In-process monitoring of a tissue-engineered oral mucosa fabricated on a micropatterned collagen scaffold: use of optical coherence tomography for quality control.

Background: We previously reported a novel technique for fabricating dermo-epidermal junction (DEJ)-like micropatterned collagen scaffolds to manufacture an ex vivo produced oral mucosa equivalent (EVPOME) for clinical translation; however, more biomimetic micropatterns are required to promote oral keratinocyte-based tissue engineering/regenerative medicine. In addition, in-process monitoring for quality control of tissue-engineered products is key to successful clinical outcomes. However, evaluating three-dimensional tissue-engineered constructs such as EVPOME is challenging. This study aimed to update our technique to fabricate a more biomimetic DEJ structure of oral mucosa and to investigate the efficacy of optical coherence tomography (OCT) in combination with deep learning for non-invasive EVPOME monitoring. Methods: A picosecond laser-textured microstructure mimicking DEJ on stainless steel was used as a negative mould to fabricate the micropatterned collagen scaffold. During EVPOME manufacturing, OCT was applied twice to monitor the EVPOME and evaluate its epithelial thickness. Findings: Our moulding system resulted in successful micropattern replication on the curved collagen scaffold. OCT imaging visualised the epithelial layer and the underlying micropatterned scaffold in EVPOME, enabling to non-invasively detect specific defects not found before the histological examination. Additionally, a gradual increase in epithelial thickness was observed over time. Conclusion: These findings demonstrate the feasibility of using a stainless-steel negative mould to create a more biomimetic micropattern on collagen scaffolds and the potential of OCT imaging for quality control in oral keratinocyte-based tissue engineering/regenerative medicine.

Correlation of preference- and profile-based quality of life of Japanese oral cancer patients during the perioperative period measured using EQ-5D-5L and FACT-H&N.

The EuroQol 5-dimension 5-level (EQ-5D-5L) instrument is among the most used preference-based quality of life (QOL) measures for cost-utility analysis. Each dimension is evaluated on five levels. The aim of this study was to clarify whether the EQ-5D-5L, which consists of only five items, correlates with profile-based QOL measures in Japanese oral cancer patients during the perioperative period. One hundred participants with oral cancer undergoing radical therapy completed QOL assessments before treatment, at treatment completion, and 1 and 3 months after treatment using the EQ-5D-5L and Functional Assessment of Cancer Therapy - Head & Neck instrument (FACT-H&N, Japanese version). To clarify how the EQ-5D-5L reflects the FACT-H&N, multiple regression analyses were performed using FACT-H&N subscales. The ceiling effect of the EQ-5D-5L was investigated. The EQ-5D-5L moderately correlated with the FACT-H&N over the entire perioperative period (rs = 0.586, P < 0.01). In the multiple regression analysis, the EQ-5D-5L was strongly reflected in the physical wellbeing subscale of the FACT-H&N, excluding social wellbeing. The pre-treatment EQ-5D-5L score was decreased owing to the impacts of the dimensions of pain/discomfort and anxiety/depression. The EQ-5D-5L did not have a ceiling effect in oral cancer patients. The EQ-5D-5L appears to generally correlate with the FACT-H&N for oral cancer patients during the perioperative period.

Correction: Myeloproliferative leukemia protein activation directly induces fibrocyte differentiation to cause myelofibrosis.

Owing to the insufficient specificity of the anti-myeloproliferative leukemia protein (MPL) antibody in the original version of this Article, Figure 6 and parts of Figures 2a, 4e, and 5a do not represent the correct information. The corrected version of Figure 6 is in this correction and those of Figures 2a, 4e, and 5a are shown in the supplemental information.

Functional characterization of the G162R and D216H genetic variants of human CYP17A1.

Cytochrome P450 17A1 (CYP17A1) is a dual-function enzyme catalyzing reactions necessary for cortisol and androgen biosynthesis. CYP17A1 is a validated drug target for prostate cancer as CYP17A1 inhibition significantly reduces circulating androgens and improves survival in castration-resistant prostate cancer. Germline CYP17A1 genetic variants with altered CYP17A1 activity manifesting as various endocrinopathies are extremely rare; however, characterizing these variants provides critical insights into CYP17A1 protein structure and function. By querying the dbSNP online database and publically available data from the 1000 genomes project (http://browser.1000genomes.org), we identified two CYP17A1 nonsynonymous genetic variants with unknown consequences for enzymatic activity and stability. We hypothesized that the resultant amino acid changes would alter CYP17A1 stability or activity. To test this hypothesis, we utilized a HEK-293T cell-based expression system to characterize the functional consequences of two CYP17A1 variants, D216H (rs200063521) and G162R (rs141821705). Cells transiently expressing the D216H variant demonstrate a selective impairment of 16α-hydroxyprogesterone synthesis by 2.1-fold compared to wild-type (WT) CYP17A1, while no effect on 17α-hydroxyprogesterone synthesis was observed. These data suggest that substrate orientations in the active site might be altered with this amino acid substitution. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to WT with a near 70% reduction in protein levels as determined by immunoblot analysis. This variant is preferentially ubiquitinated and degraded prematurely, with an enzyme half-life calculated to be ∼2.5 h, and proteasome inhibitor treatment recovers G162R protein expression to WT levels. Together, these data provide new insights into CYP17A1 structure-function and stability mechanisms.

Myeloproliferative leukemia protein activation directly induces fibrocyte differentiation to cause myelofibrosis.

Myelofibrosis (MF) may be caused by various pathogenic mechanisms such as elevation in circulating cytokine levels, cellular interactions and genetic mutations. However, the underlying mechanism of MF still remains unknown. Recent studies have revealed that fibrocytes, the spindle-shaped fibroblast-like hematopoietic cells, and the thrombopoietin (TPO)/myeloproliferative leukemia protein (MPL; TPO receptor) signaling pathway play a certain role in the development of MF. In the present study, we aimed to investigate the relationship between fibrocytes and MPL activation. We showed that TPO or a TPO receptor agonist directly induces fibrocyte differentiation using murine fibrocyte cell lines and a murine MF model. Conversely, elimination of macrophages expressing MPL by clodronate liposomes reversed the MF phenotype of the murine model, suggesting that fibrocyte differentiation induced by MPL activation contributes to the progression of MF. Furthermore, we revealed that SLAMF7high MPLhigh monocytes in human peripheral blood mononuclear cells were possible fibrocyte precursors and that these cells increased in number in MF patients not treated with ruxolitinib. Our findings confirmed a link between fibrocytes and the TPO/MPL signaling pathway, which could result in a greater understanding of the pathogenesis of MF and lead to the development of novel therapeutic interventions.

Long-term outcomes of transtrochanteric rotational osteotomy for non-traumatic osteonecrosis of the femoral head.

AIMS: Transtrochanteric rotational osteotomy (TRO) is performed for young patients with non-traumatic osteonecrosis of the femoral head (ONFH) to preserve the hip. We aimed to investigate the long-term outcomes and the risk factors for failure 15 years after this procedure. PATIENTS AND METHODS: This study included 95 patients (111 hips) with a mean age of 40 years (21 to 64) who underwent TRO for ONFH. The mean follow-up was 18.2 years (3 to 26). Kaplan-Meier survivorship analyses were performed with conversion to total hip arthroplasty (THA) and radiological failure due to secondary collapse of the femoral head or osteoarthritic changes as the endpoint. Multivariate analyses were performed to assess risk factors for each outcome. RESULTS: Survival rates at 15 years with conversion to THA and radiological failure as the endpoint were 59% (95% confidence interval (CI) 49 to 67) and 30% (95% CI 22 to 39), respectively. Necrotic type C2 ONFH (lesions extending laterally to the acetabular edge) (hazards ratio (HR) 3.9) and age > 40 years (HR 2.5) were risk factors for conversion to THA. Stage > 3a ONFH (HR 2.0) and age > 40 years (HR 1.9) were risk factors for radiological failure. CONCLUSION: The 15 year outcomes after TRO for ONFH are unfavorable because osteoarthritic changes occur after five years post-operatively. Cite this article: Bone Joint J 2017;99-B:175-83.

Gene therapy for ovarian cancer using carbonyl reductase 1 DNA with a polyamidoamine dendrimer in mouse models.

Ovarian cancer (OC) in which carbonyl reductase 1 (CBR1) is highly expressed has good prognosis. The aims of this study were to determine the optimal conditions for delivering CBR1 DNA to OC cells via a polyamidoamine (PAMAM) dendrimer and to examine the therapeutic effectiveness of using a CBR1/PAMAM dendrimer to treat OC. The ratio for mixture of the PAMAM dendrimer and CBR1 plasmid DNA was defined as the ratio of the number of moles of phosphate groups in plasmid DNA to the number of moles of amino groups in PAMAM, which was expressed as N/P ratio. Mice were intraperitoneally injected with OC cells (HRA) to create peritoneal carcinomatosis. CBR1 DNA/PAMAM dendrimer complexes were administered on alternate days after injection of HRA cells. Cells transfected with CBR1 DNA at N/P ratio of 20:1 for 48 h produced the highest level of CBR1 expression. All the mice in control group died prior to day 25. However, all the mice administered the CBR1 DNA/PAMAM dendrimer survived (P<0.001). Use of a PAMAM dendrimer allowed CBR1 DNA to be delivered to cancer cells. The results suggested that CBR1 DNA/PAMAM dendrimer complexes may represent a potent gene therapy for the treatment of advanced OC.

Effects of vibration on flexibility: a meta-analysis.

Exogenous stimulation of skeletal muscle or tendon is often used to improve range of motion. Despite substantial research efforts, however, the effects of vibration on flexibility have not been clarified. In this review, we investigated the effects of acute and chronic intervention programs which used vibration to improve flexibility in young healthy individuals. Effect size was calculated using data from a total of 600 participants in 19 studies before and after the introduction of vibration-based intervention, and a total of 324 participants in 13 studies on the additive effects of vibration compared with the identical conditions without vibration. Sub-group analyses were performed based on intervention period, type of exercise, and type of vibration. Meta-analysis showed that vibration interventions had significant effects on flexibility (standardized mean difference [SMD]=-0.79, 95% confidence interval [CI]=-1.14- -0.43; p<0.001), albeit with the possibility of heterogeneity (I(2)=75%). Another meta-analysis revealed a significant additive effect of vibration on flexibility compared with the identical condition without vibration (SMD=0.25, 95%CI=0.03-0.48; P=0.03), with small heterogeneity (I(2)=0%). The risk of publication bias was low judged from Kendall's τ statistic. We concluded that the use of vibration might lead to additive improvements in flexibility.

The effects of whole-body vibration on muscle strength and power: a meta-analysis.

Exercise with whole-body vibration (WBV) is becoming popular as an alternative to conventional training or as supplementary training. However, despite increasing research efforts in this field, additive effects of WBV on muscle performance remain unclarified. In this review, we investigated the additive effects of long-term WBV on muscle strength and power. This meta-analysis was restricted to randomized controlled trials lasting for at least 5 weeks comparing exercise with and without WBV, or comparing only WBV exposure and control. Data from a total of 314 participants in 10 studies on knee extension muscle strength, and 249 participants in 7 studies on countermovement jump height were pooled using random-effect models. Meta-analysis showed significant additional effects of WBV on muscle strength (standardized mean difference [SMD]=0.76, 95% confidence interval [CI]=0.21-1.32; p=0.007) and countermovement jump (SMD=0.87, 95% CI=0.29-1.46; p=0.003). Based on these findings, we concluded that the use of WBV would lead to greater improvements in both knee extension muscle strength and countermovement jump than under identical conditions without WBV.

Effects of resistance training with whole-body vibration on muscle fitness in untrained adults.

The effects of resistance training (RT) combined with whole-body vibration (WBV) on muscle fitness, particularly muscle hypertrophy and neuromuscular performance, are not well understood. We investigated the effects of WBV in healthy, untrained participants after a 13-week RT course by performing magnetic resonance imaging and by measuring maximal isometric (with electromyography) and isokinetic knee extension strengths, isometric lumbar extension torque, countermovement-jump, knee extension endurance, and sit-ups. Thirty-two individuals (22-49 years old) were randomly assigned to RT groups with (RT-WBV, n=16) or without WBV (RT, n=16). Following the RT course, significantly higher increases in the cross-sectional areas of m. psoas major (vs baseline values) and erector spinae muscle (vs the RT group) were observed in the RT-WBV group (+10.7%, P<0.05; +8.7%, P<0.05) compared with the RT group (+3.8%, P=0.045; 0.0%). Higher increases from baseline were also observed in maximal isometric force, concentric knee extension torque, countermovement-jump, and maximal isometric lumbar extension torque in RT-WBV (+63.5%; +76.7%, +15.0%, and +51.5%, respectively; P<0.05) than in those of RT (+25.6%, P=0.001; +17.8%, P=0.18; +11.3%, P=0.001; and +26.4%, P<0.001, respectively). The WBV-induced increases in muscle hypertrophy and isometric lumbar extension torque suggest a potential benefit of incorporating WBV into slow-velocity RT programs involving exercises of long duration.

Cytosine-phosphate-guanosine-DNA induces CD274 expression in human B cells and suppresses T helper type 2 cytokine production in pollen antigen-stimulated CD4-positive cells.

Co-stimulatory molecules are important for regulating T cell activation and immune response. CD274 [programmed death ligand 1 (PD-L1), B7-H1] has emerged as an important immune modulator that can block T cell receptor signalling. We have investigated whether PD-L1 and other co-stimulatory ligands could be expressed in human B cells stimulated by cytosine-phosphate-guanosine (CpG)-DNA. CpG-DNA strongly induced the co-inhibitory molecule ligand, PD-L1, of human B cells. Results show that nuclear factor-kappa B (NF-κB) signalling is involved directly in CpG-DNA-induced PD-L1 expression in human B cells. We sought to determine the effect of CpG-DNA-treated B cells on T helper type 2 (Th2) cytokine production in Cry j 1 (Japanese pollen antigen)-stimulated human CD4-positive cells from patients with seasonal allergic rhinitis caused by Japanese cedar pollen. CpG-DNA-treated B cells reduced Cry j 1-induced interleukin (IL)-5 and IL-13 production in CD4-positive cells. When the binding of PD-1 to PD-L1 was inhibited by PD-1-immunoglobulin (Ig), this chimera molecule reversed the previously described reductions in IL-5 and IL-13 production. In contrast, the CpG B-treated B cells increased both interferon (IFN)-γ and IL-12 production in the presence of Cry j 1-stimulated CD4-positive cells. CpG-DNA simultaneously reduced the expression of B7RP-1 [also known as inducible co-stimulator ligand (ICOSL), B7-H2] and the ligand of CD30 (CD30L). These results indicate that CpG-DNA induces co-inhibitory molecule ligand PD-L1 expression in human B cells and PD-L1 can suppress Th2 cytokine production in Cry j 1-stimulated CD4-positive cells, while CpG-DNA increased Th1 cytokine production and reduced the expression of co-stimulatory molecule ligands that can promote Th2 inflammatory responses.

BCR-ABL promotes neutrophil differentiation in the chronic phase of chronic myeloid leukemia by downregulating c-Jun expression.

The mechanism that is responsible for mature neutrophil overproduction in the chronic phase (CP) of chronic myeloid leukemia (CML), a neoplastic disease of hematopoietic stem cells carrying a constitutively active tyrosine kinase BCR-ABL, remains obscure. In this study, microarray analysis revealed that c-Jun, a monopoiesis-promoting transcription factor, was downregulated in CML neutrophils. BCR-ABL directly inhibited c-Jun expression, as c-Jun downregulation in primary CML neutrophils and in the CML blast cell lines, KCL22 and K562, was reversed by the tyrosine kinase inhibitor imatinib. We established a myeloid differentiation model in KCL22 cells using zinc-inducible CCAAT/enhancer-binding protein (C/EBP)alpha (KCL22/alpha). Myeloid differentiation was observed in C/EBP-induced KCL22/alpha cells. Imatinib-induced c-Jun upregulation promoted the monocytic differentiation of KCL22/alpha cells. c-Jun knockdown in KCL22/alpha cells by a short interfering RNA redirected their differentiation from the monocytic to the neutrophilic lineage, even after imatinib treatment. A blockade of PI3K-Akt signaling with an Akt inhibitor upregulated c-Jun and induced the monocytic differentiation of KCL22, K562, and C/EBP-induced KCL22/alpha cells. Thus, BCR-ABL downregulates c-Jun expression by activating the PI3K-Akt pathway during CML-CP, thereby allowing C/EBPs to promote neutrophil differentiation.

Serological evidence of infection with hepatitis E virus among wild Yezo-deer, Cervus nippon yesoensis, in Hokkaido, Japan.

In this study, 520 serum samples from Yezo-deer in the Hidaka district, Hokkaido, Japan were examined by enzyme-linked immunosorbent assay to investigate whether the animals were infected with hepatitis E virus (HEV). The distribution of optical density values showed a bimodal pattern and 181 samples (34.8%) were deemed to be antibody-positive against HEV. At least five (2.8%) of the positive sera gave specific bands by Western blot analysis. An age-dependent increase in prevalence of the antibodies was found among the animals. These findings indicate that Yezo-deer are a possible host for HEV infection. To avoid the risk of becoming HEV infected, the consumption of raw Yezo-deer meat must be prohibited.

Association of serum interleukin-33 level and the interleukin-33 genetic variant with Japanese cedar pollinosis.

BACKGROUND: IL-33, an IL-1-like cytokine, is a ligand for IL1RL1, which is an important effector molecule of type 2 T helper responses. Although IL-33/IL1RL1 interaction has been suggested to be important in induction of allergic airway inflammation, serum levels of IL-33 and the genetic influences of the polymorphisms of IL-33 in human allergic diseases are unclear. OBJECTIVE: The aim of this study was to examine whether the serum IL-33 level and polymorphisms in IL-33 are associated with Japanese cedar (JC) pollinosis, the most common form of allergic rhinitis, and a major public health problem, in Japan. METHODS: We performed linkage disequilibrium (LD) mapping of the gene using the HapMap database, and two selected tag single nucleotide polymorphisms were genotyped. We conducted an association study of IL-33 (JC pollinosis, n=170; normal controls, n=100) and measured the IL-33 levels in sera of the 270 subjects by ELISA. RESULTS: Serum levels of IL-33 were significantly higher in patients with JC pollinosis (P=0.0018) than in controls. In genetic association analysis, we found a positive association between the polymorphism and JC pollinosis (P=0.048). CONCLUSION: Our results support a role for IL-33 in the pathogenesis of JC pollinosis.

Effects of alpha2-adrenoceptor agonists on tetrodotoxin-resistant Na+ channels in rat dorsal root ganglion neurons.

BACKGROUND AND OBJECTIVE: When intrathecally or epidurally administered, alpha2-adrenoceptor agonists produce potent antinociception by affecting the activity of primary afferent fibres and spinal cord neurons. Recent reports have indicated that in dorsal root ganglion neurons, tetrodotoxin-resistant Na+ channels play important roles in the conduction of nociceptive sensation. We therefore investigated the effects of alpha2-adrenoceptor agonists on tetrodotoxin-resistant Na+ currents. METHODS: Using the whole-cell patch-clamp technique, we recorded tetrodotoxin-resistant Na+ currents from rat dorsal root ganglion neurons. RESULTS: Both clonidine and dexmedetomidine reduced the peak amplitude of the tetrodotoxin-resistant Na+ current concentration- and use-dependently. The concentration required for a half-maximal effect was significantly lower for dexmedetomidine (58.0 +/- 10.2 micromol) than for clonidine (257.2 +/- 30.9 micromol) at holding potential -70 mV. The current inhibitions induced by these agonists were not prevented by 1 micromol yohimbine, an alpha2-adrenoceptor antagonist. Both clonidine and dexmedetomidine shifted the inactivation curve for the tetrodotoxin-resistant Na+ current in the hyperpolarizing direction. The combinations clonidine with lidocaine and dexmedetomidine with lidocaine produced an additive blockade-type interaction on the tetrodotoxin-resistant Na+ current. CONCLUSIONS: The results suggest that a direct inhibition of tetrodotoxin-resistant Na+ channels may contribute to the antinociceptive effects of clonidine and dexmedetomidine when used as additives to regional anaesthesia.

Effects of ifenprodil on voltage-gated tetrodotoxin-resistant Na+ channels in rat sensory neurons.

BACKGROUND AND OBJECTIVE: To examine a possible mechanism for the antinociceptive action of the N-methyl-D-aspartate receptor antagonist ifenprodil, we compared its effects with those of ketamine on tetrodotoxin-resistant Na+ channels in rat dorsal root ganglion neurons, which play an important role in the nociceptive pain pathway. METHODS: Experiments were performed on dorsal root ganglion neurons from Sprague-Dawley rats, recordings of whole-cell membrane currents being made using patch-clamp technique. RESULTS: Both drugs blocked tetrodotoxin-resistant Na+ currents dose dependently, their half-maximal inhibitory concentrations being 145+/-12.1 micromol (ketamine) and 2.6+/-0.95 micromol (ifenprodil). Ifenprodil shifted the inactivation curve for tetrodotoxin-resistant Na+ channels in the hyperpolarizing direction and shifted the activation curve in the depolarizing direction. Use-dependent blockade of tetrodotoxin-resistant Na+ channels was more marked with ifenprodil than with ketamine. When paired with lidocaine, these drugs produced similar additive inhibitions of tetrodotoxin-resistant Na+ channel activity. CONCLUSIONS: The observed suppressive effects on tetrodotoxin-resistant Na+ channel activity may, at least in part, underlie the antinociceptive effects of these N-methyl-D-aspartate receptor antagonists.

Lipopolysaccharide-induced sensitization of adenylyl cyclase activity in murine macrophages.

LPS is known to modulate macrophage responses during sepsis, including cytokine release, phagocytosis, and proliferation. Although agents that elevate cAMP reverse LPS-induced macrophage functions, whether LPS itself modulates cAMP and whether LPS-induced decreases in proliferation are modulated via a cAMP-dependent pathway are not known. Murine macrophages (RAW264.7 cells) were treated with LPS in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, CaM, Gi proteins, and NF-kappaB translocation or transcription/translation. LPS effects on CaMKII phosphorylation and the expression of relevant adenylyl cyclase (AC) isoforms were measured. LPS caused a significant dose (5-10,000 ng/ml)- and time (1-8 h)-dependent increase in forskolin-stimulated AC activity that was abrogated by pretreatment with SN50 (an NF-kappaB inhibitor), actinomycin D, or cycloheximide, indicating that the effect is mediated via NF-kappaB-dependent transcription and new protein synthesis. Furthermore, LPS decreased the phosphorylation state of CaMKII, and pretreatment with a CaM antagonist attenuated the LPS-induced sensitization of AC. LPS, cAMP, or PKA activation each independently decreased macrophage proliferation. However, inhibition of NF-kappaB had no effect on LPS-induced decreased proliferation, indicating that LPS-induced decreased macrophage proliferation can proceed via PKA-independent signaling pathways. Taken together, these findings indicate that LPS induces sensitization of AC activity by augmenting the stimulatory effect of CaM and attenuating the inhibitory effect of CaMKII on isoforms of AC that are CaMK sensitive.

Purification, characterization and crystallization of human placental estrone/dehydroepiandrosterone sulfatase, a membrane-bound enzyme of the endoplasmic reticulum.

Estrone (E1)/dehydroepiandrosterone (DHEA) sulfatase (ES/DHEAS) catalyzes the hydrolysis of E1 and DHEA-sulfates releasing unconjugated steroids. ES is a component of the three-enzyme system that has been implicated in intracrine biosynthesis of estradiol, hence, proliferation of hormone dependent breast tumors. ES is bound to the membrane of the endoplasmic reticulum, presumably through multiple transmembrane and other membrane anchoring segments. The highly hydrophobic nature of the enzyme has so far prevented its purification to homogeneity in quantities sufficient for crystallization. We report here the purification, biochemical characterization and crystallization of the full-length, active form of the enzyme from the membrane bound fraction of human placenta. Our results demonstrate that the key to successful purification and growth of diffraction quality crystals of this difficult membrane bound enzyme is the exploitation of optimal solubilization and detergent conditions to protect the structural and functional integrity of the molecule, thereby preventing nonspecific aggregation and other instabilities. This work paves the way for the first structural study of a membrane bound human sulfatase and subsequent rational design of inhibitors for use as anti-tumor agents.