Sensory Qualities Point to Different Structural and Functional Skin Patterns in Chronic Pruritus Patients. A Translational Explorative Study.

Chronic pruritus (CP) is often accompanied by paresthetic sensations like warmth, burning and stinging. The aim of this study was to analyze, whether divergent sensations are linked to structural and functional skin alterations in clinically diagnosed CP patients. Clinical responses to capsaicin, histamine, and to thermal and mechanical stimulation, intraepidermal nerve fiber density, and epidermal expression of transient receptor potential (TRP)-channels were investigated in healthy controls, and in CP patients, reporting either warmth (CP-W) or neuropathic sensations (CP-N). In CP-W, pinprick hyperalgesia and increased sensitivity to capsaicin were aligned with increased epidermal TRPV1 expression, while smaller histamine axon reflex erythema matched with significantly reduced intraepidermal nerve fiber density. CP-N showed earlier onset of sensations after capsaicin stimulation, significantly increased warmth detection threshold, and higher epidermal expression of TRPV4 compared to healthy controls. The present study contributes to the neurobiological understanding of the divergence of sensory sensations in CP, indicating new treatment targets.

Isolation, culture optimization and functional characterization of stem cell neurospheres from mouse neonatal olfactory bulb and epithelium.

The olfactory epithelium contains basal cells with stem cell characteristics, which have the capacity to differentiate throughout life into olfactory receptor neurons (ORNs). Here we investigate the in vitro characteristics of stem cells taken from the olfactory bulb (OB) and the olfactory epithelium (OE) of neonatal TIS21 knock-in mice. The major aim of the study was the generation of olfactory neurospheres (ONS) derived from OB and OE of neonatal mice as a tool to further analyze the elementary processes of ORN development. Our data showed that the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) leads to a significant increase in number of ONS derived from OB but not from OE. The differentiation of ONSs led to the formation of different neuronal cell types, in particular to bipolar-shaped cells as well as putative pyramidal-neurons, astrocytes and oligodendrocytes. Immunohistochemical staining confirmed the presence of astrocytes and neurons in both types of ONSs. In order to investigate the functionality of the neurons we performed calcium imaging and patch-clamp experiments. Calcium imaging experiments revealed that the application of high potassium concentration provokes calcium transients. No excitable properties, neither sodium currents nor action potentials, were observed for the bipolar-shaped cells derived from OB and OE neurospheres, which means that these types of cells morphologically defined as putative neuronal cells, were not physiologically active. Interestingly, patch-clamp recordings performed in the pyramidal-shaped cells of OB neurospheres showed sodium and potassium currents as well as action potentials. Our study will help to establish further models in the field of olfactology.

U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III.

Formation of catalytic core of the U12-dependent spliceosome involves U6atac and U12 interaction with the 5' splice site and branch site regions of a U12-dependent intron, respectively. Beyond the formation of intermolecular helix I region between U6atac and U12 snRNAs, several other regions within these RNA molecules are predicted to form stem-loop structures. Our previous work demonstrated that the 3' stem-loop region of U6atac snRNA contains a U12-dependent spliceosome-specific targeting activity. Here, we show a detailed structure-function analysis and requirement of a substructure of U6atac 3' stem-loop in U12-dependent in vivo splicing. We show that the C-terminal RNA recognition motif of p65, a U12 snRNA binding protein, also binds to the distal 3' stem-loop of U6atac. By using a binary splice site mutation suppressor assay we demonstrate that p65 protein-binding apical stem-loop of U12 snRNA can be replaced by this U6atac distal 3' stem-loop. Furthermore, we tested the compatibility of the U6atac 3' end from phylogenetically distant species in a human U6atac background, to establish the evolutionary relatedness of these structures and in vivo function. In summary, we demonstrate that RNA-RNA and RNA-protein interactions in the minor spliceosome are highly plastic as compared to the major spliceosome.

Deep sequencing of the murine olfactory receptor neuron transcriptome.

The ability of animals to sense and differentiate among thousands of odorants relies on a large set of olfactory receptors (OR) and a multitude of accessory proteins within the olfactory epithelium (OE). ORs and related signaling mechanisms have been the subject of intensive studies over the past years, but our knowledge regarding olfactory processing remains limited. The recent development of next generation sequencing (NGS) techniques encouraged us to assess the transcriptome of the murine OE. We analyzed RNA from OEs of female and male adult mice and from fluorescence-activated cell sorting (FACS)-sorted olfactory receptor neurons (ORNs) obtained from transgenic OMP-GFP mice. The Illumina RNA-Seq protocol was utilized to generate up to 86 million reads per transcriptome. In OE samples, nearly all OR and trace amine-associated receptor (TAAR) genes involved in the perception of volatile amines were detectably expressed. Other genes known to participate in olfactory signaling pathways were among the 200 genes with the highest expression levels in the OE. To identify OE-specific genes, we compared olfactory neuron expression profiles with RNA-Seq transcriptome data from different murine tissues. By analyzing different transcript classes, we detected the expression of non-olfactory GPCRs in ORNs and established an expression ranking for GPCRs detected in the OE. We also identified other previously undescribed membrane proteins as potential new players in olfaction. The quantitative and comprehensive transcriptome data provide a virtually complete catalogue of genes expressed in the OE and present a useful tool to uncover candidate genes involved in, for example, olfactory signaling, OR trafficking and recycling, and proliferation.

Monoterpene (-)-citronellal affects hepatocarcinoma cell signaling via an olfactory receptor.

Terpenes are the major constituents of essential oils in plants. In recent years, terpenes have become of clinical relevance due to their ability to suppress cancer development. Their effect on cellular proliferation has made them promising agents in the prevention or treatment of many types of cancer. In the present study, a subset of different monoterpenes was investigated for their molecular effects on the hepatocellular carcinoma cell line Huh7. Using fluorometric calcium imaging, acyclic monoterpene (-)-citronellal was found to induce transient Ca(2+) signals in Huh7 cells by activating a cAMP-dependent signaling pathway. Moreover, we detected the (-)-citronellal-activated human olfactory receptor OR1A2 at the mRNA and protein levels and demonstrated its potential involvement in (-)-citronellal-induced calcium signaling in Huh7 cells. Furthermore, activation of OR1A2 results in phosphorylation of p38 MAPK and reduced cell proliferation, indicating an effect on hepatocellular carcinoma progression. Here, we provide for the first time data on the molecular mechanism evoked by (-)-citronellal in human hepatocellular carcinoma cells. The identified olfactory receptor could serve as a potential therapeutic target for cancer diagnosis and treatment.

Chemosensory information processing between keratinocytes and trigeminal neurons.

Trigeminal fibers terminate within the facial mucosa and skin and transmit tactile, proprioceptive, chemical, and nociceptive sensations. Trigeminal sensations can arise from the direct stimulation of intraepithelial free nerve endings or indirectly through information transmission from adjacent cells at the peripheral innervation area. For mechanical and thermal cues, communication processes between skin cells and somatosensory neurons have already been suggested. High concentrations of most odors typically provoke trigeminal sensations in vivo but surprisingly fail to activate trigeminal neuron monocultures. This fact favors the hypothesis that epithelial cells may participate in chemodetection and subsequently transmit signals to neighboring trigeminal fibers. Keratinocytes, the major cell type of the epidermis, express various receptors that enable reactions to multiple environmental stimuli. Here, using a co-culture approach, we show for the first time that exposure to the odorant chemicals induces a chemical communication between human HaCaT keratinocytes and mouse trigeminal neurons. Moreover, a supernatant analysis of stimulated keratinocytes and subsequent blocking experiments with pyrodoxalphosphate-6-azophenyl-2',4'-disulfonate revealed that ATP serves as the mediating transmitter molecule released from skin cells after odor stimulation. We show that the ATP release resulting from Javanol® stimulation of keratinocytes was mediated by pannexins. Consequently, keratinocytes act as chemosensors linking the environment and the trigeminal system via ATP signaling.

Cellular infiltrates and NFκB subunit c-Rel signaling in kidney allografts of patients with clinical operational tolerance.

BACKGROUND: Nuclear factor kappa B (NFκB) plays a potential role in tolerance by orchestrating onset and resolution of inflammation and regulatory T cell differentiation through subunit c-Rel. We characterized cellular infiltrates and expression of NFκB1, c-Rel and its upstream regulators phosphatidylinositol 3-kinase/RAC-alpha serine/threonine kinase, in allograft biopsies from patients with spontaneous clinical operational tolerance (COT). METHODS: Paraffin-fixed kidney allograft biopsies from 40 patients with COT (n=4), interstitial rejection (IR; n=12), borderline changes (BC; n=12), and long-term allograft function without rejection (NR; n=12) were used in the study. Cellular infiltrates and immunohistochemical expression of key proteins of the NFκB pathway were evaluated in the cortical tubulointerstitium and in cellular infiltrates using digital image analysis software. Results were given as mean±SEM. RESULTS: Biopsies from patients with COT exhibited a comparable amount of cellular infiltrate to IR, BC, and NR (COT, 191±81; IR, 291±62; BC, 178±45; and NR, 210±42 cells/mm) but a significantly higher proportion of forkhead box P3-positive cells (COT, 11%±1.7%; IR, 3.5%±0.70%; BC, 3.4%±0.57%; and NR, 3.7%±0.78% of infiltrating cells; P=0.02). c-Rel expression in cellular infiltrates was significantly elevated in IR, BC, and NR when analyzing the number of positive cells per mm (P=0.02) and positive cells per infiltrating cells (P=0.04). In contrast, tubular PI3K and c-Rel expression were significantly higher in IR and BC but not in NR compared with COT (P=0.03 and P=0.006, respectively). With RAC-alpha serine-threonine kinase, similar tendencies were observed (P=0.2). CONCLUSIONS: Allografts from COT patients show significant cellular infiltrates but a distinct expression of proteins involved in the NFκB pathway and a higher proportion of forkhead box P3-positive cells.