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High-content screening data derived from physiologically-relevant in vitro models promise to improve confidence in data-integrative groupings for read-across in human health safety assessments. The biological data-based read-across concept is especially applicable to bioactive chemicals with defined mechanisms of toxicity; however, the challenge of data-derived groupings for chemicals that are associated with little or no bioactivity has not been explored. In this study, we apply a suite of organotypic and population-based in vitro models for comprehensive bioactivity profiling of twenty E-Series and P-Series glycol ethers, solvents with a broad variation in toxicity ranging from relatively non-toxic to reproductive and hematopoetic system toxicants. Both E-Series and P-Series glycol ethers elicited cytotoxicity only at high concentrations (mM range) in induced pluripotent stem cell-derived hepatocytes and cardiomyocytes. Population-variability assessment comprised a study of cytotoxicity in 94 human lymphoblast cell lines from 9 populations and revealed differences in inter-individual variability across glycol ethers, but did not indicate population-specific effects. Data derived from various phenotypic and transcriptomic assays revealed consistent bioactivity trends between both cardiomyocytes and hepatocytes, indicating a more universal, rather than cell-type specific mode-of-action for the tested glycol ethers in vitro. In vitro bioactivity-based similarity assessment using Toxicological Priority Index (ToxPi) showed that glycol ethers group according to their alcohol chain length, longer chains were associated with increased bioactivity. While overall in vitro bioactivity profiles did not correlate with in vivo toxicity data on glycol ethers, in vitro bioactivity of E-series glycol ethers were indicative of and correlated with in vivo irritation scores.

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Trichloroethylene (TCE) and tetrachloroethylene (PCE) are structurally similar olefins that can cause liver and kidney toxicity. Adverse effects of these chemicals are associated with metabolism to oxidative and glutathione conjugation moieties. It is thought that CYP2E1 is crucial to the oxidative metabolism of TCE and PCE, and may also play a role in formation of nephrotoxic metabolites; however, inter-species and inter-individual differences in contribution of CYP2E1 to metabolism and toxicity are not well understood. Therefore, the role of CYP2E1 in metabolism and toxic effects of TCE and PCE was investigated using male and female wild-type [129S1/SvlmJ], Cyp2e1(-/-), and humanized Cyp2e1 [hCYP2E1] mice. To fill in existing gaps in our knowledge, we conducted a toxicokinetic study of TCE (600 mg/kg, single dose, i.g.) and a subacute study of PCE (500 mg/kg/day, 5 days, i.g.) in 3 strains. Liver and kidney tissues were subject to profiling of oxidative and glutathione conjugation metabolites of TCE and PCE, as well as toxicity endpoints. The amounts of trichloroacetic acid formed in the liver was hCYP2E1≈ 129S1/SvlmJ > Cyp2e1(-/-) for both TCE and PCE; levels in males were about 2-fold higher than in females. Interestingly, 2- to 3-fold higher levels of conjugation metabolites were observed in TCE-treated Cyp2e1(-/-) mice. PCE induced lipid accumulation only in liver of 129S1/SvlmJ mice. In the kidney, PCE exposure resulted in acute proximal tubule injury in both sexes in all strains (hCYP2E1 ≈ 129S1/SvlmJ > Cyp2e1(-/-)). In conclusion, our results demonstrate that CYP2E1 is an important, but not exclusive actor in the oxidative metabolism and toxicity of TCE and PCE.

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Nonalcoholic fatty liver disease (NAFLD) is the most prevalent pathological liver condition in developed countries. NAFLD results in severe alterations in liver function, including xenobiotic metabolism. Perchloroethylene (PERC) is a ubiquitous environmental pollutant, a known hepatotoxicant in rodents, and a probable human carcinogen. It is known that PERC disposition and metabolism are affected by NAFLD in mice; here, we examined how NAFLD changes PERC-associated liver effects. Male C57Bl6/J mice were fed a low-fat diet (LFD), high-fat diet (HFD), or methionine/folate/choline-deficient diet (MCD) to model a healthy liver, or mild and severe forms of NAFLD, respectively. After 8 weeks on diets, mice were orally administered PERC (300 mg/kg/day) or vehicle (5% aqueous Alkamuls-EL620) for 5 days. PERC-induced liver effects were exacerbated in both NAFLD groups. PERC exposure was associated with up-regulation of genes involved in xenobiotic, lipid, and glutathione metabolism, and down-regulation of the complement and coagulation cascades, regardless of the diet. Interestingly, HFD-fed mice, not MCD-fed mice, were generally more sensitive to PERC-induced liver effects. This was indicated by histopathology and transcriptional responses, where induction of genes associated with cell cycle and inflammation were prominent. Liver effects positively correlated with diet-specific differences in liver concentrations of PERC. We conclude that NAFLD alters the toxicodynamics of PERC and that NAFLD is a susceptibility factor that should be considered in future risk management decisions for PERC and other chlorinated solvents.

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Endothelial cells (ECs) play a major role in blood vessel formation and function. While there is longstanding evidence for the potential of chemical exposures to adversely affect EC function and vascular development, the hazard potential of chemicals with respect to vascular effects is not routinely evaluated in safety assessments. Induced pluripotent stem cell (iPSC)-derived ECs promise to provide a physiologically relevant, organotypic culture model that is amenable for high-throughput (HT) EC toxicant screening and may represent a viable alternative to traditional in vitro models, including human umbilical vein endothelial cells (HUVECs). To evaluate the utility of iPSC-ECs for multidimensional HT toxicity profiling of chemicals, both iPSC-ECs and HUVECs were exposed to selected positive (angiogenesis inhibitors, cytotoxic agents) and negative compounds in concentration response for either 16 or 24 h in a 384-well plate format. Furthermore, chemical effects on vascularization were quantified using EC angiogenesis on biological (Geltrex™) and synthetic (SP-105 angiogenesis hydrogel) extracellular matrices. Cellular toxicity was assessed using high-content live cell imaging and the CellTiter-Glo® assay. Assay performance indicated good to excellent assay sensitivity and reproducibility for both cell types investigated. Both iPSC-derived ECs and HUVECs formed tube-like structures on Geltrex™ and hydrogel, an effect that was inhibited by angiogenesis inhibitors and cytotoxic agents in a concentration-dependent manner. The quality of HT assays in HUVECs was generally higher than that in iPSC-ECs. Altogether, this study demonstrates the capability of ECs for comprehensive assessment of the biological effects of chemicals on vasculature in a HT compatible format.

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Substances of Unknown or Variable composition, Complex reaction products, and Biological materials (UVCBs), including many refined petroleum products, present a major challenge in regulatory submissions under the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) and US High Production Volume regulatory regimes. The inherent complexity of these substances, as well as variability in composition obfuscates detailed chemical characterization of each individual substance and their grouping for human and environmental health evaluation through read-across. In this study, we applied ion mobility mass spectrometry in conjunction with cheminformatics-based data integration and visualization to derive substance-specific signatures based on the distribution and abundance of various heteroatom classes. We used petroleum substances from four petroleum substance manufacturing streams and evaluated their chemical composition similarity based on high-dimensional substance-specific quantitative parameters including m/z distribution, drift time, carbon number range, and associated double bond equivalents and hydrogen-to-carbon ratios. Data integration and visualization revealed group-specific similarities for petroleum substances. Observed differences within a product group were indicative of batch- or manufacturer-dependent variation. We demonstrate how high-resolution analytical chemistry approaches can be used effectively to support categorization of UVCBs based on their heteroatom composition and how such data can be used in regulatory decision-making.

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An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30min or 24h and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca2+ flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30min of exposure. In contrast, after 24h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes.

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Lifestyle factors and chronic pathologic states are important contributors to interindividual variability in susceptibility to xenobiotic-induced toxicity. Nonalcoholic fatty liver disease (NAFLD) is an increasingly prevalent condition that can dramatically affect chemical metabolism. We examined the effect of NAFLD on toxicokinetics of tetrachloroethylene (PERC), a ubiquitous environmental contaminant that requires metabolic activation to induce adverse health effects. Mice (C57Bl/6J, male) were fed a low-fat diet (LFD), high-fat diet (HFD), or methionine/folate/choline-deficient diet (MCD) to model a healthy liver, steatosis, or nonalcoholic steatohepatitis (NASH), respectively. After 8 weeks, mice were orally administered a single dose of PERC (300 mg/kg) or vehicle (aqueous Alkamuls-EL620) and euthanized at various time points (1-36 hours). Levels of PERC and its metabolites were measured in blood/serum, liver, and fat. Effects of diets on liver gene expression and tissue:air partition coefficients were evaluated. We found that hepatic levels of PERC were 6- and 7.6-fold higher in HFD- and MCD-fed mice compared with LFD-fed mice; this was associated with an increased PERC liver:blood partition coefficient. Liver and serum Cmax for trichloroacetate (TCA) was lower in MCD-fed mice; however, hepatic clearance of TCA was profoundly reduced by HFD or MCD feeding, leading to TCA accumulation. Hepatic mRNA/protein expression and ex vivo activity assays revealed decreased xenobiotic metabolism in HFD- and MCD-, compared with LFD-fed, groups. In conclusion, experimental NAFLD was associated with modulation of xenobiotic disposition and metabolism and increased hepatic exposure to PERC and TCA. Underlying NAFLD may be an important susceptibility factor for PERC-associated hepatotoxicity.

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Comparative assessment of potential human health impacts is a critical step in evaluating both chemical alternatives and existing products on the market. Most alternatives assessments are conducted on a chemical-by-chemical basis and it is seldom acknowledged that humans are exposed to complex products, not individual substances. Indeed, substances of Unknown or Variable composition, Complex reaction products, and Biological materials (UVCBs) are ubiquitous in commerce yet they present a major challenge for registration and health assessments. Here, we present a comprehensive experimental and computational approach to categorize UVCBs according to global similarities in their bioactivity using a suite of in vitro models. We used petroleum substances, an important group of UVCBs which are grouped for regulatory approval and read-across primarily on physico-chemical properties and the manufacturing process, and only partially based on toxicity data, as a case study. We exposed induced pluripotent stem cell-derived cardiomyocytes and hepatocytes to DMSO-soluble extracts of 21 petroleum substances from five product groups. Concentration-response data from high-content imaging in cardiomyocytes and hepatocytes, as well as targeted high-throughput transcriptomic analysis of the hepatocytes, revealed distinct groups of petroleum substances. Data integration showed that bioactivity profiling affords clustering of petroleum substances in a manner similar to the manufacturing process-based categories. Moreover, we observed a high degree of correlation between bioactivity profiles and physico-chemical properties, as well as improved groupings when chemical and biological data were combined. Altogether, we demonstrate how novel in vitro screening approaches can be effectively utilized in combination with physico-chemical characteristics to group complex substances and enable read-across. This approach allows for rapid and scientifically-informed evaluation of health impacts of both existing substances and their chemical alternatives.

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Clinical data strongly indicate that acute kidney injury (AKI) is a critical complication in alcoholic hepatitis, an acute-on-chronic form of liver failure in patients with advanced alcoholic fibrosis. Development of targeted therapies for AKI in this setting is hampered by the lack of an animal model. To enable research into molecular drivers and novel therapies for fibrosis- and alcohol-associated AKI, we aimed to combine carbon tetrachloride (CCl4)-induced fibrosis with chronic intra-gastric alcohol feeding. Male C57BL/6J mice were administered a low dose of CCl4 (0.2ml/kg 2× week/6weeks) followed by alcohol intragastrically (up to 25g/kg/day for 3weeks) and with continued CCl4. We observed that combined treatment with CCl4 and alcohol resulted in severe liver injury, more pronounced than using each treatment alone. Importantly, severe kidney injury was evident only in the combined treatment group. This mouse model reproduced distinct pathological features consistent with AKI in human alcoholic hepatitis. Transcriptomic analysis of kidneys revealed profound effects in the combined treatment group, with enrichment for damage-associated pathways, such as apoptosis, inflammation, immune-response and hypoxia. Interestingly, Havcr1 and Lcn2, biomarkers of AKI, were markedly up-regulated. Overall, this study established a novel mouse model of fibrosis- and alcohol-associated AKI and identified key mechanistic pathways.

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Cell-based high-content screening (HCS) assays have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety assessment. The time- and cost-effectiveness of HCS assays, combined with the organotypic nature of human induced pluripotent stem cell (iPSC)-derived cells, open new opportunities to employ physiologically relevant in vitro model systems to improve screening for potential chemical hazards. In this study, we used two human iPSC types, cardiomyocytes and hepatocytes, to test various high-content and molecular assay combinations for their applicability in a multiparametric screening format. Effects on cardiomyocyte beat frequency were characterized by calcium flux measurements for up to 90 min. Subsequent correlation with intracellular cAMP levels was used to determine if the effects on cardiac physiology were G-protein-coupled receptor dependent. In addition, we utilized high-content cell imaging to simultaneously determine cell viability, mitochondrial integrity, and reactive oxygen species (ROS) formation in both cell types. Kinetic analysis indicated that ROS formation is best detectable 30 min following initial treatment, whereas cytotoxic effects were most stable after 24 h. For hepatocytes, high-content imaging was also used to evaluate cytotoxicity and cytoskeletal integrity, as well as mitochondrial integrity and the potential for lipid accumulation. Lipid accumulation, a marker for hepatic steatosis, was most reliably detected 48 h following treatment with test compounds. Overall, our results demonstrate how a compendium of assays can be utilized for quantitative screening of chemical effects in iPSC cardiomyocytes and hepatocytes and enable rapid and cost-efficient multidimensional biological profiling of toxicity.

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Exposure to the ubiquitous environmental contaminant trichloroethylene (TCE) is associated with cancer and non-cancer toxicity in both humans and rodents. Peroxisome proliferator-activated receptor-alpha (PPARα) is thought to be playing a role in liver toxicity in rodents through activation of the receptor by the TCE metabolite trichloroacetic acid (TCA). However, most studies using genetically altered mice have not assessed the potential for PPARα to alter TCE toxicokinetics, which may lead to differences in TCA internal doses and hence confound inferences as to the role of PPARα in TCE toxicity. To address this gap, male and female wild type (129S1/SvImJ), Pparα-null, and humanized PPARα (hPPARα) mice were exposed intragastrically to 400 mg/kg TCE in single-dose (2, 5 and 12 h) and repeat-dose (5 days/week, 4 weeks) studies. Interestingly, following either a single- or repeat-dose exposure to TCE, levels of TCA in liver and kidney were lower in Pparα-null and hPPARα mice as compared with those in wild type mice. Levels of trichloroethanol (TCOH) were similar in all strains. TCE-exposed male mice consistently had higher levels of TCA and TCOH in all tissues compared with females. Additionally, in both single- and repeat-dose studies, a similar degree of induction of PPARα-responsive genes was observed in liver and kidney of hPPARα and wild type mice, despite the difference in hepatic and renal TCA levels. Additional sex- and strain-dependent effects were observed in the liver, including hepatocyte proliferation and oxidative stress, which were not dependent on TCA or TCOH levels. These data demonstrate that PPARα status affects the levels of the putative PPARα agonist TCA following TCE exposure. Therefore, interpretations of studies using Pparα-null and hPPARα mice need to consider the potential contribution of genotype-dependent toxicokinetics to observed differences in toxicity, rather than attributing such differences only to receptor-mediated toxicodynamic effects.

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Herein we report the identification of a highly potent and selective CB2 agonist, RQ-00202730 (40), obtained by lead optimization of the benzimidazole scaffold. Compound 40 showed strong agonistic activity with an EC50 of 19nM and excellent selectivity (>1300-fold) over the CB1 receptor. Compound 40 displayed a dose dependent analgesic effect on TNBS-induced visceral hypersensitivity in rats by oral administration (ED50 0.66mg/kg at 2.5h after oral administration). In addition, 40 did not show a significant effect on body temperature in rats after oral administration at 300mg/kg. These findings suggest that highly selective CB2 agonists will be effective agents for IBS therapy.

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Background. The control of severe bronchial asthma, such as corticosteroid-resistant asthma, is difficult. It is also possible that immunosuppressive agents would be effective for bronchial asthma. Case Summary. A 55-year-old Japanese female presented with severe bronchial asthma controlled with tacrolimus. She had been diagnosed with bronchial asthma during childhood. Her asthma worsened, and a chest radiograph showed atelectasis of the left lung. Bronchoscopy revealed the left main bronchus to be obstructed with viscous sputum consisting of 82% neutrophils and no eosinophils. The atelectasis did not improve with corticosteroid treatment, but was ameliorated by administration of tacrolimus. Discussion. This patient had severe asthma due to neutrophilic inflammation of the airways. Tacrolimus is effective for treating severe asthma, for example, in corticosteroid-resistant cases.

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OBJECTIVE: Oxidant stress is thought to be involved in the establishment of idiopathic interstitial pneumonia (IIP). Thioredoxin 1 (TRX1) plays a role as a strong antioxidant in vivo, suggesting that TRX1 may be involved in the pathogenesis of IIPs. However, there is no report on TRX1 levels in the sera of IIPs. In addition, TRX1 expression in the lungs of non-specific interstitial pneumonia (NSIP) and cryptogenic organizing pneumonia (COP) patients also has not been reported. Here, we investigated whether or not TRX1 levels are altered in the lungs and sera of patients with idiopathic pulmonary fibrosis (IPF), NSIP, and COP. METHODS: Immunohistochemical analysis was performed to examine the expression of TRX1. TRX1 levels in sera were measured using an ELISA kit. RESULTS: TRX1 was expressed in the bronchiole-alveolar epithelium, especially with regenerative or metaplastic feature, and in alveolar macrophages in usual interstitial pneumonia (UIP) and fibrotic NSIP. TRX1 was weakly expressed in the lungs of cellular NSIP and COP. TRX1 producing cells in UIP (n=16), fibrotic NSIP (n=15), cellular NSIP (n=4), and COP (n=5) were significantly increased when compared to nonsmokers (n=7). TRX1 producing cells in UIP and fibrotic NSIP were significantly increased when compared to cellular NSIP and COP. TRX1 levels in the sera of the patients with IPF (n=32; 74.2 ± 7.5 ng/mL), fibrotic NSIP (n=7; 82.5 ± 18.4 ng/mL), cellular NSIP (n=3; 62.2 ± 3.2 ng/mL) and COP (n=17; 88.8 ± 19.7 ng/mL) were significantly higher than those of control subjects (n=74; 35.3 ± 2.7 ng/mL). Furthermore, TRX1 levels in the sera of IPF patients who later showed acute exacerbation (n=7; 106.6 ± 16.3 ng/mL) were significantly higher than those of IPF patients without acute exacerbation (n=25; 65.1 ± 7.6 ng/mL). CONCLUSION: Overproduction of TRX1 in the lungs and sera may play an important role in the pathogenesis of IIPs.

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The growth-promoting effects of fish body fluids, such as serum and embryonic extract, on fish cell cultures have been widely demonstrated. The bubble-eye variety of aquarium goldfish is characterized as having a large sac filled with fluid (sac fluid) under each eye. These sacs are believed to contain lymph, which is similar in composition to serum or blood plasma. In order to test whether the sac fluid can be used as an additive for fetal bovine serum (FBS) in growth factor supplements, we compared cell growth in media containing FBS together with different concentrations of sac fluid. A dose-dependent growth-promotion effect was observed in early passage caudal fin cells from both medaka and zebrafish. Cell-growth promotion was also confirmed in early passage medaka blastula cells and in a zebrafish embryonic cell line (ZF4). Replacement of the fluid in the eye sacs of bubble-eyes occurs within a couple of months after the sac fluid has been harvested, and the cell-growth promoting activity of the new fluid is similar to that of the fluid that was tapped initially. These findings suggest that sac fluid can be used as a growth-promoting supplement for fish cell culture. Importantly, the ability of the goldfish to replace the fluid combined with the fact that equipotent fluid can be repeatedly harvested from the eye sacs means that a sustainable source of the fluid can be obtained without needing to sacrifice the fish.

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A 85-year-old man who had a past history of gastrectomy but no subsequent chemotherapy was suffering fever and fatigue. The infiltrative shadow with cavitation on his chest X-ray film did not improve despite treatment with several antibiotics after admission. Transcutaneous needle aspiration performed for diagnosis of the cavitated lung lesion yielded Streptomyces. A definitive diagnosis of streptomyces lung infection was established. We believed that this is the first case of streptomyces infection of the lung reported in Japan. The current case was also rare because he did not have any immunosuppressive status, while almost all cases previously reported.

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PURPOSE: To evaluate the treatment outcome of patients with pulmonary tuberculosis before and after the induction of DOT. METHODS: A retrospective study of the outcome of 239 tuberculosis patients treated during January 1996 to June 2003. We reviewed clinical charts collected on all patients with positive cultures for Mycobacterium tuberculosis. The patients of non-DOT group had received a traditional unsupervised drug regimen, before we have undertaken DOT. The patients of DOT group received therapy under direct observation by nurses. We compared sputum smear conversion period, sputum culture conversion period, duration of admission and treatment, recurrence rate, treatment success (cure and completion of treatment) rate and incidence of adverse effects between DOT and non-DOT group. RESULTS: Sputum conversion period and incidence of adverse effects were not significantly different between both groups. The duration of admission and treatment of DOT group was significantly shorter than those of non-DOT group. Rate of relapse and treatment success was not different between two groups. Over-80-year-old patients treated by DOT revealed a higher culture conversion rate after 2-month treatment than those who were not treated with DOT. CONCLUSION: We could not prove the usefulness of DOT during hospitalization for tuberculosis patients from the points of treatment success rate and relapse rate.

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A 74-year-old woman had general fatigue and mild fever in August 2004. Her chest X-ray showed slight ground glass opacities in the upper and middle lung fields of both lungs. Though she was prescribed antibacterial drugs, the abnormal shadows on chest X-ray did not improve. The chest CT showed ground glass opacities and reticular shadows with thickened alveolar septa (crazy-paving appearance) in both lungs, and a clearly defined mass in the anterior mediastinum. She underwent thymo-thymectomy with wedge resection of the upper lobe of the left lung. Anterior mediastinum tumor was pathologically diagnosed as thymoma. Lung biopsy demonstrated alveoli filled with SP-A positive granular materials, and we diagnosed pulmonary alveolar proteinosis. About 1 month after operation, the shadows on chest CT showed improvement. We think there might be some relationship between thymoma and pulmonary alveolar proteinosis.

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