Specific alterations of tyrosine hydroxylase immunopositive cells in the retina of NT-4 knock out mice

AbstractTo assess the effect of NT-4 deprivation on maturation of retinal circuitry, we investigated a mouse with targeted deletion of the gene encoding nt-4 (nt-4−/−). In particular, we studied neurons immunostained by an antibody recognizing tyrosine hydroxylase (TH), the rate limiting enzyme for dopamine (DA) synthesis. We found that TH immunopositive processes were altered in the retina of nt-4−/−. Alteration of TH immunopositive processes in nt-4−/− mice resulted in changes of DA turnover, as assessed by high-pressure liquid chromatography measurements. These findings suggest that retinal NT-4 plays a role in the morphological maturation of dopaminergic retinal cells

Localization of Melatonin Receptor 1 in Mouse Retina and Its Role in the Circadian Regulation of the Electroretinogram and Dopamine Levels

Melatonin modulates many important functions within the eye by interacting with a family of G-protein-coupled receptors that are negatively coupled with adenylate cyclase. In the mouse, Melatonin Receptors type 1 (MT1) mRNAs have been localized to photoreceptors, inner retinal neurons, and ganglion cells, thus suggesting that MT1 receptors may play an important role in retinal physiology. Indeed, we have recently reported that absence of the MT1 receptors has a dramatic effect on the regulation of the daily rhythm in visual processing, and on retinal cell viability during aging. We have also shown that removal of MT1 receptors leads to a small (3–4 mmHg) increase in the level of the intraocular pressure during the night and to a significant loss (25–30%) in the number of cells within the retinal ganglion cell layer during aging. In the present study we investigated the cellular distribution in the C3H/f+/+ mouse retina of MT1 receptors using a newly developed MT1 receptor antibody, and then we determined the role that MT1 signaling plays in the circadian regulation of the mouse electroretinogram, and in the retinal dopaminergic system. Our data indicate that MT1 receptor immunoreactivity is present in many retinal cell types, and in particular, on rod and cone photoreceptors and on intrinsically photosensitive ganglion cells (ipRGCs). MT1 signaling is necessary for the circadian rhythm in the photopic ERG, but not for the circadian rhythm in the retinal dopaminergic system. Finally our data suggest that the circadian regulation of dopamine turnover does not drive the photopic ERG rhythm

Targeting the NF-κB Pathway as a Combination Therapy for Advanced Thyroid Cancer.

NF-κB signaling plays an important role in tumor cell proliferation, cell survival, angiogenesis, invasion, metastasis and drug/radiation resistance. Combination therapy involving NF-κB pathway inhibition is an attractive strategy for the treatment of advanced forms of thyroid cancer. This study was designed to test the efficacy of NF-κB pathway inhibition in combination with cytotoxic chemotherapy, using docetaxel and ionizing radiation in in vitro models of thyroid cancer. We found that while both docetaxel and ionizing radiation activated NF-κB signaling in thyroid cancer cells, there was no synergistic effect on cell proliferation and/or programmed cell death with either genetic (transduction of a dominant negative mutant form of IκBα) or pharmacologic (proteasome inhibitor bortezomib and IKKβ inhibitor GO-Y030) inhibition of the NF-κB pathway in thyroid cancer cell lines BCPAP, 8505C, THJ16T and SW1736. Docetaxel plus bortezomib synergistically decreased in vitro invasion of 8505C cells, but not in the other cell lines. Screening of a panel of clinically relevant targeted therapies for synergy with genetic NF-κB inhibition in a proliferation/cytotoxicity assay identified the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) as a potential candidate. However, the synergistic effect was confirmed only in the BCPAP cells. These results indicate that NF-κB inhibitors are unlikely to be beneficial as combination therapy with taxane cytotoxic chemotherapy, external radiation therapy or radioiodine therapy. There may be unique circumstances where NF-κB inhibitors may be considered in combination with docetaxel to reduce tumor invasion or in combination with HDAC inhibitors to reduce tumor growth, but this does not appear to be a combination therapy that could be broadly applied to patients with advanced thyroid cancer. Further research may identify which subsets of patients/tumors may respond to this therapeutic approach

A safe and effective protocol for management of post-thyroidectomy hypocalcemia.

BACKGROUND: This study evaluates the outcomes of a protocol to manage hypocalcemia after thyroidectomy (TTX). METHODS: A review of prospectively collected data was performed in 130 patients who underwent TTX after the introduction of a specific protocol. These patients were compared with a control group of 195 patients who underwent TTX the year prior when routine calcium supplementation was utilized and no specific protocol was used. RESULTS: Of the 120 patients in whom the protocol was followed, 44 (37%) patients were classified as high risk, 15 (13%) intermediate risk, and 61 (51%) low risk. The protocol had a sensitivity of 85% and a negative predictive value of 92% for predicting subsequent hypocalcemia. With the implementation of the protocol, there was significant reduction in temporary hypocalcemia events (P = .008) and intravenous calcium drip (P = .49). Also, calcium supplementation was significantly lower in the protocol group (P ≤ .001). CONCLUSIONS: This hypocalcemia protocol identifies patients who do not require additional supplementation and additional monitoring. At the same time, it identifies those who will benefit from supplementation after TTX

Comprehensive Immune Profiling of Medullary Thyroid Cancer.

Background: Despite advances in targeted kinase inhibitor development for patients with medullary thyroid cancer (MTC), most patients develop resistance and would benefit from alternative approaches. Immune-based therapies are now considered for patients with progressive MTC. This study is the first comprehensive assessment of the immune milieu, immune-suppressive molecules, and potential tumor antigens in patients with MTC. Methods: Primary and/or regionally metastatic tumor tissues from 46 patients with MTC were screened for immune infiltrates by using standard immunohistochemistry (IHC) and further analyzed by multispectral imaging for T cell and myeloid markers. RNASeq expression profiling was performed in parallel. RNASeq, targeted sequencing, and IHC techniques identified cancer-associated mutations and MTC-enriched proteins. Results: Organized immune infiltration was observed in 49% and 90% of primary and metastatic tumors, respectively. CD8(+) cells were the dominant T cell subtype in most samples, while CD163(+) macrophages were most frequent among myeloid infiltrates. PD-1(+) T cells were evident in 24% of patients. Myeloid subsets were largely major histocompatibility complex II (MHCII(−)), suggesting a dysfunctional phenotype. Expression profiling confirmed enrichment in T cell, macrophage, and inflammatory profiles in a subset of samples. PD-L1 was expressed at low levels in a small subset of patients, while the immune regulatory molecules CD155 and CD47 were broadly expressed. Calcitonin, GRP, HIST1H4E, NOMO3, and NPIPA2 were highly and specifically expressed in MTC. Mutations in tumor suppressors, PTEN and p53, and mismatch repair genes, MSH2 and MSH6, may be relevant to disease progression and antigenicity. Conclusions: This study suggests that MTC is a more immunologically active tumor that has been previously reported. Patients with advanced MTC should be screened for targetable antigens and immune checkpoints to determine their eligibility for current clinical trials. Additional studies are necessary to fully characterize the antigenic potential of MTC and may encourage the development of adoptive T cells therapies for this rare tumor

MT₁ receptor immunoreactivity in the mouse retina.

<p>A band corresponding to the molecular weight of approximately 40 kDa was observed in western blotting of retinal extract from WT mice. No band was observed when the blot was treated with antibody pre-absorbed with the blocking peptide. A band of approximately 80 kDa was also present in retinal extract from WT mice. A very faint band around 80 kDa was also present the blot treated with the blocking peptide and in the blot of retinal extract from MT₁^−/− mice. α-Tubulin expression at the molecular weight of 53 kDa is also shown. Ab = Melatonin receptor 1 antibody; BP = blocking peptide. Similar results have been obtained for at least 3 independent samples for each experimental condition (Top panel). MT₁ immunoreactivity was localized in the outer segments of the photoreceptors (A, B), and in the ganglion cells (A, C), while a weak signal is observed in the outer plexiform layer (A, white arrows). No signal was detected in the outer segments or in ganglion cells using the blocking peptide (D, E) or in retina obtained from MT₁^−/− mice (F, G). OS = outer segments; IS = inner segments; ONL = outer nuclear layer; INL = inner nuclear layer; GCL  = ganglion cell layer. Micrographs are representative of results obtained from at least four animals for each experimental condition (Bottom panel).</p

The docetaxel and bortezomib synergistically decrease thyroid cancer cell line 8505C invasion <i>in vitro</i>.

<p>Asterisks indicate a significant effect of the drug combination (p<0.05, repeated measurements one-way ANOVA, Newman-Keuls posttest). The effect of the drugs on the invasion of BCPAP and THJ16T cells was not significant. BI—Bliss independence index.</p

BCL2L11 Induction Mediates Sensitivity to Src and MEK1/2 Inhibition in Thyroid Cancer

Patients with advanced thyroid cancer, including advanced papillary thyroid cancer and anaplastic thyroid cancer (ATC), have low survival rates because of the lack of efficient therapies available that can combat their aggressiveness. A total of 90% of thyroid cancers have identifiable driver mutations, which often are components of the MAPK pathway, including BRAF, RAS, and RET-fusions. In addition, Src is a non-receptor tyrosine kinase that is overexpressed and activated in thyroid cancer, which we and others have shown is a clinically relevant target. We have previously demonstrated that combined inhibition of Src with dasatinib and the MAPK pathway with trametinib synergistically inhibits growth and induces apoptosis in BRAF- and RAS-mutant thyroid cancer cells. Herein, we identified the pro-apoptotic protein BCL2L11 (BIM) as being a key mediator of sensitivity in response to combined dasatinib and trametinib treatment. Specifically, cells that are sensitive to combined dasatinib and trametinib treatment have inhibition of FAK/Src, MEK/ERK, and AKT, resulting in the dramatic upregulation of BIM, while cells that are resistant lack inhibition of AKT and have a dampened induction of BIM. Inhibition of AKT directly sensitizes resistant cells to combined dasatinib and trametinib but will not be clinically feasible. Importantly, targeting BCL-XL with the BH3-mimeitc ABT-263 is sufficient to overcome lack of BIM induction and sensitize resistant cells to combined dasatinib and trametinib treatment. This study provides evidence that combined Src and MEK1/2 inhibition is a promising therapeutic option for patients with advanced thyroid cancer and identifies BIM induction as a potential biomarker of response

The ionizing radiation activates NF-κB signaling in BCPAP (A,C) and 8505C (B) thyroid cancer cells as measured by dual luciferase reporter assay (A, B) and p65 DNA binding ELISA (C).

<p>p65 DNA binding experiment was performed 72 h after radiation exposure. Data are normalized to non-irradiated group (at a time 0 h for panels A and B). *—p<0.05, (two-way ANOVA, Bonferroni posttest).</p