Expression and Function of the Mannose Receptor CD206 on Epidermal Dendritic Cells in Inflammatory Skin Diseases

The capability to take up mannosylated protein antigens is important for the biologic function of dendritic cells, as many glycoproteins derived from bacteria and fungi, e.g., Malassezia furfur, are mannosylated. The expression of the mannose receptor CD206 has been regarded a differentiation hallmark of immature dendritic cells, whereas monocytes and mature dendritic cells as well as epidermal Langerhans cells do not express CD206. This study describes some epidermal dendritic cells that may express CD206 under inflammatory skin conditions: Immunohistochemical and flow cytometric analysis with the CD206-specific D547 antibody confirmed that Langerhans cells from normal human skin do not express CD206. Epidermal cell suspensions from atopic dermatitis and psoriasis revealed two distinct subsets of epidermal dendritic cells: a CD1a+++/CD206– cell population (i.e., Langerhans cells) and a CD1a+/CD206++ cell population, corresponding to the previously described inflammatory dendritic epidermal cells. CD206-mediated endocytosis, assessed by dextran-fluorescein isothiocyanate uptake, was demonstrated in inflammatory dendritic epidermal cells but not in Langerhans cells. CD206-independent uptake of the fluorescent dye Lucifer yellow, a pinocytosis marker, was demonstrated in both Langerhans cells and inflammatory dendritic epidermal cells. Electron microscopic examination, known to distinguish Langerhans cells from inflammatory dendritic epidermal cells by their Birbeck granules, revealed Langerhans cells with Birbeck granules and inflammatory dendritic epidermal cells without Birbeck granules. Inflammatory dendritic epidermal cells exhibited numerous coated pits and vesicles, the latter fusing with large endosome-like structures, thus suggesting a high endocytotic activity. Immunogold staining with D547 monoclonal antibody confirmed that inflammatory dendritic epidermal cells were positive for CD206. In conclusion, inflammatory dendritic epidermal cells but not Langerhans cells are expressing CD206 in situ and use it for receptor-mediated endocytosis

Drosophila CPEB Orb2A Mediates Memory Independent of Its RNA-Binding Domain

SummaryLong-term memory and synaptic plasticity are thought to require the synthesis of new proteins at activated synapses. The CPEB family of RNA binding proteins, including Drosophila Orb2, has been implicated in this process. The precise mechanism by which these molecules regulate memory formation is however poorly understood. We used gene targeting and site-specific transgenesis to specifically modify the endogenous orb2 gene in order to investigate its role in long-term memory formation. We show that the Orb2A and Orb2B isoforms, while both essential, have distinct functions in memory formation. These two isoforms have common glutamine-rich and RNA-binding domains, yet Orb2A uniquely requires the former and Orb2B the latter. We further show that Orb2A induces Orb2 complexes in a manner dependent upon both its glutamine-rich region and neuronal activity. We propose that Orb2B acts as a conventional CPEB to regulate transport and/or translation of specific mRNAs, whereas Orb2A acts in an unconventional manner to form stable Orb2 complexes that are essential for memory to persist

Association of the Hermansky-Pudlak syndrome type-3 protein with clathrin

BACKGROUND: Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. These clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. HPS type-3 (HPS-3) results from mutations in the HPS3 gene, which encodes a 1004 amino acid protein of unknown function that contains a predicted clathrin-binding motif (LLDFE) at residues 172–176. RESULTS: Clathrin was co-immunoprecipitated by HPS3 antibodies from normal but not HPS3 null melanocytes. Normal melanocytes expressing a GFP-HPS3 fusion protein demonstrated partial co-localization of GFP-HPS3 with clathrin following a 20°C temperature block. GFP-HPS3 in which the predicted clathrin-binding domain of HPS3 was mutated (GFP-HPS3-delCBD) did not co-localize with clathrin under the same conditions. Immunoelectron microscopy of normal melanocytes expressing GFP-HPS3 showed co-localization of GFP-HPS3 with clathrin, predominantly on small vesicles in the perinuclear region. In contrast, GFP-HPS3-delCBD did not co-localize with clathrin and exhibited a largely cytoplasmic distribution. CONCLUSION: HPS3 associates with clathrin, predominantly on small clathrin-containing vesicles in the perinuclear region. This association most likely occurs directly via a functional clathrin-binding domain in HPS3. These results suggest a role for HPS3 and its protein complex, BLOC-2, in vesicle formation and trafficking

SARS-Coronavirus Replication/Transcription Complexes Are Membrane-Protected and Need a Host Factor for Activity In Vitro

SARS-coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures

Conversion of Mature Human β-Cells Into Glucagon-Producing α-Cells

Conversion of one terminally differentiated cell type into another (or transdifferentiation) usually requires the forced expression of key transcription factors. We examined the plasticity of human insulin-producing β-cells in a model of islet cell aggregate formation. Here, we show that primary human β-cells can undergo a conversion into glucagon-producing α-cells without introduction of any genetic modification. The process occurs within days as revealed by lentivirus-mediated β-cell lineage tracing. Converted cells are indistinguishable from native α-cells based on ultrastructural morphology and maintain their α-cell phenotype after transplantation in vivo. Transition of β-cells into α-cells occurs after β-cell degranulation and is characterized by the presence of β-cell–specific transcription factors Pdx1 and Nkx6.1 in glucagon+ cells. Finally, we show that lentivirus-mediated knockdown of Arx, a determinant of the α-cell lineage, inhibits the conversion. Our findings reveal an unknown plasticity of human adult endocrine cells that can be modulated. This endocrine cell plasticity could have implications for islet development, (patho)physiology, and regeneration

Constitutive expression of ftsZ overrides the whi developmental genes to initiate sporulation of Streptomyces coelicolor

The filamentous soil bacteria Streptomyces undergo a highly complex developmental programme. Before streptomycetes commit themselves to sporulation, distinct morphological checkpoints are passed in the aerial hyphae that are subject to multi-level control by the whi sporulation genes. Here we show that whi-independent expression of FtsZ restores sporulation to the early sporulation mutants whiA, whiB, whiG, whiH, whiI and whiJ. Viability, stress resistance and high-resolution electron microscopy underlined that viable spores were formed. However, spores from sporulation-restored whiA and whiG mutants showed defects in DNA segregation/condensation, while spores from the complemented whiB mutant had increased stress sensitivity, perhaps as a result of changes in the spore sheath. In contrast to the whi mutants, normal sporulation of ssgB null mutants—which fail to properly localise FtsZ—could not be restored by enhancing FtsZ protein levels, forming spore-like bodies that lack spore walls. Our data strongly suggest that the whi genes control a decisive event towards sporulation of streptomycetes, namely the correct timing of developmental ftsZ transcription. The biological significance may be to ensure that sporulation-specific cell division will only start once sufficient aerial mycelium biomass has been generated. Our data shed new light on the longstanding question as to how whi genes control sporulation, which has intrigued scientists for four decades

SARS-Coronavirus Replication Is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum

Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV), replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs) were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200–300 nm), and “vesicle packets” apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this “replication network” will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions

Effects of Deletion of Macrophage ABCA7 on Lipid Metabolism and the Development of Atherosclerosis in the Presence and Absence of ABCA1

ABCA7, a close relative of ABCA1 which facilitates cholesterol efflux to lipid-poor apoproteins, has been implicated in macrophage lipid efflux and clearance of apoptotic cells in in vitro studies. In the current study, we investigated the in vivo effects of macrophage ABCA7 deficiency on lipid metabolism and atherosclerosis. Chimeras with dysfunctional ABCA7 in macrophages and other blood cells were generated by transplantation of bone marrow from ABCA7 knockout (KO) mice into irradiated low-density lipoprotein receptor (LDLr) KO mice. Unexpectedly, macrophage ABCA7 deficiency did not significantly affect atherosclerosis susceptibility of LDLr KO mice after 10 weeks Western-type diet feeding. However, ABCA7 deficiency was associated with 2-fold (p<0.05) higher macrophage ABCA1 mRNA expression levels. Combined disruption of ABCA1 and ABCA7 in bone-marrow-derived cells increased atherosclerotic lesion development (1.5-fold (p>0.05) as compared to wild type transplanted mice. However, single deletion of ABCA1 had a similar effect (1.8-fold, p<0.05). Macrophage foam cell accumulation in the peritoneal cavity was reduced in ABCA1/ABCA7 dKO transplanted animals as compared to single ABCA1 KO transplanted mice, which was associated with increased ABCG1 expression. Interestingly, spleens of ABCA1/ABCA7 double KO transplanted mice were significantly larger as compared to the other 3 groups and showed massive macrophage lipid accumulation, a reduction in CD3+ T-cells, and increased expression of key regulators of erythropoiesis. In conclusion, deletion of ABCA7 in bone marrow-derived cells does not affect atherogenesis in the arterial wall neither in the absence or presence of ABCA1. Interestingly, combined deletion of bone marrow ABCA1 and ABCA7 causes severe splenomegaly associated with cellular lipid accumulation, a reduction in splenic CD3+ T cells, and induced markers of erythropoeisis. Our data indicate that ABCA7 may play a role in T cell proliferation and erythropoeisis in spleen

Characterization of reconstructed skin models

The aim of the present study was to evaluate tissue architecture and lipid composition of commercially available reconstructed human skin models; EpiDerm™, SkinEthic™ and Episkin™ in comparison to in-house reconstructed epidermis on a de-epidermized dermis (RE-DED) model and native tissue. For this purpose, the tissue architecture was examined using light microscopy, electron microscopy and immunohistochemistry; epidermal lipid composition was analyzed by HPTLC. Histological examination showed a completely stratified epithelium in all skin models closely resembling normal human epidermis. Low intra-batch variation in tissue architecture was observed in all skin models, but moderate to considerable inter-batch variation was noticed. In the stratum corneum extracellular space, lipid lamellae consisting of multiple alternating electron-dense and electron-lucent bands were present. Lipid analyses revealed the presence of all major epidermal lipid classes. Compared with native epidermis and RE-DED in EpiDerm, SkinEthic and Episkin models, the content of polar ceramides 5 and 6 was lower, ceramide 7 was absent, and the content of free fatty acids was very low. Evaluation of the expression and localization of a number of differentiation-specific protein markers revealed that all skin models showed an aberrant expression of keratin 6, skin-derived antileukoproteinase, small-proline-rich proteins, involucrin and transglutaminase. Although variation within batches was low, in particular keratin 6, involucrin and skin-derived antileukoproteinase expression demonstrated some inter-batch variation. In conclusion, all skin models provide a promising means for studying the effects of topically applied chemicals, although the observed deviations in tissue homeostasis and barrier properties need to be diminished. All skin models tested reproduced many of the characteristics of normal human epidermis and therefore provide a morphologically relevant in vitro means to assess skin irritation and perform other skin-related studies

Melanosome capping of keratinocytes in pigmented reconstructed epidermis - Effect of ultraviolet radiation and 3-isobutyl-1-methyl-xanthine on melanogenesis

Reconstructed pigmented epidermis was established by coseeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air-liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3 -isobutyl-1-methyl-xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: Melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied