In vitro measurements of the middle ear input immittance in temporal bones extracted from human cadavers were directly compared with similar in vivo measurements from clinically normal subjects. The results of this comparison indicate that most otoscopically normal unfixed cadaver ears have middle ear input immittances that are indistinguishable from those of live subjects in the 0.1- to 2-kHz range — As long as they have been kept from drying and the static pressures on either side of the tympanic membrane are equal. The effects of the middle ear muscles on the measured input immittance are generally small and the cadaver ears can be maintained in the frozen state for several months with little change. Tympanometry appears to be a reliable indicator of normal middle ear immittance. Cadaver middle ears are useful models of human middle ear function.

The genomic actions of thyroid hormone and steroids depend upon primary interactions of the hormones with their specific nuclear receptor proteins. Formation of nuclear co-activator or co-repressor complexes involving the liganded receptors subsequently result in transcriptional events-either activation or suppression-at genes that are specific targets of thyroid hormone or steroids. Nongenomic actions of thyroid hormone and steroids are in contrast initiated at binding sites on the plasma membrane or in cytoplasm or organelles and do not primarily require formation of intranuclear receptor protein-hormone complexes. Importantly, hormonal actions that begin nongenomically outside the nucleus often culminate in changes in nuclear transcriptional events that are regulated by both traditional intranuclear receptors as well as other nuclear transcription factors. In the case of thyroid hormone, the extranuclear receptor can be the classical "nuclear" thyroid receptor (TR), a TR isoform, or integrin αvβ3. In the case of steroid hormones, the membrane receptor is usually, but not always, the classical "nuclear" steroid receptor. This concept defines the paradigm of overlapping nongenomic and genomic hormone mechanisms of action. Here we review some examples of how extranuclear signaling by thyroid hormone and by estrogens and androgens modulates intranuclear hormone signaling to regulate a number of vital biological processes both in normal physiology and in cancer progression. We also point out that nongenomic actions of thyroid hormone may mimic effects of estrogen in certain tumors.

Angiogenesis assays allow for the evaluation of pro- or anti-angiogenic activity of endogenous or exogenous factors (stimulus or inhibitors) through investigation of their pro-or anti- proliferative, migratory, and tube formation effects on endothelial cells. To model the process of angiogenesis and the effects of biomolecules on that process, both in vitro and in vivo methods are currently used. In general, in vitro methods monitor specific stages in the angiogenesis process and are used for early evaluations, while in vivo methods more accurately simulate the living microenvironment to provide more pertinent information. We review here the current state of angiogenesis assays as well as their mechanisms, advantages, and limitations.

Thyroid hormone induces tumor cell and blood vessel cell proliferation via a cell surface receptor on heterodimeric integrin αvβ3. We investigated the role of thyroid hormone‐induced internalization of nuclear integrin αv monomer. Physiological concentration of thyroxine (free T ₄ , 10 ^–10 M), but not 3,5,3'‐triiodo‐L‐thyronine (T ₃ ), induced cellular internalization and nuclear translocation of integrin αv monomer in human non‐small‐cell lung cancer (H522) and ovarian carcinoma (OVCAR‐3) cells. T ₄ did not complex with integrin αv monomer during its internalization. The αv monomer was phosphorylated by activated ERK1/2 when it heterodimerized with integrin β3 in vitro. Nuclear αv complexed with transcriptional coactivator proteins, p300 and STAT1, and with corepressor proteins, NCoR and SMRT. Nuclear αv monomer in T ₄ ‐exposed cells, but not integrin β3, bound to promoters of specific genes that have important roles in cancer cells, including estrogen receptor‐α, cyclooxygenase‐2, hypoxia‐inducible factor‐1α , and thyroid hormone receptor β1 in chromatin immunoprecipitation assay. In summary, monomeric αv is a novel coactivator regulated from the cell surface by thyroid hormone for the expression of genes involved in tumorigenesis and angiogenesis. This study also offers a mechanism for modulation of gene expression by thyroid hormone that is adjunctive to the nuclear hormone receptor (TR)‐T ₃ pathway.—Lin, H.‐Y., Su, Y.‐F., Hsieh, M.‐T., Lin, S., Meng, M., London, D., Lin, C., Tang, H.‐Y., Hwang, J., Davis, F. B., Mousa, S. A.and Davis, P. J., Nuclear monomeric integrin αv in cancer cells is a coactivator regulated by thyroid hormone. FASEB J. 27, 3209–3216 (2013). www.fasebj.org

The PD-1 (programmed death-1)/PD-L1 (PD-ligand 1) checkpoint is a critical regulator of activated T cell-cancer cell interactions, defending tumor cells against immune destruction. Nano-diamino-tetrac (NDAT; Nanotetrac) is an anticancer/anti-angiogenic agent targeted to the thyroid hormone-tetrac receptor on the extracellular domain of integrin αvβ3. NDAT inhibits the cancer cell PI3-K and MAPK signal transduction pathways that are critical to PD-L1 gene expression. We examined actions in vitro of thyroid hormone (l-thyroxine, T4) and NDAT on PD-L1 mRNA abundance (qPCR) and PD-L1 protein content in human breast cancer (MDA-MB-231) cells and colon carcinoma (HCT116 and HT-29) cells. In MDA-MB-231 cells, a physiological concentration of T4 (10-7M total; 10-10M free hormone) stimulated PD-L1 gene expression by 38% and increased PD-L1 protein by 2.7-fold (p<0.05, all changes). NDAT (10-7M) reduced PD-L1 in T4-exposed cells by 21% (mRNA) and 39% (protein) (p<0.05, all changes). In HCT116 cells, T4 enhanced PD-L1 gene expression by 17% and protein content by 24% (p<0.05). NDAT reduced basal PD-L1 mRNA by 35% and protein by 31% and in T4-treated cells lowered mRNA by 33% and protein by 66%. In HT-29 cells, T4 increased PD-L1 mRNA by 62% and protein by 27%. NDAT lowered basal and T4-stimulated responses in PD-L1 mRNA and protein by 35-40% (p<0.05). Activation of ERK1/2 was involved in T4-induced PD-L1 accumulation. We propose that, by a nongenomic mechanism, endogenous T4 may clinically support activity of the defensive PD-1/PD-L1 checkpoint in tumor cells. NDAT non-immunologically suppresses basal and T4-induced PD-L1 gene expression and protein accumulation in cancer cells.