Carbon based fibers are considered to exhibit a carcinogenic potency when inhaled into the deep lung. Mesotheliomas develop after intraperitoneal application of multi-walled carbon nanotubes (MWCNTs) exceeding a diameter of about 37 nm, whereas carcinogenic potency decreases for diameters below this threshold. While large MWCNT diameters are associated with a rigid fiber geometry, this study examined the effects of MWCNTs with smaller diameters ranging from 10 to 30 nm. Also, a sample of single-walled carbon nanotubes (SWCNTs) exhibiting single fiber diameters significantly below 10 nm and showing a flexible geometry was included since individual SWCNT fibers can aggregate to form bundles that exhibit increased rigidity. Additionally, the carcinogenic effect of pitch-based carbon fiber fragments was investigated. Carbon fibers are industrially produced with diameters larger than 4 µm and are thus not per se respirable. However, pitch-based fibers tend to break along their longitudinal axis, resulting in respirable fragments, partially of critical WHO dimensions. Four CNT samples with a geometric mean diameter (GMD) of 30 nm, 20 nm, 10 nm, and smaller than 10 nm, as well as one fragmented carbon fiber sample (GMD 1.3 µm) were intraperitoneally injected into rats in two dosages (0.1 × 109 and 1 × 109 WHO fibers or WHO-analog nanofibers) and observed for up to 24 months. A long amosite asbestos (GMD 0.37 µm) with known fiber-specific carcinogenic effect served as a positive control (0.1 × 109 WHO fibers). A small number of mesotheliomas occurred in all fiber types, but not at all dosages. For the carbon fiber material, a possible weak carcinogenic potency is seen at the higher dosage. For the SWCNT fiber, low number of mesotheliomas likewise suggest a weak carcinogenic potency. In the case of the MWCNT fiber with a GMD of 30 nm, very low number of mesotheliomas indicate a possible very weak carcinogenic potency. No clear carcinogenic potency was observed for the MWCNTs with GMDs of 20 nm and 10 nm. Carbon fiber fragments and thin but bundled MWCNTs showed weak carcinogenic potency. Non-bundled MWCNTs with a diameter below 30 nm did not show clearcarcinogenic potency at a dose up to 1 × 109 WHO-analog nanofibers.

Background Malignant mesothelioma is an aggressive cancer that often originates in the pleural and peritoneal mesothelium. Exposure to asbestos is a frequent cause. However, studies in rodents have shown that certain multiwalled carbon nanotubes (MWCNTs) can also induce malignant mesothelioma. The exact mechanisms are still unclear. To gain further insights into molecular pathways leading to carcinogenesis, we analyzed tumors in Wistar rats induced by intraperitoneal application of MWCNTs and amosite asbestos. Using transcriptomic and epigenetic approaches, we compared the tumors by inducer (MWCNTs or amosite asbestos) or by tumor type (sarcomatoid, epithelioid, or biphasic). Results Genome-wide transcriptome datasets, whether grouped by inducer or tumor type, showed a high number of significant differentially expressed genes (DEGs) relative to control peritoneal tissues. Bioinformatic evaluations using Ingenuity Pathway Analysis (IPA) revealed that while the transcriptome datasets shared commonalities, they also showed differences in DEGs, regulated canonical pathways, and affected molecular functions. In all datasets, among highly- scoring predicted canonical pathways were Phagosome Formation , IL8 Signaling , Integrin Signaling , RAC Signaling , and TREM1 Signaling. Top-scoring activated molecular functions included cell movement , invasion of cells , migration of cells , cell transformation , and metastasis . Notably, we found many genes associated with malignant mesothelioma in humans, which showed similar expression changes in the rat tumor transcriptome datasets. Furthermore, RT-qPCR revealed downregulation of Hrasls, Nr4a1, Fgfr4, and Ret or upregulation of Rnd3 and Gadd45b in all or most of the 36 tumors analyzed. Bisulfite sequencing of Hrasls, Nr4a1, Fgfr4, and Ret revealed heterogeneity in DNA methylation of promoter regions. However, higher methylation percentages were observed in some tumors compared to control tissues. Lastly, global 5mC DNA, m6A RNA and 5mC RNA methylation levels were also higher in tumors than in control tissues. Conclusions Our findings may help better understand how exposure to MWCNTs can lead to carcinogenesis. This information is valuable for risk assessment and in the development of safe-by-design strategies.

Thymic lymphoid hyperplasia is a common age-related finding, which occurs particularly in female CD-1 mice. The main differential diagnoses are malignant lymphoma and thymoma. A systematic investigation of control groups from two carcinogenicity studies was performed including measurements of thymic size, and the immunohistochemistry (IHC) markers pan-Cytokeratin (pan-CK) for thymic epithelial cells; CD3 and CD45R/B220 for T and B lymphocytes, respectively; CD31 for endothelial cells; and F4/80 for macrophages. Thymoma can be differentiated by increased numbers of proliferating epithelial cells demonstrated by pan-CK IHC staining. Differentiation between lymphoid hyperplasia and lymphoma is more challenging as a mixture of B and T lymphocytes can be present in both findings. The present investigation showed that the thymic perivascular space is the compartment where the increased numbers of lymphocytes in hyperplasia are localized and not the medulla, as previously thought. The lymphoepithelial compartment is atrophic to the same extent in thymi diagnosed with age-related involution or lymphoid hyperplasia. Both diagnoses are thus related to variations in lymphoid cellularity of the nonepithelial perivascular space, which is continuous with the perithymic tissue. Likewise, lymphomas have a predilection to colonize the perivascular space and to spare the lymphoepithelial compartment.

TO THE EDITOR, The experimental procedure of segmental allergen challenge (SAC) in mild asthmatic subjects is an extremely valuable study tool to investigate mechanisms of bronchial asthma in patients in general and in particular for the role of eosinophils. In this procedure, BAL and bronchial mucosa can be analysed simultaneously after the induction of allergic inflammation. Older studies yielded data on different time points after the challenge with increasing numbers of eosinophils in the BAL.1 There are no data published on increased numbers of eosinophils within the bronchial mucosa 24 hours after SAC. Here, eosinophils were studied in mucosa and airway lumen of mild asthmatics undergoing segmental allergen challenge as described before.2, 3 Eosinophils and their release products were investigated in thick sections of the bronchial biopsies using advanced three-dimensional analysis. Subject data, methods and detailed results are given in the supplement. All subjects showed a clear eosinophilic response in the airway lumen and mucosa 24 hours after the challenge (Figure 1; FigureS1). The data on neutrophils were inconclusive (Figure 1; Figure S2). There was an increase of eosinophils in the mucosa and in the BAL. In the BAL, the concentrations of IL-5 and ECP were elevated (supplement). In the mucosa, ECP-stained volumes were found elevated as well as signs of eosinophil activation and degranulation (Figure 1B,C). In higher resolution, the ECP expression was associated with either cellular structures or small granules or dispersed over a large area beneath the epithelium (Figure 2A-E). In the presented study not cell associated, free ECP-positive granules were seen at baseline but only to a small extent. However, after allergen challenge the ECP-expressing volumes were massively increased (Figure 1B,C). These results might suggest that in human tissue eosinophils degranulate after a single allergen challenge and release their inflammatory mediators into the surrounding tissue. This is in contrast to animal models of asthma where eosinophilic degranulation after allergen challenge does not occur extensively.4, 5 In the present study, subjects with a strong IL-5 reaction in BAL showed a strong eosinophilic response in the lumen. However, BAL IL-5 levels did not correlate with eosinophil numbers in the mucosa or volume of ECP-positive surfaces in the mucosa (Figure S3). It is a long known fact that IL-5 levels in the BAL correlate highly with absolute numbers of eosinophils in the same compartment. In the present study, the allergic reaction is comparable to those of other studies. The missing correlation between IL-5 in the BAL and numbers of tissue eosinophils showed that the relationship between both compartments is not as simple as assumed. Here, the data for the volume of ECP-positive surfaces may give an important hint. Interestingly, the volume of ECP-positive surfaces was inversely correlated with TNF-α and IL-8. TNF-α enhances migration of eosinophils from mucosa to lumen shown in an in vitro cell culture model.6 Therefore, one possible explanation is that increased TNF-α in the BAL leads to a migration of activated eosinophils from the mucosa into the airways. In conclusion, the findings in subjects with mild asthma are in alignment with other published results and suggest that 1) human tissue eosinophils release their granules in non-provoked state, and 2) toxic content of these cells is significantly released into the surrounding tissue after a single allergen challenge, whereas the distribution and the degree of activation and degranulation of eosinophils differ widely between subjects. Many eosinophils in the airways indicate many eosinophils in the mucosa. Three-dimensional analysis in thick tissue sections using confocal microscopy is a valuable tool in the investigation of bronchial biopsies from patients suffering from bronchial asthma. We would like to thank Isabelle Bleeker for processing the biopsy samples of the classical immunohistology. None of the authors has any financial interest. AB, JH and NK planned and conducted the study. FP, AB and TT wrote the manuscript, all other authors read, corrected and approved the manuscript. TZV, KS, SR and FP established and performed the morphology and made the evaluation of tissue data. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project ( www.toxpath.org/inhand.asp ) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in most tissues and organs from the minipig used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet ( http://www.goreni.org/ ). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Myoepitheliomas of the salivary glands have been described in laboratory mice, but not in rats. A 20-week-old Wistar (Han) female rat developed a white to grey firm mass at the left side of the neck. Histologically, the mass was unencapsulated and infiltrated the adjacent tissue. The tumour parenchyma was cell rich without acinar or tubular architecture. The tumour showed a palisading basal cell pattern adjacent to blood vessels. There were areas of necrosis filled with cellular debris. The tumour cells showed strong immunohistochemical labelling for pan-cytokeratin types I and II (AE1/AE3), pan-cytokeratin (cytokeratins 1, 5, 6 and 8), cytokeratin 5, cytokeratin 14, vimentin and podoplanin, and only very slight positivity for cytokeratin 8 in small areas. There was no expression of smooth muscle actin. Based on the histological appearance and the immunohistochemistry, the tumour was diagnosed as a malignant myoepithelioma of the parotid gland originating from the parotid duct.

Background Idiopathic pulmonary fibrosis is a fatal disease with mean survival time of 3 years. Our previous work suggested a role of airway basal cells (ABC) in de novo generation of bronchial tissue/bronchiolization in progression of IPF. Recent immunohistochemistry data and single cell RNAseq analyses of IPF tissues showed airway basal cells to be shifted towards an epithelial mesenchymal transition phenotype. Histone deacetylase 6 (HDAC6) alters performance and destiny of several proteins via deacetylation and is involved in TGF-β-induced EMT (epithelial-mesenchymal transition). Objectives We got interested in the role of EMT, HDAC6 and TGF-β signaling for airway basal cell function in IPF. Methods We studied the expression of EMT markers and HDAC6 in lung tissues and isolated cells of 30 IPF patients. ABCs were harvested during bronchoscopy by bronchial brushing within the routine diagnostic work-up. Lung tissues were derived from explants. In addition, we tested the functional role of TGF-β, EMT and HDAC6 in our recently established 3D organoid assay. Sphere formation was counted by bright field microscopy and cell proliferation quantified with aid of the MTT Assay. 15 HDAC6 inhibitors were newly designed and generated by the University of Siena (Campiani lab) and tested in the 3D organoid assay. One candidate molecule was chosen for further experiments. We determined the half of maximal inhibitory effect of HDAC6 inhibitors on organoid formation [IC50 value]. Results Immunohistochemistry revealed increased levels of HDAC6 expression in IPF tissues, 3D organoids and human airway basal cells engrafted into murine lungs. Sphere formation is dependent on EMT and increased by stimuli which increase EMT such as TGF-β. Seven of the 15 newly generated HDAC6 inhibitors completely blocked sphere formation showing a concentration dependent effect. Human airway basal cells derived from IPF patients which overexpressed HDAC6 engrafted to the alveolar compartment and induced fibrosis in Rag2−/− mice. The half maximal inhibitory concentration (IC50) of the candidate molecule is 0.182 nmol in cell proliferation and 0.220 nmol in sphere counts. Conclusion Our data suggest that HDAC6 overexpression may confer hyperproliferative and profibrotic effects of airway basal cells in IPF. HDAC6 selective inhibitors abolishes bronchosphere formation in a concentration dependent manner in our 3D organoid model based on primary cell lines of IPF-ABCs and provides a new treatment strategy for IPF.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a fatal disease with a variable and unpredictable course.Objectives: To determine whether BAL cell gene expression is predictive of survival in IPF.Methods: This retrospective study analyzed the BAL transcriptome of three independent IPF cohorts: Freiburg (Germany), Siena (Italy), and Leuven (Belgium) including 212 patients. BAL cells from 20 healthy volunteers, 26 patients with sarcoidosis stage III and IV, and 29 patients with chronic obstructive pulmonary disease were used as control subjects. Survival analysis was performed by Cox models and component-wise boosting. Presence of airway basal cells was tested by immunohistochemistry and flow cytometry.Measurements and Main Results: A total of 1,582 genes were predictive of mortality in the IPF derivation cohort in univariate analyses adjusted for age and sex at false discovery rate less than 0.05. A nine-gene signature, derived from the discovery cohort (Freiburg), performed well in both replication cohorts, Siena (P < 0.0032) and Leuven (P = 0.0033). nCounter expression analysis confirmed the array results (P < 0.0001). The genes associated with mortality in BAL cells were significantly enriched for genes expressed in airway basal cells. Further analyses by gene expression, flow cytometry, and immunohistochemistry showed an increase in airway basal cells in BAL and tissues of IPF compared with control subjects, but not in chronic obstructive pulmonary disease or sarcoidosis.Conclusions: Our results identify and validate a BAL signature that predicts mortality in IPF and improves the accuracy of outcome prediction based on clinical parameters. The BAL signature associated with mortality unmasks a potential role for airway basal cells in IPF.

INTRODUCTION:Extensive vascular remodeling causing pulmonary hypertension (PH) represents a major cause of mortality in patients with congenital diaphragmatic hernia (CDH). The chemokine monocyte chemoattractant protein-1 (MCP-1) is a biomarker for the severity of PH and its activation is accompanied by pulmonary influx of monocytes and extensive vascular remodeling. MCP-1 activation can be reversed by application of rosiglitazone (thiazolidinedione). We performed this study to evaluate the role of MCP-1 for the pathogenesis of PH in experimental CDH. We hypothesized that vascular remodeling and MCP-1 activation is accompanied by pulmonary influx of fetal monocytes and can be attenuated by prenatal treatment with rosiglitazone.METHODS:In a first set of experiments pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blot (WB), and immunohistochemistry (IHC) were used to evaluate MCP-1 expression, activation, and localization. Quantification and localization of pulmonary monocytes/macrophages were carried out by IHC. In a second set of experiments nitrofen-exposed dams were randomly assigned to prenatal treatment with rosiglitazone or placebo on D18+D19. Fetal lungs were harvested on D21, divided into control, CDH+rosiglitazone, and CDH+placebo and evaluated by WB as well as IHC.RESULTS:Increased thickness of pulmonary arteries of CDH fetuses was accompanied by increased systemic and perivascular MCP-1 protein expression and significantly higher amounts of pulmonary monocytes/macrophages compared to controls (p<0.01). These effects were reversed by prenatal treatment with rosiglitazone (p<0.01 vs. CDH+P; control).CONCLUSION:Prenatal treatment with rosiglitazone has the potential to attenuate activation of pulmonary MCP-1, pulmonary monocyte influx, and vascular remodeling in experimental CDH. These results provide a basis for future research on prenatal immunomodulation as a novel treatment strategy to decrease secondary effects of PH in CDH.

Hematopoietic stem cells (HSCs) ensure a life-long regeneration of the blood system and are therefore an important source for transplantation and gene therapy. The teratoma environment supports the complex development of functional HSCs from human pluripotent stem cells, which is difficult to recapitulate in culture. This model mimics various aspects of early hematopoiesis, but is restricted by the low spontaneous hematopoiesis rate. In this study, a feasible protocol for robust hematopoiesis has been elaborated. We achieved a significant increase of the teratoma-derived hematopoietic population when teratomas were generated in the NSGS mouse, which provides human cytokines, together with co-injection of human umbilical vein endothelial cells. Since little is known about hematopoiesis in teratomas, we addressed localization and clonality of the hematopoietic lineage. Our results indicate that early human hematopoiesis is closely reflected in teratoma formation, and thus highlight the value of this model.

Here, we report findings in volunteers with bronchial asthma. Biopsies were obtained from the inner bronchial wall before and a short time again after segmental allergen provocation. In most of the baseline biopsies and in all evaluable biopsies after segmental allergen provocation, the follicular lymphoid tissue was detected by immunohistochemistry in the epithelium of these asthmatic patients. The basic occurrence of the tertiary lymphoid tissue in the bronchial mucosa of mild asthmatics was unexpected and may have consequences for the interpretation of pathophysiology, e.g., as a cause or consequence of bronchial asthma.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is a joint initiative among the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the endocrine organs (pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands and pancreatic islets) of laboratory rats and mice, with color photomicrographs illustrating examples of the lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for endocrine lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Objective The generation of hematopoietic stem cells (HSC) from human induced pluripotent stem cells (hiPS) remains challenging, although recent in vitro protocols showed promising results. The teratoma reflects a cellular environment close to human embryonic tissue. In hiPS-derived teratomas transplantable HSC were isolated. Therefore, teratomas can be applied to study early hematopoiesis. However, we lack deep understanding about teratoma tissue development. In this study, clonal development during teratoma formation and subsequent hematopoiesis were investigated by using a nucleotide barcoding technique. Gathered insights are important to estimate robustness of the model and contribute to a broader applicability. Methods The hiPS were lentivirally labelled with nucleotide barcodes and fluorescent reporters (RGB). After expansion, hiPS were used for teratoma formation in mice. Samples were analyzed by flow cytometry and sequenced by IonTorrent PGM method. Results We labelled a hiPS culture with approximately 11000 barcodes and followed clonal development through hiPS expansion and teratoma formation. Samples of hiPSC before and after expansion, teratoma cells and the CD45+ population comprised a diversity of different barcodes which indicates a high polyclonality. Although the estimated barcode pool size decreased during expansion and teratoma formation, teratomas still contained 6007 ± 1800 different barcodes (mean and SD). We further investigated if some clones of the hiPS culture preferably underwent hematopoietic differentiation. Interestingly, generation of CD45+ cells in all teratomas was highly polyclonal. Conclusion The polyclonal character of teratomas and the hematopoietic population illustrates that differentiation was rather dependent on environmental cues than on clonal preferences. This feature is important with respect to the robustness of teratoma differentiation programs. Furthermore, the model appears suited for transcription factor library screens in hiPS to address hematopoietic development or other embryonic processes.