Effect of antibiotic treatment on microbial composition and expression of antimicrobial peptides and cytokines in the chick cecum.

The aim of this study was to confirm whether the expression of innate immune molecules in the chick cecum is altered in association with changes in the composition of the intestinal microbiome that are regulated by treatment with antibiotics. Broiler chicks were administered with antibiotics (penicillin and streptomycin) daily, and the composition of the microbiota, expression of innate immune molecules, and localization of antimicrobial peptides in the chick cecum were examined at day 7 and day 14 using real-time PCR and immunohistochemistry. The oral administration of antibiotics caused an increase in the relative frequency of the Enterobacteriaceae family and a decrease in some gram-negative (Barnesiellaceae) and gram-positive bacterial (Clostridiaceae and Erysipelotrichaceae) families. The gene expression levels of immune molecules, including 4 Toll-like receptors (TLR) (TLR 2, 4, 5, and 21), inflammation-related cytokines (IL-1ß, TGFß3, TGFß4, and IL-8), and antimicrobial peptides (avian ß-defensins and cathelicidins) showed a tendency to decrease with antibiotic treatment at day 7. However, expression levels of TLR21 and some cytokines (IL-1ß, TGFß3, and IL-8) were higher in the cecum or cecal tonsils of the antibiotic-treated group than in those of the control at day 14. The immunoreactive avian ß-defensin 2 and cathelicidin 1 proteins were localized in the leukocyte-like cells in the lamina propria, and they were aggregated in the form of small islands. We conclude that the expression of innate immune molecules, including TLR, inflammation-related cytokines, and antimicrobial peptides, in the cecum are altered in association with changes in the density or composition of the luminal microbiota during the early phase of life in chicks.

Sustained effects of intralymphatic pollen-specific immunotherapy on Japanese cedar pollinosis.

BACKGROUND: Intralymphatic immunotherapy (ILIT) for allergic patients requires only a few intralymphatic injections of the allergen. However, the effectiveness and safety for Japanese cedar pollinosis are unclear. The objectives of this study were to clarify whether and how long ILIT is effective for pollinosis, and its safety. METHODS: In an open pilot investigation followed by a double-blind, placebo-controlled study, patients with Japanese cedar pollinosis received 3 intralymphatic inguinal injections of the pollen extracts before the first pollen season. The symptom medication score (SMS), nasal provocation testing and scoring visual analogue scale (VAS) were assessed after the first-third seasons. RESULTS: (1) Although mild adverse events were induced at the injected site, severe adverse events were not noted. (2) During the latter part of the first season, ILIT-treated patients (n=12) tended to show improved SMS compared to placebo-treated (n=6) without statistical significance. When assessed by nasal provocation testing and VAS scoring after the first season, the effectiveness of ILIT was significant. (3) The effects of ILIT continued until the second or third season. (4) Neither allergen-specific antibodies nor Treg/Breg cells changed in the peripheral blood. CONCLUSIONS: ILIT was safe and effective for Japanese cedar pollinosis. The clinical effects remained for 1-2 years.

Metal-ion-induced expression of gene fragments from subseafloor micro-organisms in the Kumano forearc basin, Nankai Trough.

AIMS: Using substrate-induced gene-expression (SIGEX) screening on subseafloor sediment samples from the Nankai Trough, Japan, we identified gene fragments showing an induction response to metal ions. METHODS AND RESULTS: Environmental DNA libraries in Escherichia coli host cells were tested by the addition of metal ions (Ni2+ , Co2+ , Ga3+ or Mo6+ ), followed by cell sorting of clones exhibiting green fluorescence upon co-expression of green fluorescence protein downstream of the inserted gene fragments. One clone displayed Ni2+ -specific induction, three clones displayed Ga3+ -specific induction and three clones displayed an induction response to multiple metal ions. DNA sequence analysis showed that a variety of genes showed induction responses in the screened clones. CONCLUSIONS: Using the SIGEX approach, we retrieved gene fragments with no previously identified response to metal ions that exhibited metal-ion-induced expression. This method has the potential to promote exploration of gene function through gene-induction response. SIGNIFICANCE AND IMPACT OF THE STUDY: We successfully linked gene-induction response with sequence information for gene fragments of previously unknown function. The SIGEX-based approach exhibited the potential to identify genetic function in unknown gene pools from the deep subseafloor biosphere, as well as novel genetic components for future biotechnological applications.

Innate antiviral immune response against infectious bronchitis virus and involvement of prostaglandin E2 in the uterine mucosa of laying hens.

Infectious bronchitis virus (IBV) is an enveloped RNA virus that causes deformities in eggshells. The aim of this study was to investigate the innate immune response to IBV, and to determine whether prostaglandin (PG) E2, which is synthesized during inflammation, is involved in the innate immune response in the uterine mucosa. The effects of intra-oviductal inoculation with attenuated IBV (aIBV) on the expression of viral RNA recognition receptors and innate antiviral factors were examined by real-time PCR and immunohistochemistry, and on PGE2 levels by ELISA. Then, the effects of PGE2 on the expression of innate antiviral factors in cultured uterine mucosal cells were examined. The results showed that the expression of RNA virus pattern recognition receptors (TLR3, 7, and MDA5), antimicrobial peptides (avian ß-defensins, including AvBD1, 2, 4-6 and cathelicidins, including CATH1 and 3), and interferons (IFNα, ß, γ, λ) were upregulated, and the expression of cyclooxygenase 2 (PG synthase) and the level of PGE2 were increased in the uterine mucosa following aIBV inoculation. The number of AvBD2-positive cells in the mucosa also increased in response to aIBV. In cultured mucosal cells (mainly epithelial), the expression of AvBD4, 10-13 and IFNα, ß, and λ was upregulated following incubation with 500 nM PGE2. These results suggest that the expression of viral RNA-recognition receptors, AvBDs, CATHs, and IFNs and PGE2 are induced by the IBV antigen, and that the expression of a different set of AvBDs is also induced by PGE2 in the cultured uterine mucosal cells. These antiviral factors may play a role in the protection of the uterine mucosa from IBV infection.

Relationship between Eustachian tube dysfunction and otitis media with effusion in radiotherapy patients.

OBJECTIVE: This study evaluated the relationship between radiation and Eustachian tube dysfunction, and examined the radiation dose required to induce otitis media with effusion. METHODS: The function of 36 Eustachian tubes in 18 patients with head and neck cancer were examined sonotubometrically before, during, and 1, 2 and 3 months after, intensity-modulated radiotherapy. Patients with an increase of 5 dB or less in sound pressure level (dB) during swallowing were categorised as being in the dysfunction group. Additionally, radiation dose distributions were assessed in all Eustachian tubes using three dose-volume histogram parameters. RESULTS: Twenty-two of 25 normally functioning Eustachian tubes before radiotherapy (88.0 per cent) shifted to the dysfunction group after therapy. All ears that developed otitis media with effusion belonged to the dysfunction group. The radiation dose threshold evaluation revealed that ears with otitis media with effusion received significantly higher doses to the Eustachian tubes. CONCLUSION: The results indicate a relationship between radiation dose and Eustachian tube dysfunction and otitis media with effusion.

Evaluation of efficacy and indications of surgical fixation for multiple rib fractures: a propensity-score matched analysis.

PURPOSE: The purpose of this study was to assess the effects of recent surgical rib fixation and establish its indications not only for flail chest but also for multiple rib fractures. METHODS: Between 2007 and 2015, 187 patients were diagnosed as having multiple rib fractures in our institution. After the propensity score matching was performed, ten patients who had performed surgical rib fixation and ten patients who had treated with non-operative management were included. Categorical variables were analyzed with Fischer's exact test and non-parametric numerical data were compared using the Mann-Whitney U test. Wilcoxon signed-rank test was performed for comparison of pre- and postoperative variables. All statistical data are presented as median (25-75 % interquartile range [IQR]) or number. RESULTS: The surgically treated patients extubated significantly earlier than non-operative management patients (5.5 [1-8] vs 9 [7-12] days: p = 0.019). The duration of continuous intravenous narcotic agents infusion days (4.5 [3-6] vs 12 [9-14] days: p = 0.002) and the duration of intensive care unit stay (6.5 [3-9] vs 12 [8-14] days: p = 0.008) were also significantly shorter in surgically treated patients. Under the same ventilating conditions, the postoperative values of tidal volume and respiratory rate improved significantly compared to those values measured just before the surgery. The incidence of pneumonia as a complication was significantly higher in non-operative management group (p = 0.05). CONCLUSIONS: From the viewpoints of early respiratory stabilization and intensive care unit disposition without any complications, surgical rib fixation is a sufficiently acceptable procedure not only for flail chest but also for repair of severe multiple rib fractures.

DEEP BIOSPHERE. Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor.

Microbial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~10(4) cells cm(-3). Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed.

Novel scoring system and algorithm for classifying chronic rhinosinusitis: the JESREC Study.

BACKGROUND: Chronic rhinosinusitis (CRS) can be classified into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRSwNP displays more intense eosinophilic infiltration and the presence of Th2 cytokines. Mucosal eosinophilia is associated with more severe symptoms and often requires multiple surgeries because of recurrence; however, even in eosinophilic CRS (ECRS), clinical course is variable. In this study, we wanted to set objective clinical criteria for the diagnosis of refractory CRS. METHODS: This was a retrospective study conducted by 15 institutions participating in the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC). We evaluated patients with CRS treated with endoscopic sinus surgery (ESS), and risk of recurrence was estimated using Cox proportional hazard models. Multiple logistic regression models and receiver operating characteristics curves were constructed to create the diagnostic criterion for ECRS. RESULTS: We analyzed 1716 patients treated with ESS. To diagnose ECRS, the JESREC scoring system assessed unilateral or bilateral disease, the presence of nasal polyps, blood eosinophilia, and dominant shadow of ethmoid sinuses in computed tomography (CT) scans. The cutoff value of the score was 11 points (sensitivity: 83%, specificity: 66%). Blood eosinophilia (>5%), ethmoid sinus disease detected by CT scan, bronchial asthma, aspirin, and nonsteroidal anti-inflammatory drugs intolerance were associated significantly with recurrence. CONCLUSION: We subdivided CRSwNP in non-ECRS, mild, moderate, and severe ECRS according to our algorithm. This classification was significantly correlated with prognosis. It is notable that this algorithm may give useful information to clinicians in the refractoriness of CRS before ESS or biopsy.

Continuous Vocalization during Kendo Exercises Suppresses Expiration of CO2.

One distinctive trait of kendo, the Japanese martial art of fencing, is the execution of sustained, high-effort vocalizations during actions. The purpose of this study was to determine the effect of these vocalizations on respiratory functions. First, the intensity of 3 kendo exercises was quantified by measuring oxygen uptake (V̇O2) and comparing it with V̇O2max measured during treadmill tests of 8 university kendo athletes. Respiratory variables of these 8 athletes were then analyzed using a portable breath gas analyzer during the most intensive kendo exercise, kakari-keiko, with and without vocalization. Breathing frequency (fB) increased regardless of vocalization, but in trials with vocalization, fB and ventilation were significantly lower, and expiration time was significantly longer. Components of expired gases were also affected by vocalization. Although there was no significant difference in oxygen uptake, vocalization yielded a reduction in carbon dioxide output (V̇CO2) and an increase in fraction of end-tidal carbon dioxide (FetCO2). We thus conclude that these vocalizations greatly affect expiration breathing patterns in kendo. Moreover, repetition of kakari-keiko caused a reduction in V̇CO2 and an increase in FetCO2 and CO2 storage. We consider the possibility that the sustained high-effort vocalizations of kendo also increase cerebral blood flow.

Effect of reduced coenzyme Q10 (ubiquinol) supplementation on blood pressure and muscle damage during kendo training camp: a double-blind, randomized controlled study.

AIM: This randomized controlled study was designed to examine the effects of reduced coenzyme Q10 (ubiquinol; CoQ10) supplementation on blood pressure (BP) and exercise-induced muscle damage in kendo athletes during a 4-day kendo training camp. METHODS: In a double-blinded manner, 32 young kendo athletes were randomly assigned to supplement with either placebo or CoQ10 (600 mg) daily for 11 days from 1 week prior to camp to end of camp. BP was measured every morning after waking up during the training camp. Blood samples were taken at 3 time points; 1 week and 1 day prior and upon completion of training camp at 17:30. Statistical analysis was performed by repeated-measures analysis of variance followed by Bonferroni/Dunn post-hoc tests. RESULTS: Before the training camp started, there were no differences in diastolic BP between these groups. However, after kendo training started, diastolic BP in the CoQ10 group was significantly lower than that in the placebo group (P<0.05). Plasma creatine kinase (CK) and myoglobin (Mb) concentrations were significantly increased in both groups during the camp (P<0.05), whereas there were no significant differences in CK and Mb between CoQ10 and placebo groups (CK: P=0.82, Mb: P=0.69). CONCLUSION: Oral supplementation with reduced form of CoQ10 (ubiquinol; Kaneka QHTM) showed a significant hypotensive effect in young male kendo athletes during a 4-day kendo training camp, although it did not significantly ameliorate kendo exercise-induced muscle damage.

Dose-saving isolation procedure in percutaneous ethanol sclerotherapy for venous malformations.

OBJECTIVES: To evaluate the feasibility and effectiveness of an isolation technique during ethanol injection sclerotherapy for venous malformations (VMs) in the head and neck region. METHODS: The subjects were 23 patients with 35 VM lesions in the head and neck, treated between 1999 and 2012. The mean lesion area was 3.75 ± 3.09 cm(2) (±standard deviation). We confirmed the contour of the lesions to be treated on a fully filled image on direct injection cisternography, and observed patterns of communicating drainage to systemic veins. The cisterns were evacuated by squeezing and were isolated by manual compression of the communicators. Ethanol (94.5%) with a contrast agent was then injected into both isolable and unisolable lesions, up to a total volume of 1 mL/cm(2), avoiding complications. We investigated the relationship between lesion size and injected ethanol dose, and also dose per unit area. RESULTS: Both manual evacuation by compression and isolation were performed in 20 (57.1%) isolable lesions, but not in 15 unisolable lesions. The mean injected ethanol dose was 0.65 ± 0.31 mL/cm(2) overall, 0.70 ± 0.32 in isolable and 0.59 ± 0.30 in unisolable lesions (NS). However, the injected ethanol dose was significantly lower for lesions sized >6 cm. Complete to near-complete shrinkage was observed in all isolable lesions, and in 60% of unisolable lesions (P < 0.05). Clinical outcome seemed unrelated to the injected ethanol dose or the dose per unit area. There was one case of recurrence and one complication in the unisolable lesions. No further relapses or complications were observed during the follow-up period of 38.6 ± 12.3 months. CONCLUSIONS: Clinical outcome was related to the isolability not to the injected dose. The isolation appears useful for improving the safety and effectiveness of ethanol sclerotherapy for VM.

Exercise lowers postprandial glucose but not fasting glucose in type 2 diabetes: a meta-analysis of studies using continuous glucose monitoring.

Exercise has repeatedly been shown to improve glycemic control as assessed by glycated hemoglobin. However, changes in glycated hemoglobin do not provide information regarding which aspects of glycemic control have been altered. The purpose of this systematic review was to examine the effect of exercise as assessed by continuous glucose monitoring systems (CGMS) in type 2 diabetes. Databases (PubMed, Medline, EMBASE) were searched up to February 2013. Eligible studies had participants with type 2 diabetes complete standardized exercise protocols and used CGMS to measure changes in glycemic control. Randomized controlled trials, crossover trials and studies with pre-post designs were included. Average glucose concentration, daily time spent in hyperglycemia or hypoglycemia, and fasting glucose concentration were compared between exercise and control conditions. Eleven studies met the inclusion criteria and were included in the review. Eight studies had short-term (≤2 weeks) exercise interventions, whereas three studies had a longer-term intervention (all >2 months). The types of exercises utilized included aerobic, resistance and a combination of the two. The eight short-term studies were included in quantitative analysis. Exercise significantly decreased average glucose concentrations (-0.8 mmol/L, p < 0.01) and daily time spent in hyperglycemia (-129 minutes, p < 0.01), but did not significantly affect daily time spent in hypoglycemia (-3 minutes, p = 0.47) or fasting glucose (-0.3 mmol/L, p = 0.13). The four randomized crossover trials had similar findings compared to studies with pre-post designs. Exercise consistently reduced average glucose concentrations and time spent in hyperglycemia despite not significantly affecting outcomes such as fasting glucose and hypoglycemia.

Hemodynamics of 8 different configurations of stenting for bifurcation aneurysms.

BACKGROUND AND PURPOSE: SACE is performed for complex aneurysms. There are several configurations of stent placement for bifurcation aneurysms. We investigated hemodynamics among 8 different configurations of stent placement, which may relate to the recanalization rate. MATERIALS AND METHODS: We created a silicone block model of a patient-specific asymmetric bifurcation aneurysm. Enterprise closed-cell stents were deployed in the model as various configurations. 3D images of these stents were obtained by micro-CT. We performed CFD simulations for a no-stent model and 8 stent models: a single stent from a proximal vessel to a right or left distal vessel, a horizontal stent, a kissing-Y stent with a uniformly narrowed structure, a nonoverlapping-Y stent, a virtual-Y stent with no narrowed structure (fusion of 2 single stents), and 2 different crossing-Y stents with a focally narrowed structure. Hemodynamic parameters were evaluated. RESULTS: Cycle-averaged velocity and WSS in the aneurysm were reduced because of stent placement in the following order: single stent (19% reduction in cycle-averaged velocity) < nonoverlapping-Y stent (29%) < virtual-Y stent (32%) < horizontal stent (39%) < kissing-Y stent (48%) < crossing-Y stent (54%). Kissing- and crossing-Y stents redirected impingement flow into the distal vessels because of lowered porosity of stents due to narrowed structures. CONCLUSIONS: Among 8 different configurations of stent placement, kissing- and crossing-Y stents showed the strongest reduction in flow velocity in the aneurysm because of lowered porosity of stents and redirection of impingement flow. This may be a desirable reconstruction of flow hemodynamics and may decrease recanalization rates in SACE.

Structural basis for the altered drug sensitivities of non-small cell lung cancer-associated mutants of human epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) has an essential role in multiple signaling pathways, including cell proliferation and migration, through extracellular ligand binding and subsequent activation of its intracellular tyrosine kinase (TK) domain. The non-small cell lung cancer (NSCLC)-associated EGFR mutants, L858R and G719S, are constitutively active and oncogenic. They display sensitivity to TK inhibitors, including gefitinib and erlotinib. In contrast, the secondary mutation of the gatekeeper residue, T790M, reportedly confers inhibitor resistance on the oncogenic EGFR mutants. In this study, our biochemical analyses revealed that the introduction of the T790M mutation confers gefitinib resistance on the G719S mutant. The G719S/T790M double mutant has enhanced activity and retains high gefitinib-binding affinity. The T790M mutation increases the ATP affinity of the G719S mutant, explaining the acquired drug resistance of the double mutant. Structural analyses of the G719S/T790M double mutant, as well as the wild type and the G719S and L858R mutants, revealed that the T790M mutation stabilizes the hydrophobic spine of the active EGFR-TK conformation. The Met790 side chain of the G719S/T790M double mutant, in the apo form and gefitinib- and AMPPNP-bound forms, adopts different conformations that explain the accommodation of these ligands. In the L858R mutant structure, the active-site cleft is expanded by the repositioning of Phe723 within the P-loop. Notably, the introduction of the F723A mutation greatly enhanced the gefitinib sensitivity of the wild-type EGFR in vivo, supporting our hypothesis that the expansion of the active-site cleft results in enhanced gefitinib sensitivity. Taken together, our results provide a structural basis for the altered drug sensitivities caused by distinct NSCLC-associated EGFR mutations.