IL-17A stimulates the expression of inflammatory cytokines via celecoxib-blocked prostaglandin in MC3T3-E1 cells

Objective

The prostaglandins (PGs) released from osteoblasts can alter the process of bone remodelling. Recently, we showed that compressive force induced the expression of pro-inflammatory cytokine interleukin (IL)-17s and their receptors in osteoblastic MC3T3-E1 cells and that IL-17A was expressed most highly. Consequently, in the current study we examined the effect of IL-17A and/or celecoxib on PGE₂ production and the expression of cyclooxygenases (COXs) and inflammatory cytokines in MC3T3-E1 cells. We also examined the effects of PGE₂ and cyclohexamide on the expression of inflammatory cytokines.

Methods

Cells were cultured with or without IL-17A (0.1, 1.0, or 10 ng/ml) in the presence or absence of 10 μM celecoxib, a specific inhibitor of COX-2, for up to 72 h. Cells were pretreated with or without 10 μg/ml cycloheximide, protein synthesis inhibitor, for 30 min, and then cultured with 10 ng/ml IL-17A for 24 h. Cells were also cultured with or without 1.5 ng/ml PGE₂ for 24 h. PGE₂ production was determined by ELISA. The expression of COX-1, COX-2, IL-1α, IL-6, IL-8, IL-11, and TNF-α mRNAs and proteins was determined by real-time PCR and ELISA, respectively.

Results

The expression of COX-2, IL-1α, IL-6, IL-8, IL-11, and TNF-α, as well as PGE₂ production increased in the presence of IL-17A, whereas COX-1 expression did not change. Celecoxib blocked the stimulatory effect of IL-17A on the expression of COX-2, IL-1α, IL-6, IL-8, and IL-11 as well as PGE₂ production, whereas it did not block TNF-α expression. Cycloheximide pretreatment suppressed the expression of IL-17-induced inflammatory cytokines. The expression of IL-1α, IL-6, IL-8, and IL-11 increased by the addition of PGE₂, whereas TNF-α expression was not affected.

Conclusion

These results suggest that IL-17A stimulates the expression of bone resorption-related inflammatory cytokines through an autocrine mechanism involving celecoxib-blocked PGs, mainly PGE₂, in osteoblasts.     

Use of quantitative PCR to evaluate methods of bacteria sampling in periodontal patients

Periodontal disease is associated with specific periodontal pathogens and may persist as gingivitis or progress to more severe disease. The bacteria involved in disease initiation and progression have not been identified. We used quantitative polymerase chain reaction (PCR) to compare the levels of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, and total bacteria detected by different sampling methods. On the basis of the results of clinical examinations, 57 patients were divided into 3 groups: healthy group (group A), gingivitis group (group B), and periodontitis group (group C). Bacterial samples were collected from saliva, mouthwash, and by paper-point sampling of gingival crevicular fluid (GCF), and the samples were analyzed with quantitative PCR targeting 16S rRNA. The numbers of total bacteria in samples of GCF, saliva, and mouthwash were 10? to 10?, 10?, and 10?, respectively, per milliliter. The number of P. gingivalis in GCF samples was lower than 10 in group A; however, in groups B and C, the values were 103 and 10?, respectively, indicating that the number of P. gingivalis increased with worsening clinical status. Findings were similar in the samples of saliva and mouthwash. The numbers of T. forsythia showed a pattern similar to that of P. gingivalis in all 3 samples. These results suggest that saliva and mouthwash samples are clinically useful for bacterial testing of periodontal diseases by quantitative PCR. In addition, mouthwash sampling is more feasible and straightforward than saliva sampling.     

Tracheal chondrosarcoma

Tracheal chondrosarcomas are rare, with only 14 cases previously documented. We report a case of chondrosarcoma of the trachea and review other published cases. Our patient was a 34-year-old man who began to feel shortness of breath 8 months after initial hemoptysis. Transverse and coronal computed tomography demonstrated a tumor that was obstructing nearly 80% of the tracheal lumen at the thoracic inlet. A bronchoscopic biopsy specimen led to an initial diagnosis of chondroma. Five tracheal rings from the third to the seventh including the tumor were completely resected, and an end-to-end anastomosis was performed. The histological diagnosis of the surgical specimen showed it to be a chondrosarcoma. The patient was alive without recurrence at 6 years 4 months after surgery. Because recurrence after incomplete resection and malignant transformation can occur with chondromas, radical resection of the trachea is recommended for all cartilaginous tumors of the trachea.     

Effects of flavoxate hydrochloride on voltage-dependent Ba<Superscript>2+</Superscript> currents in human detrusor myocytes at different experimental temperatures

The inhibitory effects of flavoxate hydrochloride (piperidinoethyl-3-methylflavone-8-carboxylate; hereafter referred as flavoxate) on voltage-dependent nifedipine-sensitive inward Ba²⁺ currents (I _Ba) in human detrusor myocytes were investigated at different temperatures using conventional whole-cell patch-clamp techniques. When the bath-solution temperature was increased from 22°C to 30°C, I _Ba peak amplitude was enhanced by approximately twice at several test potentials. Neither the I _Ba threshold nor the membrane potentials for the I _Ba maximum peak amplitude was affected by the temperature change. The concentration-response curves of flavoxate at both 30°C (K _i  = 5.1 μM) and 37°C (K _i  = 4.6 μM) were slightly shifted to the left in comparison with that at 22°C (K _i  = 10.3 μM). Similar results were also obtained in the presence of nifedipine (K _i  = 14 nM at 22°C vs. K _i  = 2.5 nM at 30°C and K _i  = 2.1 nM at 37°C). Altering the bath-solution temperature from 22°C to 30°C shifted the steady-state inactivation curve of I _Ba at −90 mV to the left. At 30°C, the steady-state inactivation curve of I _Ba in the presence of flavoxate was also shifted to the left in comparison with that in the absence of flavoxate. Either 3-isobutyl-1-methylxanthine (IBMX) or theophylline, a phosphodiesterase inhibitor, caused little effects on I _Ba, although cyclic nucleotides (dibutyryl cAMP and 8-Br-cGMP) inhibited I _Ba. These results suggest that the inhibitory actions of flavoxate on I _Ba in human detrusor myocytes were slightly changed at different experimental temperatures and that flavoxate directly blocked voltage-dependent L-type Ca²⁺ channels, not through the inhibition of phosphodiesterase activity pathway.     

Behavioural characterisation of rats exposed neonatally to bisphenol-A: responses to a novel environment and to methylphenidate challenge in a putative model of attention-deficit hyperactivity disorder

Neonatal exposure of rats to bisphenol-A, an endocrine disruptor, has recently been proposed as a possible animal model of attention-deficit hyperactivity disorder (ADHD), because such rats exhibit motor hyperactivity. To strengthen the face validity of this animal model, the present study replicated the original experiments and additionally analysed both changes in habituation to a novel environment and behavioural responses to methylphenidate, the two phenomena known to be altered in ADHD. Single intracisternal administration of bisphenol-A (20 and 40 microg) into 5-day-old male Wistar rats impaired habituation to a novel environment in the light, but not the dark, phase at 4 weeks of age. Thus, habituation as assessed by time-dependent decrease of locomotor activity, rearing, sniffing and grooming was significantly reduced in bisphenol-A-pretreated rats. Methylphenidate (1 and 3 mg/kg, i.p.) dose-dependently enhanced locomotor activity in both vehicle-pretreated and bisphenol-A-pretreated rats during both the dark and the light phases. Thus, the effects of methylphenidate did not differ between bisphenol-A-pretreated and vehicle-pretreated rats. Apart from a slight methylphenidate-induced increase in rearing and sniffing in bisphenol-A (20 microg)-pretreated rats, the overall effects of methylphenidate on rearing, sniffing and grooming were similar in both vehicle- and bisphenol-A-pretreated rats. It is concluded that neonatal exposure of rats to bisphenol-A is an animal model with limited face validity for ADHD, because the motor hyperactivity and reduced habituation to a novel environment are not accompanied by altered responses to methylphenidate.     

Long-term control or possible cure? Treatment of stage D2 prostate cancer under chemotherapy using cisplatin and estramustine phosphate followed by maximal androgen blockade

Metastatic prostate cancer (PC) is incurable by androgen deprivation therapy alone, due to the presence of androgen-independent/supersensitive cells in hormone-naive PC. A 67-year-old man was diagnosed with PC (Gleason score, 5 + 4) with multiple bone metastases. He was treated by chemohormonal therapy with cisplatin and estramustine phosphate (EMP) followed by maximal androgen blockade, and showed a complete response. As of the time of writing, no clinical or prostate-specific antigen recurrence has been observed for over 15 years, despite cessation of the treatment. This is the first report to indicate a possible cure of metastatic PC by chemohormonal therapy combined with appropriate anti-tumor drugs targeted to both androgen-independent and -dependent clones before the hormone-refractory state.     

On/off-pump beating coronary artery bypass grafting after thoracoplasty

Off-pump coronary artery bypass grafting (CABG) [OPCAB] or on-pump beating CABG (conventional CABG) was performed in 3 post-thoracoplasty patients. Considering their poor respiratory function after thoracoplasty, OPCAB is considered more suitable than conventional CABG with cardio-pulmonary bypass in such cases with severe coronary lesions. However, because the sternum inclines or the pleural cavity may be polluted in these patients, special care is necessary for the operation. In such cases, it may be impossible to bypass to the circumflex artery in OPCAB, and is necessary to consider the use of percutaneous cardiopulmonary support (PCPS), cardio-pulmonary bypass or hybrid therapy before the operation.     

Increased IL‐6 Expression in Osteoclasts Is Necessary But Not Sufficient for the Development of Paget's Disease of Bone

Measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) and mutation of the SQSTM1 (p62) gene contribute to the increased OCL activity in Paget's disease (PD). OCLs expressing MVNP display many of the features of PD OCLs. Interleukin‐6 (IL‐6) production is essential for the pagetic phenotype, because transgenic mice with MVNP targeted to OCLs develop pagetic OCLs and lesions, but this phenotype is absent when MVNP mice are bred to IL‐6^–/– mice. In contrast, mutant p62 expression in OCL precursors promotes receptor activator of NF‐κB ligand (RANKL) hyperresponsivity and increased OCL production, but OCLs that form have normal morphology, are not hyperresponsive to 1,25‐dihydroxyvitamin D₃ (1,25‐(OH)₂D₃), nor produce elevated levels of IL‐6. We previously generated p62^P394L knock‐in mice (p62KI) and found that although OCL numbers were increased, the mice did not develop pagetic lesions. However, mice expressing both MVNP and p62KI developed more exuberant pagetic lesions than mice expressing MVNP alone. To examine the role of elevated IL‐6 in PD and determine if MVNP mediates its effects primarily through elevation of IL‐6, we generated transgenic mice that overexpress IL‐6 driven by the tartrate‐resistant acid phosphatase (TRAP) promoter (TIL‐6 mice) and produce IL‐6 at levels comparable to MVNP mice. These were crossed with p62KI mice to determine whether IL‐6 overexpression cooperates with mutant p62 to produce pagetic lesions. OCL precursors from p62KI/TIL‐6 mice formed greater numbers of OCLs than either p62KI or TIL‐6 OCL precursors in response to 1,25‐(OH)₂D₃. Histomorphometric analysis of bones from p62KI/TIL‐6 mice revealed increased OCL numbers per bone surface area compared to wild‐type (WT) mice. However, micro‐quantitative CT (µQCT) analysis did not reveal significant differences between p62KI/TIL‐6 and WT mice, and no pagetic OCLs or lesions were detected in vivo. Thus, increased IL‐6 expression in OCLs from p62KI mice contributes to increased responsivity to 1,25‐(OH)₂D₃ and increased OCL numbers, but is not sufficient to induce Paget's‐like OCLs or bone lesions in vivo. © 2014 American Society for Bone and Mineral Research.     

Complete response to treatment with hepatic arterial infusion of cisplatin powder (IA-call) in a case of hepatocellular carcinoma with multiple lung metastases]

A 74-year-old woman was admitted to our hospital to treat her hepatocellular carcinoma (stage IVB) with multiple lung metastases. She was treated with 3 times of hepatic arterial infusion of cisplatin powder (IA-call). After the treatment, liver mass and lung tumors were disappeared and high levels of tumor markers (AFP and PIVKA-II) were markedly decreased. These data revealed that a complete response (CR) was obtained for her. She has still been maintained in CR for 2 years since the first treatment.     

Stress fracture of the olecranon in an adult baseball player

Stress fractures of the olecranon caused by repetitive stress force have infrequently been reported as a cause of elbow pain in adult athletes, engaged in throwing and pitching sports. We diagnosed as a stress fracture of the olecranon by clinical and radiographic findings and treated surgically. The patient returned to playing baseball at a competitive level and was asymptomatic 4 months after the first operation. However, the patient re-injured the olecranon and a second surgical treatment was performed almost 1 year after the first operation. After the second surgery, the patient returned to playing baseball at a competitive level and was free from elbow symptoms. We presented a stress fracture of the olecranon in a semi-professional adult baseball player and suggested that surgical treatment is necessary.