent upon the induction of COX-2 mRNA expression by U/S and offer a mechanistic insight into how U/S accelerates fracture repair.

Influence of cell surface glycoprotein gC produced by pseudorabies virus on cytopathic effect.

The wild-type pseudorabies virus (WT-PRV) produced a round-type cytopathic effect (CPE) in PK-15 cell line of porcine kidney origin, while PRVgCs lacking in gC-transmembrane-anchor region and PRVgC-defecting in gC gene produced a syncytium-type CPE. The mouse embryo cell line (BALB/3T3 clone A31) were transfected with recombinant plasmid of pcDNA3 which incorporated with gC gene. The transfected A31/gC cells were stably expressing gC. Only a round-type CPE was observed in these cells infected with WT-PRV, while a syncytium-type CPE was observed in the cells infected with each of the PRVgCs and PRVgC-. Any viruses described above induced a syncytium-type CPE in A31/pcDNA cells transfected with a plasmid without gC gene. By WT-PRV infection, PK-15 cells generated about 2- or 8-fold more gC than the A31/gC and A31/pcDNA cells when gC was measured by hemagglutination test. Flowcytometric analysis revealed that amount of gC on the cell surface of A31/gC and PK-15 cells increased after infection with WT-PRV. Round-type CPE was observed with the increase of gC. These results suggest that the type of CPE formation induced by PRV is dominated by the amount of gC on the infected cell surface.

Efficacy of a mixed glycoprotein vaccine against pseudorabies in pregnant sows.

A pseudorabies virus (PRV) glycoprotein-mixed vaccine was prepared by heparin-affinity chromatography from PRV-infected PK-15 cell lysates. In our previous study, the trial vaccine was induced protection with suppression of virus shedding in one-month-old pigs and generation of cytotoxic T lymphocyte (CTL) response in mice. In this study, the effect of the trial vaccine on suppression of both virus shedding and reproductive failure in pregnant sows was examined. Three sows were vaccinated twice until one week before mating. Each of them was infected intranasally with 10(6) TCID50 of PRV on day 28, 54, or 85 after mating, respectively. Three other sows were also mated and challenged at the same time as the respective control. The vaccinated sows produced virus-neutralizing antibodies. Sows with high level of VN antibody lowered the level and period of virus shedding after challenge. The maximum level of shed-virus titers in vaccinated sows were 10(1.25) to 10(3) times lower than controls. Vaccinated sows shed virus for 1 or 5 days, while controls shed for 8, 9, or 12 days. No abortion or stillbirth was observed in vaccinated sows during pregnancy. On the other hand, control sow challenged at a late stage of pregnancy showed abortion and stillbirth. The results obtained here indicate that our trial vaccine is effective to prevent reproductive failure by pseudorabies virus.

EGG phosphatidylcholine combined with vitamin B12 improved memory impairment following lesioning of nucleus basalis in rats.

We investigated the effects of egg phosphatidylcholine (PC) combined with vitamin B12 on memory in the Morris water maze task, and on choline and acetylcholine (ACh) concentrations in the brain of rats. Animals with nucleus basalis Magnocellularis (NBM) lesion received intragastric administration of egg PC or vitamin B12, or both for 18 days. Memory acquisition and retention were remarkably impaired in NBM lesioned rats compared with in sham-operated control. NBM lesioned group had lower choline and ACh concentrations than control group in the frontal cortex. High dose of egg PC alone significantly increased choline concentration, but did not change ACh concentration in the frontal cortex. High dose of vitamin B12 alone did not change choline and ACh concentrations in the brain. Either egg PC or vitamin B12 did not improve memory acquisition and retention. However, low dose of egg PC combined with vitamin B12 significantly increased ACh concentration and improved memory acquisition and retention in the NBM lesioned rats. We concluded that egg PC combined with vitamin B12 improved the memory impairment of NBM lesioned rats through the action on the cholinergic neurons.

Bilateral adrenal lymphoma with neoplastic angioendotheliosis.

We report a case of bilateral lymphoma of the adrenal glands. A 61-year-old man was admitted to our hospital in clinical shock with anuria. Endocrine examination indicated a diagnosis of Addison's disease. Large tumors were found bilaterally in the suprarenal area, and a needle biopsy was performed, and the diagnosis was malignant lymphoma. Treatment with chemotherapy achieved a moderate response. He was discharged, but died 1 month later. On autopsy, microscopic examination showed extensive intravascular infiltration of lymphoma cells. We considered this to be a case of bilateral adrenal lymphoma with neoplastic angioendotheliosis.

Special folding pathway to tetramer only through the micelle state of the corticotropin-releasing factor.

The ovine corticotropin-releasing factor (CRF), a peptide hormone of 41 residues stimulating the secretion of adrenocorticotropic hormone, was thermodynamically investigated. By means of size exclusion chromatography and/or ultrafiltration, the CRF solution could be separated into random coil monomers and highly alpha-helical tetramers, which seem to have amphipathic helix bundle structure. Circular dichroism measurements along with diluting or concentrating the CRF solution revealed that there exists the micelle state above the concentration of 0.1 mM, which would be the critical micelle concentration (cmc). The micelle state was also proved by binding ability for 8-anilino-1-naphthalenesulfonate and endothermic change by dilution across the cmc. The tetramer showed the cooperative thermal transition at about 55 degrees C in the buffer solution (pH 7.5), so that it would have native-protein-like folding. On the other hand, the micelle undergoes gradual change to dissociated state by heating, regardless of the similar alpha-helicity to the tetramer. Above the cmc the equilibrium between the tetramer and the micelle takes place as well as that between the monomer and the micelle. Whereas, the direct conversion between the tetramer and the monomer scarcely occurred below the cmc. The titration experiment with 2,2,2-trifluoroethanol (TFE) revealed that the cmc decreases with increasing the concentration of TFE. This tendency is the same as that of general surfactants. Most of experimental results can be well explained by this three-phase model involving the monomer, the tetramer, and the micelle. The lack of the equilibrium between the monomer and the tetramer indicates that the folding pathway of the tetramer is the transformation only through the micelle state and not from the monomer. This pathway resembles the collapse model among the phenomenological models for thermodynamic protein folding. By the mathematical consideration for the dissociation of micelle, we have demonstrated that the expected content of undegradable k-mer is 2/(k + 1), which agreed well with the observed tetramer content of CRF (40%).

Genetic controls of susceptibility and resistance to 4-nitroquinoline 1-oxide-induced tongue carcinomas in rats.

We analyzed the incidence of infiltrative mass-type tongue carcinomas (IMTC) induced in 550 rats by continuous oral administration of 0.001% 4-nitroquinoline 1-oxide solution for 180 days. The study included various crosses of susceptible Dark-Agouti rats (DA) and resistant Wistar/Furth rats (WF). DA showed a 93.6% incidence of IMTC measuring more than 5 mm in their largest diameter, while WF showed only a 4% incidence. Reciprocal F1 and F2 hybrids mated by DA and WF showed 47.5% and 45.8% incidences, respectively. Meanwhile, reciprocal backcrossed hybrids to DA and WF showed 73.7%, and 24.6% incidences, respectively. Segregation of the incidences suggests that there are two autosomal dominant genes, one linked to the susceptibility of DA and the other to the resistance of WF.

Effect of egg white on serum cholesterol concentration in young women.

In a previous study we observed favorable effects of egg white on serum lipids in rats and mice. The present study was designed to elucidate these effects in 24 female university students with moderate hypercholesterolemia. About 30% of total protein was supplied with egg white, tofu or cheese. The experiment was conducted for a complete menstruation cycle of each subject. Lipid intake was about 30% of total energy intake. The energy intake of each subject was constant throughout the experiment. Body weight was measured every morning. Daily activity was measured by a pedometer. Blood was withdrawn after an overnight fast on the first, 15th and last days and serum lipids were measured. Body weight was measured every morning. Daily activity was measured by a pedometer. Blood was withdrawn after an overnight fast on the first, 15th and last days and serum lipids were measured. Body weight and daily activity were maintained in all the groups throughout the experiment. The egg white group showed a similar decrease in the total cholesterol (Total-C) concentration but a greater increase of high-density lipoprotein cholesterol (HDL-C) concentration as compared to the tofu group and a greater decrease in Total-C and low-density lipoprotein cholesterol (LDL-C) concentrations and a greater increase in the HDL-C concentration as compared to the cheese group (p < 0.05). The results indicate the favorable effects of egg white in the control of hypercholesterolemia.

Effects of dietary phosphatidylcholine on memory in memory deficient mice with low brain acetylcholine concentration.

Data concerning the effect of phosphatidylcholine (PCh) administration on the improvement of memory in senile dementia of Alzheimer type are inconsistent, probably due to the different conditions under which studies were conducted. Animal studies provide a good model, but data on this is limited. We studied the effect of PCh on memory in memory deficient mice (Dull mice) with low brain acetylcholine (ACh) concentration and normal mice. Mice were fed 24% casein diet (control) or this diet supplemented with 2 or 8% egg yolk PCh from gestation (Experiment 1) and after weaning (Experiment 2). Memory acquisition and retention were studied by step-down type passive avoidance performance at 8 and 10 weeks old, respectively. Control group of Dull mice had poorer memories than that of the normal mice in Experiments 1 and 2. On the 2% PCh diet, Dull mice improved memory acquisition and retention in Experiment 1 and retention in Experiment 2. On the 8% PCh diet in Dull mice there was improvement of memory and retention in Experiment 1, but no effect was observed in Experiment 2 (P > 0.05). In the normal mice, the 2% PCh diet did not affect memory acquisition and retention, however on the 8% PCh diet, there was no or adverse effect. These results suggest that dietary supplementation of egg yolk PCh improves memory of Dull mice, particularly when given from gestation and that the 2% PCh diet elicits better response than the 8% PCh diet.

Effect of age on left ventricular geometric patterns in hypertensive patients.

OBJECTIVES: To investigate the effect of age on left ventricular structure and geometry in hypertensive patients, we studied the relationship between age and echocardiographic variables in patients with uncomplicated essential hypertension. PATIENTS AND METHODS: We divided 168 patients with hypertension into three groups according to age: young (<40 years), middle-aged (40-59 years) and an elderly group (> or = 60 years). They were further categorized according to relative wall thickness and the left ventricular mass index. We then evaluated the prevalence of left ventricular geometric patterns in these patients according to age. RESULTS: The left ventricular end-diastolic dimension decreased with age, both in normotensive control subjects and in hypertensive patients. The magnitude of this decrease was similar for both. The relative wall thickness and left ventricular mass index were greater in the hypertensive patients than in the normotensive control subjects, and these increased with age both in the controls and the hypertensives. The differences between normotensives and hypertensives in these variables remained unchanged with age. The prevalence of a normal left ventricle (normal relative wall thickness and left ventricular mass index) in the hypertensive patients decreased with age. Conversely, the prevalence of concentric remodeling (increased relative wall thickness with normal left ventricular mass index) and concentric hypertrophy (increased relative wall thickness and left ventricular mass index) increased with age. CONCLUSIONS: These results demonstrate that age significantly affects left ventricular structure both in normotensive control subjects and in hypertensive patients. Thus, the differences in left ventricular geometric patterns with age may have important implications in assessing left ventricular structure and geometric patterns in hypertensive patients.

Detection of perioperative coronary vasospasm on iodine-123-MIBG, thallium-201 and iodine-123-BMIPP myocardial SPECT images.

A 64-yr-old woman, who had no history of anginal attack, underwent cholecystectomy under general anesthesia. A few hours after successful surgery, the EKG showed T-wave inversions with QT-interval prolongations and torsades de pointes. The [123I]MIBG scintigraphic findings revealed marked denervation in the anteroseptal and inferoposterior myocardium, whereas 201TI myocardial SPECT showed only slightly reduced tracer uptake in those areas. The [123I]BMIPP scans showed abnormal fatty acid metabolism in the anteroseptal myocardium. Coronary angiogram detected no fixed stenosis, but coronary vasoconstriction in the left anterior descending artery was induced by intracoronary injection of acetylcholine. In our patient, the findings of cardiac imaging with [123I]MIBG, 201TI and [123I]BMIPP led to coronary angiography and the final diagnosis of probable perioperative coronary vasospasm.

Vasospastic angina in two sisters.

Vasospastic angina was observed in two sisters. The 52-year-old younger sister presented with rest angina at midnight and in the early morning. The coronary arteriogram showed no significant organic stenosis. Vasospasm to the left anterior descending and right coronary arteries was induced by the intracoronary administration of acetylcholine. The 57-year-old elder sister complained of rest and effort angina. Her coronary arteriogram was also normal. Vasospasm to the left circumflex and right coronary arteries was provoked by acetylcholine. In both cases, human leukocyte antigen DQw3 was negative. In the present cases, genetic factors may partly contribute to the mechanism of vasospastic angina.

[Malignant fibrous histiocytoma of the chest wall in a nonagenarian].

We report a case of malignant fibrous histiocytoma of the chest wall observed in a 94-year-old woman. She noticed appetite loss and general edema a week before admission. The patient was diagnosed as having congestive heart failure due to valvular heart disease on the basis of echocardiographic findings and became symptom-free by treatment with vasodilators and diuretics. However, chest roentgenogram disclosed a extrapleural mass in the left mid-lateral chest. About 2 months after admission, she experienced left lateral chest pain for the first time. The chest CT scan revealed a 5 x 5 x 2 cm mass, adjacent to the lateral-posterior chest wall and projecting into the thoracic cavity and rib osteolysis. Gallium-67 citrate scintigram showed abnormal isotope accumulation in the left middle chest. Biopsy was not done. The therapeutic approach was mainly pain relief, and no tumor resection, chemotherapy, or radiotherapy was performed. The mass increased in size, and increasingly extended into the thoracic cavity on follow-up CT scans. Furthermore, marked invasion of the tumor to subcutis and subscapula was found. She died of cachexia and respiratory failure 34 weeks after admission. Histologic examination revealed malignant fibrous histiocytoma.