Differential Regulation of Circadian Clock Genes by UV-B Radiation and 1,25-Dihydroxyvitamin D: A Pilot Study during Different Stages of Skin Photocarcinogenesis.

BACKGROUND: Increasing evidence points at an important physiological role of the timekeeping system, known as the circadian clock (CC), regulating not only our sleep-awake rhythm but additionally many other cellular processes in peripheral tissues. It was shown in various cell types that environmental stressors, including ultraviolet B radiation (UV-B), modulate the expression of genes that regulate the CC (CCGs) and that these CCGs modulate susceptibility for UV-B-induced cellular damage. It was the aim of this pilot study to gain further insights into the CCs' putative role for UV-B-induced photocarcinogenesis of skin cancer. METHODS: Applying RT-PCR, we analyzed the expression of two core CCGs (brain and muscle ARNT-like 1 (Bmal1) and Period-2 (Per2)) over several time points (0-60 h) in HaCaT cells with and without 1,25-dihydroxyvitamin D (D3) and/or UV-B and conducted a cosinor analysis to evaluate the effects of those conditions on the circadian rhythm and an extended mixed-effects linear modeling to account for both fixed effects of experimental conditions and random inter-individual variability. Next, we investigated the expression of these two genes in keratinocytes representing different stages of skin photocarcinogenesis, comparing normal (Normal Human Epidermal Keratinocytes-NHEK; p53 wild type), precancerous (HaCaT keratinocytes; mutated p53 status), and malignant (Squamous Cell Carcinoma SCL-1; p53 null status) keratinocytes after 12 h under the same conditions. RESULTS: We demonstrated that in HaCaT cells, Bmal1 showed a robust circadian rhythm, while the evidence for Per2 was limited. Overall expression of both genes, but especially for Bmal1, was increased following UV-B treatment, while Per2 showed a suppressed overall expression following D3. Both UVB and 1,25(OH)2D3 suggested a significant phase shift for Bmal1 (p < 0.05 for the acrophase), while no specific effect on the amplitude could be evidenced. Differential effects on the expression of BMAL1 and Per2 were found when we compared different treatment modalities (UV-B and/or D3) or cell types (NHEK, HaCaT, and SCL-1 cells). CONCLUSIONS: Comparing epidermal keratinocytes representing different stages of skin photocarcinogenesis, we provide further evidence for an independently operating timekeeping system in human skin, which is regulated by UV-B and disturbed during skin photocarcinogenesis. Our finding that this pattern of circadian rhythm was differentially altered by treatment with UV-B, as compared with treatment with D3, does not support the hypothesis that the expression of these CCGs may be regulated via UV-B-induced synthesis of vitamin D but might be introducing a novel photoprotective property of vitamin D through the circadian clock.

Cross-talk of Aryl Hydrocarbon Receptor (AHR)- and Vitamin D Receptor (VDR)-signaling in Human Keratinocytes.

BACKGROUND/AIM: Vitamin D receptor (VDR), activated upon binding of 1,25(OH)2D3, was described as a tumor suppressor in the skin. New biological functions of non-classical vitamin D derivatives were recently identified, that are mediated via binding to alternate receptors, including the aryl hydrocarbon receptor (AHR) and that indicate functional interaction between AHR and VDR signaling in various human tissues. We aimed to gain further insights into the cross-talk of VDR and AHR signaling in skin photo-carcinogenesis. MATERIALS AND METHODS: Using real-time quantitative PCR, we analyzed in vitro effects of the complete carcinogen UVB and of 1,25(OH)2D3 on the expression of members of the AHR and VDR pathways in human keratinocytes revealing characteristics of different stages of skin photo-carcinogenesis. RESULTS: In precancerous HaCaT keratinocytes, induction of a target gene of AHR-mediated transcription (CYP1A1) was markedly stronger after treatment with UVB, as compared to treatment with 1,25(OH)2D3. In contrast, in SCL-1 cells (that reveal the complete phenotype of malignant transformation), expression of CYP1A1 was higher after treatment with 1,25(OH)2D3 as compared to treatment with UVB. The classical VDR target CYP24A1 was up-regulated by 1,25(OH)2D3, but not by UVB, in both cell lines. However, the combined treatment with UVB strongly enhanced the 1,25(OH)2D3-mediated up-regulation of CYP24A1 exclusively in SCL-1, but not in HaCaT cells. CONCLUSION: There is a differential regulation of VDR and AHR target genes by UVB and 1,25(OH)2D3 in HaCaT and SCL-1 cells, that points to a complex and highly orchestrated network of vitamin D derivatives (and other photoproducts) and its relevance for photo-carcinogenesis.

IL-17C and IL-17RE Promote Wound Closure in a Staphylococcus aureus-Based Murine Wound Infection Model.

The epithelial cytokine interleukin-17C (IL-17C) mediates inflammation through the interleukin 17 receptor E (IL-17RE). Prior studies showed a detrimental role of IL-17C in the pathogenesis of immune-mediated skin diseases (e.g., psoriasis). Here, we examined the role of IL-17C/IL-17RE in wound closure in a Staphylococcus aureus wound infection model. We demonstrate that wound closure is significantly delayed in IL-17RE (Il-17re-/-)- and 17C (Il-17c-/-)-deficient mice. There was no significant difference between WT, Il-17re-/-, and Il-17c-/- mice in the absence of infection. Deficiency for IL-17RE and IL-17C did not significantly affect the elimination of bacteria. IL-17C expression was increased in the epidermis of human S. aureus-infected skin. Our results indicate that the IL-17C/IL-17RE axis contributes to the closure of infected wounds but does not contribute to the elimination of S. aureus.

A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1.

Legionella pneumophila is an important cause of pneumonia. It invades alveolar macrophages and manipulates the immune response by interfering with signaling pathways and gene transcription to support its own replication. MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression and are involved in defense against bacterial infections. Several pathogens have been shown to exploit the host miRNA machinery to their advantage. We therefore hypothesize that macrophage miRNAs exert positive or negative control over Legionella intracellular replication. We found significant regulation of 85 miRNAs in human macrophages upon L. pneumophila infection. Chromatin immunoprecipitation and sequencing revealed concordant changes of histone acetylation at the putative promoters. Interestingly, a trio of miRNAs (miR-125b, miR-221, and miR-579) was found to significantly affect intracellular L. pneumophila replication in a cooperative manner. Using proteome-analysis, we pinpointed this effect to a concerted downregulation of galectin-8 (LGALS8), DExD/H-box helicase 58 (DDX58), tumor protein P53 (TP53), and then MX dynamin-like GTPase 1 (MX1) by the three miRNAs. In summary, our results demonstrate a new miRNA-controlled immune network restricting Legionella replication in human macrophages.IMPORTANCE Cases of Legionella pneumophila pneumonia occur worldwide, with potentially fatal outcome. When causing human disease, Legionella injects a plethora of virulence factors to reprogram macrophages to circumvent immune defense and create a replication niche. By analyzing Legionella-induced changes in miRNA expression and genomewide chromatin modifications in primary human macrophages, we identified a cell-autonomous immune network restricting Legionella growth. This network comprises three miRNAs governing expression of the cytosolic RNA receptor DDX58/RIG-I, the tumor suppressor TP53, the antibacterial effector LGALS8, and MX1, which has been described as an antiviral factor. Our findings for the first time link TP53, LGALS8, DDX58, and MX1 in one miRNA-regulated network and integrate them into a functional node in the defense against L. pneumophila.

Transcriptional analysis identifies potential biomarkers and molecular regulators in pneumonia and COPD exacerbation.

Lower respiratory infections, such as community-acquired pneumonia (CAP), and chronic obstructive pulmonary disease (COPD) rank among the most frequent causes of death worldwide. Improved diagnostics and profound pathophysiological insights are urgent clinical needs. In our cohort, we analysed transcriptional networks of peripheral blood mononuclear cells (PBMCs) to identify central regulators and potential biomarkers. We investigated the mRNA- and miRNA-transcriptome of PBMCs of healthy subjects and patients suffering from CAP or AECOPD by microarray and Taqman Low Density Array. Genes that correlated with PBMC composition were eliminated, and remaining differentially expressed genes were grouped into modules. One selected module (120 genes) was particularly suitable to discriminate AECOPD and CAP and most notably contained a subset of five biologically relevant mRNAs that differentiated between CAP and AECOPD with an AUC of 86.1%. Likewise, we identified several microRNAs, e.g. miR-545-3p and miR-519c-3p, which separated AECOPD and CAP. We furthermore retrieved an integrated network of differentially regulated mRNAs and microRNAs and identified HNF4A, MCC and MUC1 as central network regulators or most important discriminatory markers. In summary, transcriptional analysis retrieved potential biomarkers and central molecular features of CAP and AECOPD.

THP-1-derived macrophages render lung epithelial cells hypo-responsive to Legionella pneumophila - a systems biology study.

Immune response in the lung has to protect the huge alveolar surface against pathogens while securing the delicate lung structure. Macrophages and alveolar epithelial cells constitute the first line of defense and together orchestrate the initial steps of host defense. In this study, we analysed the influence of macrophages on type II alveolar epithelial cells during Legionella pneumophila-infection by a systems biology approach combining experimental work and mathematical modelling. We found that L. pneumophila-infected THP-1-derived macrophages provoke a pro-inflammatory activation of neighboring lung epithelial cells, but in addition render them hypo-responsive to direct infection with the same pathogen. We generated a kinetic mathematical model of macrophage activation and identified a paracrine mechanism of macrophage-secreted IL-1ß inducing a prolonged IRAK-1 degradation in lung epithelial cells. This intercellular crosstalk may help to avoid an overwhelming inflammatory response by preventing excessive local secretion of pro-inflammatory cytokines and thereby negatively regulating the recruitment of immune cells to the site of infection. This suggests an important but ambivalent immunomodulatory role of macrophages in lung infection.

Legionella pneumophila infection activates bystander cells differentially by bacterial and host cell vesicles.

Extracellular vesicles from eukaryotic cells and outer membrane vesicles (OMVs) released from gram-negative bacteria have been described as mediators of pathogen-host interaction and intercellular communication. Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. The differential effect of bacterial and host cell vesicles in L. pneumophila infection is unknown so far. We infected THP-1-derived or primary human macrophages with L. pneumophila and isolated supernatant vesicles by differential centrifugation. We observed an increase of exosomes in the 100 k pellet by nanoparticle tracking analysis, electron microscopy, and protein markers. This fraction additionally contained Legionella LPS, indicating also the presence of OMVs. In contrast, vesicles in the 16 k pellet, representing microparticles, decreased during infection. The 100 k vesicle fraction activated uninfected primary human alveolar epithelial cells, A549 cells, and THP-1 cells. Epithelial cell activation was reduced by exosome depletion (anti-CD63, or GW4869), or blocking of IL-1ß in the supernatant. In contrast, the response of THP-1 cells to vesicles was reduced by a TLR2-neutralizing antibody, UV-inactivation of bacteria, or - partially - RNase-treatment of vesicles. Taken together, we found that during L. pneumophila infection, neighbouring epithelial cells were predominantly activated by exosomes and cytokines, whereas myeloid cells were activated by bacterial OMVs.

Genome-wide Chromatin Profiling of Legionella pneumophila-Infected Human Macrophages Reveals Activation of the Probacterial Host Factor TNFAIP2.

BACKGROUND: Legionella pneumophila is a causative agent of severe pneumonia. Infection leads to a broad host cell response, as evident, for example, on the transcriptional level. Chromatin modifications, which control gene expression, play a central role in the transcriptional response to L. pneumophila METHODS: We infected human-blood-derived macrophages (BDMs) with L. pneumophila and used chromatin immunoprecipitation followed by sequencing to screen for gene promoters with the activating histone 4 acetylation mark. RESULTS: We found the promoter of tumor necrosis factor α-induced protein 2 (TNFAIP2) to be acetylated at histone H4. This factor has not been characterized in the pathology of L. pneumophila TNFAIP2 messenger RNA and protein were upregulated in response to L. pneumophila infection of human-BDMs and human alveolar epithelial (A549) cells. We showed that L. pneumophila-induced TNFAIP2 expression is dependent on the NF-κB transcription factor. Importantly, knock down of TNFAIP2 led to reduced intracellular replication of L. pneumophila Corby in A549 cells. CONCLUSIONS: Taken together, genome-wide chromatin analysis of L. pneumophila-infected macrophages demonstrated induction of TNFAIP2, a NF-κB-dependent factor relevant for bacterial replication.

MicroRNAs Constitute a Negative Feedback Loop in Streptococcus pneumoniae-Induced Macrophage Activation.

Streptococcus pneumoniae causes high mortality as a major pneumonia-inducing pathogen. In pneumonia, control of innate immunity is necessary to prevent organ damage. We assessed the role of microRNAs (miRNAs) as regulators in pneumococcal infection of human macrophages. Exposure of primary blood-derived human macrophages with pneumococci resulted in transcriptional changes in several gene clusters and a significant deregulation of 10 microRNAs. Computational network analysis retrieved miRNA-146a as one putatively important regulator of pneumococci-induced host cell activation. Its induction depended on bacterial structural integrity and was completely inhibited by blocking Toll-like receptor 2 (TLR-2) or depleting its mediator MyD88. Furthermore, induction of miRNA-146a release did not require the autocrine feedback of interleukin 1ß and tumor necrosis factor α released from infected macrophages, and it repressed the TLR-2 downstream mediators IRAK-1 and TRAF-6, as well as the inflammatory factors cyclooxygenase 2 and interleukin 1ß. In summary, pneumococci recognition induces a negative feedback loop, preventing excessive inflammation via miR-146a and potentially other miRNAs.

Expression of insulin-like growth factor-1 receptor in conventional cutaneous squamous cell carcinoma with different histological grades of differentiation.

BACKGROUND: Insulin-like growth factor-1 receptor (IGF-1R) is a key regulator of cell transformation and controls the expression of genes that governs cell cycling and cell survival. The aim of this pilot study was to gain insight into the expression pattern of IGF-1R in conventional cutaneous squamous cell carcinoma (CSCC) using immunohistochemical analysis. MATERIALS AND METHODS: Five cases of normal human paraffin-embedded skin sections, 4 cases of actinic keratosis, and 28 cases of paraffin-embedded sections of different histological subtypes of CSCC were selected for immunohistochemical analysis. RESULTS: In normal skin, IGF-1R expression was detected in the epidermal basal cell layer. In actinic keratosis, IGF-1R was expressed in the lower part of the epidermis. IGF-1R was detected in the cell surface membrane of well-differentiated CSCC. In moderately differentiated CSCC, IGF-1R was expressed predominantly in the cytoplasm. Interestingly, IGF-1R was expressed in the nuclei of tumor cells of poorly differentiated CSCC. CONCLUSIONS: The strong and differential expression of IGF-1R in different histological degrees of CSCC indicates a possible role for IGF-insulin receptor in the carcinogenesis and differentiation of this disease and identifies IGF-1R as an interesting target for prevention and treatment of CSCC that deserves further investigation.

ORAI1 Ca(2+) channels control endothelin-1-induced mitogenesis and melanogenesis in primary human melanocytes.

UV radiation of the skin triggers keratinocytes to secrete endothelin-1 (ET-1) that binds to endothelin receptors on neighboring melanocytes. Melanocytes respond with a prolonged increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which is necessary for proliferation and melanogenesis. A major fraction of the Ca(2+) signal is caused by entry through Ca(2+)-permeable channels of unknown identity in the plasma membrane. ORAI Ca(2+) channels are molecular determinants of Ca(2+) release-activated Ca(2+) (CRAC) channels and are expressed in many tissues. Here, we show that ORAI1-3 and their activating partners stromal interaction molecules 1 and 2 (STIM1 and STIM2) are expressed in human melanocytes. Although ORAI1 is the predominant ORAI isoform, STIM2 mRNA expression exceeds STIM1. Inhibition of ORAI1 by 2-aminoethoxydiphenyl borate (2-APB) or downregulation of ORAI1 by small interfering RNA (siRNA) reduced Ca(2+) entry and CRAC current amplitudes in activated melanocytes. In addition, suppression of ORAI1 caused reduction in the ET-1-induced cellular viability, melanin synthesis, and tyrosinase activity. Our results imply a role for ORAI1 channels in skin pigmentation and their potential involvement in UV-induced stress responses of the human skin.

How risky are pinholes in gloves? A rational appeal for the integrity of gloves for isolators.

Isolators provide a high degree of protection for the product and/or the environment and operators in pharmaceutical production, as well as for analytical and sterility testing. Gloves allow for performing testing and for easy access to the process. Due to their nature-thin plastic, highly flexible-and their risk of puncture or rupture, they are regarded as one of the main potential sources of contamination. Glove integrity testing is therefore a main issue and has been addressed by many regulations such as those imposed by the USP, U.S. Food and Drug Administration, and Pharmaceutical Inspection Convention. This paper presents a short overview of different glove integrity test procedures and their ability to detect leaking gloves. Additionally, extensive microbiological tests have been performed to give more evidence and cross-correlation to physical testing. Most of the physical tests have limitations either in detecting pinholes and/or they are difficult to implement for routine testing. Microbiological tests are only applicable for evaluation and validation purposes, but not for routine testing, because they are time-consuming and do not allow immediate action. Routine visual verification of gloves by trained personnel turns out to be a very reliable technique. Additional microbiological tests supported by microbiological environmental monitoring helped to develop a new concept presented here on how to handle gloves with pinholes. It is proposed not to automatically consider a pinhole in a glove as a breach in isolator integrity, but to consider any action in view of controlling and monitoring the effective bioload on the outside of the gloves. With the combination of semi-automatic physical testing with independent protocol, visual inspection, and control of bioload through microbiological environmental monitoring potential contamination, risks can be minimized and maximum safety maintained. LAY ABSTRACT: Isolators are enclosure designs to protect critical handling and process steps in pharmaceutical environments. They provide a high degree of protection for product and/or environment and operators against particles, potentially hazardous active principles, and microbial load. Gloves mounted on windows and doors of the isolator allow for manipulation, performing testing, and access to the process. Due to their nature and their use with risk of puncture or rupture, they are regarded as a potential source for contamination. Glove integrity testing has therefor been addressed by regulations such as those imposed by the USP and the Food and Drug Administration. This paper presents a short overview of various glove integrity test procedures and their ability to detect leaking gloves. Most of the tests have limitations either in detecting pinholes and/or they are difficult to implement for routine testing. Routine visual verification of gloves by trained personnel turns out to be a very reliable technique. Additional microbiological tests led to a new concept presented here on how to handle gloves with pinholes and how to take action. With this approach, risks can be minimized and maximum safety maintained by controlling and monitoring the effective bioload on the outside of the gloves.

Exploring synthetic avenues for the effective synthesis of selenium- and tellurium-containing multifunctional redox agents.

Various human illnesses, including several types of cancer and infectious diseases, are related to changes in the cellular redox homeostasis. During the last decade, several approaches have been explored which employ such disturbed redox balances for the benefit of therapy. Compounds able to modulate the intracellular redox state of cells have been developed, which effectively, yet also selectively, appear to kill cancer cells and a range of pathogenic microorganisms. Among the various agents employed, certain redox catalysts have shown considerable promise since they are non-toxic on their own yet develop an effective, often selective cytotoxicity in the presence of the 'correct' intracellular redox partners. Aminoalkylation, amide coupling and multicomponent reactions are suitable synthetic methods to generate a vast number of such multifunctional catalysts, which are chemically diverse and, depending on their structure, exhibit various interesting biological activities.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a new tool in diagnostic investigation of nail disorders?

The incidence and prevalence of onychomycosis are rising worldwide. Common diagnostic techniques often lack sensitivity or specificity. Differentiation between non-infectious nail disorders is frequently not possible. The aim of this study was to establish a better diagnostic routine procedure based on modern mass spectrometric peptide analysis techniques. One hundred and fifty-five nail samples from 145 patients with clinically suspected onychomycosis (n = 96, 62%) and without onychomycosis [e.g. nail psoriasis or nail dystrophy resulting from eczema (n = 59, 38%)] were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting in comparison with standard techniques. We demonstrated that MALDI-TOF MS represents a precise, robust and fast tool in diagnostic investigation of nail disorders, which is superior to common standard methods.

Mitofilin and titin as target antigens in melanoma-associated retinopathy.

Melanoma-associated retinopathy (MAR) is a rare paraneoplastic syndrome in patients with melanoma. Since the onset of MAR symptoms is often associated with tumor progression or recrudescence of metastases, MAR-related symptoms are prognostic relevant. The pathomechanism underlying MAR is supposed to result from antibody production against yet unknown melanoma-associated antigens that are also expressed in retinal tissue, leading to the destruction of retinal cells and resulting in defective signal transduction. Only a 35 kDa protein in Müller glial cells, a 22 kDa neuronal antigen and retinal transducin have been identified as MAR-associated antigens to date. To identify additional antigens potentially involved in the pathogenesis of MAR, we screened a retina cDNA phage library for reactivity with antibodies in the sera from 9 patients with MAR or subclinical MAR using the serological analysis of recombinantly expressed clones (SEREX) approach. Six sera from melanoma patients without evidence of MAR and 10 sera from healthy donors served as controls. Mitofilin and titin were identified as antigens against which antibodies were found exclusively in sera of MAR patients, but not in the sera of MM patients without MAR or healthy donors. This is the first study to demonstrate that titin is highly expressed from retinal tissue and melanoma. The fact that none of the MAR-associated antigens detected to date by their capacity to elicit a humoral immune response is located on the cell surface questions a major pathogenetic role of the respective antibodies and suggests that cellular, rather than humoral mechanisms are operative in the primary immune attack against the retina in MAR.

Effect of carrier materials on the resistance of spores of Bacillus stearothermophilus to gaseous hydrogen peroxide.

The testing of the H2O2 decontamination process using spores of Bacillus stearothermophilus has gained widespread acceptance. Usually, commercially available Biological Indicators (BIs) with a specified resistance to H2O2 are challenged to qualify the process. The question arises whether the resistance of test spores is dependant on the type of carrier material and whether the resistance is representative for the system under test. The objective of the study is to quantify the effect of different carrier materials on the resistance of spores of Bacillus stearothermophilus to H2O2. Materials from which isolators were built, as well as those used in disposables during daily work were investigated. These materials were inoculated with 106 spores of Bacillus stearothermophilus (ATCC 7953). The spore resistance was tested to a well defined H2O2 decontamination cycle by determining the D-value using the "Fractional Negative" method. This paper reports on the effect of different carrier materials to the resistance of the test organism against H2O2. Various materials have significantly increased resistance of the spores and should be avoided in isolator systems. If commercially available BIs are used for process qualification, the resistance of the BI used, the fluctuation in resistance caused by isolator materials, the required log reduction, and at least the bioload of isolator surfaces need to be known.