Probes for INS

Central activation of fibroblast growth factor (FGF) receptors regulates peripheral glucose homeostasis and reduces food intake in preclinical models of obesity and diabetes. The current work was undertaken to advance our understanding of the receptor expression, as sites of ligand action by FGF19, FGF21, and FGF1 in the mammalian brain remains unresolved. Recent advances in automated RNAscope in situ hybridization and droplet digital PCR (ddPCR) technology allowed us to interrogate central FGFR/beta klotho (Klb) system at the cellular level in the mouse, with relevant comparisons to nonhuman primate and human brain. FGFR1-3 gene expression was broadly distributed throughout the CNS in Mus musculus, with FGFR1 exhibiting the greatest heterogeneity. FGFR4 expression localized only in the medial habenula and subcommissural organ of mice. Likewise, Klb mRNA was restricted to the suprachiasmatic nucleus (SCh) and select midbrain and hindbrain nuclei. ddPCR in the rodent hypothalamus confirmed that, although expression levels are indeed low for Klb, there is nonetheless a bonafide subpopulation of Klb+ cells in the hypothalamus. In NHP and human midbrain and hindbrain, Klb + cells are quite rare, as is expression of FGFR4. Collectively, these data provide the most robust central map of the FGFR/Klb system to date and highlight central regions that may be of critical importance to assess central ligand effects with pharmacological dosing, such as the putative interactions between the endocrine FGFs and FGFR1/Klb, or FGF19 with FGFR4.

Although 2014 World Health Organization criteria require unequivocal glandular and squamous differentiation for a diagnosis of cervical adenosquamous carcinoma, in practice, adenosquamous carcinoma diagnoses are often made in tumors that lack unequivocal squamous and/or glandular differentiation. Considering the ambiguous etiologic, morphological, and clinical features and outcomes associated with adenosquamous carcinomas, we sought to redefine these tumors. We reviewed slides from 59 initially diagnosed adenosquamous carcinomas (including glassy cell carcinoma and related lesions) to confirm an adenosquamous carcinoma diagnosis only in the presence of unequivocal malignant glandular and squamous differentiation. Select cases underwent immunohistochemical profiling as well as human papillomavirus (HPV) testing by in situ hybridization. Of the 59 cases originally classified as adenosquamous carcinomas, 34 retained their adenosquamous carcinoma diagnosis, 9 were reclassified as pure invasive stratified mucin-producing carcinomas, 10 as invasive stratified mucin-producing carcinomas with other components (such as HPV-associated mucinous, usual-type, or adenosquamous carcinomas), and 4 as HPV-associated usual or mucinous adenocarcinomas with benign-appearing squamous metaplasia. Two glassy cell carcinomas were reclassified as poorly differentiated usual-type carcinomas based on morphology and immunophenotype. There were significant immunophenotypic differences between adenosquamous carcinomas and pure invasive stratified mucin-producing carcinomas with regard to HPV (p < 0.0001), PAX8 (p = 0.038; more in adenosquamous carcinoma), p40 (p < 0.0001; more in adenosquamous carcinoma), p63 (p = 0.0018; more in adenosquamous carcinoma) and MUC6 (p  < 0.0001; less in adenosquamous carcinoma), HNF-1beta (p = 0.0023), vimentin (p = 0.0003), p53 (p = 0.0004), and CK7 (p = 0.0002) expression. Survival outcomes were similar between all groups. Adenosquamous carcinomas should be diagnosed only in the presence of unequivocal malignant glandular and squamous differentiation. The two putative glassy cell carcinomas studied did not meet our criteria for adenosquamous carcinoma, and categorizing them as such should be reconsidered.

Abstract

Background

Laryngeal squamous cell carcinoma (LSCC) is strongly associated with tobacco use, but recent reports suggest an increasing incidence of LSCC in patients without traditional risk factors, suggesting an alternative etiology of tumorigenesis. The purpose of this study is to characterize this non-smoking population and to compare immunohistochemical markers in tumor specimens from non-smokers and smokers with LSCC.

Methods

A retrospective chart review of patients with LSCC at Johns Hopkins Hospital (JHH) was performed. A tissue microarray (TMA) was constructed with tumor specimen from non-smokers with stage and age-matched smokers and stained for a variety of immunologic and molecular targets.

Results

In the JHH cohort of 521 patients, 12% (n = 63) were non-smokers. Non-smokers were more likely to be <45 years old at time of diagnosis (OR 4.13, p = 0.001) and to have glottic tumors (OR 2.46, p = 0.003). The TMA was comprised of tumors from 34 patients (14 non-smokers, 20 smokers). Only 2 patients (6%) were human-papillomavirus (HPV) positive by high-risk RNA in situ hybridization (ISH). There was no correlation between smoking status and p16 (p = 0.36), HPV-ISH positivity (p = 0.79), phosphatase and tensin homolog (PTEN, p = 0.91), p53 (p = 0.14), or programmed death-ligand 1 (PD-L1, p = 0.27) expression.

Conclusions

Non-smokers with LSCC are more likely to be younger at the time of diagnosis and have glottic tumors than smokers with LSCC. In TMA analysis of stage and age-matched specimens from smoker and non-smokers with LSCC, the pattern of expression for common molecular and immunologic markers is similar. Further, HPV does not appear to be a major causative etiology of LSCC in either smokers or non-smokers in our cohort of patients.

In the arcuate nucleus of the hypothalamus (ARH) satiety signaling (anorexigenic) pro-opiomelanocortin (POMC)-expressing and hunger signaling (orexigenic) agouti-related peptide (AgRP)-expressing neurons are key components of the neuronal circuits that control food intake and energy homeostasis. Here, we assessed whether the catecholamine noradrenalin directly modulates the activity of these neurons in mice. Perforated patch clamp recordings showed that noradrenalin changes the activity of these functionally antagonistic neurons in opposite ways, increasing the activity of the orexigenic NPY/AgRP neurons and decreasing the activity of the anorexigenic POMC neurons. Cell type-specific transcriptomics and pharmacological experiments revealed that the opposing effect on these neurons is mediated by the activation of excitatory α1A - and β- adrenergic receptors in NPY/AgRP neurons, while POMC neurons are inhibited via α2A - adrenergic receptors. Thus, the coordinated differential modulation of the key hypothalamic neurons in control of energy homeostasis assigns noradrenalin an important role to promote feeding.

Light Sheet Fluorescence Microscopy (LSFM) of whole organs, in particular the brain, offers a plethora of biological data imaged in 3D. This technique is however often hindered by cumbersome non-automated analysis methods. Here we describe an approach to fully automate the analysis by integrating with data from the Allen Institute of Brain Science (AIBS), to provide precise assessment of the distribution and action of peptide-based pharmaceuticals in the brain. To illustrate this approach, we examined the acute central nervous system effects of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide. Peripherally administered liraglutide accessed the hypothalamus and brainstem, and led to activation in several brain regions of which most were intersected by projections from neurons in the lateral parabrachial nucleus. Collectively, we provide a rapid and unbiased analytical framework for LSFM data which enables quantification and exploration based on data from AIBS to support basic and translational discovery.