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Fibrillin-rich microfibrils (FRMs) constitute integral components of the dermal elastic fibre network with a distinctive ultrastructural 'beads-on-a-string' appearance that can be visualised using atomic force microscopy and characterised by measurement of their length and inter-bead periodicity. Their deposition within the dermis in photoprotected skin appears to be contingent on skin ethnicity, and influences the ultrastructure of papillary - but not reticular - dermal FRMs. Truncation and depletion of FRMs at the dermal-epidermal junction of skin occurs early in photoageing in people with lightly pigmented skin; a process of accelerated skin ageing that arises due to chronic sun exposure. Accumulation of ultraviolet radiation (UVR)-induced damage, either by the action of enzymes, oxidation or direct photon absorption, results in FRM remodelling and changes to ultrastructure. In the current study, the direct effect of UVR exposure on FRM ultrastructure was assayed by isolating FRMs from the papillary and reticular dermis of photoprotected buttock skin of individuals of either black African or white Northern European ancestry and exposing them to solar-simulated radiation (SSR). Exposure to SSR resulted in significant reduction in inter-bead periodicity for reticular dermis-derived FRMs across both cohorts. In contrast, papillary dermal FRMs exhibited significantly increased inter-bead periodicity, with the magnitude of damage greater for African FRMs, as compared to Northern European FRMs. Our data suggest that FRMs of the dermis should be considered as two distinct populations that differentially accrue damage in response to SSR. Furthermore, papillary dermal FRMs derived from black African subjects show greater change following UVR challenge, when extracted from skin. Future studies should focus on understanding the consequences of UVR exposure in vivo, regardless of skin ethnicity, on the molecular composition of FRMs and how this UVR-induced remodelling may affect the role FRMs play in skin homeostasis.

The dermal elastic fibre network is the primary effector of skin elasticity, enabling it to extend and recoil many times over the lifetime of the individual. Fibrillin-rich microfibrils (FRMs) constitute integral components of the elastic fibre network, with their distribution showing differential deposition in the papillary dermis across individuals of diverse skin ethnicity. Despite these differential findings in histological presentation, it is not known if skin ethnicity influences FRM ultrastructure. FRMs are evolutionarily highly conserved from jellyfish to man and, regardless of tissue type or species, isolated FRMs have a characteristic 'beads-on-a-string' ultrastructural appearance, with an average inter-bead distance (or periodicity) of 56 nm. Here, skin biopsies were obtained from the photoprotected buttock of healthy volunteers (18-27 years; African: n = 5; European: n = 5), and FRMs were isolated from the superficial papillary dermis and deeper reticular dermis and imaged by atomic force microscopy. In the reticular dermis, there was no significant difference in FRM ultrastructure between European and African participants. In contrast, in the more superficial papillary dermis, inter-bead periodicity was significantly larger for FRMs extracted from European participants than from African participants by 2.20 nm (p < .001). We next assessed whether these differences in FRM ultrastructure were present during early postnatal development by characterizing FRMs from full-thickness neonatal foreskin. Analysis of FRM periodicity identified no significant difference between neonatal cohorts (p = .865). These data suggest that at birth, FRMs are developmentally invariant. However, in adults of diverse skin ethnicity, there is a deviation in ultrastructure for the papillary dermal FRMs that may be acquired during the passage of time from child to adulthood. Understanding the mechanism by which this difference in papillary dermal FRMs arises warrants further study.

Langerhans cells (LCs) are sentinels of skin's immune system, their loss from epidermis contributing to UVR suppression of cell-mediated immunity (CMI). Omega-3 polyunsaturated fatty acids show potential to reduce UVR suppression of CMI in mice and humans, potentially through modulation of LC migration. Our objectives were to examine whether eicosapentaenoic acid (EPA) ingestion influences UV-mediated effects on epidermal LC numbers and levels of immunomodulatory mediators including prostaglandin (PG)D2 , which is expressed by LC. In a double-blind randomised controlled study, healthy individuals took 5-g EPA-rich (n=40) or control (n=33) lipid for 12 weeks; UVR-exposed and unexposed skin samples were taken pre- and postsupplementation. Epidermal LC numbers were assessed by immunofluorescence for CD1a, and skin blister fluid PG and cytokines were quantified by LC-MS/MS and Luminex assay, respectively. Presupplementation, UVR reduced mean (SEM) LC number/mm2 from 913 (28) to 322 (40) (P

Excessive ultraviolet radiation (UVR) exposure of the skin is associated with adverse clinical outcomes. Although both exogenous sunscreens and endogenous tissue components (including melanins and tryptophan-derived compounds) reduce UVR penetration, the role of endogenous proteins in absorbing environmental UV wavelengths is poorly defined. Having previously demonstrated that proteins which are rich in UVR-absorbing amino acid residues are readily degraded by broadband UVB-radiation (containing UVA, UVB and UVC wavelengths) here we hypothesised that UV chromophore (Cys, Trp and Tyr) content can predict the susceptibility of structural proteins in skin and the eye to damage by physiologically relevant doses (up to 15.4 J/cm2) of solar UVR (95% UVA, 5% UVB). We show that: i) purified suspensions of UV-chromophore-rich fibronectin dimers, fibrillin microfibrils and β- and γ-lens crystallins undergo solar simulated radiation (SSR)-induced aggregation and/or decomposition and ii) exposure to identical doses of SSR has minimal effect on the size or ultrastructure of UV chromophore-poor tropoelastin, collagen I, collagen VI microfibrils and α-crystallin. If UV chromophore content is a factor in determining protein stability in vivo, we would expect that the tissue distribution of Cys, Trp and Tyr-rich proteins would correlate with regional UVR exposure. From bioinformatic analysis of 244 key structural proteins we identified several biochemically distinct, yet UV chromophore-rich, protein families. The majority of these putative UV-absorbing proteins (including the late cornified envelope proteins, keratin associated proteins, elastic fibre-associated components and β- and γ-crystallins) are localised and/or particularly abundant in tissues that are exposed to the highest doses of environmental UVR, specifically the stratum corneum, hair, papillary dermis and lens. We therefore propose that UV chromophore-rich proteins are localised in regions of high UVR exposure as a consequence of an evolutionary pressure to express sacrificial protein sunscreens which reduce UVR penetration and hence mitigate tissue damage., Graphical abstract, Highlights Major structural proteins such as collagen I and tropoelastin are UVA-resistant. In contrast, proteins which are rich in Cys, Trp and Tyr residues are UV-susceptible. These proteins are concentrated in UV exposed tissues. UV-chromophore (Cys, Trp, Tyr)-rich proteins may act as endogenous sunscreens.

Serum response factor (SRF) recruits members of two families of signal-regulated coactivators, the extracellular signal-regulated kinase (ERK)-regulated ternary complex factors (TCFs) and the actin-regulated myocardin-related transcription factors (MRTFs), to its target genes through its DNA-binding domain. Whether coactivator association is required for SRF function in vivo and whether particular SRF functions reflect specific coupling to one or the other signal pathway have remained largely unexplored. We show that SRF is essential for thymocyte positive selection and thymic T(reg) and NK T-cell development but dispensable for early thymocyte development and negative selection. Expression of wild-type SRF, or mutants lacking the N-terminal phosphorylation sites or C-terminal transcriptional activation domain, restores positive selection in SRF null thymocytes. In contrast, SRF.V194E, which cannot recruit TCF or MRTF family members, is inactive, although it is recruited to target genes. Fusion of a TCF C-terminal activation domain to SRF.V194E effectively restores ERK-dependent single-positive (SP) thymocyte development. The resulting SP thymocytes exhibit normal surface marker expression and proliferation following T-cell receptor cross-linking. Thus, ERK signaling through the TCF pathway to SRF is necessary and sufficient for SRF function in thymocyte positive selection.

Regulatory T cells (Treg) play an important role in immune regulation. Their development in the thymus requires TCR activation and recognition of peptide-MHC, although the downstream signals controlling commitment to the lineage are unclear. To compare the requirements for positive selection and Treg development, we studied knockout and transgenic mice defective in Raf signaling and the ERK effector SRF accessory protein 1 (SAP-1), a member of the ternary complex factor family of Ets domain transcription factors. Although SAP-1 deficient mice display a severe defect in thymocyte positive selection, Treg development was unimpaired as assessed by expression of Foxp3 and the activation markers CD25, GITR, CTLA4, and CD103 in the CD4+ cell population. In contrast, inhibition of Raf signaling by the interfering dominant negative Raf derivative reduced both Foxp3+ and Foxp3− CD4+ populations. In SAP-1-deficient CD4+CD25+ Treg cells, TCR crosslinking efficiently induced ERK activation, but transcriptional induction of the immediate early gene Egr-1 was impaired. Nevertheless, neither deletion of SAP-1 nor expression of a dominant negative Raf derivative affected the ability of CD4+CD25+ Treg cells to suppress CD4+CD25− cell proliferation in vitro. Finally the suppressive activity of CD4+CD25+ Treg cells lacking SAP-1 in an in vivo colitis model was not significantly impaired. The signaling requirements for development of Treg cells in the thymus are thus distinct from those required for “conventional” T cell positive selection, and ERK signaling to SAP-1 is not required for the suppressive activity of Treg cells.

International audience; Chemokine signaling is important for the seeding of different sites by hematopoietic stem cells (HSCs) during development. Serum response factor (SRF) controls multiple genes governing adhesion and migration, mainly by recruiting members of the myocardin-related transcription factor (MRTF) family of G-actin-regulated cofactors. We used vav-iCre to inactivate MRTF-SRF signaling early during hematopoietic development. In both Srf- and Mrtf-deleted animals, hematopoiesis in fetal liver and spleen is intact but does not become established in fetal bone marrow. Srf-null HSC progenitor cells (HSC/Ps) fail to effectively engraft in transplantation experiments, exhibiting normal proximal signaling responses to SDF-1, but reduced adhesiveness, F-actin assembly, and reduced motility. Srf-null HSC/Ps fail to polarize in response to SDF-1 and cannot migrate through restrictive membrane pores to SDF-1 or Scf in vitro. Mrtf-null HSC/Ps were also defective in chemotactic responses to SDF-1. Srf-null HSC/Ps exhibit substantial deficits in cytoskeletal gene expression. MRTF-SRF signaling is thus critical for expression of genes required for the response to chemokine signaling during hematopoietic development.

T cell activation is triggered by several hours of contact with peptide-major histocompatibility (MHC) complexes on the surface of antigen-presenting cells (APCs). The nature and location of the sustained signal transduction pathways required for T cell activation are unknown. We show here that the production of phosphatidylinositol(3,4,5)triphosphate (PIP3) was dynamically sustained for hours as T cells responded to antigen. In addition, sustained elevation of PIP3 was essential for T cell proliferation. There was PIP3 accumulation in the T cell-APC contact zone and at the antipodal pole of the cell. The immune synapse is thus not the sole site of sustained signal transduction in activated T cells.

Adenovirus sensitivity, TCD50 value and mean time to cytopathic effect (CPE) were compared in Madin-Darby canine kidney (MDCK), rhesus monkey kidney (RhMK), human lung carcinoma (A549) and in newly identified continuous human lung (CHL) cell cultures. High-titer stock and positive original specimens were inoculated in culture. Patients specimens were also placed in shell-vial cultures. A549 and CHL cells demonstrated comparable sensitivity, however the former generally produced CPE earlier. Sensitivity was respectively reduced and poor in RhMK and MDCK cultures, as expected in cells of nonhuman origin. The performance of CHL for adenoviruses and other pathogens makes them a useful addition in diagnostic virology laboratories.

Monoclonal antibody pools directed against group B coxsackievirus and echovirus antigens (Chemicon, Temecula, California) were evaluated as tools in the identification of enteroviral aseptic meningitis. Cerebrospinal fluid (CSF) and serial dilutions of stock coxsackievirus were inoculated into tube and shell vial Rhesus monkey kidney (RhMk) cell cultures. Positive cellular fluorescence was observed only in conjunction with cytopathic effect (CPE). The time from inoculation to CPE was similar with both tube and shell vial cultures.

The ternary complex factors (TCFs; SAP-1, Elk-1, and Net) are serum response factor cofactors that share many functional properties and are coexpressed in many tissues. SAP-1, the predominant thymus TCF, is required for thymocyte positive selection. In this study, we assessed whether the different TCFs are functionally equivalent. Elk-1 deletion, but not the hypomorphic Netδ mutation, exacerbated the SAP-1 positive selection phenotype, but triply deficient thymocytes were no more defective than SAP-1−/− Elk-1−/− cells. Inactivation of the other TCFs did not affect SAP-1–independent processes, including β-selection, regulatory T cell selection, and negative selection, although reduced marginal zone B cells were observed in SAP-1−/− Elk-1−/− animals. Ectopic expression of Elk-1, but not Net, rescued positive selection of SAP-1−/− thymocytes; thus, SAP-1 and Elk-1 are functionally equivalent in this system, and the SAP-1 null selection phenotype reflects only its high expression in the thymus. Array analysis of TCR-stimulated double-positive cells identified SAP-1–dependent inducible genes whose transcription was further impaired in SAP-1−/− Elk-1−/− cells; thus, these genes, which include Egr-1 and Egr-2, represent candidate mediators of positive selection. Chromatin immunoprecipitation revealed subtly different promoter targeting between the different TCFs. Ectopic expression of Egr-1 restored positive selection in SAP-1 null thymocytes, establishing it (and possibly other Egr family members) as the major effector for ERK–SAP-1 signaling in thymocyte positive selection.

In an effort to reduce the number of unreadable MRC-5 cell culture monolayers (i.e., monolayers that are obscured by an opaque film or are detached from their coverslips), urine specimens were subjected to low-speed (500 g for 10 min at 4°C) centrifugation prior to their inoculation into shell vials for the detection of cytomegalovirus (CMV). A total of 182 urine specimens obtained from patients suffering from ARC or AIDS were subjected to routine centrifugation culture and to preinoculation centrifugation. Cytomegalovirus recovery among the two specimen-treatment procedures was determined by counting the number of immunofluorescent MRC 5-positive cells. Eleven of 19 positive specimens were in accord in both systems. However, routinely treated (i.e., without preinoculation centrifugation) specimens were difficult to read specifically among those specimens that were cloudy or opaque. Cytome-galovirus was recovered in 18 of the 19 positive specimens that were subjected to the preinoculation centrifugation procedure. Only 12 of 19 positive specimens were identified following direct inoculation of untreated urine into shell vials. The additional preinoculation centrifugation step is appropriate for the removal of obfuscating amorphous substances from the assay system without compromising CMV detection rates.

The aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) on protease and catabolic cytokine and receptor gene expression in normal and degenerate human nucleus pulposus cells in alginate culture.Cells isolated from normal and degenerate nucleus pulposus regions of human intervertebral discs were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-alpha or IL-1beta for 48 hours prior to RNA extraction. Quantitative real-time polymerase chain reaction was used to assess the effect of TNF-alpha or IL-beta stimulation on the expression of matrix metalloproteinase (MMP)-3, -9 and -13, TNF-alpha, TNF receptor 1 (TNF-R1), TNF receptor 2 (TNF-R2), IL-1alpha, IL-1beta, IL-1 receptor 1 (IL-1R1) and IL-1 receptor antagonist (IL-1Ra).MMP-3 and MMP-9 gene expressions were upregulated to a greater level by IL-1beta than TNF-alpha. MMP-13 was upregulated by each cytokine to a similar extent. TNF-alpha and TNF-R2 expressions were upregulated by both TNF-alpha and IL-beta, whereas TNF-R1 expression was not significantly affected by either cytokine. IL-1beta and IL-1Ra expressions were significantly upregulated by TNF-alpha, whereas IL-1alpha and IL-1R1 were unchanged.TNF-alpha does not induce MMP expression to the same degree as stimulation by IL-1beta, but it does act to upregulate IL-1beta expression as well as TNF-alpha and TNF-R2. The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix. These results support the hypothesis that, while TNF-alpha may be an important initiating factor in matrix degeneration, IL-1beta plays a greater role in established pathological degradation.