The expression of activation antigens, namely CD25, CD69, CD71, and HLA‐DR on T cells from 15 healthy individuals stimulated with different mitogens and specific antigens was evaluated by immunofluorescence assay and flow cytometric analysis and compared with cell proliferation as a function of [³H]thymidine incorporation. CD69 was the earliest expressed antigen on stimulated cells, while HLA‐DR was the latest. Regardless of the stimulus used, lymphocytes expressing CD25 and CD71 were always more numerous than cells expressing CD69 and HLA‐DR. Variations in the proportion of CD4⁺ and CD8⁺ T cells expressing each activation marker were observed with different antigenic stimuli. The expression of each activation marker showed overall agreement with the [³H]thymidine incorporation assay in discriminating between positive and negative immune response. However, no correlation was observed between the percentage of CD25‐, CD69‐, CD71‐, and HLA‐DR‐positive T cells and the amount of [³H]thymidine incorporation. Moreover, low doses of mitogens and antigens as well as short time of stimulation were sufficient to induce T cells to express activation antigens but not to proliferate. Our data show that results obtained by flow cytometry and [³H]thymidine incorporation may differ qualitatively, at least under certain conditions; this suggests that the 2 assays are complementary, and when combined, may gives a clearer understanding of events leading to efficient cell‐mediated immune response. Cytometry 27:71–76, 1997.

Introduction: Carcinoma-associated fibroblasts (CAFs) play a pivotal role in cancer progression by contributing to invasion, metastasis and angiogenesis. Solid tumors possess a unique microenvironment characterized by local hypoxia, which induces gene expression changes and biological features leading to poor outcomes. Hypoxia Inducible Factor 1 (HIF-1) is the main transcription factor that mediates the cell response to hypoxia through different mechanisms that include the regulation of genes strongly associated with cancer aggressiveness. Among the HIF-1 target genes, the G-protein estrogen receptor (GPER) exerts a stimulatory role in diverse types of cancer cells and in CAFs. Methods: We evaluated the regulation and function of the key angiogenic mediator vascular endothelial growth factor (VEGF) in CAFs exposed to hypoxia. Gene expression studies, Western blotting analysis and immunofluorescence experiments were performed in CAFs and breast cancer cells in the presence of cobalt chloride (CoCl₂₎ or cultured under low oxygen tension (2% O₂), in order to analyze the involvement of the HIF-1α/GPER signaling in the biological responses to hypoxia. We also explored the role of the HIF-1α/GPER transduction pathway in functional assays like tube formation in human umbilical vein endothelial cells (HUVECs) and cell migration in CAFs. Results: We first determined that hypoxia induces the expression of HIF-1α and GPER in CAFs, then we ascertained that the HIF-1α/GPER signaling is involved in the regulation of VEGF expression in breast cancer cells and in CAFs exposed to hypoxia. We also assessed by ChIP assay that HIF-1α and GPER are both recruited to the VEGF promoter sequence and required for VEGF promoter stimulation upon hypoxic condition. As a biological counterpart of these findings, conditioned medium from hypoxic CAFs promoted tube formation in HUVECs in a HIF-1α/GPER dependent manner. The functional cooperation between HIF-1α and GPER in CAFs was also evidenced in the hypoxia-induced cell migration, which involved a further target of the HIF-1α/GPER signaling like connective tissue growth factor (CTGF). Conclusions: The present results provide novel insight into the role elicited by the HIF-1α/GPER transduction pathway in CAFs towards the hypoxia-dependent tumor angiogenesis. Our findings further extend the molecular mechanisms through which the tumor microenvironment may contribute to cancer progression.

(1) Background: Acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent for the coronavirus disease (COVID-19) that has led to a pandemic that began in March 2020. The role of the SARS-CoV-2 components on innate and adaptive immunity is still unknown. We investigated the possible implication of pathogen-associated molecular patterns (PAMPs)–pattern recognition receptors (PRRs) interaction. (2) Methods: We infected Calu-3/MRC-5 multicellular spheroids (MTCSs) with a SARS-CoV-2 clinical strain and evaluated the activation of RNA sensors, transcription factors, and cytokines/interferons (IFN) secretion, by quantitative real-time PCR, immunofluorescence, and ELISA. (3) Results: Our results showed that the SARS-CoV-2 infection of Calu-3/MRC-5 multicellular spheroids induced the activation of the TLR3 and TLR7 RNA sensor pathways. In particular, TLR3 might act via IRF3, producing interleukin (IL)-1α, IL-1β, IL-4, IL-6, and IFN-α and IFN-β, during the first 24 h post-infection. Then, TLR3 activates the NFκB transduction pathway, leading to pro-inflammatory cytokine secretion. Conversely, TLR7 seems to mainly act via NFκB, inducing type 1 IFN, IFN-γ, and IFN-λ3, starting from the 48 h post-infection. (4) Conclusion: We showed that both TLR3 and TLR7 are involved in the control of innate immunity during lung SARS-CoV-2 infection. The activation of TLRs induced pro-inflammatory cytokines, such as IL-1α, IL-1β, IL-4, and IL-6, as well as interferons. TLRs could be a potential target in controlling the infection in the early stages of the disease.

Monoclonal antibodies (MAbs) of predefined specificity have been generated by utilizing a synthetic peptide reflecting amino acid positions 10-17 of the Hu-rasT24 gene product as immunogen. These MAbs, designated RAP-1 through RAP-5 (RA, ras; P, peptide), have been shown to react with the ras gene product p21. Since the Hu-ras reactive determinants (positions 10-17) have been predicted to be within the tertiary structure of the p21 molecule, it was not unexpected that denaturation of cell extracts or tissue sections with Formalin or glutaraldehyde enhanced binding of the RAP MAbs. When paraffin-embedded Formalin-fixed tissue sections and the avidin-biotin complex immunoperoxidase method were used, the RAP MAbs clearly defined enhanced ras p21 expression in the majority of human colon and mammary carcinomas. The majority of all abnormal ducts and lobules from fibroadenoma and fibrocystic disease patients were negative, as were all normal mammary and colonic epithelia examined. The findings reported here form the basis for quantitative radioimmunoassays for a ras translational product and provide a means to evaluate ras p21 expression within individual cells of normal tissues and benign, "premalignant," and malignant lesions.