Jiella pelagia sp. nov., isolated from the phosphonate-amended seawater of the northwestern Pacific Ocean.

A novel Gram-stain-negative, aerobic, rod-shaped bacterium, designated as HL-NP1T, was isolated from the surface water of the northwestern Pacific Ocean after enrichment cultivation using the organic phosphorous compound of 2-aminoethylphosphonate. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Jiella, with the highest similarity to Jiella pacifica 40Bstr34T (98.7 %). The complete genome sequence of strain HL-NP1T comprised a circular chromosome of 5.58 Mbp and two circular plasmids of 0.15 and 0.22 Mbp. Comparison of the genome sequences between strains HL-NP1T and J. pacifica 40Bstr34T revealed that average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values (88.0, 86.4 and 33.9 %, respectively) were below the recommended cut-off levels for delineating bacterial species. Strain HL-NP1T showed optimal growth at 30 °C, pH 6.5-7.0, with 2.0-2.5 % (w/v) NaCl. The sole respiratory quinone was ubiquinone-10. The predominant fatty acid was summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c). The polar lipids comprised diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, an unidentified aminolipid and four unidentified lipids. The G+C content of the genomic DNA was 65.1 %. Based on phylogenetic, genotypic, phenotypic and chemotaxonomic data, strain HL-NP1T is proposed to represent a novel species of the genus Jiella, for which the name Jiella pelagia sp. nov. is proposed. The type strain is HL-NP1T (= KCCM 90499T = JCM 35838T).

Clonal evolution of long-term expanding head and neck cancer organoid: Impact on treatment response for personalized therapeutic screening.

OBJECTIVES: In biobanking based on patient-derived organoids (PDO), the genetic stability of organoid lines is critical for the clinical relevance of PDO with parental tumors. However, data on mutational heterogeneity and clonal evolution of PDO and their effects on treatment response are insufficient. METHODS: To investigate whether head and neck cancer organoids (HNCOs) could maintain the genetic characteristics of their original tumors and elucidate the clonal evolution process during a long-term passage, we performed targeted sequencing, covering 377 cancer-related genes and adopted a sub-clonal fraction model. To explore therapeutic response variability between an early and late passage (>passage 6), we generated dose-response curves for drugs and radiation using two HNCO lines. RESULTS: Using 3D ex vivo organoid culture protocol, we successfully established 27 HNCOs from 39 patients with an overall success rate of 70% (27/39). Their mutational profiles were highly concordant, with three of the HNCOs analyzed showing greater than 70% concordance. Only one HNCO displayed less than 50% concordance. However, many of these organoid lines displayed clonal evolution during serial passaging, although major cancer driver genes and VAF distributions were shared between early and later passages. We also found that all late passages of HNCOs tended to be more sensitive to radiation than early passages, similar to drug response results. CONCLUSIONS: We report the establishment of HNCO lines derived from 27 patients and demonstrate their genetic concordance with corresponding parental tumors. Furthermore, we show serial changes in mutational profiles of HNCO along with long passage culture and the impact of these clonal evolutions on response to radiotherapy.

Roseovarius pelagicus sp. nov., a facultatively anaerobic bacterium with potential for degrading polypropylene, isolated from Arctic seawater.

A Gram-stain-negative, facultatively anaerobic, non-motile, rod-shaped bacterial strain, designated HL-MP18T, was isolated from Arctic seawater after a prolonged incubation employing polypropylene as the sole carbon source. Phylogenetic analyses of the 16S rRNA gene sequence revealed that strain HL-MP18T was affiliated to the genus Roseovarius with close relatives Roseovarius carneus LXJ103T (96.8 %) and Roseovarius litorisediminis KCTC 32327T (96.5 %). The complete genome sequence of strain HL-MP18T comprised a circular chromosome of 3.86 Mbp and two circular plasmids of 0.17 and 0.24 Mbp. Genomic comparisons based on average nucleotide identity and digital DNA-DNA hybridization showed that strain HL-MP18T was consistently discriminated from its closely related taxa in the genus Roseovarius. Strain HL-MP18T showed optimal growth at 25 °C, pH 7.0 and 2.5 % (w/v) sea salts. The major cellular fatty acids were C18 : 1 ω6c and/or C18 : 1 ω7c (49.6 %), C19 : 0 cyclo ω8c (13.5 %), and C16 : 0 (12.8 %). The major respiratory quinone was ubiquinone-10. The polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and three unidentified lipids. The genomic DNA G+C content of the strain was 59.2 mol%. The phylogenetic, genomic, phenotypic and chemotaxonomic results indicate that strain HL-MP18T is distinguishable from the recognized species of the genus Roseovarius. Therefore, we propose that strain HL-MP18T represents a novel species belonging to the genus Roseovarius, for which the name Roseovarius pelagicus sp. nov. is proposed. The type strain is HL-MP18T (=KCCM 90405T=JCM 35639T).

Maribacter litopenaei sp. nov., isolated from the intestinal tract of the Pacific white shrimp Litopenaeus vannamei.

A Gram-stain-negative, aerobic, rod-shaped bacterial strain, designated HL-LV01T, was isolated from the intestinal tract content of the Pacific white shrimp Litopenaeus vannamei. The 16S rRNA gene sequence of strain HL-LV01T showed that the strain was clearly a member of the genus Maribacter. According to the phylogenetic analyses, strain HL-LV01T was most closely related to the species Maribacter flavus KCTC 42508T with 98.2 % sequence similarity. The average nucleotide identity and digital DNA-DNA hybridization values between strain HL-LV01T and M. flavus KCTC 42508T were 80.6 % and 23.0 %, respectively, indicating different genomic species in the genus Maribacter. Strain HL-LV01T showed optimal growth at 35 °C, pH 7.0, and 2.5 % (w/v) sea salts. The major cellular fatty acids were iso-C15 : 0 (32.5 %), iso-C17 : 0 3-OH (22.3 %), and iso-C15 : 1 G (15.5 %). The major respiratory quinone was menaquinone-6. The polar lipids consisted of phosphatidylethanolamine, three unidentified aminolipids, and seven unidentified lipids. The genomic DNA G+C content of the strain was 39.8 mol%. The comprehensive phylogenetic, genomic, phenotypic, and chemotaxonomic results indicate that strain HL-LV01T is distinct from validly published species of the genus Maribacter. Hence, we propose strain HL-LV01T as a novel species belonging to the genus Maribacter, for which the name Maribacter litopenaei sp. nov. is proposed. The type strain is HL-LV01T (= KCCM 90498T = JCM 35709T).

A novel 3D pillar/well array platform using patient-derived head and neck tumor to predict the individual radioresponse.

Predicting individual radiotherapy (RT) response is valuable in managing head and neck squamous cell carcinoma (HNSCC). We assessed the feasibility of our novel 3D culture platform to measure radioresponse using patient-derived cells (PDCs) from HNSCC patients. Cells from the FaDu line and tumor samples from 39 HNSCC patients were cultivated serially in MatrigelTM on a 3D pillar/well array culture system. The 3D tumor models were exposed to 0 to 8 Gy of radiation dose, and the radioresponse index (RTauc, area under the dose-response curve) was measured quantitatively with Calcein AM staining of live tumor cells. Calcein AM fluorescence showed reduced density and the number of FaDu colonies as radiation increased, implying a dose-dependent effect on cell viability in the 3D pillar/well culture system. 3D tumor models using PDCs were established successfully from 39 HNSCC patient tumor samples, maintaining original genomic and pathological characteristics. These 3D tumor models were exposed to ionizing radiation on a 3D pillar/well array, with a mean period of 12 days from tumor harvest to the measurement of RTauc. The RTauc of all PDCs varied from 3.5 to 9.4, and the lower 40th percentile (Z-score = -0.26) was considered a good radioresponse group with a threshold RTauc of 4.6. The good radioresponse group showed fewer adverse features than others. As of the last follow-up, recurrence-free survival was better in the good radioresponse group (p = 0.037). 3D pillar/well array platforms using PDC could rapidly quantify radioresponse index in patients with HNSCC, showing potential as a novel prognosticator.

Dental students' perceptions on a simulated practice using patient-based customised typodonts during the transition from preclinical to clinical education.

PURPOSE/OBJECTIVES: Dental students experience difficulties during the transition from preclinical to clinical curriculum. In order to help the students to adapt to the clinical education programme, a simulated practice using patient-based customised models was introduced in this study to prepare for their first clinical practice. METHODS: This study included 45 third-year predoctoral students (D3 students) who were about to perform the preparation of a single crown abutment on their first patient. After practicing abutment preparation using simulated models and providing the actual treatment to their own patient, the students were surveyed to investigate their perceptions on the simulated practice using the 3D-printed customised typodont model. The statistical analysis of the quantitative data and the thematic analysis of the qualitative data were conducted. RESULTS: Regarding this simulation, more than 80% of the students gave positive feedback on their practice of (a) operative positions and postures, (b) finger rest, (c) occlusal reduction, (d) axial reduction and (e) proximal reduction. Student responses on the open-ended questions about how they perceived the usefulness of this simulation were categorised as "First clinical case," "Patient-based model" and "Realistic simulation environment." In addition, a number of improvements of the simulation were also suggested by the students including the typodont and the manikin. CONCLUSIONS: This study gives insights into the significance of simulated practice using patient-based customised typodonts as a transitional education tool and its direction of development in the field of restorative treatments accompanied by irreversible tooth preparations.

A rapid quantification of invasive phenotype in head and neck squamous cell carcinoma: A novel 3D pillar array system.

BACKGROUND: The widely used in vitro invasion assays for head and neck squamous cell carcinoma (HNSCC) are wound healing, transwell, and organotypic assays. However, these are still lab-intensive and time-consuming tasks. For the rapid detection and high throughput screening of invasiveness in 3D condition, we propose a novel spheroid invasion assay using commercially available pillar platform system. MATERIALS AND METHODS: Using the pillar-based spheroid invasion assay, migration and invasion was evaluated in three patient-derived cells (PDCs) of HNSCC. Immunofluorescence of live cells was used for the quantitative measurement of migratory and invaded cells attached to the pillar. Expression of epithelial-mesenchymal transition (EMT)-related gene (snai1/2) was measured by qRT-PCR. We also tested the impact of drug treatments (cisplatin, docetaxel) on the changes in the invasive phenotype. RESULTS: All PDCs successfully formed spheroid at 4 days and can be measured invasiveness within 7 days. Intriguingly, one PDC (#1) obtained from the advanced stage showed robust migration, invasion and higher transcription of snai1/2, compared with the other two PDCs. Furthermore, the invasion ratio of the control spheroids was about 70% while the invasion ratios of drug-treated spheroids were lower than 50%, and the difference showed statistical significance (p < 0.01). CONCLUSION: The presented spheroid invasion assay using pillar array could be useful for the evaluation of cancer cell behavior and physiology in response to diverse therapeutic drugs.

Caveolin-1, Ring finger protein 213, and endothelial function in Moyamoya disease.

BACKGROUND: Moyamoya disease is a unique cerebrovascular occlusive disease of unknown etiology. Ring finger protein 213 (RNF213) was identified as a susceptibility gene for Moyamoya disease in East Asian countries. However, the pathogenesis of Moyamoya disease remains unclear. METHODS: We prospectively analyzed clinical data for 139 patients with Moyamoya disease (108 bilateral Moyamoya disease, 31 unilateral Moyamoya disease), 61 patients with intracranial atherosclerotic stroke, and 68 healthy subjects. We compared the genetic (RNF213 variant) and protein biomarkers for caveolae (caveolin-1), angiogenesis (vascular endothelial growth factor (VEGF) and receptor (VEGFR2), and antagonizing cytokine (endostatin)) and endothelial dysfunction (asymmetric dimethylarginine (ADMA), and nitric oxide and its metabolites (nitrite and nitrate)) between patients with Moyamoya disease and intracranial atherosclerotic stroke. We then performed path analysis to evaluate whether a certain protein biomarker mediates the association between genes and Moyamoya disease. RESULTS: Caveolin-1 level was decreased in patients with Moyamoya disease and markedly decreased in RNF213 variant carriers. Circulating factors such as VEGF and VEGFR2 did not differ among the groups. Markers for endothelial dysfunction were significantly higher in patients with intracranial atherosclerotic stroke but normal in those with Moyamoya disease. Path analysis showed that the presence of the RNF213 variant was associated with caveolin-1 levels that could lead to Moyamoya disease. The level of combined marker of Moyamoya disease (caveolin-1) and intracranial atherosclerotic stroke (ADMA, an endothelial dysfunction marker) predicted Moyamoya disease with good sensitivity and specificity. CONCLUSION: Our results suggest that Moyamoya disease is a caveolae disorder but is not related to endothelial dysfunction or dysregulation of circulating cytokines.

Stroke Serum Priming Modulates Characteristics of Mesenchymal Stromal Cells by Controlling the Expression miRNA-20a.

Transplantation of mesenchymal stem cells (MSCs) expanded with fetal bovine serum (FBS) has some limitations, including the requirement of a long culture period to obtain a sufficient amount of stem cells. Priming of MSCs with serum from patients with ischemic stroke (stroke serum) increased the proliferation rate and the neurorestorative capacity of MSCs. We hypothesized that this novel priming method increases the proliferation rate of MSCs via the regulation of microRNAs (miRs). Thus, we investigated miR profiling in stroke serum-primed MSCs and tested whether the regulation of certain miRs may affect the proliferation rate of rat MSCs. The proliferation rate of MSCs cultured with stroke serum was higher than that of MSCs cultured with normal serum or FBS. Using miR microarray analysis, we compared the miR expression profiles between MSCs cultured in FBS and in stroke serum. Among miRs associated with cell proliferation, miR-20a was most significantly increased. Similarly, miR-20a was increased in MSCs obtained from the bone marrow of stroke rats compared with MSCs from normal rats. Furthermore, the deregulation of miR-20a by the transfection of MSCs with pre-miR-20a or anti-miR-20a was significantly correlated with the increased proliferation rate of MSCs. The overexpression of miR-20a in MSCs cultured in FBS improved the proliferation rate, while the knockdown of endogenous miR-20a decreased the proliferation rate. In addition, miR-20a promoted proliferation by suppressing the expression of p21 cyclin-dependent kinase inhibitor 1 (CDKN1A). A dual-luciferase reporter assay showed that CDKN1A is a target of miR-20a. Our findings indicate that stroke serum priming upregulated the expression of miR-20a, which promoted MSC proliferation by regulating the cell cycle inhibitor p21 CDKN1A, and suggest the possible roles of priming methods in modulating the characteristics of MSCs by controlling the expression of miR in MSCs.

Optimal Route for Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Transplantation to Protect Against Neonatal Hyperoxic Lung Injury: Gene Expression Profiles and Histopathology.

The aim of this study was to determine the optimal route of mesenchymal stem cell (MSC) transplantation. To this end, gene expression profiling was performed to compare the effects of intratracheal (i.t.) versus intravenous (i.v.) MSC administration. Furthermore, the therapeutic efficacy of each route to protect against neonatal hyperoxic lung injury was also determined. Newborn Sprague-Dawley rats were exposed to hyperoxia (90% oxygen) from birth for 14 days. Human umbilical cord blood-derived MSCs labeling with PKH26 were transplanted through either the i.t. (5×10(5)) or i.v. (2×10(6)) route at postnatal day (P) 5. At P14, lungs were harvested for histological, biochemical and microarray analyses. Hyperoxic conditions induced an increase in the mean linear intercept and mean alveolar volume (MAV), indicative of impaired alveolarization. The number of ED-1 positive cells was significantly decreased by both i.t. and i.v. transplantations. However, i.t. administration of MSCs resulted in a greater decrease in MAV and ED-1 positive cells compared to i.v. administration. Moreover, the number of TUNEL-positive cells was significantly decreased in the i.t. group, but not in the i.v. group. Although the i.t. group received only one fourth of the number of MSCs that the i.v. group did, a significantly higher number of donor cell-derived red PKH 26 positivity were recovered in the i.t. group. Hyperoxic conditions induced the up regulation of genes associated with the inflammatory response, such as macrophage inflammatory protein-1 α, tumor necrosis factor-α and inter leukin-6; genes associated with cell death, such as p53 and caspases; and genes associated with fibrosis, such as connective tissue growth factor. In contrast, hyperoxic conditions induced the dwon-regulation of vascular endothelial growth factor and hepatocyte growth factor. These hyperoxia-induced changes in gene expression were decreased in the i.t. group, but not in the i.v. group. Thus, local i.t. MSC transplantation was more effective than systemic i.v. MSC administration in protecting against neonatal hyperoxic lung injury.

Long-term (postnatal day 70) outcome and safety of intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells in neonatal hyperoxic lung injury.

PURPOSE: This study was performed to evaluate the long-term effects and safety of intratracheal (IT) transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in neonatal hyperoxic lung injury at postnatal day (P)70 in a rat model. MATERIALS AND METHODS: Newborn Sprague Dawley rat pups were subjected to 14 days of hyperoxia (90% oxygen) within 10 hours after birth and allowed to recover at room air until sacrificed at P70. In the transplantation groups, hUCB-MSCs (5×105) were administered intratracheally at P5. At P70, various organs including the heart, lung, liver, and spleen were histologically examined, and the harvested lungs were assessed for morphometric analyses of alveolarization. ED-1, von Willebrand factor, and human-specific nuclear mitotic apparatus protein (NuMA) staining in the lungs and the hematologic profile of blood were evaluated. RESULTS: Impaired alveolar and vascular growth, which evidenced by an increased mean linear intercept and decreased amount of von Willebrand factor, respectively, and the hyperoxia-induced inflammatory responses, as evidenced by inflammatory foci and ED-1 positive alveolar macrophages, were attenuated in the P70 rat lungs by IT transplantation of hUCB-MSCs. Although rare, donor cells with human specific NuMA staining were persistently present in the P70 rat lungs. There were no gross or microscopic abnormal findings in the heart, liver, or spleen, related to the MSCs transplantation. CONCLUSION: The protective and beneficial effects of IT transplantation of hUCB-MSCs in neonatal hyperoxic lung injuries were sustained for a prolonged recovery period without any long-term adverse effects up to P70.

Neuroprotective effects of L-carnitine against oxygen-glucose deprivation in rat primary cortical neurons.

PURPOSE: Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD). METHODS: Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO) and propidium iodide (PI) were counted, and lactate dehydrogenase (LDH) activity and reactive oxygen species (ROS) levels were measured. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 µM, 10 µM, and 100 µM) on OGD-induced neurotoxicity. RESULTS: Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 µM and 100 µM of L-carnitine compared with the untreated OGD group (P<0.05). The application of L-carnitine at 100 µM significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P<0.05). CONCLUSION: L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.

Intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells attenuates Escherichia coli-induced acute lung injury in mice.

BACKGROUND: Human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuate hyperoxic neonatal lung injury primarily through anti-inflammatory effects. We hypothesized that intratracheal transplantation of human UCB-derived MSCs could attenuate Escherichia coli (E. coli)-induced acute lung injury (ALI) in mice by suppressing the inflammatory response. METHODS: Eight-week-old male ICR mice were randomized to control or ALI groups. ALI was induced by intratracheal E. coli instillation. Three-hours after E. coli instillation, MSCs, fibroblasts or phosphate-buffered saline were intratracheally administered randomly and survival was analyzed for 7 days post-injury. Lung histology including injury scores, myeloperoxidase (MPO) activity, and protein levels of interleukin (IL)-1α, IL-1ß, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-2 as well as the wet-dry lung ratio and bacterial counts from blood and bronchoalveolar lavage (BAL) were evaluated at 1, 3, and 7 days post-injury. Levels of inflammatory cytokines in the lung were also profiled using protein macroarrays at day 3 post-injury which showed peak inflammation. RESULTS: MSC transplantation increased survival and attenuated lung injuries in ALI mice, as evidenced by decreased injury scores on day 3 post-injury and reduced lung inflammation including increased MPO activity and protein levels of IL-1α, IL-1ß, IL-6, TNF-α, and MIP-2 on day 3 and 7 post-injury. Inflammatory cytokine profiles in the lungs at day 3 post-injury were attenuated by MSC transplantation. MSCs also reduced the elevated lung water content at day 3 post-injury and bacterial counts in blood and BAL on day 7 post-injury. CONCLUSIONS: Intratracheal transplantation of UCB-derived MSCs attenuates E. coli-induced ALI primarily by down-modulating the inflammatory process and enhancing bacterial clearance.

Intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells dose-dependently attenuates hyperoxia-induced lung injury in neonatal rats.

Intratracheal transplantation of human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuates the hyperoxia-induced neonatal lung injury. The aim of this preclinical translation study was to optimize the dose of human UCB-derived MSCs in attenuating hyperoxia-induced lung injury in newborn rats. Newborn Sprague-Dawley rats were randomly exposed to hyperoxia (95% oxygen) or normoxia after birth for 14 days. Three different doses of human UCB-derived MSCs, 5 × 10(3) (HT1), 5 × 10(4) (HT2), and 5 × 10(5) (HT3), were delivered intratracheally at postnatal day (P) 5. At P14, lungs were harvested for analyses including morphometry for alveolarization, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, myeoloperoxidase activity, mRNA level of tumor necross factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, and transforming growth factor-ß (TGF-ß), human glyceradehyde-3-phosphate dehydrogenase (GAPDH), and p47(phox), and collagen levels. Increases in TUNEL-positive cells were attenuated in all transplantation groups. However, hyperoxia-induced lung injuries, such as reduced alveolarization, as evidenced by increased mean linear intercept and mean alveolar volume, and increased collagen levels were significantly attenuated in both HT2 and HT3, but not in HT1, with better attenuation in HT3 than in HT2. Dose-dependent human GAPDH expression, indicative of the presence of human RNA in lung tissue, was observed only in the transplantation groups, with higher expression in HT3 than in HT2, and higher expression in HT2 than in HT1. Hyperoxia-induced inflammatory responses such as increased myeloperoxidase acitivity, mRNA levels of TNF-α, IL-1ß, IL-6, and TGF-ß of the lung tissue, and upregulation of both cytosolic and membrane p47(phox), indicative of oxidative stress, were significantly attenuated in both HT2 and HT3 but not in HT1. These results demonstrate that intratracheal transplantation of human UCB-derived MSCs with appropriate doses may attenuate hyperoxia-induced lung injury through active involvement of these cells in modulating host inflammatory responses and oxidative stress in neonatal rats.

Human umbilical cord blood-derived mesenchymal stem cells attenuate hyperoxia-induced lung injury in neonatal rats.

Recent evidence suggests mesenchymal stem cells (MSCs) can downmodulate bleomycin-induced lung injury, and umbilical cord blood (UCB) is a promising source for human MSCs. This study examined whether intratracheal or intraperitoneal transplantation of human UCB-derived MSCs can attenuate hyperoxia-induced lung injury in immunocompetent newborn rats. Wild-type Sprague-Dawley rats were randomly exposed to 95% oxygen or air from birth. In the transplantation groups, a single dose of PKH26-labeled human UCB-derived MSCs was administered either intratracheally (2 x 10(6) cells) or intraperitoneally (5 x 10(5) cells) at postnatal day (P) 5. At P14, the harvested lungs were examined for morphometric analyses of alveolarization and TUNEL staining, as well as the myeoloperoxidase activity, the level of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and transforming growth factor (TGF)-beta mRNA, alpha-smooth muscle actin (SMA) protein, and collagen levels. Differentiation of MSCs to the respiratory epithelium was also evaluated both in vitro before transplantation and in vivo after transplantation. Despite one fourth dosage of MSCs, significantly more PKH26-labeled donor cells were recovered with intratracheal administration than with intraperitoneal administration both during normoxia and hyperoxia. The hyperoxia-induced increase in the number of TUNEL-positive cells, myeloperoixdase activity, and the level of IL-6 mRNA were significantly attenuated with both intratracheal and intraperitoneal MSCs transplantation. However, the hyperoxia-induced impaired alveolarization and increased the level of TNF-alpha and TGF-beta mRNA, alpha-SMA protein, and collagen were significantly attenuated only with intratracheal MSCs transplantation. MSCs differentiated into respiratory epithelium in vitro and a few PKH26-positive donor cells were colocalized with pro surfactant protein C in the damaged lungs. In conclusion, intratracheal transplantation of human UCB-derived MSCs is more effective than intraperitoneal transplantation in attenuating the hyperoxia-induced lung injury in neonatal rats.

Alpha-phenyl-N-tert-butylnitrone attenuates hyperoxia-induced lung injury by down-modulating inflammation in neonatal rats.

This study was done to determine whether alpha -phenyl-N-tert-butylnitrone (PBN), a spin-trapping agent possessing significant anti-inflammatory capabilities, could attenuate hyperoxia-induced lung injury, and if so, whether this protective effect is mediated by the down-modulation of inflammation in neonatal rats. Newborn Sprague-Dawley rat pups were subjected to 14 days of hyperoxia (> 90% oxygen) within 10 hours after birth. PBN treatment, given 100 mg/kg intraperitoneally daily throughout the experiment, significantly attenuated hyperoxia-induced lung pathology, such as decreased radial alveolar count, increased mean linear intercept, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling-positive cells. Hyperoxia-induced activation of nicotinamide adenine dinucleotide phosphate oxidase that is responsible for superoxide anion production, as evidenced by up-regulation and membrane translocation of p67phox, and the inflammatory responses, such as increased mRNA expression of tumor necrosis factor-alpha, interleukin-6, and transforming growth factor-beta, were also significantly attenuated with PBN treatment. In summary, a spin-trapping agent PBN significantly attenuated hyperoxia-induced lung injury by down-regulating the inflammatory responses in neonatal rats.

Activation of nicotinic acetylcholine receptor prevents the production of reactive oxygen species in fibrillar beta amyloid peptide (1-42)-stimulated microglia.

Recent studies have reported that the cholinergic anti-inflammatory pathway regulates peripheral inflammatory responses via alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) and that acetylcholine and nicotine regulate the expression of proinflammatory mediators such as TNF-alpha and prostaglandin E2 in microglial cultures. In a previous study we showed that ATP released by beta-amyloid-stimulated microglia induced reactive oxygen species (ROS) production, in a process involving the P2X(7) receptor (P2X(7)R), in an autocrine fashion. These observations led us to investigate whether stimulation by nicotine could regulate fibrillar beta amyloid peptide (1-42) (fAbeta1-42)-induced ROS production by modulating ATP efflux-mediated Ca(2+) influx through P2X(7)R. Nicotine inhibited ROS generation in fAbeta(1-42)-stimulated microglial cells, and this inhibition was blocked by mecamylamine, a non-selective nAChR antagonist, and a-bungarotoxin, a selective alpha7 nAChR antagonist. Nicotine inhibited NADPH oxidase activation and completely blocked Ca(2+) influx in fAbeta(1-42)-stimulated microglia. Moreover, ATP release from fAbeta(1-42)-stimulated microglia was significantly suppressed by nicotine treatment. In contrast, nicotine did not inhibit 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP)-induced Ca(2+) influx, but inhibited ROS generation in BzATP-stimulated microglia, indicating an inhibitory effect of nicotine on a signaling process downstream of P2X(7)R. Taken together, these results suggest that the inhibitory effect of nicotine on ROS production in fAbeta1-42-stimulated microglia is mediated by indirect blockage of ATP release and by directly altering the signaling process downstream from P2X(7)R.

Cross talk between P2 purinergic receptors modulates extracellular ATP-mediated interleukin-10 production in rat microglial cells.

Previously we demonstrated that ATP released from LPS-activated microglia induced IL-10 expression in a process involving P2 receptors, in an autocrine fashion. Therefore, in the present study we sought to determine which subtype of P2 receptor was responsible for the modulation of IL-10 expression in ATP-stimulated microglia. We found that the patterns of IL-10 production were dose-dependent (1, 10, 100, 1,000 microM) and bell-shaped. The concentrations of ATP, ATP-gammaS, ADP, and ADP-betaS that showed maximal IL-10 release were 100, 10, 100, and 100 microM respectively. The rank order of agonist potency for IL-10 production was 2'-3'-O-(4-benzoyl)-benzoyl ATP (BzATP)=dATP>2-methylthio-ADP (2-meSADP). On the other hand, 2-methylthio-ATP (2-meSATP), alpha,beta-methylene ATP (alpha,beta-meATP), UTP, and UDP did not induce the release of IL-10 from microglia. Further, we obtained evidence of crosstalk between P2 receptors, in a situation where intracellular Ca(2+) release and/or cAMP-activated PKA were the main contributors to extracellular ATP-(or ADP)-mediated IL-10 expression, and IL-10 production was down-regulated by either MRS2179 (a P2Y(1) antagonist) or 5'-AMPS (a P2Y(11) antagonist), indicating that both the P2Y(1) and P2Y(11) receptors are major receptors involved in IL-10 expression. In addition, we found that inhibition of IL-10 production by high concentrations of ATP-gammaS (100 microM) was restored by TNP-ATP (an antagonist of the P2X(1), P2X(3), and P2X(4) receptors), and that IL-10 production by 2-meSADP was restored by 2meSAMP (a P2Y(12) receptor antagonist) or pertussis toxin (PTX; a Gi protein inhibitor), indicating that the P2X(1), P2X(3), P2X(4)receptor group, or the P2Y(12) receptor, negatively modulate the P2Y(11) receptor or the P2Y(1) receptor, respectively.

Free radical production in CA1 neurons induces MIP-1alpha expression, microglia recruitment, and delayed neuronal death after transient forebrain ischemia.

Several studies report microglial accumulation and activation in the CA1 area in response to transient forebrain ischemia (TFI). Here we examine the possibility that free radicals and chemokines mediate the transient activation of microglia. Free radicals are produced primarily in CA1 pyramidal neurons within 2 h of TFI. Administration of trolox, a vitamin E analog, led to the inhibition of free radical production and recruitment of microglia in the CA1 area. In addition, intrahippocampal injection of Fe2+ triggered free radical production in CA1 neurons, followed by the recruitment and activation of microglial cells into this area. TFI-induced expression of macrophage inflammatory protein-1alpha (MIP-1alpha) was increased in CA1 neurons before microglial recruitment, and blocked by trolox. Moreover, the MIP-1alpha level was upregulated in cultured hippocampal neurons exposed to Fe2+, suggesting an essential role of free radicals in TFI-induced expression of MIP-1alpha. Intracerebroventricular injection of vMIP-2 (viral macrophage inflammatory protein-2), a broad-spectrum peptide antagonist of chemokine receptors, attenuated microglial recruitment and delayed CA1 neuronal degeneration after TFI. Our data suggest that free radicals produced in CA1 neurons contribute to the recruitment and activation of microglia and neurodegeneration through MIP-1alpha expression.

ATP released from beta-amyloid-stimulated microglia induces reactive oxygen species production in an autocrine fashion.

Present study demonstrated that fibrillar beta-amyloid peptide (fAbeta1-42) induced ATP release, which in turn activated NADPH oxidase via the P2X7 receptor (P2X7R). Reactive oxygen species (ROS) production in fAbeta1-42- treated microglia appeared to require Ca2+ influx from extracellular sources, because ROS generation was abolished to control levels in the absence of extracellular Ca2+. Considering previous observation of superoxide generation by Ca2+ influx through P2X7R in microglia, we hypothesized that ROS production in fAbeta-stimulated microglia might be mediated by ATP released from the microglia. We therefore examined whether fAbeta1-42-induced Ca2+ influx was mediated through P2X7R activation. In serial experiments, we found that microglial pretreatment with the P2X7R antagonists Pyridoxal-phosphate-6-azophenyl-2',4'- disulfonate (100 microM) or oxidized ATP (100 microM) inhibited fAbeta-induced Ca2+ influx and reduced ROS generation to basal levels. Furthermore, ATP efflux from fAbeta1-42- stimulated microglia was observed, and apyrase treatment decreased the generation of ROS. These findings provide conclusive evidence that fAbeta-stimulated ROS generation in microglial cells is regulated by ATP released from the microglia in an autocrine manner.