Malignant peritoneal mesotheliomas of rats induced by multiwalled carbon nanotubes and amosite asbestos: transcriptome and epigenetic profiles.

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.

Immunohistochemical Characterization of Proliferative Lesions in the Thymus of Aging CD-1 Mice From Two Studies on the RITA Database, With Special Reference to the Perivascular Space.

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.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Minipig.

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.

BAL Cell Gene Expression Is Indicative of Outcome and Airway Basal Cell Involvement in Idiopathic Pulmonary Fibrosis.

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.

Prenatal treatment with rosiglitazone attenuates vascular remodeling and pulmonary monocyte influx in experimental congenital diaphragmatic hernia.

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.

Isolated aggregates of lymphoid cells in the inner bronchial wall in asthma patients.

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.

Human Teratoma-Derived Hematopoiesis Is a Highly Polyclonal Process Supported by Human Umbilical Vein Endothelial Cells.

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.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Endocrine System.

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.