Efficacy of Treatments for Cholestatic Pruritus: A Systemic Review and Meta-analysis.

Cholestatic itch is a disabling symptom that may be secondary to liver or biliary diseases. Management of cholestatic pruritus is complex. A systematic review and meta-analysis on the efficacy of treatments for cholestatic pruritus were performed. PubMed and Cochrane Library were searched using the algorithm "(hepatitis OR cholestatic OR liver) AND (pruritus OR itch) AND (management OR treatment OR treatments)" for 1975-2019. Of the 2,264 articles identified, 93 were included in a systematic review and 15 in a meta-analysis (studies evaluating pruritus with a visual analogue scale). Some treatments act by reducing levels of pruritogens in the enterohepatic cycle, others modify the metabolism or secretion of these pruritogens, or act on pruritus pathways. A further possible treatment is albumin dialysis. However, due to many heterogeneities in the reviewed studies it is difficult to identify and recommend an optimum treatment. Only 15 studies were included in the meta-analysis, due to the small number of randomized studies using a visual analogue scale.

Comparative transcriptional profiling of human Merkel cells and Merkel cell carcinoma.

Merkel cell carcinoma is believed to be derived from Merkel cells after infection by Merkel cell polyomavirus (MCPyV) and other poorly understood events. Transcriptional profiling using cDNA microarrays was performed on cells from MCPy-negative and MCPy-positive Merkel cell carcinomas and isolated normal Merkel cells. This microarray revealed numerous significantly upregulated genes and some downregulated genes. The extensive list of genes that were identified in these experiments provides a large body of potentially valuable information of Merkel cell carcinoma carcinogenesis and could represent a source of potential targets for cancer therapy.

Effect of human skin explants on the neurite growth of the PC12 cell line.

The skin is a densely innervated organ. After a traumatic injury, such as an amputation, burn or skin graft, nerve growth and the recovery of sensitivity take a long time and are often incomplete. The roles played by growth factors and the process of neuronal growth are crucial. We developed an in vitro model of human skin explants co-cultured with a rat pheochromocytoma cell line differentiated in neuron in presence of nerve growth factor (NGF). This model allowed the study of the influence of skin explants on nerve cells and nerve fibre growth, probably through mediators produced by the explant, in a simplified manner. The neurite length of differentiated PC12 cells co-cultured with skin explants increased after 6 days. These observations demonstrated the influence of trophic factors produced by skin explants on PC12 cells.

Characterization of neurons from adult human skin-derived precursors in serum-free medium : a PCR array and immunocytological analysis.

Adult stem cells could be small sources of neurons or other cellular types for regenerative medicine and tissue engineering. Recently, pluripotent stem cells have been extracted from skin tissue, which opened a new accessible source for research. To routinely obtain a high yield of functional neurons from adult human skin stem cells with defined serum-free medium, stem cells from abdominal skin were cultured in serum-free medium. To differentiate them, we used a defined medium containing growth factors. Differentiated cells were identified using the following methods: (i) Oil-red-O staining for adipocytes, immunocytochemistry with antibodies recognising (ii) neurofilaments and PGP9.5 for neural differentiation, (iii) glial fibrillary acidic protein (GFAP) for glial differentiation, (iv) Ki-67 for proliferative cells, (v) FM1-43 staining to analyse vesicle trafficking in neuronal cells and (vi) a PCR array was used. Stem cells were floating in spheres and were maintained in culture for 4 months or more. They expressed nestin and Oct 4 and were proliferative. We induced specific differentiation into adipocytes, glial and neuronal cells. The yields of differentiated neurons were high and reproducible. They were maintained for long time (1 month) in the culture medium. Furthermore, these neurons incorporated FM1-43 dye, which indicates a potent acquisition of synaptic features in neurons. Stem cells from adult human skin could be valuable and reproducible tools/source to obtain high numbers of functional specific cellular types, such as neurons, for tissue engineering. In this work, the possibility to obtain a high yield of differentiated neurons, with the ability of endocytosis and vesicle cell trafficking, was shown.

Effects of the re-innervation of organotypic skin explants on the epidermis.

The nervous system takes part in skin homeostasis and interacts with skin cells. In in vitro organotypic skin models, these interactions are lost owing to the absence of nerve endings. We have developed an in vitro organotypic skin model based on a re-innervated human skin explant using primary sensory neurons from the dorsal root ganglia of rats. After 10 days of co-culture between skin explant and neurons, a dense network of nerve fibres was observed. The epidermis and dermis presented nerve fibres associated with cellular body from sensory neurons introduced in the co-culture. Epidermal thickness, cell density and quality of re-innervated skin explant were all higher when skin explants were re-innervated by sensory neurons at 10 days of culture. Proliferation of epidermal cell was not modified, but the apoptosis was significantly diminished. Hence, this innovative model of co-cultured skin explants and neurons allows better epidermal integrity and could be useful for studies concerning interactions between the skin and its peripheral nervous system.

Immunohistochemical localization of hepatopancreatic phospholipase in gastropods mollusc, Littorina littorea and Buccinum undatum digestive cells.

BACKGROUND: Among the digestive enzymes, phospholipase A2 (PLA2) hydrolyzes the essential dietary phospholipids in marine fish and shellfish. However, we know little about the organs that produce PLA2, and the ontogeny of the PLA2-cells. Accordingly, accurate localization of PLA2 in marine snails might afford a better understanding permitting the control of the quality and composition of diets and the mode of digestion of lipid food. RESULTS: We have previously producted an antiserum reacting specifically with mSDPLA2. It labeled zymogen granules of the hepatopancreatic acinar cells and the secretory materials of certain epithelial cells in the depths of epithelial crypts in the hepatopancreas of snail. To confirm this localization a laser capture microdissection was performed targeting stained cells of hepatopancreas tissue sections. A Western blot analysis revealed a strong signal at the expected size (30 kDa), probably corresponding to the PLA2. CONCLUSIONS: The present results support the presence of two hepatopancreatic intracellular and extracellular PLA2 in the prosobranchs gastropods molluscs, Littorina littorea and Buccinum undatum and bring insights on their localizations.

Immunohistochemical localization of hepatopancreatic phospholipase A2 in Hexaplex trunculus digestive cells.

BACKGROUND: Mammalian sPLA2-IB localization cell are well characterized. In contrast, much less is known about aquatic primitive ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes and the mode of digestion of lipid food. RESULTS: The marine snail digestive phospholipase A2 (mSDPLA2) has been previously purified from snail hepatopancreas. The specific polyclonal antibodies were prepared and used for immunohistochimical and immunofluorescence analysis in order to determine the cellular location of mSDPLA2. Our results showed essentially that mSDPLA2 was detected inside in specific vesicles tentatively named (mSDPLA2+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion in the snail might occur in specific granules inside the digestive cells. CONCLUSION: The cellular location of mSDPLA2 suggests that intracellular phospholipids digestion, like other food components digestion of snail diet, occurs in these digestive cells. The hepatopancreas of H. trunculus has been pointed out as the main region for digestion, absorption and storage of lipids.

CD8 positive T cells express IL-17 in patients with chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a progressive and irreversible chronic inflammatory disease of the lung. The nature of the immune reaction in COPD raises the possibility that IL-17 and related cytokines may contribute to this disorder. This study analyzed the expression of IL-17A and IL-17F as well as the phenotype of cells producing them in bronchial biopsies from COPD patients. METHODS: Bronchoscopic biopsies of the airway were obtained from 16 COPD subjects (GOLD stage 1-4) and 15 control subjects. Paraffin sections were used for the investigation of IL-17A and IL-17F expression in the airways by immunohistochemistry, and frozen sections were used for the immunofluorescence double staining of IL-17A or IL-17F paired with CD4 or CD8. In order to confirm the expression of IL-17A and IL-17F at the mRNA level, a quantitative RT-PCR was performed on the total mRNA extracted from entire section or CD8 positive cells selected by laser capture microdissection. RESULTS: IL-17F immunoreactivity was significantly higher in the bronchial biopsies of COPD patients compared to control subjects (P < 0.0001). In the submucosa, the absolute number of both IL-17A and IL-17F positive cells was higher in COPD patients (P < 0.0001). After adjusting for the total number of cells in the submucosa, we still found that more cells were positive for both IL-17A (P < 0.0001) and IL-17F (P < 0.0001) in COPD patients compared to controls. The mRNA expression of IL-17A and IL-17F in airways of COPD patients was confirmed by RT-PCR. The expression of IL-17A and IL-17F was co-localized with not only CD4 but also CD8, which was further confirmed by RT-PCR on laser capture microdissection selected CD8 positive cells. CONCLUSION: These findings support the notion that Th17 cytokines could play important roles in the pathogenesis of COPD, raising the possibility of using this mechanism as the basis for novel therapeutic approaches.

Development of an in vitro coculture of primary sensitive pig neurons and keratinocytes for the study of cutaneous neurogenic inflammation.

Cutaneous neurogenic inflammation (CNI) is often associated with skin disorders. Activated sensory neurons secrete neuropeptides, such as substance P (SP), which initiate or aggravate inflammation in the skin. The discovery of new molecules acting on these neurons is hampered by the difficulty of reproducing the interactions between nerve endings and skin in vitro. We developed an in vitro model based on the coculture of porcine primary keratinocytes and sensory neurons, which mimics skin innervation. To test the relevance of this model, we compared the effects of different substances on CNI by measuring SP secretion in vitro using a sensitive enzyme immunoassay. Collectively, our results indicate that the use of porcine cells could be very useful to perform an in vitro model of CNI. By adding capsaicin, which induces the secretion of SP by neurons, to the culture, we show that our model mimics CNI in vitro, allowing us to screen for molecules that inhibit this inflammatory response. Such a model can be used to test the effects of different substances on CNI and may be useful for dermatological or cosmetic applications. Based on our screen, we found that extracts of Laminaria digitata and Vernonia sublutea inhibit CNI.

Effects of sangre de drago in an in vitro model of cutaneous neurogenic inflammation.

Sangre de drago (SD) is a viscous bright red resin collected from Croton lechleri trees that grow in the South American jungle. This sap is used extensively in the native pharmacopoeia to treat skin disorders. Its effectiveness as an inhibitor of neurogenic inflammation has been recently demonstrated. To understand the underlying mechanisms of these effects, we examined the ability of SD to reduce substance P (SP) release in an in vitro model of cutaneous neurogenic inflammation (CNI). This model is based on an enzyme immunoassay of SP (an inducer of CNI) in a porcine co-culture of dorsal root ganglion neurons and keratinocytes. After incubation with different concentrations of SD, we noted an immediate and significant dose-dependent decrease in basal SP release, with average values of 32% at 1% SD (v/v) and 26% at 0.1% (v/v). On the other hand, pretreatment (72 or 1 h) of the co-culture with 1% SD (v/v) was sufficient to induce a 111% (72 h) or 65% (1 h) inhibition of capsaicin-induced SP release, while 0.1% SD (v/v) triggered a 109% (72 h) or 30% (1 h) inhibition. We conclude that sangre de drago is a potent inhibitor of CNI through direct inhibition of neuropeptide release by sensory afferent nerves.

Rat Merkel cells are mechanoreceptors and osmoreceptors.

Merkel cells (MCs) associated with nerve terminals constitute MC-neurite complexes, which are involved in slowly-adapting type I mechanoreception. Although MCs are known to express voltage-gated Ca2+ channels and hypotonic-induced membrane deformation is known to lead to Ca2+ transients, whether MCs initiate mechanotransduction is currently unknown. To answer to this question, rat MCs were transfected with a reporter vector, which enabled their identification.Their properties were investigated through electrophysiological studies. Voltage-gated K+, Ca2+ and Ca2+-activated K+ (KCa)channels were identified, as previously described. Here, we also report the activation of Ca2+ channels by histamine and their inhibition by acetylcholine. As a major finding, we demonstrated that direct mechanical stimulations induced strong inward Ca2+ currents in MCs. Depolarizations were dependent on the strength and the length of the stimulation. Moreover, touch-evoked currents were inhibited by the stretch channel antagonist gadolinium. These data confirm the mechanotransduction capabilities of MCs. Furthermore, we found that activation of the osmoreceptor TRPV4 in FM1-43-labeled MCs provoked neurosecretory granule exocytosis. Since FM1-43 blocks mechanosensory channels, this suggests that hypo-osmolarity activates MCs in the absence of mechanotransduction. Thus, mechanotransduction and osmoreception are likely distinct pathways.

Merkel cells as putative regulatory cells in skin disorders: an in vitro study.

Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca(2+)-independent, as inhibition of Ca(2+) channels or culture in the absence of Ca(2+) failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated.

The epidermis: a sensory tissue.

The skin is an efficient barrier which protects our bodies from the external environment but it is also an important site for the perception of various stimuli. Sensory neurones of the peripheral nervous system send many primary afferent fibres to the skin. They pass through the dermis and penetrate the basement membrane to innervate epidermal cells or remain as free endings. Nerve fibres are clearly involved in somatosensation. However, they are not always so numerous, for example in distal parts of the limbs, and some kinds of sensors can be at a distance of hundreds of micrometers from each other. The skin can detect patterns at a very fine and smaller scale, which suggests that nerve terminals are helped by epidermal sensors. All epidermal cells (keratinocytes, melanocytes, Langerhans cells and Merkel cells) express sensor proteins and neuropeptides regulating the neuro-immuno-cutaneous system. Hence, they must play a part in the epidermal sensory system. This review will consider the epidermal components of this forefront sensory system and the stimulations they perceive. The epidermis can be considered a true sensory tissue where sensor proteins and neurone-like properties enable epidermal cells to participate in the skin surface perception through interactions with nerve fibres.

Merkel cells.

Merkel cells are post-mitotic cells scattered throughout the epidermis of vertebrates. They are particularly interesting because of the close connections that they develop with sensory nerve endings and the number of peptides they can secrete. These features suggest that they may make an important contribution to skin homeostasis and cutaneous nerve development. However, these cells remain mysterious because they are difficult to study. They have not been successfully cultured and cannot be isolated, severely hampering molecular biology and functional analysis. Merkel cells probably originate in the neural crest of avians and mammalians, and their "spontaneous" appearance in the epidermis may be caused by a neuron-independent epidermal differentiation process. Their functions are still unclear: they take part in mechanoreception or at least interact with neurons, but little is known about their interactions with other epidermal cells. This review provides a new look at these least-known cells of the skin. The numerous peptides they synthesize and release may allow them to communicate with many cells other than neurons, and it is plausible that Merkel cells play a key role in skin physiology and physiopathology.