Transferability and reproducibility of exposed air-liquid interface co-culture lung models.

BACKGROUND: The establishment of reliable and robust in vitro models for hazard assessment, a prerequisite for moving away from animal testing, requires the evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface (ALI) are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as a monoculture and, to increase the physiologic relevance of the model, also as a co-culture with macrophages (either derived from the THP-1 monocyte cell line or from human blood monocytes). The lung model was exposed to NMs using the VITROCELL® Cloud12 system at physiologically relevant dose levels. RESULTS: Overall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production. CONCLUSION: The transferability and reproducibility of a lung co-culture model and its exposure to aerosolized particles at the ALI were evaluated and recommendations were provided for performing inter-laboratory comparison studies. Although the results are promising, optimizations of the lung model (including more sensitive read-outs) and/or selection of higher deposited doses are needed to enhance its predictive value before it may be taken further towards a possible OECD guideline.

Effects of antipsychotics on intravenous sedation with midazolam and propofol during dental treatment for patients with intellectual disabilities.

BACKGROUND: Some patients with intellectual disabilities (ID) are prescribed antipsychotic drugs for symptomatic treatment of behavioural disorders. Nevertheless, it can still prove difficult to perform dental treatments safely for some patients with ID. In such cases, treatment under intravenous sedation (IVS) is one option. Sedative, hypnotic and α-blocking effects of antipsychotic drugs may cause adverse events, such as severe hypotension, among patients who take antipsychotic drugs regularly. This study aimed to investigate the effects of oral antipsychotic medication on cardiovascular function during IVS. Accordingly, we compared mean blood pressure (MBP) and heart rate (HR) between patients who regularly take antipsychotic drugs and patients who do not. METHODS: Thirty-seven patients with ID were enrolled in this study. All participants were outpatients of Special Care Dentistry of general hospital and received dental treatment under IVS performed with a combination of midazolam and propofol. Eighteen patients regularly took antipsychotics (medication group), and 19 patients were not currently taking antipsychotics (non-medication group). MBP, HR, dose, and effect-site concentration of intravenous sedative medications were measured at three points: 'before IVS', 'at optimal sedation', and 'during dental treatment'. RESULTS: The magnitude of reduction of MBP was significantly smaller in the medication group than in the non-medication group (P < 0.023). However, there were no differences in MBP, HR, dose, and effect-site concentration of midazolam and propofol between groups at any point. CONCLUSION: These results suggest that antipsychotic medication may not have clinically significant adverse effects on cardiovascular fluctuations during dental treatment under IVS for persons with ID.

The airborne mycobiome and associations with mycotoxins and inflammatory markers in the Norwegian grain industry.

Grain dust exposure is associated with respiratory symptoms among grain industry workers. However, the fungal assemblage that contribute to airborne grain dust has been poorly studied. We characterized the airborne fungal diversity at industrial grain- and animal feed mills, and identified differences in diversity, taxonomic compositions and community structural patterns between seasons and climatic zones. The fungal communities displayed strong variation between seasons and climatic zones, with 46% and 21% of OTUs shared between different seasons and climatic zones, respectively. The highest species richness was observed in the humid continental climate of the southeastern Norway, followed by the continental subarctic climate of the eastern inland with dryer, short summers and snowy winters, and the central coastal Norway with short growth season and lower temperature. The richness did not vary between seasons. The fungal diversity correlated with some specific mycotoxins in settled dust and with fibrinogen in the blood of exposed workers, but not with the personal exposure measurements of dust, glucans or spore counts. The study contributes to a better understanding of fungal exposures in the grain and animal feed industry. The differences in diversity suggest that the potential health effects of fungal inhalation may also be different.

Circulating miRNAs as molecular markers of occupational grain dust exposure.

Dust from grain and feed production may cause adverse health effects in exposed workers. In this study we explored circulating miRNAs as potential biomarkers of occupational grain dust exposure. Twenty-two serum miRNAs were analyzed in 44 grain dust exposed workers and 22 controls. Exposed workers had significantly upregulated miR-18a-5p, miR-124-3p and miR-574-3p, and downregulated miR-19b-3p and miR-146a-5p, compared to controls. Putative target genes for the differentially expressed miRNAs were involved in a range of Kyoto Encyclopedia of Genes and Genomes signaling pathways, and 'Pathways in cancer' and 'Wnt signaling pathway' were common for all the five miRNAs. MiRNA-diseases association analysis showed a link between the five identified miRNAs and several lung diseases terms. A positive correlation between miR-124-3p, miR-18a-5p, and miR-574-3p and IL-6 protein level was shown, while miR-19b-3p was inversely correlated with CC-16 and sCD40L protein levels. Receiver-operating characteristic analysis of the five miRNA showed that three miRNAs (miR-574-3p, miR-124-3p and miR-18a-5p) could distinguish the grain dust exposed group from the control group, with miR-574-3p as the strongest predictor of grain dust exposure. In conclusion, this study identified five signature miRNAs as potential novel biomarkers of grain dust exposure that may have potential as early disease markers.

The Inhalable Mycobiome of Sawmill Workers: Exposure Characterization and Diversity.

Exposure to fungal spores has been associated with respiratory symptoms and allergic alveolitis among sawmill workers, but the complexity of sawmill workers' fungal exposure has been poorly studied. We characterized the fungal diversity in air samples from sawmill workers' breathing zones and identified differences in the richness, diversity, and taxonomic composition between companies, departments, wood types, and seasons. Full-shift personal inhalable dust samples (n = 86) collected from 11 industrial sawmill, sorting mill, and planer mill companies processing spruce and/or pine were subjected to DNA metabarcoding using the fungal internal transcribed spacer (ITS) region 2. The workers were exposed to a higher total number of operational taxonomic units (OTUs) in summer than in winter and when processing spruce than when processing pine. Workers in the saw department had the richest fungal exposure, followed by workers in the planing department and sorting of dry timber department. Sawmills explained 11% of the variation in the fungal community composition of the exposure, followed by season (5%) and department (3%). The fungal compositions of the exposures also differed between seasons, sawmills, wood types, and departments at the taxonomic level, ranging from the phylum to the species level. The differences in exposure diversity suggest that the potential health effects of fungal inhalation may also be different; hence, a risk assessment based on the fungal diversity differences should be performed. This study may serve as a basis for establishing a fungal profile of signature species that are specific for sawmills and that can be measured quantitatively in future risk assessments of sawmill workers.IMPORTANCE To gain more knowledge about exposure-response relationships, it is important to improve exposure characterization by comprehensively identifying the temporal and spatial fungal composition and diversity of inhalable dust at workplaces. The variation in the diverse fungal communities to which individuals are exposed in different seasons and sawmills suggests that variations in exposure-related health effects between seasons and companies can be expected. More importantly, the distinct fungal profiles between departments across companies indicate that workers in different job groups are differently exposed and that health risks can be department specific. DNA metabarcoding provides insight into a broad spectrum of airborne fungi that may serve as a basis for obtaining important knowledge about the fungi to which workers are exposed.

A novel mutation of the GATA site in the erythroid cell-specific regulatory element of the ABO gene in a blood donor with the Am B phenotype.

The Am and Bm phenotypes are characterized by weak expression of the A or B antigens, respectively, by red blood cells with a normal expression by the saliva of secretors. Deletion of the regulatory element in the first intron of the ABO gene and disruption of the GATA motif in the element were found to be responsible. In this study, we identified a novel mutation within the GATA motif (G>C substitution at position c.28 + 5830) in the regulatory element of the A allele that might diminish transcription activity causing the generation of the Am B phenotype.

Pim-2 kinase is an important target of treatment for tumor progression and bone loss in myeloma.

Pim-2 kinase is overexpressed in multiple myeloma (MM) cells to enhance their growth and survival, and regarded as a novel therapeutic target in MM. However, the impact of Pim-2 inhibition on bone disease in MM remains unknown. We demonstrated here that Pim-2 expression was also upregulated in bone marrow stromal cells and MC3T3-E1 preosteoblastic cells in the presence of cytokines known as the inhibitors of osteoblastogenesis in MM, including interleukin-3 (IL-3), IL-7, tumor necrosis factor-α, transforming growth factor-ß (TGF-ß) and activin A, as well as MM cell conditioned media. The enforced expression of Pim-2 abrogated in vitro osteoblastogenesis by BMP-2, which suggested Pim-2 as a negative regulator for osteoblastogenesis. Treatment with Pim-2 short-interference RNA as well as the Pim inhibitor SMI-16a successfully restored osteoblastogenesis suppressed by all the above inhibitory factors and MM cells. The SMI-16a treatment potentiated BMP-2-mediated anabolic signaling while suppressing TGF-ß signaling. Furthermore, treatment with the newly synthesized thiazolidine-2,4-dione congener, 12a-OH, as well as its prototypic SMI-16a effectively prevented bone destruction while suppressing MM tumor growth in MM animal models. Thus, Pim-2 may have a pivotal role in tumor progression and bone loss in MM, and Pim-2 inhibition may become an important therapeutic strategy to target the MM cell-bone marrow interaction.

Functional characterization of 21 CYP2C19 allelic variants for clopidogrel 2-oxidation.

Genetic variations in cytochrome P450 2C19 (CYP2C19) contribute to interindividual variability in the metabolism of therapeutic agents such as clopidogrel. Polymorphisms in CYP2C19 are associated with large interindividual variations in the therapeutic efficacy of clopidogrel. This study evaluated the in vitro oxidation of clopidogrel by 21 CYP2C19 variants harboring amino acid substitutions. These CYP2C19 variants were heterologously expressed in COS-7 cells, and the kinetic parameters of clopidogrel 2-oxidation were estimated. Among the 21 CYP2C19 variants, 12 (that is, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.14, CYP2C19.16, CYP2C19.19, CYP2C19.22, CYP2C19.24 and CYP2C19.25) showed no or markedly low activity compared with the wild-type protein CYP2C19.1B. This comprehensive in vitro assessment provided insights into the specific metabolic activities of CYP2C19 proteins encoded by variant alleles, and this may to be valuable when interpreting the results of in vivo studies.

Visualization of the trochlear nerve in the cistern with use of high-resolution turbo spin-echo multisection motion-sensitized driven equilibrium.

BACKGROUND AND PURPOSE: The trochlear nerve is so thin that it is rarely observed with MR imaging. Therefore, we used high-resolution MSDE to reliably visualize the cisternal segments of the trochlear nerve. MATERIALS AND METHODS: Participants were 10 healthy young adults (mean age, 24 years), and 20 trochlear nerves were examined. HR-MRC, BS-MRC, and HR-MSDE were performed. A neuroradiologist judged the visibility of the trochlear nerves as 1 of 4 grades ("Excellent," "Good," "Fair," and "Not") in each MR imaging sequence. The findings were then statistically analyzed with the χ(2) test. RESULTS: Of all 20 trochlear nerves, 6 with HR-MRC, 13 with BS-MRC, and 18 with HR-MSDE were judged as "Excellent." CSF flow-related artifacts and vessels in the cistern and cerebellar tentorium in HR-MRC tended to prevent the neuroradiologists from identifying the trochlear nerve. Vessels in the cistern and cerebellar tentorium in BS-MRC also tended to prevent the neuroradiologists from identifying the trochlear nerve. Compared with other sequences, HR-MSDE visualized the trochlear nerve more often. The χ(2) test revealed statistically significant differences among the 3 MR imaging sequences (P < .01). The scan time of HR-MSDE was approximately 1.5-2.2 times longer than that of the other sequences. CONCLUSIONS: HR-MSDE is able to clearly visualize the trochlear nerve and has the same or better ability to delineate the trochlear nerve compared with other MR imaging sequences, though its long scan time does not yet yield practical use.

The serine/threonine kinase Pim-2 is a novel anti-apoptotic mediator in myeloma cells.

Bone marrow stromal cells (BMSCs) and osteoclasts (OCs) confer multiple myeloma (MM) cell survival through elaborating factors. We demonstrate herein that IL-6 and TNF family cytokines, TNFα, BAFF and APRIL, but not IGF-1 cooperatively enhance the expression of the serine/threonine kinase Pim-2 in MM cells. BMSCs and OCs upregulate Pim-2 expression in MM cells largely via the IL-6/STAT3 and NF-κB pathway, respectively. Pim-2 short interfering RNA reduces MM cell viability in cocultures with BMSCs or OCs. Thus, upregulation of Pim-2 appears to be a novel anti-apoptotic mechanism for MM cell survival. Interestingly, the mammalian target of rapamycin inhibitor rapamycin further suppresses the MM cell viability in combination with the Pim-2 silencing. The Pim inhibitor (Z)-5-(4-propoxybenzylidene) thiazolidine-2, 4-dione and the PI3K inhibitor LY294002 cooperatively enhance MM cell death. The Pim inhibitor suppresses 4E-BP1 phosphorylation along with the reduction of Mcl-1 and c-Myc. Pim-2 may therefore become a new target for MM treatment.

Isolation and characterisation of prolactin-releasing peptide in chicks and its effect on prolactin release and feeding behaviour.

Prolactin (PRL)-releasing peptides (PrRP) have been identified in mammals, amphibians and fishes, and these animals have several PrRPs that consist of different numbers of amino acids such as 20, 31 and 37. In the present study, we identified the cDNA encoding chicken prepro-PrRP, which can generate putative PrRPs, and cloned and sequenced it. Sequences for the coding region suggested the occurrence of putative PrRPs of 20, 31 and 32 amino acid residues. The amino acid sequence of chicken PrRP20 showed 100%, 95% and 70% identity with those of PrRP20s from teleosts, Xenopus laevis and mammals, respectively. On the other hand, chicken PrRP31 showed approximately 90% and 52-55% homology to PrRP31s of X. laevis and mammals, respectively. Native chicken PrRPs were purified from an acid extract of chick brain by a Sep-Pak C18 cartridge (Waters Corp., Milford, MA, USA), affinity chromatography using anti-salmon PrRP serum, and reverse phase high-performance liquid chromatography (HPLC) on an ODS-120T column (TOSOH, Tokyo, Japan). The existence of chicken PrRP20 and PrRP31 in the brain was demonstrated by comparing them with the synthetic peptides using HPLC and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Chicken PrRP31 increased plasma PRL concentration when administered peripherally, whereas central administration decreased the concentration, suggesting that chicken PrRP31 has a distinct effect on PRL secretion between tissues in chicks. On the other hand, plasma growth hormone concentration decreased with both peripheral and central administrations of chicken PrRP31. Furthermore, central administration of chicken PrRP31 increased food intake in chicks compared to those observed in mammals and fishes. Taken together with the results indicating that chicken PrRP20 did not show endocrine and behavioural effects, we showed that chicken PrRP has a similar amino acid sequence to teleosts, Xenopus laevis and mammals, although the actions were variable among vertebrates.

Impact of superselective transarterial infusion therapy of high-dose cisplatin on maxillary cancer with orbital invasion.

BACKGROUND AND PURPOSE: We have been performing the superselective transarterial infusion of high-dose cisplatin for advanced maxillary cancer since 1998 and the local control rate, disease free survival rate, and organ preservation have improved markedly compared with our former therapy. This study evaluates the effectiveness of superselective transarterial infusion therapy by using high-dose cisplatin on maxillary cancer with orbital invasion. MATERIALS AND METHODS: We treated 23 patients with maxillary cancer by using superselective transarterial infusion therapy with high-dose cisplatin and concomitant radiation therapy for 10 years. Of all patients, 15 showed orbital invasion, with 11 of these tumors fed by both internal maxillary and ophthalmic arteries. In all patients, we performed superselective transarterial infusion therapy via the internal maxillary artery and/or the other feeding branches from the external carotid artery. After the operation, we determined whether a pCR had occurred by checking for the presence of viable cells. In addition, we calculated the overall survival rate, preservation rate of the eyeball, and disease-free survival rate. RESULTS: For all 23 patients, pCR and overall survival rates were 95.7% and 78.4%, respectively. To date, 2 of these patients died of lung metastasis without local recurrence. For the 15 patients with orbital invasion, the respective pCR and disease-free survival rates were 93.3% and 87.5%. Eyeballs were preserved in all patients, and local recurrence occurred in only 1 patient, at the inferior wall of the maxillary sinus (not in the orbit). CONCLUSIONS: Superselective transarterial infusion therapy with high-dose cisplatin remarkably improved the local control rate and disease-free survival rate of maxillary cancer. Even in patients with orbital invasion, a high local control rate was achieved, with preservation of the eyeball, through infusion only into branches of the external carotid artery.