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Skin irritation evaluation is an important endpoint for the safety assessment of cosmetic ingredients required by various regulatory authorities for notification and/or import of test substances. The present study was undertaken to investigate possible protocol adaptations of the currently validated in vitro skin irritation test methods based on reconstructed human epidermis (RhE) for the testing of plant extracts and natural botanicals. Due to their specific physico-chemical properties, such as lipophilicity, sticky/buttery-like texture, waxy/creamy foam characteristics, normal washing procedures can lead to an incomplete removal of these materials and/or to mechanical damage to the tissues, resulting in an impaired prediction of the true skin irritation potential of the materials. For this reason different refined washing procedures were evaluated for their ability to ensure appropriate removal of greasy and sticky substances while not altering the normal responses of the validated RhE test method. Amongst the different procedures evaluated, the use of a SDS 0.1% PBS solution to remove the sticky and greasy test material prior to the normal washing procedures was found to be the most suitable adaptation to ensure efficient removal of greasy and sticky in-house controls without affecting the results of the negative control. The predictive capacity of the refined SDS 0.1% washing procedure, was investigated by using twelve oily and viscous compounds having known skin irritation effects supported by raw and/or peer reviewed in vivo data. The normal washing procedure resulted in 8 out of 10 correctly predicted compounds as compared to 9 out of 10 with the refined washing procedures, showing an increase in the predictive ability of the assay. The refined washing procedure allowed to correctly identify all in vivo skin irritant materials showing the same sensitivity as the normal washing procedures, and further increased the specificity of the assay from 5 to 6 correct predictions out of 7 non irritants as compared to the normal washing procedures. In addition, when exposed to non-irritant oily and viscous materials, tissues rinsed with 0.1% SDS generally showed increased viabilities accompanied by decreased variabilities as compared to the normal washing procedures. Similar results were obtained when testing typical in-house natural botanical ingredients. In conclusion, the use of a refined washing procedure making use of SDS 0.1% in PBS was found a suitable procedure to ensure efficient removal of greasy and sticky materials, leading to an increased predictive capacity and decreased variability of the tissue responses while maintaining its sensitivity and not affecting untreated tissues morphology and viability., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The epidermis can be considered as a sensory organ. Sensory neurons of the peripheral nervous system send many primary afferent fibers to the skin, creating a dynamic communication with epidermal cells. However, little is known about the functional interactions between the sensory fibers and the keratinocytes. It is therefore difficult to reproduce these interactions in vitro. We have developed an in vitro model based on the coculture of primary human keratinocytes and the dorsal root ganglion cell line F-11. F-11 cells have been classically used to mimic authentic peptidergic nociceptive neurons. We first investigated the morphological and functional characteristics of F-11 cells cultured in basal keratinocyte medium and then analyzed the influence of keratinocytes on these properties. We found that F-11 cells survived and differentiated well in this medium. Therefore, the addition of neurotrophins did not enhance their survival or differentiation. These neurons expressed sensory neuron markers and were able to release neuropeptides after capsaicin activation. We noted that neuropeptides release were obtained even at a low calcium concentration and that axonal outgrowth was not influenced by external calcium (Ca(2+)) levels. These properties were reproduced when F-11 cells were cocultured with keratinocytes, but they had no significant influence on axonal development or neuropeptide release. In this study, we describe for the first time the culture of F-11 neurons with another cell type. This coculture model in which keratinocytes and neurons are maintained in low Ca(2+) concentrations may be a useful in vitro alternative for studying and characterizing the close communication between keratinocytes and sensory neurons., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Currently, the cosmetics industry relies on the results of in vitro genotoxicity tests to assess the safety of chemicals. Although the cytokinesis-block micronucleus (CBMN) test for the detection of cells that have divided once is routinely used and currently accepted by regulatory agencies, it has some limitations. Reconstituted human epidermis (RHE) is widely used in safety assessments because its physiological properties resemble those of the skin, and because it allows testing of substances such as hydrophobic compounds. Thus, the micronucleus test is being adapted for application in RHE-reconstructed tissues. Here we investigated whether two different reconstructed epidermis models (EPI/001 from Straticell, and RHE/S/17 from Skinethic) are suitable for application of the micronucleus test. We found that acetone does not modify micronucleus frequency, cell viability, and model structure, compared with non-treated RHE. Treatment of the EPI/001 model with mitomycin C and vinblastine resulted in a dose-dependent increase of micronucleus frequency as well as a decrease of tissue viability and of binucleated cell rate, while no changes of the epidermal structure were observed. The number of binucleated cells obtained with the RHE/S/17 model was too small to permit micronucleus testing. These results indicate that the proliferative rate of the tissue used is a critical parameter in performing the micronucleus test on a 3D model., (© 2012 Elsevier B.V. All rights reserved.)

In adult animals, bone marrow is the major site of blood cell production, which is controlled by interactions between the local stroma and blood cell progenitors. The endosteal/subendosteal environment comprises bone-lining and adjacent reticular cells and sustains haemopoietic stem cell (HSC) self-renewal, proliferation and differentiation. We have questioned the specific role of each of these stroma cells in controlling HSC fate. We have isolated two distinct stroma-cell populations containing subendosteal reticulocytes (F-RET) and osteoblasts (F-OST) from periosteum-free fragments of murine femurs by a two-step collagenase-digestion procedure. Both populations produce similar extracellular matrix (collagen I, laminin, fibronectin, decorin), except for collagen IV, which is low in F-OST. They also express osteogenic markers: osteopontin, osteonectin, bone sialoprotein and alkaline phosphatase (ALP). The quantity and activity of ALP are however higher in F-OST. When co-cultured with bone marrow mononuclear cells or lineage-negative haemopoietic progenitors, F-OST stroma induces low proliferation and high maintenance of early haemopoietic progenitors, whereas F-RET stroma induces high short-term proliferation and differentiation. Analysis by reverse transcription/polymerase chain reaction has revealed higher levels of Jagged-1 expression by F-OST cells than by the F-RET population. Thus, two adjacent stroma cells (subendosteal and endosteal) play distinct roles in controlling the stem-cell capacity and fate of HSC and probably contribute distinctly to HSC niche formation.

The coelome-associated lympho-myeloid tissues, including the omentum, are derived from early embryo haemopoietic tissue of the splanchnopleura, and produce B lymphocytes and macrophages. They are reactive in pathologies involving coelomic cavities, in which they can expand in situ the cells of inflammatory infiltrates. We have addressed the question of the role of the adult omentum in permanent basal production of early lymphopoietic progenitors (pro-B/pre-B cells), through characterisation of omentum cells ex vivo, and study of their in vitro differentiation. We have shown that the murine omentum produces early haemopoietic progenitors throughout life, including B-cell progenitors prior to the Ig gene recombination expressing RAG-1 and lambda5, as well as macrophages. Their production is stroma-dependent. The omentum stroma can supply in vitro the cytokines (SDF-1alpha, Flt3 ligand and IL-7) and the molecular environment required for generation of these two cell lineages. Omentum haemopoietic progenitors are similar to those observed in foetal blood cell production, rather than to progenitors found in the adult haemopoietic tissue in the bone marrow--in terms of phenotype expression and differentiation capacity. We conclude that a primitive pattern of haemopoiesis observed in the early embryo is permanently preserved and functional in the adult omentum, providing production of cells engaged in nonspecific protection of abdominal intestinal tissue and of the coelomic cavity.

We examined gap junction communication in an in vitro model of hematopoiesis, using the murine bone marrow stroma cell line S-17, and primary cultures of murine marrow-derived blood cell precursors. S-17 cells express several connexins, the major one being connexin 43. Connexin expression and formation of functional gap junctions is modulated by stroma cell density. Transfection of S-17 cells with a vector containing connexin 43 sense or anti-sense sequences increased or decreased, respectively, connexin 43 synthesis and intercellular dye coupling. Under these conditions, modulation of gap junction-mediated communication modified the growth pattern of stroma itself, as well as the ability of the stroma to sustain hematopoiesis. Increased connexin 43 expression was associated with a delay in differentiation of blood cells, resulting in increased production of hematopoietic precursors, while decreased connexin 43 expression elicited an accelerated differentiation of myeloid blood cell precursor cells. These results suggest that connexin-mediated coupling in the stroma modulates the ratio between proliferation and differentiation of hematopoietic precursors. We therefore propose that increased gap junction communication in the stroma elicits an enhanced production of immature bone marrow cells through the delay in their terminal differentiation, inducing consequently an extended proliferation period of blood cell precursors.

In addition to the steady-state production of all blood cells, bone marrow can respond to an increased requirement for one or several cell lineages. The hormonal controls involved may act directly on blood cell progenitors or indirectly through modification of the haemopoietic environment. Intercellular gap junctions formed by connexins (Cx) provide direct communication among adjacent cells and the functional integration of multicellular systems. Since haemopoietic stroma is determinant for blood cell production, we have questioned whether gap-junction-dependent controls of haemopoiesis are sensitive to hormones and vitamins. We have analysed the expression, synthesis, cell distribution and formation of functional gap junctions in the murine bone-marrow stroma cell line S-17, and between stromal cells and blood cell progenitors. Nine Cxs were identified by reverse transcription/polymerase chain reaction, and only Cx43 by Western blot and immunofluorescence. All of the studied parameters were sensitive to intrinsic controls dependent upon the pattern of cell growth and modulated by exogenous controls mediated by retinol and steroids. Positive or negative modulation was specific for different Cxs. FACS analysis showed communication among the stromal cells and between stromal cells and myeloid (Mac1+) but not lymphoid (B220+) progenitors. Calcein transfer modulation did not correspond to the modulation of Cx43 expression and formation of connexons, suggesting the participation of other Cxs. Thus, functional gap junctions among haemopoietic stroma cells and between stroma and haematopoietic cells in the bone marrow may be modulated in response to hormonal stimuli, potentially controlling overall blood cell production.

Coelomic cavities are relatively isolated from the systemic circulation of blood cells. Resident cell populations have a proper phenotype and kinetics, maintaining their steady-state populations and their responsiveness to local inflammatory reactions, in which the number and quality of coelomic cells can be greatly increased and modified. We have addressed the question of whether the increase in cell infiltrate in the inflamed abdominal cavity is sustained by the proliferation of myeloid cells in the omentum, and if so what are the characteristics of the progenitor cells involved and how the omentum controls their proliferation and differentiation. In the omentum under normal conditions and with inflammation due to schistosomal infection we found that pluripotent early myeloid progenitors were capable of giving rise to all the myeloid lineages in clonogenic assays, but not to the totipotent blood stem cells. Besides the major haemopoietins (GM-CSF, M-CSF, G-CSF, IL-5), the omentum stroma constitutively expressed SDF-1 alpha, the chemokine which elicits homing of circulating early haemopoietic progenitors. While normal omentum stroma produced LIF, its expression was substituted by SCF in inflamed tissues. In the first situation a slow steady-state renewal of progenitors is potentially favoured, while their intense expansion may be predominant in the latter one. We propose that the increase in cells in the abdominal cavity in inflammatory reactions is due to the enhanced input and expansion of early myeloid progenitors sustaining the in situ production of abdominal cell populations, rather than to the input of systemic circulating inflammatory cells.