Probes for INS

The lateral parabrachial nucleus (lPBN), located in the pons, is a well-recognized anorexigenic center harboring, amongst others, the calcitonin gene-related peptide (CGRP)-expressing neurons that play a key role. The receptor for the orexigenic hormone ghrelin (the growth hormone secretagogue receptor, GHSR) is also abundantly expressed in the lPBN and ghrelin delivery to this site has recently been shown to increase food intake and alter food choice. Here we sought to explore whether GHSR-expressing cells in the lPBN (GHSRlPBN cells) contribute to feeding control, food choice and body weight gain in mice offered an obesogenic diet, involving studies in which GHSRlPBN cells were silenced. We also explored the neurochemical identity of GHSRlPBN cells. To silence GHSRlPBN cells, Ghsr-IRES-Cre male mice were bilaterally injected intra-lPBN with a Cre-dependent viral vector expressing tetanus toxin-light chain. Unlike control wild-type littermates that significantly increased in body weight on the obesogenic diet (i.e., high-fat high-sugar free choice diet comprising chow, lard and 9% sucrose solution), the heterozygous mice with silenced GHSRlPBN cells were resistant to diet-induced weight gain with significantly lower food intake and fat weight. The lean phenotype appeared to result from a decreased food intake compared to controls and caloric efficiency was unaltered. Additionally, silencing the GHSRlPBN cells altered food choice, significantly reducing palatable food consumption. RNAscope and immunohistochemical studies of the lPBN revealed considerable co-expression of GHSR with glutamate and pituitary adenylate cyclase-activating peptide (PACAP), and much less with neurotensin, substance P and CGRP. Thus, the GHSRlPBN cells are important for diet-induced weight gain and adiposity, as well as in the regulation of food intake and food choice. Most GHSRlPBN cells were found to be glutamatergic and the majority (76%) do not belong to the well-characterized anorexigenic CGRP cell population.

Interleukins (IL)-17, IL-22, and IL-31 play roles in human atopic dermatitis (AD), but scant information is available on canine AD. Histopathological assessment for interleukin expression is a challenge due to a lack of canine specific antibodies. To evaluate the mRNA and protein expression of IL-17 and IL-22, and mRNA expression of IL-31 and their receptors in the skin of healthy and atopic dogs, seventeen atopic (10 with and 7 without an active infection) and 13 healthy privately owned dogs were sampled. RNAscope In situ hybridization (ISH) for IL-17, IL-22, IL-31, and their receptors was performed on archived canine skin samples. Simultaneously, indirect immunofluorescence (IIF) was performed for IL-17 and IL-22. RNAscope ISH probes were validated by RT-PCR and RNAscope ISH on cytospin preparations of peripheral blood mononuclear cells from atopic dogs. IL-17, IL-22, IL-31, and their receptors were successfully detected by RNAscope ISH and by IIF (IL-17 and IL-22) in both atopic and healthy canine skin. There was no significant difference in the expression of interleukins and their receptors between healthy and atopic skin with or without active infection. Data from both methodologies were similar. The role and the relationship among those proteins in atopic skin is unclear from this study results. Data from IIF and ISH were overlapping and support each other. Fresh skin samples taken at different times during the development of atopic dermatitis might better assess the role that interleukins and their receptors play in AD.

ObjectiveDetermine the presence and prognostic value of human papillomavirus (HPV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCPyV), and cell cycle proteins in head and neck squamous cell carcinoma (HNSCC) of non-smokers and non-drinkers (NSND).MethodsClinical characteristics and tumors of 119 NSND with HNSCC were retrospectively collected and analyzed on tissue microarrays. RNAscope in situ hybridization (ISH) was used to screen for the presence of HPV and MCPyV mRNA. Immunohistochemistry was performed for expression of p16 as surrogate marker for HPV, Large T-antigen for MCPyV, and cell cycle proteins p53 and pRb. Positive virus results were confirmed with polymerase chain reaction. For EBV, EBV encoded RNA ISH was performed. Differences in 5-year survival between virus positive and negative tumors were determined by log rank analysis.ResultsAll oropharyngeal tumors (OPSCC) (n = 10) were HPV-positive, in addition to one oral (OSCC) and one nasopharyngeal tumor (NPSCC). The other three NPSCC were EBV-positive. MCPyV was not detected. Patients with HPV or EBV positive tumors did not have a significantly better 5-year disease free or overall survival. Over 70% of virus negative OSCC showed mutant-type p53 expression.ConclusionIn this cohort, all OPSCC and NPSCC showed HPV or EBV presence. Besides one OSCC, all other oral (n = 94), hypopharyngeal (n = 1), and laryngeal (n = 9) tumors were HPV, EBV, and MCPyV negative. This argues against a central role of these viruses in the ethiopathogenesis of tumors outside the oro- and nasopharynx in NSND. So, for the majority of NSND with virus negative OSCC, more research is needed to understand the carcinogenic mechanisms in order to consider targeted therapeutic options.

DNA vaccine evaluation in small animals is hampered by low immunogenicity when the vaccines are delivered using a needle and syringe. To overcome this technical hurdle we tested the possibility that a device developed for human intradermal medicine delivery might be adapted to successfully deliver a DNA vaccine to small animals. Disposable syringe jet injection (DSJI) does not currently exist for small animals. However, a commercialized, human intradermal device used to to administer medicines to the human dermis in a 0.1 mL volume was evaluated in Syrian hamsters. Here, we found that hantavirus DNA vaccines administered to hamsters using DSJI were substantially more immunogenic than the same vaccines delivered by needle/syringe or particle mediated epidermal delivery (gene gun) vaccination. By adjusting how the device was used we could deliver vaccine to either subcutaneous tissues, or through the skin into the muscle. RNA and/or antigen expression was detected in epidermal, subepidermal and fibroblast cells. We directly compared six optimized and non-optimized hantavirus DNA vaccines in hamsters. Optimization, including codon-usage and mRNA stability, did not necessarily result in increased immunogenicity for all vaccines tested; however, optimization of the Andes virus (ANDV) DNA vaccine protected vaccinated hamsters from lethal disease. This is the first time active vaccination with an ANDV DNA vaccine has shown protective efficacy in the hamster model. The adaptation of a human intradermal jet injection device for use as a method of subcutaneous and intramuscular jet injection of DNA vaccines will advance the development of nucleic acid based medical countermeasures for diseases modeled in hamsters.

Human papillomavirus (HPV) infection has been recognized as a cause of head and neck squamous cell carcinomas (HNSCC). Laryngeal squamous cell carcinoma (LSCC) is one of the most common pathologic types of HNSCC. Clinical trials show that there are differences in response to immunotherapy according to HPV status. It was reported that a high level of programmed cell death-ligand 1 (PD-L1) is correlated with better survival in HPV-positive head and neck cancer. In this study, we investigated the expression of PD-L1 in HPV-positive and HPV-negative LSCC to determine its prevalence and prognostic value. 52 cases of LSCC were collected from Tangshan Head and Neck Disease Pathology Research Base. PCR-reverse dot blot hybridization and RNAscope in situ hybridization were used to detect HPV status. PD-L1 expression was evaluated by immunohistochemistry and all cases were followed up for survival. SPSS24.0 was used for data entry and statistical analysis. Kaplan-Meier method and Log-rank time series analysis were used for single factor analysis. Multivariate analysis was performed using Cox proportional hazard regression model, and HR and 95% CI were calculated. Of the 52 LSCC patients, 32.7% (17/52) were HPV-positive by RNAscope in situ hybridization, and 51.9% (27/52) of patients were positive for PD-L1 expression by immunohistochemistry. Regression analysis showed that with a median follow-up period of 69 months, smoking and late stage were associated with poor overall survival (OS), whereas HPV positivity and PD-L1 expression showed a better overall survival outcome. Smoking status, tumor stage, HPV status, and PD-L1 expression in tumor cells may represent useful prognostic biomarkers in patients with LSCC. IJCEP

Purpose : Human corneal epithelial stem cells or limbal stem cells (LSCs), have been recognized to locate in corneal limbus for three decades. However, the molecular identity and definitive markers of LSCs are still elusive. This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC population at single cell resolution. Methods : Single cells of human limbal basal epithelium were isolated from young donor corneas. Single-cell RNA-Sequencing was performed using 10x Genomics platform, followed by clustering cell types through the graph-based visualization method Uniform Manifold Approximation and Projection (UMAP) and unbiased computational informatic analysis. Tissue RNA in situ hybridization with RNAscope, immunofluorescent staining and multiple functional assays were performed using ex vivo donor corneal tissues and in vitro culture models of primary human limbal epithelial cells (HLECs). Results : Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters belonging to three lineages. A smallest cluster (0.4% of total cells) was identified as LSCs based on their quiescent and undifferentiated states with enriched top expressed genes known as markers of putative epithelial stem cells. TSPAN7 and SOX17 are discovered and validated as new LSC markers based on their exclusive expression pattern and spatial localization in limbal basal epithelium by RNAscope and immunofluorescent staining, as well as their functional role in cell growth and tissue regeneration models with RNA interference in cultures. Interestingly, five cell types/states mapping a developmental trajectory of LSC from quiescence to proliferation and differentiation are uncovered by Monocle3 and CytoTRACE pseudotime analysis. The transcription factor networks linking novel signaling pathways are revealed to maintain LSC stemness. Conclusions : This human corneal single-cell transcriptomics identifies the LSC population and uncovers novel cell types mapping the differentiation trajectory in heterogenous limbal basal epithelium. The findings provide insight into LSC concept and lay the foundation for understanding the corneal homeostasis and diseases.

Growth factors are essential for maintenance of intestinal health. We previously showed that exogenous neuregulin-4 (NRG4) promotes colonocyte survival during cytokine challenge and is protective against acute models of intestinal inflammation. However, the function(s) of endogenous NRG4 are not well understood. Using NRG4-/- mice, we tested the role of endogenous NRG4 in models of colitis skewed toward either adaptive (interleukin-10 receptor [IL-10R] neutralization) or innate (dextran sulfate sodium [DSS]) immune responses.NRG4-/- and wild-type cage mate mice were subjected to chronic IL-10R neutralization colitis and acute DSS colitis. Disease was assessed by histological examination, inflammatory cytokine levels, fecal lipocalin-2 levels, and single cell mass cytometry immune cell profiling. Homeostatic gene alterations were evaluated by RNA sequencing analysis from colonic homogenates, with real-time quantitative polymerase chain reaction confirmation in both tissue and isolated epithelium.During IL-10R neutralization colitis, NRG4-/- mice had reduced colonic inflammatory cytokine expression, histological damage, and colonic CD8+ T cell numbers vs wild-type cage mates. Conversely, in DSS colitis, NRG4-/- mice had elevated cytokine expression, fecal lipocalin-2 levels, and impaired weight recovery. RNA sequencing showed a loss of St3gal4, a sialyltransferase involved in immune cell trafficking, in NRG4-null colons, which was verified in both tissue and isolated epithelium. The regulation of St3gal4 by NRG4 was confirmed with ex vivo epithelial colon organoid cultures from NRG4-/- mice and by induction of St3gal4 in vivo following NRG4 treatment.NRG4 regulates colonic epithelial ST3GAL4 and thus may allow for robust recruitment of CD8+ T cells during adaptive immune responses in colitis. On the other hand, NRG4 loss exacerbates injury driven by innate immune responses.

In newborn humans, and up to approximately 2 y of age, calvarial bone defects can naturally regenerate. This remarkable regeneration potential is also found in newborn mice and is absent in adult mice. Since previous studies showed that the mouse calvarial sutures are reservoirs of calvarial skeletal stem cells (cSSCs), which are the cells responsible for calvarial bone regeneration, here we hypothesized that the regenerative potential of the newborn mouse calvaria is due to a significant amount of cSSCs present in the newborn expanding sutures. Thus, we tested whether such regenerative potential can be reverse engineered in adult mice by artificially inducing an increase of the cSSCs resident within the adult calvarial sutures. First, we analyzed the cellular composition of the calvarial sutures in newborn and in older mice, up to 14-mo-old mice, showing that the sutures of the younger mice are enriched in cSSCs. Then, we demonstrated that a controlled mechanical expansion of the functionally closed sagittal sutures of adult mice induces a significant increase of the cSSCs. Finally, we showed that if a calvarial critical size bone defect is created simultaneously to the mechanical expansion of the sagittal suture, it fully regenerates without the need for additional therapeutic aids. Using a genetic blockade system, we further demonstrate that this endogenous regeneration is mediated by the canonical Wnt signaling. This study shows that controlled mechanical forces can harness the cSSCs and induce calvarial bone regeneration. Similar harnessing strategies may be used to develop novel and more effective bone regeneration autotherapies.

Epithelial cell adhesion molecule (EpCAM) is frequently used to distinguish carcinoma from background mesothelial cells during cytologic examination of body cavity fluids. Previously, the authors identified one malignant mesothelioma case with strong and diffuse membranous EpCAM staining, making it indistinguishable from carcinoma.In this study, the authors evaluated all available effusion specimens from patients with malignant mesothelioma, including the above-mentioned index case, obtained at Stanford Health Care, from 2011 to 2021 (N = 17) as well as control cases (N = 5). Analyses included an immunohistochemistry (IHC) assay for EpCAM and claudin-4, a multiplexed immunofluorescent (IF) assay for EpCAM, and an RNA in situ hybridization assay targeting EpCAM.The authors detected EpCAM positivity of variable intensity and percentage in four malignant mesothelioma cases (23.5%; although only two showed positivity for the epithelial-specific IHC marker MOC31 in ≥40% of cells) and claudin-4 negativity in all cases, with two cases displaying focal and weak claudin-4 staining in <1% of cells. Multiplexed IF staining on the cases with EpCAM IHC positivity showed strong, membranous EpCAM staining in one of four cases. RNA in situ hybridization also was used to assess the correlation between EpCAM positivity by IHC/IF and RNA expression levels. Strong EpCAM RNA expression was detected in the three malignant mesothelioma cases.The current findings revealed that a subset of epithelioid malignant mesothelioma cases mimic or exhibit the immunophenotypic features of carcinoma when evaluating for EpCAM only. Additional biomarker testing, such as claudin-4, may help avoid this potential pitfall to yield accurate diagnoses.

Opioid peptides are well-known modulators of the central control of reproduction. Among them, dynorphin coexpressed in kisspeptin (KP) neurons of the arcuate nucleus (ARC) has been thoroughly studied for its autocrine effect on KP release through κ opioid receptors. Other studies have suggested a role for β-endorphin (BEND), a peptide cleaved from the pro-opiomelanocortin precursor, on food intake and central control of reproduction. Similar to KP, BEND content in the ARC of sheep is modulated by day length and BEND modulates food intake in a dose-dependent manner. Because KP levels in the ARC vary with photoperiodic and metabolic status, a photoperiod-driven influence of BEND neurons on neighboring KP neurons is plausible. The present study aimed to investigate a possible modulatory action of BEND on KP neurons located in the ovine ARC. Using confocal microscopy, numerous KP appositions on BEND neurons were found but there was no photoperiodic variation of the number of these interactions in ovariectomized, estradiol-replaced ewes. By contrast, BEND terminals on KP neurons were twice as numerous under short days, in ewes having an activated gonadotropic axis, compared to anestrus ewes under long days. Injection of 5 μg BEND into the third ventricle of short-day ewes induced a significant and specific increase of activated KP neurons (16% vs. 9% in controls), whereas the percentage of overall activated (c-Fos positive) neurons, was similar between both groups. These data suggest a photoperiod-dependent influence of BEND on KP neurons of the ARC, which may influence gonadotropin-releasing hormone pulsatile secretion and inform KP neurons about metabolic status.

In the quest for "missing proteins" (MPs), the proteins encoded by the human genome still lacking evidence of existence at the protein level, novel approaches are needed to detect this challenging group of proteins. The current count stands at 1,343 MPs, and it is likely that many of these proteins are expressed at low levels, in rare cell or tissue types, or the cells in which they are expressed may only represent a small minority of the tissue. Here, we used an integrated omics approach to identify and explore MPs in human ovaries. By taking advantage of publicly available transcriptomics and antibody-based proteomics data in the Human Protein Atlas (HPA), we selected 18 candidates for further immunohistochemical analysis using an exclusive collection of ovarian tissues from women and patients of reproductive age. The results were compared with data from single-cell mRNA sequencing, and seven proteins (CTXN1, MRO, RERGL, TTLL3, TRIM61, TRIM73, and ZNF793) could be validated at the single-cell type level with both methods. We present for the first time the cell type-specific spatial localization of 18 MPs in human ovarian follicles, thereby showcasing the utility of the HPA database as an important resource for identification of MPs suitable for exploration in specialized tissue samples. The results constitute a starting point for further quantitative and qualitative analysis of the human ovaries, and the novel data for the seven proteins that were validated with both methods should be considered as evidence of existence of these proteins in human ovary.

Twenty-five to fifty percent of patients undergoing chemotherapy will develop anticipatory nausea and vomiting (ANV), in which symptoms occur in anticipation of treatment. ANV is triggered by environmental cues and shows little response to traditional antiemetic therapy, suggesting that unique neural pathways mediate this response. Understanding the underlying neural mechanisms of this disorder is critical to the development of novel therapeutic interventions. The purpose of the present study was to identify brain areas activated during ANV and characterize sex differences in both the behavior and the brain areas activated during ANV. We used a rat model of ANV by pairing a novel context with the emetic drug lithium chloride (LiCl) to produce conditioned nausea behaviors in the LiCl-paired environment. We quantitated gaping, an analog of human vomiting, after acute or repeated LiCl in a unique environment. To identify brain regions associated with gaping, we measured c-fos activation by immunochemical staining after these same treatments. We found that acute LiCl activated multiple brain regions including the supraoptic nucleus of the hypothalamus, central nucleus of the amygdala, nucleus of the solitary tract and area postrema, none of which were activated during ANV. ANV activated c-fos expression in the frontal cortex, insula and paraventricular nucleus of the hypothalamus of males but not females. These data suggest that therapies such as ondansetron which target the area postrema are not effective in ANV because it is not activated during the ANV response. Further studies aimed at characterizing the neural circuits and cell types that are activated in the conditioned nausea response will help identify novel therapeutic targets for the treatment of this condition, improving both quality of life and outcomes for patients undergoing chemotherapy.