Severe acute respiratory syndrome coronavirus 2: virus mutations in specific European populations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is being intensively studied, particularly its evolution, in the increasingly available sequences between countries/continents with classical phylogenetic tree representation. More recently, certain protein mutations have been correlated with specific functional impacts. Our clinical data from patients suggest that clinical symptoms differ between European countries. Among other factors, SARS-CoV-2 mutations could explain these disparities. Our analyses point to an association of diverse mutations, including co-evolving ones, in a few SARS-CoV-2 proteins within specific countries. We therefore suggest combining clinical information from patients and the determination of the associated SARS-CoV-2 genome to better understand the specific symptoms.

Relationships between cell division, expression of growth factors and microcirculation in the thyroids of Tg-A2aR transgenic mice and patients with Graves' disease.

Tissue heterogeneity and nodule formation are hallmarks of thyroid growth. This is accounted for by the clonality theory that acknowledges different individual cellular abilities to respond to trophic stimuli. In order to test the hypothesis that functional and mitotic properties of thyrocytes could be influenced by paracrine interactions with neighbour endothelial cells, studies were conducted in both mouse and human goitre models. In the first part of the study, homogenous goitres in C57 black mice were compared with heterogeneous goitres in transgenic hyperthyroid mice expressing the A2 adenosine receptor (Tg-A2aR). The second part of the study concentrated on comparing human thyroid tIssue of control individuals and of patients with Graves' disease. The rate of cell division was evaluated by immunohistochemical detection of cells positive for proliferating cell nuclear antigen (PCNA). Their spatial distribution was then correlated with immunohistochemical cellular expression of growth- and vasoactive-related factors (fibroblast growth factor-2, transforming growth factor-beta, endothelin-1, vascular endothelial growth factor, nitric oxide synthase III), and with microcirculation expansion. Observations were made on digitalised images of histological serial sections. The nearest-neighbour method was used to distinguish between random or clustered distribution. PCNA-positive cells were both randomly and uniformly distributed in homogenous goitres from C57 black mice, and were clustered in tIssue areas identified as papillary and hyperplastic zones in heterogeneous goitres from Tg-A2aR mice. However, they were absent in the so-called compact cellular zones featuring resting cells. Moreover, whereas papillary and hyperplastic zones were highly vascularised, compact zones were nearly free of microvessels. Spatial distribution of dividing cells was positively correlated with the expression of growth-related factors. A similar pattern was observed in the thyroids of patients with Graves' disease. In accordance with the recent demonstration of the presence of angiofollicular units in the thyroid, these data strongly support the hypothesis that functional and mitotic properties of each single thyrocyte, likely to be responsible for growth heterogeneity of hyperplastic glands, may be adjusted at tIssue level by specific interactions with neighbour endothelial cells that, in turn, could alter the mitotic rate of thyrocytes through paracrine signals.

Differential patterns of cell cycle regulatory proteins expression in transgenic models of thyroid tumours.

Cell cycle proteins regulate the transitions from G1 to S and G2 to M phases. In higher eukaryotes, their function is controlled by intracellular cascades regulated by extracellular growth factors. We have studied in previously described transgenic mouse models for thyroid proliferative diseases the expression of the key proteins regulating the cell cycle by Western blotting and immunohistochemistry, and have correlated the observations with the known actions of the transgenes on the signal transduction cascades. In the adenosine A2a receptor model, the cyclic AMP pathway, upstream of the Rb family cell division block, is constitutively activated. In the model expressing HPV 16 E7 protein, the Rb-like proteins are inhibited. Cyclin-dependent kinases cdk4, cdk2 and cdc2, and the associated cyclins D, E and A have been studied. Cyclin D3 appears as the major cyclin D subtype expressed in mouse thyroid epithelial cells in normal and transgenic mice. In the adenosine A2aR model, all cell cycle proteins tested were accumulated. In the E7 model, all cell cycle proteins except for D-type cyclins and cdk4 were also accumulated. A similar pattern was observed in thyroids coexpressing both transgenes, suggesting a dominant effect of E7 over the consequences of the cAMP cascade activation. The cyclin-dependent kinase inhibitors p21cip1/waf1 and p27kip1 were not downregulated in these proliferating thyroids which suggest other roles than the inhibition of the cell cycle progression.

Early occurrence of metastatic differentiated thyroid carcinomas in transgenic mice expressing the A2a adenosine receptor gene and the human papillomavirus type 16 E7 oncogene.

We report here the characterization of a transgenic mouse model (Tg-A2aR/Tg-E7) resulting from the coexpression of two oncogenic transgenes in the thyroid. The two transgenes (Tg-A2aR and Tg-E7) were placed under control of the thyroid specific thyroglobulin gene promoter, and directed the expression of either the A2a adenosine receptor that constitutively activates the cAMP pathway, or the E7 protein of the human papillomavirus type 16, that binds and inactivates the retinoblastoma susceptibility gene product (Rb1). Transgenic mice expressing both transgenes were generated by interbreeding the Tg-A2aR and Tg-E7 transgenic lines, generated and characterized previously (Ledent et al., 1992, 1995). These mice develop a larger goiter than that of the two parental lines, and a severe hyperthyroidism comparable to that observed in the Tg-A2aR parental line. The main feature of the Tg-A2aR/Tg-E7 mice is the rapid occurrence of malignant lesions, and the dissemination of malignant thyroid tissue through the blood stream, generating multiple differentiated and functional metastases in the lung. These metastases appeared as early as 2 months after birth and their frequency increased to 75% over 3 months. They were associated with the presence of large vascular lakes in the thyroid. Electron microscopy of the malignant cells revealed nuclear features similar to those of human thyroid papillary carcinoma. These mice, in which two oncogenes are co-expressed in the thyroid, represent the first genetic animal model developing metastatic differentiated carcinomas of the thyroid with a high frequency.

Transgenic models for proliferative and hyperfunctional thyroid diseases.

Mouse transgenic models that develop thyroid diseases were generated. All transgenes were driven by the thyroid specific promoter of the thyroglobulin gene. The tissue specificity of the promoter was investigated by using the bacterial chloramphenicol acetyl transferase gene as reporter. The expression of an adenosine A2a receptor resulted in the permanent activation of the cAMP cascade. As a consequence, the transgenic mice developed severe hyperthyroidism and a large goiter, demonstrating in vivo the role of the cAMP cascade in the promotion of both function and proliferation of the thyroid cell. These mice constitute a model for autonomous hyperfunctional adenoma and non-autoimmune familial hyperthyroidism, where mutant thyrotropin receptors stimulate the cAMP cascade constitutively. In another transgenic model, the function of the retinoblastoma susceptibility gene product RB1 (and of related proteins) was inhibited by expression of the E7 oncoprotein of human papillomavirus type 16. The result was the development of a differentiated and normofunctional colloid goiter, with the progressive development of differentiated malignant lesions. This model suggests the essential role of RB1 and related proteins in the negative control of proliferation that characterizes thyroid cells in the adult. Other transgenic models of thyroid diseases are discussed.