Topical Ectoine Application in Children and Adults to Treat Inflammatory Diseases Associated with an Impaired Skin Barrier: A Systematic Review.

INTRODUCTION: Inflammatory skin diseases are a significant burden on affected patients. Inflammation is caused by various stress factors to the epidermis resulting in, e.g., dryness, redness, and pruritus. Emollients are used in basic therapy to restore the natural skin barrier and relieve symptoms. A systematic review was performed to evaluate the efficacy and safety of ectoine-containing topical formulations in inflammatory skin diseases characterized by an impaired skin barrier. METHODS: A systematic review was carried out in PubMed, the Cochrane Library, and Microsoft Academic up to October 2021. Inclusion criteria were ectoine-containing topical formulations (creams, emollients) used for (adjuvant) therapy of inflammatory skin diseases. Clinical studies of any design published in any language were included. RESULTS: A total of 230 references were screened for eligibility, of which six were selected for inclusion in the review (five studies on atopic dermatitis and one study on prevention and management of retinoid dermatitis). The application of topical formulations containing 5.5-7.0% ectoine positively influenced skin dryness and, consequently, pruritus and dermatitis-specific scores in patients with atopic dermatitis. Especially in infants and children, who belong to the most frequently affected group, the formulations were well-tolerated when applied for up to 4 weeks. In studies where ectoine was used as an adjuvant therapy, application was associated with a decreased need for pharmacological therapy and also improved the effectiveness of, e.g., topical corticosteroids. In patients undergoing isotretinoin therapy, ectoine was as effective as dexpanthenol in reducing retinoid dermatitis or improving symptoms. CONCLUSION: Ectoine is an effective natural substance with an excellent tolerability and safety profile, representing a beneficial alternative as basic therapy or to increase the efficacy of the pharmacological treatment regimen for patients with inflammatory skin diseases, including infants and children.

Endosomal recognition of Lactococcus lactis G121 and its RNA by dendritic cells is key to its allergy-protective effects.

BACKGROUND: Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown. We examined the ability of Lactococcus lactis G121 to prevent allergic inflammatory reactions. OBJECTIVE: We sought to identify the ligands and pattern recognition receptors through which L lactis G121 confers allergy protection. METHODS: L lactis G121-induced cytokine release and surface expression of costimulatory molecules by untreated or inhibitor-treated (bafilomycin and cytochalasin D) human monocyte-derived dendritic cells (moDCs), bone marrow-derived mouse dendritic cells (BMDCs), and moDC/naive CD4+ T-cell cocultures were analyzed by using ELISA and flow cytometry. The pathology of ovalbumin-induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy. RESULTS: L lactis G121-treated murine BMDCs and human moDCs released TH1-polarizing cytokines and induced TH1 T cells. Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of TH1-polarizing cytokines, costimulatory molecule expression, and T-cell activation on L lactis G121 challenge. In vivo allergy protection mediated by L lactis G121 was dependent on endosomal acidification in dendritic cells (DCs). Toll-like receptor (Tlr) 13-/- BMDCs showed a weak response to L lactis G121 and were unresponsive to its RNA. The TH1-polarizing activity of L lactis G121-treated human DCs was blocked by TLR8-specific inhibitors, mediated by L lactis G121 RNA, and synergistically enhanced by activation of nucleotide-binding oligomerization domain-containing protein (NOD) 2. CONCLUSION: Bacterial RNA is the main driver of L lactis G121-mediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification. In mice L lactis G121 RNA signals through TLR13; however, the most likely intracellular receptor in human subjects is TLR8.

Allergy protection by cowshed bacteria - recent findings and future prospects.

Since the first publication in 1999, numerous epidemiologic studies provided strong evidence that frequent contact to a traditional farm environment in early life protects children from the development of allergic airway diseases. These consistent findings prompted enormous efforts to identify and characterize the potential causative factors and the underlying immunologic mechanisms in experimental studies. The cumulating evidence for the role of the cowshed-associated bacterial flora led to enhanced efforts not only to identify the relevant species but also to examine their specific immunomodulatory capacity, the bacterial components involved, and particularly the cellular and molecular mechanisms of their interaction with the immune system. We review here the methods applied to identify relevant bacterial species, the species which emerged thereof, and the similarities and differences in their mode of action as revealed so far. We further consider the impact of the current knowledge on worthwhile clinical application and reflect on the required next steps to foster the translation of the encouraging scientific progress which has been made in recent years.

A ternary complex consisting of AICD, FE65, and TIP60 down-regulates Stathmin1.

The ternary complex consisting of AICD/FE65/TIP60 is thought to play a role in gene expression and was suggested to have a crucial impact in Alzheimer's disease. AICD is the intracellular subdomain of the amyloid precursor protein (APP) and able to bind the adapter protein FE65 and the histone acetyltransferase TIP60 setting up a nuclear dot-like phenotype. Within this work we readdressed the generation of the complex as a function of its compartments. Subsequently, we studied the proteome of AFT expressing cells vs. controls and identified Stathmin1 significantly down-regulated in AFT cells. Stathmin1 functions as an important regulatory protein of microtubule dynamics and was found associated with neurofibrillary tangles in brains of Alzheimer's disease patients. We validated our results using an independent label-free mass spectrometry based method using the same cell culture model. In a reversal model with diminished APP expression, caused by simultaneous knock-down of all three members of the APP family, we further confirmed our results, as Stathmin1 was regulated in an opposite fashion. We hypothesize that AICD-dependent deregulation of Stathmin1 causes microtubule disorganization, which might play an important role for the pathophysiology of Alzheimer's disease.

Stepwise isolation of human peripheral erythrocytes, T lymphocytes, and monocytes for blood cell proteomics.

PURPOSE: Density gradient centrifugation and magnetic- or fluorescence-activated cell sorting are common and robust techniques for the isolation of different types of blood cells. In this article, we give detailed description of a stepwise application of these methods as one isolation strategy for enrichment of different cell types from one blood sample. EXPERIMENTAL DESIGN: The workflow targeted erythrocytes, monocytes, and T lymphocytes. Pancoll® density gradient centrifugation was used together with subsequent MACS™ isolation. Purity of monocytes and T lymphocytes was controlled by fluorescence-activated cell sorting analysis, and cells were used for carrier-ampholine-based 2D-PAGE to confirm compatibility of the procedure to standard proteomic applications. RESULTS: Gradient centrifugation resulted in an average of 125 µL of packed erythrocytes per milliliter blood. MACS™ sorting reached purities of 90 ± 2% (monocytes) and 93 ± 2% (T lymphocytes), with an average yield of 12 × 10(4) monocytes or T lymphocytes. 2D-PAGE of isolated cells showed well-separated spot patterns. CONCLUSIONS AND CLINICAL RELEVANCE: A combined isolation holds substantial advantages especially in clinical studies, as it allows for the comparison of findings not only between individuals, but also between different cell types derived from one donor. Our approach ensured high reproducibility, yields, and purities of cells as required for reliable proteome analysis.

Flow cytometry confirms reticulate evolution and reveals triploidy in Central European Diphasiastrum taxa (Lycopodiaceae, Lycophyta).

BACKGROUND AND AIMS: Interspecific Diphasiastrum hybrids have been assumed to be homoploid and to produce well-formed spores serving sexual reproduction. If this were the case, forms intermediate between hybrids and parents or hybrid swarms should be expected. The purpose of this study was: (1) to check whether homoploidy consistently applies to the three hybrids throughout their Central European range; (2) to examine whether their genome sizes confirm their parentage as assumed by morphology; and (3) to perform a screening for detection of ploidy levels other than diploid and variation in DNA content due to backcrossing. METHODS: Flow cytometry was used first to measure the relative DNA values [with 4',6-diamidino-2-phenylindole (DAPI) staining] and ploidy level as a general screening, and secondly to determine the absolute DNA 2C values [with propidium iodide (PI) staining] in a number of selected samples with the main focus on the hybrids. KEY RESULTS: A considerable variation of DNA 2C values (5·26-7·52 pg) was detected between the three European Diphasiastrum species. The values of the diploid hybrids are highly constant without significant variation between regions. They are also intermediate between their assumed parents and agree closely with those calculated from their putative parents. This confirms their hybrid origin, assumed parentage and homoploid status. Considerably higher DNA amounts (9·48-10·30 pg) were obtained for three populations, suggesting that these represent triploid hybrids, an interpretation that is strongly supported by their morphology. CONCLUSIONS: Diploid hybrids have retained their genetic and morphological identites throughout their Central European range, and thus no indications for diploid backcrossing were found. The triploid hybrids have probably originated from backcrossing between a diploid gametophyte of a hybrid (derived from a diplospore) and a haploid gametophyte of a diploid parental species. By repeated crossing events, reticulate evolution patterns arise that are similar to those known for a number of ferns.

Arabinogalactan isolated from cowshed dust extract protects mice from allergic airway inflammation and sensitization.

BACKGROUND: Extract from cowshed dust (CDE) is a source of immunomodulating substances. We have previously shown that such substances protect from experimental allergic disorders in a mouse model of asthma. OBJECTIVE: The objective of this study was to identify immunomodulatory molecules in extracts of dust from an allergy protective farming environment. METHODS: Polysaccharides were isolated from CDE and plants by chromatography and precipitation with specific reagents. Polysaccharides were then characterized by nuclear magnetic resonance spectroscopy. Subsequently, the allergy-protective potential of isolated polysaccharides was tested in a mouse model of asthma. RESULTS: The authors demonstrate that plant arabinogalactans are contained in CDE in high concentrations. The source of this arabinogalactan is fodder, in particular a prevalent grass species known as Alopecurus pratensis. Treatment of murine dendritic cells with grass arabinogalactan resulted in autocrine IL-10 production. Interestingly, these dendritic cells were not able to induce an allergic immune response. Furthermore, intranasal application of grass arabinogalactan protected mice from developing atopic sensitization, allergic airway inflammation and airway hyperreactivity in a mouse model of allergic asthma. This allergy-protective effect is specific for grass arabinogalactan because control experiments with arabinogalactan from gum arabic and larch revealed that these molecules do not show allergy-protective properties. This is likely because of structural differences because we were able to show by nuclear magnetic resonance spectroscopy that although they are predominantly composed of arabinose and galactose, the molecules differ in structure. CONCLUSIONS: The authors conclude that grass arabinogalactans are important immunomodulatory substances that contribute to the protection from allergic airway inflammation, airway hyperresponsiveness, and atopic sensitization in a mouse model of asthma.

Modulation of dendritic cell function by cowshed dust extract.

We have shown previously that inhalation of cowshed dust extract (CDE) resulted in decreased airway reactivity, eosinophilic inflammation and sensitization in a mouse model of allergic asthma. Our data suggested down-regulation of allergic immune response rather than activation of a Th1 response towards the model allergen. However, the precise mechanism of allergy protection is not yet understood in detail. To gain deeper insight into CDE-induced immune modulation, we have analysed the effects of CDE on dendritic cell biology. Dendritic cells were generated from murine bone marrow cells (BMDC). Cells were stimulated with CDE and subsequently used to sensitize mice via the airways. Our results showed that cells were not able to prime mice for allergic immune response when they were treated with CDE 2 days before pulsing with allergen, whereas cells that were stimulated with CDE simultaneously to OVA pulsing induced a fully developed allergic immune response. Surprisingly, CDE-treated cells that were not able to prime mice for allergic immune response exhibit an activated phenotype with high expression of the co-stimulatory surface molecule CD86. Moreover, CDE-treated cells transiently produced high amounts of cytokines such as IL-10, IL-12p70 and TNF-alpha. Interestingly, blocking of autocrine-produced IL-10 in vitro partially restored the allergy-inducing capacity of CDE-exposed cells. Thus, we conclude that prolonged exposure to CDE reduces the allergy-inducing capacity of dendritic cells. Furthermore, we present evidence that an autocrine IL-10 dependent mechanism seems to be involved in down-regulation of dendritic cell function due to stimulation with CDE.

Anti-BDCA-4 (neuropilin-1) antibody can suppress virus-induced IFN-alpha production of plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (PDC) in human blood are the main source of virus-induced interferon (IFN)-alpha. They exhibit a lineage-negative phenotype but all express BDCA-4, which is homologous to the neuronal receptor neuropilin-1. Specific staining with anti-BDCA-4 antibody is used for positive isolation of PDC from blood by magnetic cells sorting. Here, it is demonstrated that these positively selected PDC showed reduced or completely abolished IFN-alpha release compared to unstained PDC, which were negatively selected by magnetic depletion of lineage-positive blood mononuclear cells. In addition, treatment of these unstained PDC with anti-BDCA-4 mAb also resulted in at least two-fold lower or reduced virus-induced IFN-alpha production. It is shown that the antibody not only affects cell survival or block virus attachment but also reduces IFN-alpha release induced by non-viral CpG oligodeoxynucleotides. In conclusion, data suggest an immunoregulatory role for BDCA-4 on PDC as demonstrated for IFN-alpha response to virus.

Synergistically upregulated interleukin-10 production in cocultures of monocytes and T cells after stimulation with respiratory syncytial virus.

BACKGROUND: Respiratory syncytial virus (RSV) is known as a causal factor of severe bronchiolitis in young children. It has also been detected in patients with chronic obstructive pulmonary disease (COPD), a disease that is associated with an increased number of T cells in the bronchial mucosa. Here, we investigated the potential direct interaction between RSV and T cells and its impact on cytokine response. METHODS: Purified human peripheral blood T cells were stimulated with RSV in vitro and analyzed by flow cytometry and fluorescence microscopy. Cytokine expression and release were measured in T cell cultures and in cocultures with peripheral blood monocytes as well as with alveolar macrophages from bronchoalveolar lavage fluid by quantitative real-time PCR and ELISA. RESULTS: It was shown that RSV adhered to the surface of T cells. Stimulation of purified T cells with RSV led to a significant increase in interleukin (IL)-10 mRNA expression after 24 h. Moreover, in cocultures of T cells with monocytes or alveolar macrophages, IL-10 production was synergistically upregulated 24 h after stimulation with RSV. CONCLUSION: These results suggest that RSV can cause an excessive IL-10 response leading to downregulation of antiviral defense mechanisms and reduced elimination of respiratory pathogens when antigen-presenting cells and T cells are simultaneously present on the site of infection. This effect may possibly contribute to high frequencies of respiratory pathogens found in patients with chronic inflammatory airway diseases associated with increased local T cell influx such as COPD.