Duloxetine suppresses BMP-4-induced release of osteoprotegerin via inhibition of the SMAD signaling pathway in osteoblasts.

Duloxetine, a selective serotonin-norepinephrine reuptake inhibitor, is currently recommended for the treatment of chronic painful disorders such as fibromyalgia, chronic musculoskeletal pain, and diabetic peripheral neuropathy. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates osteoprotegerin (OPG) production in osteoblast-like MC3T3-E1 cells, and that p70 S6 kinase positively regulates OPG synthesis. The present study aimed to investigate the effect of duloxetine on BMP-4-stimulated OPG synthesis in these cells. Duloxetine dose-dependently suppressed OPG release stimulated by BMP-4. Fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), reduced BMP-4-stimulated OPG release, whereas a selective and specific norepinephrine reuptake inhibitor, reboxetine, failed to affect OPG release. In addition, another SSRI sertraline also inhibited BMP-4-stimulated OPG release. On the other hand, siRNA of SMAD1 reduced the OPG release stimulated by BMP-4, indicating the involvement of the SMAD1/5/8 pathway in OPG release. Rapamycin inhibited BMP-4-stimulated p70 S6 kinase phosphorylation, and compound C suppressed the SMAD1/5/8 phosphorylation stimulated by BMP-4. Duloxetine did not affect BMP-4-induced phosphorylation of p70 S6 kinase but suppressed SMAD1/5/8 phosphorylation. Both fluvoxamine and sertraline also inhibited BMP-4-elicited phosphorylation of SMAD1/5/8. These results strongly suggest that duloxetine suppresses BMP-4-stimulated OPG release via inhibition of the Smad1/5/8 signaling pathway in osteoblasts.

Duloxetine strengthens osteoblast activation by prostaglandin E1: Upregulation of p38 MAP kinase.

Duloxetine, a serotonin-norepinephrine reuptake inhibitor, is currently recommended as a useful medicine to chronic pain including low back pain. However, as the analogy of classical selective serotonin reuptake inhibitors, there is a concern to deteriorate osteoporosis with remaining to clarify the exact mechanism of duloxetine in bone metabolism. We have previously reported that prostaglandin E1 (PGE1) induces the synthesis of both osteoprotegerin (OPG) and interleukin-6 (IL-6), essential regulators of bone metabolism, in osteoblast-like MC3T3-E1 cells. Based upon them, we herein investigated the mechanism whereby the effect of duloxetine on the synthesis of OPG and IL-6 induced by PGE1 in these cells. Duloxetine enhanced the release from MC3T3-E1 cells of both OPG and IL-6 stimulated by PGE1. However, reboxetine, a selective and specific inhibitor of norepinephrine reuptake, failed to affect the PGE1-induced release of OPG or IL-6. Oppositely, fluvoxamine and sertraline, agents belonging to the class of selective serotonin reuptake inhibitor, upregulated the PGE1-stimulated release of both OPG and IL-6. Duloxetine amplified the expression of OPG mRNA and IL-6 mRNA stimulated by PGE1. Duloxetine strengthened the PGE1-induced p38 MAP kinase phosphorylation, which was amplified by fluvoxamine as well. SB203880, an inhibitor of p38 MAP kinase, suppressed the amplifying effects by duloxetine or fluvoxamine on the PGE1-stimulated release of OPG and IL-6. These results strongly suggest that duloxetine could strengthen osteoblast activation by PGE1 through the upregulation of p38 MAP kinase, leading to increasing the synthesis of OPG and IL-6.

Effect of Postoperative Administration of Pregabalin for Post-thoracotomy Pain: A Randomized Study.

OBJECTIVE: The present study was performed to evaluate the effect of postoperative administration of pregabalin in patients who reported moderate-to-severe pain after epidural analgesia following thoracotomy. DESIGN: An open-label, randomized, controlled, parallel-group study. SETTING: A single center in Japan. PARTICIPANTS: Consecutive patients (aged≥20 years) who reported moderate-to-severe pain after effectual 2-day epidural analgesia post-thoracotomy for lung cancer from February 2012 to March 2013. INTERVENTIONS: Patients were assigned to 2 groups: control (control treatment: acetaminophen, 400 mg, and codeine phosphate powder, 20 mg) or pregabalin (pregabalin, 75 mg, plus control treatment). The 12-week study period included 2-week study treatment and 10-week follow-up. MEASUREMENTS AND MAIN RESULTS: For efficacy, the primary endpoint was the visual analog scale (VAS) scores for pain at rest and with coughing at week 2, and secondary endpoints were the VAS scores for pain and the neuropathic pain questionnaire at week 12. Fifty patients were randomized (25 per group). At week 2, the VAS scores for pain at rest (mean [SD]) were 29.5 (21.9) in the control group and 16.3 (15) in the pregabalin group (p = 0.02); for pain with coughing, the scores were 45.2 (20.9) and 28.8 (25.9), respectively (p = 0.02). VAS scores improved more in the pregabalin group than in the control group over the 12 weeks. Patients free from possible neuropathic pain were 48% of the control group and 88% of the pregabalin group, respectively (p = 0.001). CONCLUSIONS: Postoperative administration of pregabalin effectively reduced post-thoracotomy pain.

Long-term effects of second cochlear implantation with sequential bilateral cochlear implantation in Japanese children.

OBJECTIVE: This study aimed to longitudinally evaluate speech perception ability and sound-field thresholds with the first, second, or bilateral cochlear implants (CIs) and MAP parameters of second CI in children. METHODS: Eighteen children who underwent bilateral cochlear implantation at Kyoto University Hospital were included. We evaluated speech perception under quiet and noisy conditions using the first, second, or bilateral CIs, CI-aided sound-field thresholds using the first or second CI, and MAP parameter values (C-levels, T-levels, and dynamic range) of the second CI of more than 5 years after the second implantation. RESULTS: Patients with a second CI after 7 years of age had significantly worse speech perception ability with the second CI even long after the surgery than those with a second CI before 7 years of age. CI-aided sound-field thresholds using the first or second CI were similar, regardless of the second implantation timing. Speech perception in noise with bilateral CIs was enhanced by the addition of a second CI, even after 7 years of age. Patients undergoing second cochlear implantation before 3.5 years of age showed significantly higher C-levels and wider dynamic ranges in the second CI MAP parameters. CONCLUSIONS: When the second implantation was performed after 7 years of age, the second CI effects were limited even with long-term use, which is attributed to unstable MAP parameters. The second CI-aided sound-field threshold contributed to the better outcome of bilateral CIs in noise, even if the second implantation was performed at age of ≥7 years.

LXR/CD38 activation drives cholesterol-induced macrophage senescence and neurodegeneration via NAD+ depletion.

Although dysregulated cholesterol metabolism predisposes aging tissues to inflammation and a plethora of diseases, the underlying molecular mechanism remains poorly defined. Here, we show that metabolic and genotoxic stresses, convergently acting through liver X nuclear receptor, upregulate CD38 to promote lysosomal cholesterol efflux, leading to nicotinamide adenine dinucleotide (NAD+) depletion in macrophages. Cholesterol-mediated NAD+ depletion induces macrophage senescence, promoting key features of age-related macular degeneration (AMD), including subretinal lipid deposition and neurodegeneration. NAD+ augmentation reverses cellular senescence and macrophage dysfunction, preventing the development of AMD phenotype. Genetic and pharmacological senolysis protect against the development of AMD and neurodegeneration. Subretinal administration of healthy macrophages promotes the clearance of senescent macrophages, reversing the AMD disease burden. Thus, NAD+ deficit induced by excess intracellular cholesterol is the converging mechanism of macrophage senescence and a causal process underlying age-related neurodegeneration.

Rapamycin-encapsulated nanoparticle delivery in polycystic kidney disease mice.

Rapamycin slows cystogenesis in murine models of polycystic kidney disease (PKD) but failed in clinical trials, potentially due to insufficient drug dosing. To improve drug efficiency without increasing dose, kidney-specific drug delivery may be used. Mesoscale nanoparticles (MNP) selectively target the proximal tubules in rodents. We explored whether MNPs can target cystic kidney tubules and whether rapamycin-encapsulated-MNPs (RapaMNPs) can slow cyst growth in Pkd1 knockout (KO) mice. MNP was intravenously administered in adult Pkd1KO mice. Serum and organs were harvested after 8, 24, 48 or 72 h to measure MNP localization, mTOR levels, and rapamycin concentration. Pkd1KO mice were then injected bi-weekly for 6 weeks with RapaMNP, rapamycin, or vehicle to determine drug efficacy on kidney cyst growth. Single MNP injections lead to kidney-preferential accumulation over other organs, specifically in tubules and cysts. Likewise, one RapaMNP injection resulted in higher drug delivery to the kidney compared to the liver, and displayed sustained mTOR inhibition. Bi-weekly injections with RapaMNP, rapamycin or vehicle for 6 weeks resulted in inconsistent mTOR inhibition and little change in cyst index, however. MNPs serve as an effective short-term, kidney-specific delivery system, but long-term RapaMNP failed to slow cyst progression in Pkd1KO mice.

Successful treatment with spinal cord stimulation for pain due to eosinophilic granulomatosis with polyangiitis: a case report.

BACKGROUND: Although most patients of eosinophilic granulomatosis with polyangiitis (EGPA) experience a reduction in pain within several weeks to months of the initiation of immunotherapies, some suffer from residual neuropathic symptoms for a long time. CASE PRESENTATION: A 28-year-old woman diagnosed with EGPA visited. She had been treated with steroid pulse therapy, intravenous immunoglobulin, and mepolizumab (antiinterleukin-5 agent). Her symptoms other than peripheral neuropathy improved, but posterior lower thigh pain and weakness of the lower legs worsened. At the initial visit, she used crutches and complained of numb pain in both posterior lower thighs, especially the left one. She also presented with left foot drop and reported a decreased tactile sensation on the lateral sides of both lower thighs. We performed spinal cord stimulation (SCS) at the L1 level on both sides. Her pain remarkably decreased, her tactile sensation improved, her muscle strength increased, and she was able to walk without crutches. CONCLUSIONS: We herein report the first case of lower extremity pain being successfully treated with SCS in an EGPA patient who did not respond well to drug therapy. Because the cause of pain in EGPA is neuropathy induced by vasculitis, there is ample ability for SCS to improve this pain. When pain is neuropathic, whatever the cause, SCS may be worth trying, even for pain from disorders other than EGPA.

Infection control and COVID-19 outbreak training at elderly care facilities.

Fatalities caused by pneumonia and underlying diseases from COVID-19 infection show the highest relative frequency among elderly people. Long-term care facilities for elderly people have continued to be the areas most vulnerable to COVID-19. We examined the effectiveness of training for infection control and COVID-19 at elderly care facilities. After sending questionnaires to all long-term elderly care facilities in Ibaraki prefecture, Japan during January 18-29, 2022, we received useful responses from 98 facilities. Using logistic regression, we regressed a dummy variable for outbreak experience to dummy variables representing routine but partial training, routine training for all staff members, long-term care facilities for elderly people, numbers of nurses, and numbers of residents. Outbreak experiences of two types were inferred, as represented by a dummy variable for a COVID-19 outbreak at the facility, and by a dummy variable for outbreak experience at the facility before COVID-19 was found. Multivariate analysis indicated routine training for all staff members as the most effective, in fact the only effective, countermeasure against COVID-19 outbreak.

Accelerated cystogenesis by dietary protein load is dependent on, but not initiated by kidney macrophages.

Background: Disease severity of autosomal dominant polycystic kidney disease (ADPKD) is influenced by diet. Dietary protein, a recognized cyst-accelerating factor, is catabolized into amino acids (AA) and delivered to the kidney leading to renal hypertrophy. Injury-induced hypertrophic signaling in ADPKD results in increased macrophage (MФ) activation and inflammation followed by cyst growth. We hypothesize that the cystogenesis-prompting effects of HP diet are caused by increased delivery of specific AA to the kidney, ultimately stimulating MФs to promote cyst progression. Methods: Pkd1flox/flox mice with and without Cre (CAGG-ER) were given tamoxifen to induce global gene deletion (Pkd1KO). Pkd1KO mice were fed either a low (LP; 6%), normal (NP; 18%), or high (HP; 60%) protein diet for 1 week (early) or 6 weeks (chronic). Mice were then euthanized and tissues were used for histology, immunofluorescence and various biochemical assays. One week fed kidney tissue was cell sorted to isolate tubular epithelial cells for RNA sequencing. Results: Chronic dietary protein load in Pkd1KO mice increased kidney weight, number of kidney infiltrating and resident MФs, chemokines, cytokines and cystic index compared to LP diet fed mice. Accelerated cyst growth induced by chronic HP were attenuated by liposomal clodronate-mediated MФ depletion. Early HP diet fed Pkd1KO mice had larger cystic kidneys compared to NP or LP fed counterparts, but without increases in the number of kidney MФs, cytokines, or markers of tubular injury. RNA sequencing of tubular epithelial cells in HP compared to NP or LP diet group revealed increased expression of sodium-glutamine transporter Snat3, chloride channel Clcnka, and gluconeogenesis marker Pepck1, accompanied by increased excretion of urinary ammonia, a byproduct of glutamine. Early glutamine supplementation in Pkd1KO mice lead to kidney hypertrophy. Conclusion: Chronic dietary protein load-induced renal hypertrophy and accelerated cyst growth in Pkd1KO mice is dependent on both infiltrating and resident MФ recruitment and subsequent inflammatory response. Early cyst expansion by HP diet, however, is relient on increased delivery of glutamine to kidney epithelial cells, driving downstream metabolic changes prior to inflammatory provocation.

Tramadol regulates the activation of human platelets via Rac but not Rho/Rho-kinase.

Tramadol is a useful analgesic which acts as a serotonin and noradrenaline reuptake inhibitor in addition to µ-opioid receptor agonist. Cytoplasmic serotonin modulates the small GTPase activity through serotonylation, which is closely related to the human platelet activation. We recently reported that the combination of subthreshold collagen and CXCL12 synergistically activates human platelets. We herein investigated the effect and the mechanism of tramadol on the synergistic effect. Tramadol attenuated the synergistically stimulated platelet aggregation (300 µM of tramadol, 64.3% decrease, p<0.05). Not morphine or reboxetine, but duloxetine, fluvoxamine and sertraline attenuated the synergistic effect of the combination on the platelet aggregation (30 µM of fluvoxamine, 67.3% decrease, p<0.05; 30 µM of sertraline, 67.8% decrease, p<0.05). The geranylgeranyltransferase inhibitor GGTI-286 attenuated the aggregation of synergistically stimulated platelet (50 µM of GGTI-286, 80.8% decrease, p<0.05), in which GTP-binding Rac was increased. The Rac1-GEF interaction inhibitor NSC23766 suppressed the platelet activation and the phosphorylation of p38 MAPK and HSP27 induced by the combination of collagen and CXCL12. Tramadol and fluvoxamine almost completely attenuated the levels of GTP-binding Rac and the phosphorylation of both p38 MAPK and HSP27 stimulated by the combination. Suppression of the platelet aggregation after the duloxetine administration was observed in 2 of 5 patients in pain clinic. These results suggest that tramadol negatively regulates the combination of subthreshold collagen and CXCL12-induced platelet activation via Rac upstream of p38 MAPK.

Risk Factors of Hyperglycemia After Nerve Blockade with Dexamethasone in Non-Diabetes Mellitus Patients: A Cohort Study.

BACKGROUND: Glucocorticoids (GCs) are expected to inhibit the synthesis and release of proinflammatory cytokines, which induces local pain. Serious side effects or complications are considered rare with single-dose GC use. However, the amount of systemic absorption and the side effects induced by local GC injections are not well understood. OBJECTIVES: We measured the changes in glucose levels after single-does dexamethasone injection with nerve blockade using a continuous glucose monitoring system (CGMS) in non-diabetes mellitus (DM) patients and investigated the risk factors for hyperglycemia. STUDY DESIGN: This is a cohort study. SETTING: This study was conducted at Gifu University Hospital in Japan. METHODS: Forty-six non-DM patients who underwent elective lumbar or sacral nerve root pulsed radiofrequency or lumbar medial branch of the posterior primary rami conventional radiofrequency with dexamethasone (0.1 mg/kg) were analyzed. The patients underwent monitoring of their interstitial glucose using a CGMS. Hyperglycemia was defined as a blood glucose level >= 200 mg/dL. The area under the curve (AUC) where the blood glucose level was over 200 mg/dL was calculated and analyzed. The risk factors of hyperglycemia were determined using an applied ordinal regression model analysis with the AUC as the objective variable and 4 factors (age, body mass index, glucose level just before GC injection, and glycosylated hemoglobin) as explanatory variables. The blood glucose levels were predicted by a nonlinear regression model. RESULTS: The AUC and maximum glucose level were higher on the first day than after the second day. None of the 4 factors were predictors of hyperglycemia. The glucose level before the procedure was associated with the predicted blood glucose level on the first day (P = 0.042). However, the 95% upper confidence limit of the maximum predicted blood glucose level was less than the safety margin. The predicted blood glucose levels returned to the usual level after the second day. LIMITATIONS: First, GCs are metabolized by cytochrome p450 3A4, and it is possible that the inhibition of this pathway decreases the clearance of GCs. Some of our patients were taking medications that influence this cytochrome pathway. Second, we cannot eliminate the possibility of stress-induced hyperglycemia. Finally, we were unable to record the exact meal timing and calories the patients had consumed. CONCLUSIONS: The blood glucose levels were higher than usual on the first day following a local dexamethasone injection, but the levels were not critical in most cases. Because we cannot predict which patients will develop hyperglycemia, we must determine whether or not GCs can be safely administered and inform patients about potential complications.

Amplification by tramadol of PGD2-induced osteoprotegerin synthesis in osteoblasts: Involvement of µ-opioid receptor and 5-HT transporter.

Tramadol, a weak µ-opioid receptor (MOR) agonist with inhibitory effects on the reuptake of serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine, is an effective analgesic to chronic pains. Osteoprotegerin produced by osteoblasts is essential for bone remodeling to suppress osteoclastic bone resorption. We previously reported that prostaglandin D2 (PGD2) induces osteoprotegerin synthesis whereby p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) are involved in osteoblast-like MC3T3-E1 cells. Herein, we investigated the mechanism underlying the effect of tramadol on the PGD2-induced osteoprotegerin synthesis in these cells. Tramadol enhanced the PGD2-induced release and mRNA expression of osteoprotegerin. Naloxone, a MOR antagonist, reduced the amplification by tramadol of the PGD2-stimulated osteoprotegerin release. Not the selective norepinephrine reuptake inhibitor reboxetine but the selective serotonin reuptake inhibitors fluvoxamine and sertraline upregulated the PGD2-induced osteoprotegerin release, which was further amplified by morphine. Tramadol enhanced PGD2-stimulated phosphorylation of p38 MAP kinase and SAPK/JNK, but not p44/p42 MAP kinase. Both SB203580 and SP600125 suppressed the tramadol effect to enhance the PGD2-stimulated osteoprotegerin release. Tramadol enhanced the PGE2-induced osteoprotegerin release as well as PGD2. These results suggest that tramadol amplifies the PGD2-induced osteoprotegerin synthesis at the upstream of p38 MAP kinase and SAPK/JNK in the involvement of both MOR and 5-HT transporter in osteoblasts.

Acetaminophen reduces osteoprotegerin synthesis stimulated by PGE2 and PGF2α in osteoblasts: attenuation of SAPK/JNK but not p38 MAPK or p44/p42 MAPK.

Acetaminophen is one of the most widely used analgesic and antipyretic medicines, whose long-period use has reportedly been associated with an increased risk of bone fracture. However, the mechanism underlying this undesired effect remains to be investigated. The homeostatic control of bone tissue depends on the interaction between osteoblasts and osteoclasts. Osteoprotegerin produced by osteoblasts is known to play an essential role in suppressing osteoclast induction. We have previously reported that prostaglandin (PG) E2 and PGF2α induce osteoprotegerin synthesis through p38 mitogen-activated protein kinase (MAPK), p44/p42 MAPK and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of acetaminophen on the osteoprotegerin synthesis induced by PGE2 and PGF2α in MC3T3-E1 cells. Acetaminophen significantly suppressed the osteoprotegerin release stimulated by PGE2 and PGF2α. The PGE2-induced expression of osteoprotegerin mRNA was also reduced by acetaminophen. Acetaminophen markedly downregulated the phosphorylation of SAPK/JNK stimulated by PGE2 and PGF2α, but not those of p38 MAPK or p44/p42 MAPK. SP600125, an inhibitor of SAPK/JNK, suppressed the levels of PGE2- and PGF2α-upregulated osteoprotegerin mRNA expression. Taken together, these results strongly suggest that acetaminophen reduces the PGE2- and PGF2α-stimulated synthesis of osteoprotegerin in osteoblasts, and that the suppressive effect is exerted via attenuation of SAPK/JNK. These findings provide a molecular basis for the possible effect of acetaminophen on bone tissue metabolism.

Mechanisms of tumor necrosis factor-alpha-induced interleukin-6 synthesis in glioma cells.

BACKGROUND: Interleukin (IL)-6 plays a pivotal role in a variety of CNS functions such as the induction and modulation of reactive astrogliosis, pathological inflammatory responses and neuroprotection. Tumor necrosis factor (TNF)-alpha induces IL-6 release from rat C6 glioma cells through the inhibitory kappa B (IkappaB)-nuclear factor kappa B (NFkappaB) pathway, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). The present study investigated the mechanism of TNF-alpha-induced IL-6 release in more detail than has previously been reported. METHODS: Cultured C6 cells were stimulated by TNF-alpha. IL-6 release from the cells was measured by an enzyme-linked immunosorbent assay, and the phosphorylation of IkappaB, NFkappaB, the MAP kinase superfamily, and signal transducer and activator of transcription (STAT)3 was analyzed by Western blotting. Levels of IL-6 mRNA in cells were evaluated by real-time reverse transcription-polymerase chain reaction. RESULTS: TNF-alpha significantly induced phosphorylation of NFkappaB at Ser 536 and Ser 468, but not at Ser 529 or Ser 276. Wedelolactone, an inhibitor of IkappaB kinase, suppressed both TNF-alpha-induced IkappaB phosphorylation and NFkappaB phosphorylation at Ser 536 and Ser 468. TNF-alpha-stimulated increases in IL-6 levels were suppressed by wedelolactone. TNF-alpha induced phosphorylation of STAT3. The Janus family of tyrosine kinase (JAK) inhibitor I, an inhibitor of JAK 1, 2 and 3, attenuated TNF-alpha-induced phosphorylation of STAT3 and significantly reduced TNF-alpha-stimulated IL-6 release. Apocynin, an inhibitor of NADPH oxidase that suppresses intracellular reactive oxygen species, significantly suppressed TNF-alpha-induced IL-6 release and mRNA expression. However, apocynin failed to affect the phosphorylation of IkappaB, NFkappaB, p38 MAP kinase, SAPK/JNK or STAT3. CONCLUSION: These results strongly suggest that TNF-alpha induces IL-6 synthesis through the JAK/STAT3 pathway in addition to p38 MAP kinase and SAPK/JNK in C6 glioma cells, and that phosphorylation of NFkappaB at Ser 536 and Ser 468, and NADPH oxidase are involved in TNF-alpha-stimulated IL-6 synthesis.

Effects of bilateral cochlear implants in children: Timing of second surgery and the significance of wearing bilateral cochlear implants in Japan.

OBJECTIVE: This study aims to evaluate the speech perception with first, second, or bilateral cochlear implants (CI) and to reveal the effects of wearing bilateral CI in children. METHODS: After reviewing the medical records, a total of 19 children who underwent bilateral cochlear implantation serially between 2012 and 2015 at Kyoto University Hospital (tertiary referral center) were included in this study. All patients had no delay in language development. The study group comprised nine boys and ten girls, and their age ranged from 3 years 8 months to 12 years 5 months when they underwent the tests in this study. The mean and median ages were 8 years 6 months and 9 years 2 months, respectively. We measured the appropriate signal/noise ratio (SNR) to test speech perception of Japanese language in noise by testing the hearing ability of unilateral CI patients with or without noise and by surveying the sound environment in a classroom of a mainstream elementary school. Speech perception in quiet and noise and the left-right localization ability were examined using first, second, or bilateral cochlear implants in all patients. RESULTS: Considering the results of hearing ability tests with noise and the SNR of the elementary school classrooms, we decided to use SNR of +10 dB to evaluate the speech perception ability in noise. The speech perception ability using the second CI was significantly worse in patients undergoing second cochlear implantation after 7 years old than in those who underwent surgery before 3.5 years old. Moreover, patients undergoing second cochlear implantation before 7 years old showed significantly better left-right localization of high-frequency sound. CONCLUSIONS: Second cochlear implantation before 7 years old is a critical factor in acquiring beneficial speech perception ability with the second CI and sound localization ability with the bilateral CI.

Tramadol enhances PGF2α-stimulated osteoprotegerin synthesis in osteoblasts.

Osteoprotegerin (OPG) synthesized by osteoblasts is currently considered a crucial regulator to suppress the formation and function of osteoclasts. We previously showed that the synthesis of OPG is stimulated by prostaglandin F2α (PGF2α) in the involvement of p38 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. We also found that Rho-kinase is involved in the signaling of PGF2α upstream of p38 MAPK in these cells. Tramadol is widely used to treat chronic pain, such as low back pain associated with osteoporosis. We investigated whether or not tramadol affects the OPG release induced by PGF2α in osteoblast-like MC3T3-E1 cells. The levels of OPG in the conditioned medium were measured by an enzyme-linked immunosorbent assay. The mRNA expression of OPG was determined with real-time reverse transcription polymerase chain reaction. The phosphorylation of target protein was determined with a Western blot analysis. PGF2α induced the release and the mRNA expression of OPG, which tramadol significantly enhanced. Morphine, a selective µ-opioid receptor (MOR) agonist, also enhanced the PGF2α-induced OPG release. In addition, naloxone, a MOR antagonist, suppressed the enhancement by tramadol or morphine of the PGF2α-induced OPG synthesis. Tramadol upregulated the phosphorylation of SAPK/JNK and p38 MAPK stimulated by PGF2α but not that of p44/p42 MAPK or myosin phosphatase targeting protein (MYPT), a substrate of Rho-kinase. The inhibitors of both p38 MAPK and SAPK/JNK, SB203580 and SP600125, respectively, reduced the tramadol amplification of OPG release stimulated by PGF2α. The present results strongly suggest that tramadol enhances the synthesis of OPG stimulated by PGF2α through MOR in osteoblasts, and that the amplifying effect is exerted at upstream of p38 MAPK and SAPK/JNK but downstream of Rho-kinase.

Identification of ALDH1A3 as a Viable Therapeutic Target in Breast Cancer Metastasis-Initiating Cells.

The development of efficacious therapies targeting metastatic spread of breast cancer to the brain represents an unmet clinical need. Accordingly, an improved understanding of the molecular underpinnings of central nervous system spread and progression of breast cancer brain metastases (BCBM) is required. In this study, the clinical burden of disease in BCBM was investigated, as well as the role of aldehyde dehydrogenase 1A3 (ALDH1A3) in the metastatic cascade leading to BCBM development. Initial analysis of clinical survival trends for breast cancer and BCBM determined improvement of breast cancer survival rates; however, this has failed to positively affect the prognostic milestones of triple-negative breast cancer (TNBC) brain metastases (BM). ALDH1A3 and a representative epithelial-mesenchymal transition (EMT) gene signature (mesenchymal markers, CD44 or Vimentin) were compared in tumors derived from BM, lung metastases (LM), or bone metastases (BoM) of patients as well as mice after injection of TNBC cells. Selective elevation of the EMT signature and ALDH1A3 were observed in BM, unlike LM and BoM, especially in the tumor edge. Furthermore, ALDH1A3 was determined to play a role in BCBM establishment via regulation of circulating tumor cell adhesion and migration phases in the BCBM cascade. Validation through genetic and pharmacologic inhibition of ALDH1A3 via lentiviral shRNA knockdown and a novel small-molecule inhibitor demonstrated selective inhibition of BCBM formation with prolonged survival of tumor-bearing mice. Given the survival benefits via targeting ALDH1A3, it may prove an effective therapeutic strategy for BCBM prevention and/or treatment.

Glioma-initiating cells at tumor edge gain signals from tumor core cells to promote their malignancy.

Intratumor spatial heterogeneity facilitates therapeutic resistance in glioblastoma (GBM). Nonetheless, understanding of GBM heterogeneity is largely limited to the surgically resectable tumor core lesion while the seeds for recurrence reside in the unresectable tumor edge. In this study, stratification of GBM to core and edge demonstrates clinically relevant surgical sequelae. We establish regionally derived models of GBM edge and core that retain their spatial identity in a cell autonomous manner. Upon xenotransplantation, edge-derived cells show a higher capacity for infiltrative growth, while core cells demonstrate core lesions with greater therapy resistance. Investigation of intercellular signaling between these two tumor populations uncovers the paracrine crosstalk from tumor core that promotes malignancy and therapy resistance of edge cells. These phenotypic alterations are initiated by HDAC1 in GBM core cells which subsequently affect edge cells by secreting the soluble form of CD109 protein. Our data reveal the role of intracellular communication between regionally different populations of GBM cells in tumor recurrence.

Targeting glioma-initiating cells via the tyrosine metabolic pathway.

OBJECTIVE: Despite an aggressive multimodal therapeutic regimen, glioblastoma (GBM) continues to portend a grave prognosis, which is driven in part by tumor heterogeneity at both the molecular and cellular levels. Accordingly, herein the authors sought to identify metabolic differences between GBM tumor core cells and edge cells and, in so doing, elucidate novel actionable therapeutic targets centered on tumor metabolism. METHODS: Comprehensive metabolic analyses were performed on 20 high-grade glioma (HGG) tissues and 30 glioma-initiating cell (GIC) sphere culture models. The results of the metabolic analyses were combined with the Ivy GBM data set. Differences in tumor metabolism between GBM tumor tissue derived from within the contrast-enhancing region (i.e., tumor core) and that from the peritumoral brain lesions (i.e., tumor edge) were sought and explored. Such changes were ultimately confirmed at the protein level via immunohistochemistry. RESULTS: Metabolic heterogeneity in both HGG tumor tissues and GBM sphere culture models was identified, and analyses suggested that tyrosine metabolism may serve as a possible therapeutic target in GBM, particularly in the tumor core. Furthermore, activation of the enzyme tyrosine aminotransferase (TAT) within the tyrosine metabolic pathway influenced the noted therapeutic resistance of the GBM core. CONCLUSIONS: Selective inhibition of the tyrosine metabolism pathway may prove highly beneficial as an adjuvant to multimodal GBM therapies.

Comparative effects of ultra-short-acting beta1-blockers on voltage-gated tetrodotoxin-resistant Na+ channels in rat sensory neurons.

BACKGROUND AND OBJECTIVE: To examine a possible mechanism for their antinociceptive actions, we compared the effects of two clinically used ultra-short-acting beta1-blockers, landiolol and esmolol, on tetrodotoxin-resistant sodium (TTX-r Na) channels in rat dorsal root ganglion neurons, which are important for nociception. METHODS: In small (<30 microm) dorsal root ganglion neurons from Sprague-Dawley rats, recordings of whole-cell membrane currents were made using the patch-clamp technique. To examine the effects of landiolol and esmolol on TTX-r Na currents, whole-cell membrane Na currents were evoked every 10 s by stepping for 50 ms from a holding potential of -70 to -10 mV. Each drug was applied at stepwise-increased concentrations every 2 min. The voltage dependence of the steady-state inactivation of the TTX-r Na current was investigated by using a conventional double-pulse protocol. To test for use-dependent blockade of TTX-r Na channels by beta-blockers, trains of depolarizing pulses (to -10 from a holding potential of -70 mV) were applied at one of three frequencies (0.2, 5 or 20 Hz) in the absence or presence of drug (landiolol 8 mmol l, esmolol 140 micromol l). RESULTS: Esmolol blocked TTX-r Na currents in a dose-dependent and use-dependent manner, but a very high concentration of landiolol was required to block TTX-r Na channel activities. The half-maximal inhibitory concentrations (IC50) for the TTX-r Na current were (holding potential, -70 mV) landiolol 7.66 +/- 0.62 mmol l (n = 6) and esmolol 145 +/- 7.5 micromol l (n = 6), and the Hill coefficients were landiolol 1.06 +/- 0.09 (n = 6) and esmolol 0.96 +/- 0.05 (n = 6). CONCLUSION: Esmolol, but not landiolol, may have useful effects against pain related to TTX-r Na channel activity.