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Papain is commonly used in food, pharmaceutical, textile, and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Dermatophagoides pteronyssinus 1 from house dust mite exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction (TJ) proteins were tested in vitro in human primary keratinocytes. Using C57BL/6 wild-type and Toll-like receptor 4 (TLR4)-deficient mice, we analyzed the sensitization potential of papain, its effects on the skin barrier, and immune cell recruitment. Our results show that papain affects the skin barrier by increasing transepidermal water loss, degrading TJ proteins and inducing vasodilation. When topically applied, papain exhibited a high epicutaneous inflammatory potential by recruiting neutrophils, mast cells, and CD3-positive cells and by induction of a TH2-biased antibody response. However, its high potency for specific sensitization via the skin was TLR4 independent and, in spite of its capacity to degrade epidermal TJ proteins, does not rely on its enzymatic function. From our data, we conclude that papain has all features to act as a strong allergen via the skin.

Background and Aims: The Mounier-Kuhn syndrome (MKS) is a rare disease characterized by a pathological dilation of the trachea and the bronchial system. The etiology of the disorder remains elusive, but genetic alterations and degradation of elastic fibers are thought to be involved in the pathogenesis. No causative treatment is available although transplantation is an option for end-stage disease. Here, we describe a patient suffering from MKS who received a double lung transplant at our department., Methods: Since a familial clustering of MKS is discussed in the literature, we performed a chromosomal analysis and an array-comparative genomic hybridization (CGH) to search for genetic abnormalities. At the time of transplantation, we collected samples from the bronchi and performed hematoxylin and eosin (HE), Elastic von-Gieson (EVG) and immunohistochemical stains of the explanted MKS bronchus, a control bronchus and of the inflammatory infiltrates. Specimens of main bronchi from the donor lung harvested for transplant served as control. Bronchial smears were taken from both main bronchi of the recipient for microbiological cultures., Results: No genetic alterations could be found in chromosomal analysis and in array-CGH. Histological analysis revealed a strong reduction of elastic fibers in the submucosal connective tissue and a diffuse inflammatory infiltrate, mainly comprised of CD4+ cells. In addition, immunohistochemistry showed increased matrix metalloproteinases (MMPs) protein expression of MMP-1, 2, 3 and 9., Conclusions: Based on our findings, we hypothesize that MKS is a chronic inflammatory disease characterized by an MMP-mediated degradation of submucosal elastic fibers., (© 2014 John Wiley & Sons Ltd.)

Autophagy is the central cellular mechanism for delivering organelles and cytoplasm to lysosomes for degradation and recycling of their molecular components. To determine the contribution of autophagy to melanocyte (MC) biology, we inactivated the essential autophagy gene Atg7 specifically in MCs using the Cre-loxP system. This gene deletion efficiently suppressed a key step in autophagy, lipidation of microtubule-associated protein 1 light chain 3 beta (LC3), in MCs and induced slight hypopigmentation of the epidermis in mice. The melanin content of hair was decreased by 10-15% in mice with autophagy-deficient MC as compared with control animals. When cultured in vitro, MCs from mutant and control mice produced equal amounts of melanin per cell. However, Atg7-deficient MCs entered into premature growth arrest and accumulated reactive oxygen species (ROS) damage, ubiquitinated proteins, and the multi-functional adapter protein SQSTM1/p62. Moreover, nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent expression of NAD(P)H dehydrogenase, quinone 1, and glutathione S-transferase Mu 1 was increased, indicating a contribution of autophagy to redox homeostasis in MCs. In summary, the results of our study suggest that Atg7-dependent autophagy is dispensable for melanogenesis but necessary for achieving the full proliferative capacity of MCs.

After spinal cord injury (SCI), secondary damage caused by oxidative stress, inflammation, and ischemia leads to neurological deterioration. In recent years, therapeutic approaches to trauma have focused on modulating this secondary cascade. There is increasing evidence that the success of cell-based SCI therapy is due mainly to secreted factors rather than to cell implantation per se. This study investigated peripheral blood mononuclear cells as a source of factors for secretome- (MNC-secretome-) based therapy. Specifically, we investigated whether MNC-secretome had therapeutic effects in a rat SCI contusion model and its possible underlying mechanisms. Rats treated with MNC-secretome showed substantially improved functional recovery, attenuated cavity formation, and reduced acute axonal injury compared to control animals. Histological evaluation revealed higher vascular density in the spinal cords of treated animals. Immunohistochemistry showed that MNC-secretome treatment increased the recruitment of CD68(+) cells with concomitant reduction of oxidative stress as reflected by lower expression of inducible nitric oxide synthase. Notably, MNC-secretome showed angiogenic properties ex vivo in aortic rings and spinal cord tissue, and experiments showed that the angiogenic potential of MNC-secretome may be regulated by CXCL-1 upregulation in vivo. Moreover, systemic application of MNC-secretome activated the ERK1/2 pathway in the spinal cord. Taken together, these results indicate that factors in MNC-secretome can mitigate the pathophysiological processes of secondary damage after SCI and improve functional outcomes in rats., (Copyright © 2015. Published by Elsevier Inc.)

Background: Pulmonary metastases are common in patients with primary colorectal cancer (CRC). Heat-shock protein 27 (Hsp27) is upregulated in activated fibroblasts during wound healing and systemically elevated in various diseases. Cancer-associated fibroblasts (CAFs) are also thought to play a role as prognostic and predictive markers in various malignancies including CRC. Surprisingly, the expression of Hsp27 has never been assessed in CAFs. Therefore we aimed to investigate the expression level of Hsp27 in CAFs and its clinical implications in patients with CRC lung metastases., Methods: FFPE tissue samples from 51 pulmonary metastases (PMs) and 33 paired primary tumors were evaluated for alpha-SMA, CD31, Hsp27 and vimentin expression by immunohistochemistry and correlated with clinicopathological variables. 25 liver metastases served as control group. Moreover, serum samples (n=10) before and after pulmonary metastasectomy were assessed for circulating phospho-Hsp27 and total Hsp27 by ELISA., Results: Stromal expression of Hsp27 was observed in all PM and showed strong correlation with alpha-SMA (P<0.001) and vimentin (P<0.001). Strong stromal Hsp27 was associated with higher microvessel density in primary CRC and PM. Moreover, high stromal Hsp27 and αSMA expression were associated with decreased recurrence-free survival after pulmonary metastasectomy (P=0.018 and P=0.008, respectively) and overall survival (P=0.031 and P=0.017, respectively). Serum levels of phospho- and total Hsp27 dropped after metastasectomy to levels comparable to healthy controls., Conclusions: Herein we describe for the first time that Hsp27 is highly expressed in tumor stroma of CRC. Stromal α-SMA and Hsp27 expressions correlate with the clinical outcome after pulmonary metastasectomy. Moreover, serum Hsp27 might pose a future marker for metastatic disease in CRC.

Aims: Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model., Methods and Results: BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells., Conclusion: MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases., (© The Author 2013. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: High dose ionizing radiation (IR) induces potent toxic cell effects mediated by either direct DNA damage or the production of reactive oxygen species (ROS). IR-induced modulations in multiple biological processes have been proposed to be partly regulated by radiosensitive microRNA (miRNA). In order to gain new insights into the role of miRNAs in the regulation of biological processes after IR, we have investigated changes in mRNA and miRNA expression after high dose IR., Results: IR induced changes in the mRNA and miRNA profiles of human peripheral blood mononuclear cells (PBMCs). When comparing non-irradiated and irradiated samples, we detected a time-dependent increase in differentially expressed mRNAs and miRNAs, with the highest differences detectable 20 hours after exposure. Gene ontology analysis revealed that very early events (up to 4 hours) after irradiation were specifically associated with p53 signaling and apoptotic pathways, whereas a large number of diverse cellular processes were deregulated after 20 hours. Transcription factor analysis of all up-regulated genes confirmed the importance of p53 in the early post-irradiation phase. When analyzing miRNA expression, we found 177 miRNAs that were significantly regulated in the late post-irradiation phase. Integrating miRNA and target gene expression data, we found a significant negative correlation between miRNA-mRNA and identified hepatic leukemia factor (HLF) as a transcription factor down-regulated in the response to IR. These regulated miRNAs and the HLF target genes were involved in modulating radio-responsive pathways, such as apoptosis, the MAKP signaling pathway, endocytosis, and cytokine-cytokine interactions., Conclusion: Using a large dataset of mRNA and miRNA expression profiles, we describe the interplay of mRNAs and miRNAs in the regulation of gene expression in response to IR at a posttranscriptional level and their involvement in the modulation of radiation-induced biological pathways.

Objective: The molecular determinants of chronic thromboembolic pulmonary hypertension (CTEPH) and idiopathic pulmonary arterial hypertension (iPAH) remain poorly understood. The receptor for advanced glycation endproducts (RAGE) and its ligands: HMGB1 and S100A9 are involved in inflammatory disorders. We sought to investigate the role of the RAGE axis in patients with CTEPH undergoing pulmonary endarterectomy (PEA), iPAH undergoing lung transplantation (LuTX). The high pulmonary vascular resistance in CTEPH/iPAH results in pressure overload of the right ventricle. We compared sRAGE measurements to that of patients with aortic valve stenosis (AVS) - pressure overload of the left ventricle., Methods: We enrolled patients with CTEPH(26), iPAH(15), AVS(15) and volunteers(33). Immunohistochemistry with antibodies to RAGE and HMGB1 was performed on PEA specimens and lung tissues. We employed enzyme-linked immunosorbent assays to determine the concentrations of sRAGE, esRAGE, HMGB1 and S100A9 in serum of volunteers and patients with CTEPH, iPAH, AVS before and after PEA, LuTX and aortic valve replacement (AVR)., Results: In endarterectomised tissues from patients with CTEPH RAGE and HMGB1 were identified in myofibroblasts (α-SMA+vimentin+CD34-), recanalizing vessel-like structures of distal myofibrotic tissues and endothelium of neointima. RAGE was differentially expressed in prototypical Heath Edwards lesions in iPAH. We found significantly increased serum concentrations of sRAGE, esRAGE and HMGB1 in CTEPH. In iPAH, sRAGE and esRAGE were significantly higher than in controls. Serum concentrations of sRAGE were significantly elevated in iPAH(p<0.001) and CTEPH(p = 0.001) compared to AVS. Serum sRAGE was significantly higher in iPAH compared to CTEPH(p = 0.042) and significantly reduced in AVS compared to controls(p = 0.001). There were no significant differences in sRAGE serum concentrations before and after surgical therapy for CTEPH, iPAH or AVS., Conclusions: Our data suggest a role for the RAGE pathway in the pathophysiology of CTEPH and iPAH. PEA improves the local control of disease but may not influence the systemic inflammatory mechanisms in CTEPH patients through the RAGE pathway.

Background: The human lung is considered a nonsterile organ, and surgical interventions therefore take place in a more or less contaminated operating field. Nevertheless, infectious complications of the pleural cavity are low after major lung resections. Antimicrobial peptides (AMPs) are part of the innate immunity and display a broad capacity to kill pathogens. We hypothesized that the pleural space must have a high natural antimicrobial barrier and that AMPs might effectively protect the pleural cavity., Methods: Pleural effusions were collected after lung operations. Antimicrobial activity of the fluids against gram-positive and gram-negative pathogens was analyzed by microdilution assays. AMPs were determined by enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and immunohistochemical analysis. The impact of proinflammatory triggers on AMP release from pleural mesothelial cells was evaluated., Results: Antimicrobial activity assays revealed high bactericidal properties of postoperative pleural drainage fluids. They effectively killed gram-negative pathogens (Escherichia coli, Pseudomonas aeruginosa) as well as gram-positive pathogens (Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes). A variety of AMPs was detected at constantly high concentrations in the pleural fluids. They mainly derived from leukocytes and pleural epithelium. Although proinflammatory cytokine levels were elevated in the postoperative pleural fluids, AMP expression could not be augmented by Toll-like receptor (TLR) triggering or by the proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)α., Conclusions: We provide the first evidence of a high abundance of AMPs in postoperative pleural fluids. Our findings might explain the broad protection against infectious complications of the pleural space after major lung operations., (Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Antimicrobial peptides (AMPs), an important part of the innate immune system, are crucial for defense against invading microorganisms. Whereas AMPs have been extensively studied in adult skin, little is known about the impact of AMPs in the developing human skin. We therefore compared the expression and regulation of AMPs in fetal, neonatal, and adult keratinocytes (KCs) in vitro. The constitutive expression of human β-defensin-2 (HBD-2), HBD-3, S100 protein family members, and cathelicidin was significantly higher in KCs from fetal skin than in KCs from postnatal skin. The capacity to further increase AMP production was comparable between prenatal and postnatal KCs. Analysis of skin equivalents (SEs) revealed a strong constitutive expression of S100 proteins in fetal but not in neonatal and adult SEs. The elevated AMP levels correlated with reduced H3K27me3 (tri-methyl-lysine 27 on histone H3) levels and increased expression of the histone demethylase JMJD3. Knockdown of JMJD3 in fetal KCs elevated H3K27me3 levels and significantly downregulated the expression of HBD-3, S100A7, S100A8, S100A9, and cathelicidin. Our data indicate a crucial contribution of histone modifications in the regulation of AMP expression in the skin during ontogeny. The elevated AMP expression in prenatal skin might represent an essential defense strategy of the unborn.

There is evidence of continued stimulation of foliage photosynthesis in trees exposed to elevated atmospheric CO2 concentrations; however, this is mostly without a proportional growth response. Consequently, we lack information on the fate of this extra carbon (C) acquired. By a steady application of a (13)CO2 label in a free air CO2 enrichment (FACE) experiment, we traced the fate of C in 37-m-tall, ca. 110-year-old Picea abies trees in a natural forest in Switzerland. Hence, we are not reporting tree responses to elevated CO2 (which would require equally (13)C labeled controls), but are providing insights into assimilate processing in such trees. Sunlit needles and branchlets grow almost exclusively from current assimilates, whereas shaded parts of the crowns also rely on stored C. Only 2.5 years after FACE initiation, tree rings contained 100% new C. Stem-respiratory CO2 averaged 50% of new C over the entire FACE period. Fine roots and mycorrhizal fungi contained 49-56 and 26-43% new C, respectively, after 2.5 years. The isotopic signals in soil CO2 arrived 12 days after the onset of FACE, yet it contained only ca. 15% new C thereafter. We conclude that new C first feeds into fast turnover C pools in the canopy and becomes increasingly mixed with older C sources as one moves away (downward) from the crown. We speculate that enhanced C turnover (its metabolic cost) along the phloem path, as evidenced by basipetal isotope signal depletion, explains part of the 'missing carbon' in trees that assimilated more C under elevated CO2.

The pursuit of targeting multiple pathways in the ischemic cascade of cerebral stroke is a promising treatment option. We examined the regenerative potential of conditioned medium derived from rat and human apoptotic mononuclear cells (MNC), rMNC (apo sec) and hMNC (apo sec), in experimental stroke. We performed middle cerebral artery occlusion on Wistar rats and administered apoptotic MNC-secretomes intraperitoneally in two experimental settings. Ischemic lesion volumes were determined 48 hours after cerebral ischemia. Neurological evaluations were performed after 6, 24 and 48 hours. Immunoblots were conducted to analyze neuroprotective signal-transduction in human primary glia cells and neurons. Neuronal sprouting assays were performed and neurotrophic factors in both hMNC (apo sec) and rat plasma were quantified using ELISA. Administration of rat as well as human apoptotic MNC-secretomes significantly reduced ischemic lesion volumes by 36% and 37%, respectively. Neurological examinations revealed improvement after stroke in both treatment groups. Co-incubation of human astrocytes, Schwann cells and neurons with hMNC (apo sec) resulted in activation of several signaling cascades associated with the regulation of cytoprotective gene products and enhanced neuronal sprouting in vitro. Analysis of neurotrophic factors in hMNC (apo sec) and rat plasma revealed high levels of brain derived neurotrophic factor (BDNF). Our data indicate that apoptotic MNC-secretomes elicit neuroprotective effects on rats that have undergone ischemic stroke.

Recently, a role of the receptor for advanced glycation endproducts (RAGE) in myasthenia gravis was described. RAGE and its ligand high mobility group box 1 (HMGB1) play key roles in autoimmunity and cancer. To test whether these molecules are involved in patients with thymic abnormalities we applied immunohistochemical analysis in 33 cases of thymic epithelial tumors, comprising 27 thymomas and 6 thymic carcinomas, and 21 nonneoplastic thymuses. Both molecules were detected in neoplastic epithelial cells: RAGE staining was most intense in WHO type B2 thymomas and thymic carcinomas (p<0.001). HMGB1 nuclear staining was strongest in A and AB, and gradually less in B1 = B2>B3>thymic carcinoma (p<0.001). Conversely, HMGB1 cytoplasmic staining intensities were as follows: A and AB (none), B1 (strong), B2 (moderate), B3 and thymic carcinoma (weak); (p<0.001). Fetal thymic tissue showed a distinct expression of RAGE and HMGB1 in subcapsular cortical epithelial cells which was found in 50% of myasthenic patients. Furthermore RAGE and HMGB1 were expressed in thymocytes, macrophages, Hassall's corpuscles, thymic medulla, and germinal center cells in myasthenic patients. Immunohistochemistry results were complemented by systemic measurements (immunosorbent assay): serum levels of soluble RAGE were significantly reduced in patients with epithelial tumors (p = 0.008); and in invasive tumors (p = 0.008). Whereas RAGE was equally reduced in thymic hyperplasia and epithelial tumors (p = 0.003), HMGB1 was only elevated in malignancies (p = 0.036). Results were most pronounced in thymic carcinomas. Thus, RAGE and HMGB1 are involved in the (patho-)physiology of thymus, as evidenced by differentiated thymic and systemic expression patterns that may act as diagnostic or therapeutic targets in autoimmune disease and cancer.

The quantity of cells with paracrine effects for use in myocardial regeneration therapy is limited. This study investigated the effects of catheter-based endomyocardial delivery of secretome of 2.5 × 10(9) apoptotic peripheral blood mononuclear cells (APOSEC) on porcine chronic post-myocardial infarction (MI) left ventricular (LV) dysfunction and on gene expression. Closed-chest reperfused MI was induced in pigs by 90-min occlusion followed by reperfusion of the mid-LAD (day 0). At day 30, animals were randomized to receive porcine APOSEC (n = 8) or medium solution (control; n = 8) injected intramyocardially into the MI border zone using 3D NOGA guidance. At day 60, cardiac MRI with late enhancement and diagnostic NOGA (myocardial viability) were performed. Gene expression profiling of the infarct core, border zone, and normal myocardium was performed using microarray analysis and confirmed by quantitative real-time PCR. Injection of APOSEC significantly decreased infarct size (p < 0.05) and improved cardiac index and myocardial viability compared to controls. A trend towards higher LV ejection fraction was observed in APOSEC vs. controls (45.4 ± 5.9% vs. 37.4 ± 8.9%, p = 0.052). Transcriptome analysis revealed significant downregulation of caspase-1, tumor necrosis factor and other inflammatory genes in APOSEC-affected areas. rtPCR showed higher expression of myogenic factor Mefc2 (p < 0.05) and downregulated caspase genes (p < 0.05) in APOSEC-treated pigs. In conclusion, overexpression of MEF2c and repression of caspase was related to decreased infarct size and improved cardiac function in secretome-treated animals. Altered gene expression 1-month post-APOSEC treatment proved the long-acting effects of cell-free therapy with paracrine factors., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background and Purpose: AT₁ receptor antagonists decrease body weight gain in models of murine obesity. However, fewer data are available concerning the anti-obesity effects of these antagonists, given as a treatment after obesity had been established., Experimental Approach: In spontaneously hypertensive rats, obesity was established by cafeteria diet (CD) feeding for 19 weeks. Rats were then were treated with telmisartan (8 mg·kg⁻¹·d⁻¹) or amlodipine (10 mg·kg⁻¹·d⁻¹; serving as blood pressure control) or telmisartan + amlodipine (2 + 10 mg·kg⁻¹·d⁻¹; to control for dose-dependency) for 17 weeks. Rats receiving only chow (C(chow)) or CD-fed rats treated with vehicle (C(CD)) served as controls., Key Results: The CD feeding induced obesity, hyperphagia, hyperlipidaemia, and leptin and insulin resistance. Telmisartan reduced the CD-induced increase in body weight and abdominal fat mass. Whereas energy intake was higher rather than lower, the respiratory ratio was lower. After telmisartan, leptin-induced energy intake was reduced and respiratory ratio was increased compared with C(CD) rats. Telmisartan also decreased plasma levels of triglycerides, free fatty acids and low-density lipoprotein. Amlodipine alone or the combination telmisartan + amlodipine did not affect body weight and eating behaviour. Telmisartan, but not amlodipine and telmisartan + amlodipine, improved glucose utilization. The decrease in BP reduction was almost the same in all treatment groups., Conclusions and Implications: Telmisartan exerted anti-obesity effects and restored leptin sensitivity, given as a treatment to rats with obesity. Such effects required high doses of telmisartan and were independent of the decrease in blood pressure., (© 2013 The British Pharmacological Society.)

Despite intense efforts, the exact phenotype of the epidermal Langerhans cell (LC) precursors during human ontogeny has not been determined yet. These elusive precursors are believed to migrate into the embryonic skin and to express primitive surface markers, including CD36, but not typical LC markers such as CD1a, CD1c and CD207. The aim of this study was to further characterize the phenotype of LC precursors in human embryonic epidermis and to compare it with that of LCs in healthy adult skin. We found that epidermal leukocytes in first trimester human skin are negative for CD34 and heterogeneous with regard to the expression of CD1c, CD14 and CD36, thus contrasting the phenotypic uniformity of epidermal LCs in adult skin. These data indicate that LC precursors colonize the developing epidermis in an undifferentiated state, where they acquire the definitive LC marker profile with time. Using a human three-dimensional full-thickness skin model to mimic in vivo LC development, we found that FACS-sorted, CD207(-) cord blood-derived haematopoietic precursor cells resembling foetal LC precursors but not CD14(+)CD16(-) blood monocytes integrate into skin equivalents, and without additional exogenous cytokines give rise to cells that morphologically and phenotypically resemble LCs. Overall, it appears that CD14(-) haematopoietic precursors possess a much higher differentiation potential than CD14(+) precursor cells.

The skin is exposed to environmental insults such as UV light that cause oxidative damage to macromolecules. A centerpiece in the defense against oxidative stress is the Nrf2 (nuclear factor (erythroid-derived-2)-like 2)-mediated transcriptional upregulation of antioxidant and detoxifying enzymes and the removal of oxidatively damaged material. Autophagy has an important role in the intracellular degradation of damaged proteins and entire organelles, but its role in the epidermis has remained elusive. Here, we show that both UVA and UVA-oxidized phospholipids induced autophagy in epidermal keratinocytes. Oxidative stressors induced massive accumulation of high-molecular-weight protein aggregates containing the autophagy adaptor protein p62/SQSTM1 in autophagy-deficient (autophagy-related 7 (ATG7) negative) keratinocytes. Strikingly, even in the absence of exogenous stress, the expression of Nrf2-dependent genes was elevated in autophagy-deficient keratinocytes. Furthermore, we show that autophagy-deficient cells contained significantly elevated levels of reactive oxidized phospholipids. Thus, our data demonstrate that autophagy is crucial for both the degradation of proteins and lipids modified by environmental UV stress and for limiting Nrf2 activity in keratinocytes. Lipids that promote inflammation and tissue damage because of their reactivity and signaling functions are commonly observed in aged and diseased skin, and thus targeting autophagy may be a promising strategy to counteract the damage promoted by excessive lipid oxidation.

Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SEC(PBMC)). Six mm punch biopsy wounds were set on the backs of C57BL/6J-mice and SEC(PBMC) containing emulsion or controls were applied daily for three days. Morphology and neo-angiogenesis were analyzed by H&E-staining and CD31 immuno-staining, respectively. In vitro effects on diverse skin cells were investigated by migration assays, cell cycle analysis, and tube formation assay. Signaling pathways were analyzed by Western blot analysis. Application of SEC(PBMC) on 6 mm punch biopsy wounds significantly enhanced wound closure. H&E staining of the wounds after 6 days revealed that wound healing was more advanced after application of SEC(PBMC) containing emulsion. Furthermore, there was a massive increase in CD31 positive cells, indicating enhanced neo-angiogenesis. In primary human fibroblasts (FB) and keratinocytes (KC) migration but not proliferation was induced. In endothelial cells (EC) SEC(PBMC) induced proliferation and tube-formation in a matrigel-assay. In addition, SEC(PBMC) treatment of skin cells led to the induction of multiple signaling pathways involved in cell migration, proliferation and survival. In summary, we could show that emulsions containing the secretome of PBMC derived from healthy individuals accelerates wound healing in a mouse model and induce wound healing associated mechanisms in human primary skin cells. The formulation and use of such emulsions might therefore represent a possible novel option for the treatment of non-healing skin ulcers.

Tight junction (TJ) proteins are involved in a number of cellular functions, including paracellular barrier formation, cell polarization, differentiation, and proliferation. Altered expression of TJ proteins was reported in various epithelial tumors. Here, we used tissue samples of human cutaneous squamous cell carcinoma (SCC), its precursor tumors, as well as sun-exposed and non-sun-exposed skin as a model system to investigate TJ protein alteration at various stages of tumorigenesis. We identified that a broader localization of zonula occludens protein (ZO)-1 and claudin-4 (Cldn-4) as well as downregulation of Cldn-1 in deeper epidermal layers is a frequent event in all the tumor entities as well as in sun-exposed skin, suggesting that these changes result from chronic UV irradiation. In contrast, SCC could be distinguished from the precursor tumors and sun-exposed skin by a frequent complete loss of occludin (Ocln). To elucidate the impact of down-regulation of Ocln, we performed Ocln siRNA experiments in human keratinocytes and uncovered that Ocln downregulation results in decreased epithelial cell-cell adhesion and reduced susceptibility to apoptosis induction by UVB or TNF-related apoptosis-inducing ligand (TRAIL), cellular characteristics for tumorigenesis. Furthermore, an influence on epidermal differentiation was observed, while there was no change of E-cadherin and vimentin, markers for epithelial-mesenchymal transition. Ocln knock-down altered Ca(2+)-homeostasis which may contribute to alterations of cell-cell adhesion and differentiation. As downregulation of Ocln is also seen in SCC derived from other tissues, as well as in other carcinomas, we suggest this as a common principle in tumor pathogenesis, which may be used as a target for therapeutic intervention.

Background: Defects in keratinocyte differentiation and skin barrier are important features of inflammatory skin diseases like atopic dermatitis. Mast cells and their main mediator histamine are abundant in inflamed skin and thus may contribute to disease pathogenesis., Methods: Human primary keratinocytes were cultured under differentiation-promoting conditions in the presence and absence of histamine, histamine receptor agonists and antagonists. The expression of differentiation-associated genes and epidermal junction proteins was quantified by real-time PCR, Western blot, and immunofluorescence labeling. The barrier function of human skin models was tested by the application of biotin as tracer molecule., Results: The addition of histamine to human keratinocyte cultures and organotypic skin models reduced the expression of the differentiation-associated proteins keratin 1/10, filaggrin, and loricrin by 80-95%. Moreover, the addition of histamine to skin models resulted in the loss of the granular layer and thinning of the epidermis and stratum corneum by 50%. The histamine receptor H1R agonist, 2-pyridylethylamine, suppressed keratinocyte differentiation to the same extent as did histamine. Correspondingly, cetirizine, an antagonist of H1R, virtually abrogated the effect of histamine. The expression of tight junction proteins zona occludens-1, occludin, claudin-1, and claudin-4, as well as that of desmosomal junction proteins corneodesmosin and desmoglein-1, was down-regulated by histamine. The tracer molecule biotin readily penetrated the tight junction barrier of skin cultures grown in the presence of histamine, while their diffusion was completely blocked in nontreated controls., Conclusions: Our findings suggest a new mechanism by which mast cell activation and histamine release contribute to skin barrier defects in inflammatory skin diseases., (© 2012 John Wiley & Sons A/S.)

Toll-like receptors (TLRs) initiate innate immune responses and direct subsequent adaptive immunity. They play a major role in cutaneous host defense against micro-organisms and in the pathophysiology of several inflammatory skin diseases. To understand the role of TLRs in the acquisition of immunological competence, we conducted a comprehensive study to evaluate TLR expression and function in the developing human skin before and after birth and compared it with adults. We found that prenatal skin already expresses the same spectrum of TLRs as adult skin. Strikingly, many TLRs were significantly higher expressed in prenatal (TLRs 1-5) and infant and child (TLRs 1 and 3) skin than in adult skin. Surprisingly, neither dendritic cell precursors in prenatal skin nor epidermal Langerhans cells and dermal dendritic cells in adult skin expressed TLRs 3 and 6, whereas the staining pattern and intensity of both TLRs in fetal basal keratinocytes was almost comparable to those of adults. Stimulation of primary human keratinocytes from fetal, neonatal and adult donors with selected TLR agonists revealed that the synthetic TLR3 ligand poly (I:C) specifically, mimicking viral double-stranded RNA, induced a significantly enhanced secretion of CXCL8/IL8, CXCL10/IP-10 and TNFα in fetal and neonatal keratinocytes compared with adult keratinocytes. This study demonstrates quantitative age-specific modifications in TLR expression and innate skin immune reactivity in response to TLR activation. Thus, antiviral innate immunity already in prenatal skin may contribute to protect the developing human body from viral infections in utero in a scenario where the adaptive immune system is not yet fully functional.

The adult human skin harbors a variety of leukocytes providing immune surveillance and host defense, but knowledge about their ontogeny is scarce. In this study we investigated the number and phenotype of leukocytes in prenatal human skin (dermal dendritic cells (DDCs), macrophages, T cells (including FoxP3(+) regulatory T cells), and mast cells) to unravel their derivation and to get a clue as to their putative function in utero. By flow cytometry and immunofluorescence, we found a distinction between CD206(+)CD1c(+)CD11c(+) DDCs and CD206(+)CD209(+)CD1c(-) skin macrophages by 9 weeks estimated gestational age (EGA). T cells appear at the end of the first trimester, expressing CD3 intracytoplasmatically. During midgestation, CD3(+)FoxP3(-) and CD3(+)FoxP3(+) cells can exclusively be found in the dermis. Similarly, other leukocytes such as CD117(+) (c-kit) mast cells were not identified before 12-14 weeks EGA and only slowly acquire a mature phenotype during gestation. Our data show at which time point during gestation antigen-presenting cells, T cells, and mast cells populate the human dermis and provide a step forward to a better understanding of the development of the human skin immune system.

Although epicardial blood flow can be restored by an early intervention in most cases, a lack of adequate reperfusion at the microvascular level is often a limiting prognostic factor of acute myocardial infarction (AMI). Our group has recently found that paracrine factors secreted from apoptotic peripheral blood mononuclear cells (APOSEC) attenuate the extent of myocardial injury. The aim of this study was to determine the influence of APOSEC on microvascular obstruction (MVO) in a porcine AMI model. A single dose of APOSEC was intravenously injected in a closed chest reperfused infarction model. MVO was determined by magnetic resonance imaging and cardiac catheterization. Role of platelet function and vasodilation were monitored by means of ELISA, flow cytometry, aggregometry, western blot and myographic experiments in vitro and in vivo. Treatment of AMI with APOSEC resulted in a significant reduction of MVO. Platelet activation markers were reduced in plasma samples obtained during AMI, suggesting an anti-aggregatory capacity of APOSEC. This finding was confirmed by in vitro tests showing that activation and aggregation of both porcine and human platelets were significantly impaired by co-incubation with APOSEC, paralleled by vasodilator-stimulated phosphoprotein (VASP)-mediated inhibition of platelets. In addition, APOSEC evidenced a significant vasodilatory capacity on coronary arteries via p-eNOS and iNOS activation. Our data give first evidence that APOSEC reduces the extent of MVO during AMI, and suggest that modulation of platelet activation and vasodilation in the initial phase after myocardial infarction contributes to the improved long-term outcome in APOSEC treated animals.

Silver has been used successfully for decades as an antibacterial agent and has become a standard treatment for burns and bacterial skin infections. Silver-containing creams, particularly silver sulfadiazine (SSD), possess effective activities against bacteria and fungi. However, there is serious concern that silver ions applied to denuded skin might be absorbed in significant amounts, thus introducing the risk of silver deposition, potentially leading to internal organ injury. In view of these facts we compared the percutaneous absorption and the antimicrobial potency of SSD with a new composition, nanoscalic silver (NSAg). In a murine model topical application of NSAg resulted in significantly lower percutaneous absorption and internal organ deposition compared to SSD. Strikingly, antimicrobial activity of NSAg used as a 0.1% formulation was comparable not only with 0.1% SSD against different bacterial strains including methicillin-resistant Staphylococcus aureus, but also against different yeast and dermatophyte species., From the Clinical Editor: Nanoscale silver (NSAg) was demonstrated to have significantly lower percutaneous absorption and less accumulation in multiple organs when applied to denuded skin. Its antimicrobial activity against MRSA was not only comparable to silver sulfadiazine, but the formulation was also effective against different yeast and dermatophyte species., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The thymic epithelium plays critical roles in the positive and negative selection of T cells. Recently, it was proposed that autophagy in thymic epithelial cells is essential for the induction of T cell tolerance to self antigens and thus for the prevention of autoimmune diseases. Here we have tested this hypothesis using mouse models in which autophagy was blocked specifically in epithelial cells expressing keratin 14 (K14), including the precursor of thymic epithelial cells. While the thymic epithelial cells of mice carrying the floxed Atg7 gene (ATG7 f/f) showed a high level of autophagy, as determined by LC3 Western blot analysis and fluorescence detection of the recombinant green fluorescent protein (GFP)-LC3 reporter protein on autophagosomes, autophagy in the thymic epithelium was efficiently suppressed by deletion of the Atg7 gene using the Cre-loxP system (ATG7 f/f K14-Cre). Suppression of autophagy led to the massive accumulation of p62/sequestosome 1 (SQSTM1) in thymic epithelial cells. However, the structure of the thymic epithelium as well as the organization and the size of the thymus were not altered in mutant mice. The ratio of CD4 to CD8-positive T cells, as well as the frequency of activated (CD69+) CD4 T cells in lymphoid organs, did not differ between mice with autophagy-competent and autophagy-deficient thymic epithelium. Inflammatory infiltrating cells, potentially indicative of autoimmune reactions, were present in the liver, lung, and colon of a similar fraction of ATG7 f/f and ATG7 f/f K14-Cre mice. In contrast to previously reported mice, that had received an autophagy-deficient thymus transplant, ATG7 f/f K14-Cre mice did not suffer from autoimmunity-induced weight loss. In summary, the results of this study suggest that autophagy in the thymic epithelium is dispensable for negative selection of autoreactive T cells.

Rationale: Acute myocardial infarction (AMI) followed by ventricular remodeling is the major cause of congestive heart failure and death in western world countries., Objective: Of relevance are reports showing that infusion of apoptotic leucocytes or anti-lymphocyte serum after AMI reduces myocardial necrosis and preserves cardiac function. In order to corroborate this therapeutic mechanism, the utilization of an immunosuppressive agent with a comparable mechanism, such as anti-thymocyte globulin (ATG) was evaluated in this study., Methods and Results: AMI was induced in rats by ligation of the left anterior descending artery. Initially after the onset of ischemia, rabbit ATG (10 mg/rat) was injected intravenously. In vitro and in vivo experiments showed that ATG induced a pronounced release of pro-angiogenic and chemotactic factors. Moreover, paracrine factors released from ATG co-incubated cell cultures conferred a down-regulation of p53 in cardiac myocytes. Rats that were injected with ATG evidenced higher numbers of CD68+ macrophages in the ischemic myocardium. Animals injected with ATG evidenced less myocardial necrosis, showed a significant reduction of infarct dimension and an improvement of post-AMI remodeling after six weeks (infarct dimension 24.9% vs. 11.4%, p<0.01). Moreover, a higher vessel density in the peri-infarct region indicated a better collateralization in rats that were injected with ATG., Conclusions: These data indicate that ATG, a therapeutic agent successfully applied in clinical transplant immunology, triggered cardioprotective effects after AMI that salvaged ischemic myocardium by down-regulation of p53. This might have raised the resistance against apoptotic cell death during ischemia. The combination of these mechanisms seems to be causative for improved cardiac function and less ventricular remodeling after experimental AMI.

Heart failure following acute myocardial infarction (AMI) is a major cause of morbidity and mortality. Our previous observation that injection of apoptotic peripheral blood mononuclear cell (PBMC) suspensions was able to restore long-term cardiac function in a rat AMI model prompted us to study the effect of soluble factors derived from apoptotic PBMC on ventricular remodelling after AMI. Cell culture supernatants derived from irradiated apoptotic peripheral blood mononuclear cells (APOSEC) were collected and injected as a single dose intravenously after myocardial infarction in an experimental AMI rat model and in a porcine closed chest reperfused AMI model. Magnetic resonance imaging (MRI) and echocardiography were used to quantitate cardiac function. Analysis of soluble factors present in APOSEC was performed by enzyme-linked immunosorbent assay (ELISA) and activation of signalling cascades in human cardiomyocytes by APOSEC in vitro was studied by immunoblot analysis. Intravenous administration of a single dose of APOSEC resulted in a reduction of scar tissue formation in both AMI models. In the porcine reperfused AMI model, APOSEC led to higher values of ejection fraction (57.0 vs. 40.5%, p < 0.01), a better cardiac output (4.0 vs. 2.4 l/min, p < 0.001) and a reduced extent of infarction size (12.6 vs. 6.9%, p < 0.02) as determined by MRI. Exposure of primary human cardiac myocytes with APOSEC in vitro triggered the activation of pro-survival signalling-cascades (AKT, Erk1/2, CREB, c-Jun), increased anti-apoptotic gene products (Bcl-2, BAG1) and protected them from starvation-induced cell death. Intravenous infusion of culture supernatant of apoptotic PBMC attenuates myocardial remodelling in experimental AMI models. This effect is probably due to the activation of pro-survival signalling cascades in the affected cardiomyocytes.

Undifferentiated transcription factor-1 (UTF-1) and reduced expression protein-1 (REX-1) are used as markers for the undifferentiated state of pluripotent stem cells. Because no highly specific cytochemical marker for epidermal stem cells has yet been identified, we investigated the expression pattern of these markers in human epidermis and skin tumours by immunohistochemistry and in keratinocyte cell cultures. Both presumed stem cell markers were widely expressed in the epidermis and skin appendages. Distinct expression was found in the matrix cells of the hair shaft. Differentiation of human primary keratinocytes (KC) in vitro strongly downregulated UTF-1 and REX-1 expression. In addition, REX-1 was upregulated in squamous cell carcinomas, indicating a possible role of this transcription factor in malignant tumour formation. Our data point to a role for these proteins not only in maintaining KC stem cell populations, but also in proliferation and differentiation of matrix cells of the shaft and also suprabasal KC., (© 2011 The Authors. International Journal of Experimental Pathology © 2011 International Journal of Experimental Pathology.)

Congestive heart failure developing after acute myocardial infarction (AMI) is a major cause of morbidity and mortality. Clinical trials of cell-based therapy after AMI evidenced only a moderate benefit. We could show previously that suspensions of apoptotic peripheral blood mononuclear cells (PBMC) are able to reduce myocardial damage in a rat model of AMI. Here we experimentally examined the biochemical mechanisms involved in preventing ventricular remodelling and preserving cardiac function after AMI. Cell suspensions of apoptotic cells were injected intravenously or intramyocardially after experimental AMI induced by coronary artery ligation in rats. Administration of cell culture medium or viable PBMC served as controls. Immunohistological analysis was performed to analyse the cellular infiltrate in the ischaemic myocardium. Cardiac function was quantified by echocardiography. Planimetry of the infarcted hearts showed a significant reduction of infarction size and an improvement of post AMI remodelling in rats treated with suspensions of apoptotic PBMC (injected either intravenously or intramoycardially). Moreover, these hearts evidenced enhanced homing of macrophages and cells staining positive for c-kit, FLK-1, IGF-I and FGF-2 as compared to controls. A major finding in this study further was that the ratio of elastic and collagenous fibres within the scar tissue was altered in a favourable fashion in rats injected with apoptotic cells. Intravenous or intramyocardial injection of apoptotic cell suspensions results in attenuation of myocardial remodelling after experimental AMI, preserves left ventricular function, increases homing of regenerative cells and alters the composition of cardiac scar tissue. The higher expression of elastic fibres provides passive energy to the cardiac scar tissue and results in prevention of ventricular remodelling.

Although it is widely accepted that filaggrin (FLG) deficiency contributes to an abnormal barrier function in ichthyosis vulgaris and atopic dermatitis, the pathomechanism of how FLG deficiency provokes a barrier abnormality in humans is unknown. We report here that the presence of FLG mutations in Caucasians predicts dose-dependent alterations in epidermal permeability barrier function. Although FLG is an intracellular protein, the barrier abnormality occurred solely via a paracellular route in affected stratum corneum. Abnormal barrier function correlated with alterations in keratin filament organization (perinuclear retraction), impaired loading of lamellar body contents, followed by nonuniform extracellular distribution of secreted organelle contents, and abnormalities in lamellar bilayer architecture. In addition, we observed reductions in corneodesmosome density and tight junction protein expression. Thus, FLG deficiency provokes alterations in keratinocyte architecture that influence epidermal functions localizing to the extracellular matrix. These results clarify how FLG mutations impair epidermal permeability barrier function., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.

Background: The World Health Organization (WHO) has recommended guidelines for a HIV drug resistance (HIVDR) survey for resource-limited countries. Eligibility criteria for patients include age below 25 years in order to focus on the prevalence of transmitted HIVDR (tHIVDR) in newly-infected individuals. Most of the participating sites across Africa have so far reported tHIVDR prevalences of below 5%. In this study we investigated whether the rate of HIVDR in patients <25 years is representative for HIVDR in the rest of the therapy-naïve population., Methods and Findings: HIVDR was determined in 88 sequentially enrolled ART-naïve patients from Mwanza, Tanzania (mean age 35.4 years). Twenty patients were aged <25 years and 68 patients were aged 25-63 years. The frequency of HIVDR in the study population was 14.8% (95%; CI 0.072-0.223) and independent of NVP-resistance induced by prevention of mother-to-child transmission programs. Patients >25 years had a significantly higher HIVDR frequency than younger patients (19.1%; 95% CI 0.095-0.28) versus 0%, P = 0.0344). In 2 out of the 16 patients with HIVDR we found traces of antiretrovirals (ARVs) in plasma., Conclusions: ART-naïve patients aged over 25 years exhibited significantly higher HIVDR than younger patients. Detection of traces of ARVs in individuals with HIVDR suggests that besides transmission, undisclosed misuse of ARVs may constitute a significant factor in the generation of the observed high HIVDR rate. The current WHO tHIVDR survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may therefore result in substantial underestimation of the prevalence of HIVDR in the therapy-naïve population. Similar studies should be performed also in other areas to test whether the so far reported optimistic picture of low HIVDR prevalence in young individuals is really representative for the rest of the ART-naïve HIV-infected population.

Background: In human epidermal keratinocytes the expression of hsp27 is closely related to differentiation in vitro and in situ., Objective: We aimed to gain further insight into the role of hsp27 in epidermal differentiation by specific inhibition through siRNA and inhibition of p38-MAPK, the key enzyme of hsp27 phosphorylation., Methods: Normal human keratinocytes (KC) and organotypic skin cultures (SE-skin equivalents) were used. Expression and phosphorylation of hsp27 was inhibited in these models by siRNA and SB203580, a specific inhibitor of p38-MAPK, respectively. Modification of morphology and expression of hsp27 and other differentiation associated proteins was investigated by immunofluorescence, western blot, and RT-PCR., Results: Inhibition of p38-MAPK resulted in a downregulation of hsp27 in KC and SE. Additionally, in the presence of SB203580 Ca(2+) induced expression of pro-filaggrin and loricrin was inhibited at the protein level and expression of filaggrin, keratin 10, and transglutaminase 1 at the mRNA level. Addition of SB203580 to SE, as well as hsp27 knockdown in this model resulted in identical patterns of irregular differentiation, disturbance of epidermal layers, and delayed expression of K10., Conclusion: These results provide evidence that the expression of hsp27 and its phosphorylation by p38-MAPK are required for keratinocyte differentiation and for the formation of a regularly stratified epidermis., (Copyright © 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

The female urogenital tract requires an efficient defense against bacteria, potentially derived from the adjacent intestinal tract. We have thus sought to identify the factors that protect against Escherichia coli (E. coli) in the female genital tract. Vaginal fluid from healthy human donors consistently killed E. coli in vitro and vaginal epithelium strongly expressed and secreted psoriasin. Psoriasin was constitutively produced in an organotypic vaginal epithelium model, and exposure of these cells to supernatants of E. coli cultures led to an enhanced psoriasin expression. Secreted psoriasin in vaginal fluids accounted for approximately 2.5-3% of total protein. Fractionation of vaginal fluids by high performance liquid chromatography (HPLC) showed that psoriasin co-eluted with a peak of E. coli killing activity. Our data show that normal vaginal fluid contains a powerful intrinsic antimicrobial defense against E. coli and that psoriasin contributes to the innate immune response of the female genital tract.

Antimicrobial peptides (AMPs) have a central role in the innate immune system of the skin. Epidermal keratinocytes (KCs) express numerous such peptides either constitutively or in response to exposure to microbial compounds. Here, we investigated the regulation of S100A8 (calgranulin A) and S100A9 (calgranulin B), which form an antimicrobial heterodimeric complex also known as calprotectin, in KCs. Culture supernatants of gram-negative bacteria, but not of gram-positive bacteria nor of the yeast Candida albicans, triggered the expression of S100A8 and S100A9. To identify pathogen-associated molecular patterns (PAMPs) responsible for the upregulation of S100A8 and S100A9, KCs were stimulated with ligands for Toll-like receptors (TLRs). Quantitative real-time PCR (qRT-PCR) analysis revealed that the TLR5 ligand flagellin increased the mRNA expression of both S100A8 and S100A9. Supernatant from wild-type (WT) Escherichia coli, but not from a flagellin-deficient E. coli strain (ΔFliC), induced S100A8 and S100A9 protein production in KCs. Moreover, small interfering RNA-mediated knockdown of TLR5 expression suppressed the ability of KCs to upregulate S100A8 and S100A9 mRNA expression in response to E. coli supernatant. Like in cell culture, stimulation of human skin explants with E. coli induced the expression of S100A8 and S100A9. Our data suggest that bacterial flagellin induces the upregulation of S100A8/S100A9 via a TLR5-dependent mechanism in epidermal KCs.

Retinoids are regulators of keratinocyte differentiation in the epidermis and important therapeutics in dermatology. The formation of the most active retinoid, all-trans-retinoic acid (RA) by oxidation of retinal is catalyzed by aldehyde dehydrogenases (ALDH), of which ALDH1A3 has been shown to be most efficient. Here we investigated the expression of ALDH1A3 in epidermal cultures. Three alternatively spliced mRNAs of ALDH1A3 were detected in skin cultures with the conventionally spliced mRNA being predominant. Among a panel of ALDH genes, only ALDH1A3 was upregulated by RA in primary keratinocytes. RA increased the expression of ALDH1A3 also in organotypic human skin cultures and in an epidermal explant in vitro whereas no upregulation was detected in dermal fibroblasts and HeLa cells. Our results indicate that the regulation of the retinoic acid metabolism in the epidermis involves transcriptional activation of ALDH1A3, possibly representing a positive feedback loop, which enhances the effect of exogenous RA., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Bacterial ghosts (BGs) as non-living bacterial envelopes devoid of cytoplasmic content with preserved and intact inner and outer membrane structures of their living counterparts have been used to study the ability of their surface components for the induction of antimicrobial peptides and pro-inflammatory cytokines in human primary keratinocytes (KCs). Quantitative real-time PCR analysis revealed that incubation of KCs with BGs generated from wild-type Escherichia coli induced the mRNA expression of antimicrobial psoriasin (S100A7c) in a BGs particle concentration-dependent manner. Using immunoblot analysis we showed that BGs generated from the flagellin-deficient (ΔFliC) E. coli strain NK9375 were as effective as its isogenic wild-type (wt) E. coli strain NK9373 to induce psoriasin expression when normalized to BG particles being taken up by KCs. However, results obtained from endocytic activity of KCs reflect that internalization of BGs is greatly dependent on the presence of flagellin on the surface of BGs. Moreover, BGs derived from wt E. coli NK9373 strongly induced the release of the pro-inflammatory cytokines IL-6 and IL-8, compared to ΔFliC E. coli NK9375 BGs. Taken together, obtained data demonstrate that non-living BGs possessing all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat have the capacity to induce the expression of innate immune modulators and that these responses are partially dependent on the presence of flagellin., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.

Aims: Serum levels of the soluble growth stimulation gene-2 (sST2) are elevated in heart and pulmonary diseases. However, the relationship of the sST2/interleukin (IL)-33 axis and its triggers as well as its organ distribution is still not known. This study was thus designed to investigate the cellular origin and regulation of sST2 and IL-33 in vitro and in vivo., Methods and Results: sST2 and IL-33 gene expression and protein secretion were analysed in pooled organ-specific cDNAs and in primary cell cultures, respectively, by RT-PCR and ELISA technology. The strongest sST2 mRNA expression was detected in heart and lung tissues, which correlated with spontaneous secretion of sST2 protein in vitro. The inflammatory cytokines IL-1alpha, IL-1beta, and tumour necrosis factor alpha as well as supernatants of lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells led to an enhanced secretion of sST2 in cultured cardiac myocytes and lung alveolar epithelial cells. These cytokines enhanced sST2 secretion via an NFkappaB-dependent mechanism. In addition, LPS stimulation in humans in vivo induced a short-term inflammatory response that was followed by a massive enhancement of sST2 secretion., Conclusion: These results identify the primary sources and inflammatory triggers for the enhancement of sST2 secretion and demonstrate a relationship between inflammation and the secretion of a bioactive member of the IL-1R family, both in vitro and in vivo.

The hepatocyte growth factor/scatter factor (HGF/SF) tyrosine kinase (TK) receptor c-Met plays a crucial role in the development of the invasive phenotype of tumors and thus represents an attractive candidate for targeted therapies in a variety of malignancies, including human malignant melanoma (MM). In contrast to what has been shown previously, we were not able to detect any genetic alterations, either in the juxtamembrane- or in the TK-domain of c-Met, in the studied MM cell lines. Nevertheless, c-Met was constitutively active in these cell lines without exogenous HGF/SF stimulation. The active receptor was localized to the adhesion sites of the cells. Addition of the c-Met TK inhibitor SU11274 specifically decreased the phosphotyrosine signal at the focal adhesions sites, which was accompanied by a decrease in cell proliferation as well as an increase in apoptotic cells. In addition, non-apoptotic concentrations of SU11274 significantly reduced the in vitro migratory capacity of MM cells in the modified Boyden-chamber assay. Administration of SU11274 significantly decreased primary tumor growth as well as the capacity for liver colony formation of MM cells in SCID mice. Our study provides the first evidence for an in vivo antitumor activity of SU11274 in a human melanoma xenograft model, and suggests c-Met as a valid target for the therapy of MM. Consequently, SU11274 treatment might represent a useful strategy for controlling melanoma progression and metastasis in patients with MM.

Aging is a multifactorial process where deterioration of body functions is driven by stochastic damage while counteracted by distinct genetically encoded repair systems. To better understand the genetic component of aging, many studies have addressed the gene and protein expression profiles of various aging model systems engaging different organisms from yeast to human. The recently identified small non-coding miRNAs are potent post-transcriptional regulators that can modify the expression of up to several hundred target genes per single miRNA, similar to transcription factors. Increasing evidence shows that miRNAs contribute to the regulation of most if not all important physiological processes, including aging. However, so far the contribution of miRNAs to age-related and senescence-related changes in gene expression remains elusive. To address this question, we have selected four replicative cell aging models including endothelial cells, replicated CD8(+) T cells, renal proximal tubular epithelial cells, and skin fibroblasts. Further included were three organismal aging models including foreskin, mesenchymal stem cells, and CD8(+) T cell populations from old and young donors. Using locked nucleic acid-based miRNA microarrays, we identified four commonly regulated miRNAs, miR-17 down-regulated in all seven; miR-19b and miR-20a, down-regulated in six models; and miR-106a down-regulated in five models. Decrease in these miRNAs correlated with increased transcript levels of some established target genes, especially the cdk inhibitor p21/CDKN1A. These results establish miRNAs as novel markers of cell aging in humans.

The antimicrobial defense of the skin is partially mediated by RNase 7, an abundant ribonuclease of the stratum corneum (SC). Here, we investigated the expression and regulation of members of the RNase A family and of the endogenous RNase inhibitor (RI) protein in epidermal keratinocytes (KCs). Reverse transcription-PCR screening revealed that KCs expressed not only RNase 7 but also RNase 5, which was shown earlier to kill the yeast Candida albicans, as well as RNase 1, RNase 4, and RI. The mRNA and protein levels of RNase 5, RNase 7, and RI increased during KC differentiation. When RNase 5 and RNase 7 were incubated with RI in vitro, not only their ribonucleolytic activities but also their antimicrobial activities were strongly suppressed. Immunochemical analyses revealed that SC contains RNase 5, whereas RI was not detectable. Unlike recombinant RNase 5, recombinant RI was degraded when exposed to SC extract. The addition of aprotinin prevented the degradation of RI, indicating that serine proteases of the SC cleave RI. Taken together, this study adds RNase 5 to the list of antimicrobial factors present in the SC and suggests that proteases contribute indirectly to the defense function of the SC by releasing the RI-mediated inhibition of RNase 5 and RNase 7.

Objectives: Acanthamoebae can cause infections of several organs, including eye, skin, lung and brain. Except for Acanthamoeba keratitis, these infections are linked to immunodeficiency. Treatment is generally problematic, due to the lack of sufficiently effective and also easily manageable drugs. In a previous study we discovered that miltefosine (hexadecylphosphocholine) is highly active against Acanthamoeba spp. in vitro. The aim of the current study was to evaluate the suitability of miltefosine for the topical treatment of Acanthamoeba infections., Methods: Storage life and time dependency, susceptibilities of opportunistic bacterial and fungal pathogens, and synergistic and adverse effects of combinations with other anti-Acanthamoeba substances were determined. Moreover, an organotypic skin equivalent was adapted for investigating the penetration of acanthamoebae into the epidermis and the human tissue tolerability of miltefosine., Results: It was shown that miltefosine can be stored as a 2 mM stock solution and also as a 50 microM dilution over a period of 12 months at 4 degrees C without any loss of activity. Efficacies against staphylococci and Candida albicans were established. Acanthamoebae were able to penetrate the skin equivalent within 24 h. This penetration was prevented by treatment with miltefosine, while miltefosine treatment was well tolerated by the skin equivalent., Conclusions: Miltefosine has been approved for oral and topical treatment of leishmaniasis and may also be a promising candidate for the topical treatment of Acanthamoeba infections.

Background: Acute myocardial infarction (AMI) is followed by post AMI cardiac remodelling, often leading to congestive heart failure. Homing of c-kit+ endothelial progenitor cells (EPC) has been thought to be the optimal source for regenerating infarcted myocardium., Methods: Immune function of viable peripheral blood mononuclear cells (PBMC) was evaluated after co-culture with irradiated apoptotic PBMC (IA-PBMC) in vitro. Viable PBMC, IA-PBMC and culture supernatants (SN) thereof were obtained after 24 h. Reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were utilized to quantify interleukin-8 (IL-8), vascular endothelial growth factor, matrix metalloproteinase-9 (MMP9) in PBMC, SN and SN exposed fibroblasts. Cell suspensions of viable- and IA-PBMC were infused in an experimental rat AMI model. Immunohistological analysis was performed to detect inflammatory and pro-angiogenic cells within 72 h post-infarction. Functional data and determination of infarction size were quantified by echocardiography and Elastica van Gieson staining., Results: The IA-PBMC attenuated immune reactivity and resulted in secretion of pro-angiogenic IL-8 and MMP9 in vitro. Fibroblasts exposed to viable and IA-PBMC derived SN caused RNA increment of IL-8 and MMP9. AMI rats that were infused with IA-PBMC cell suspension evidenced enhanced homing of endothelial progenitor cells within 72 h as compared to control (medium alone, viable-PBMC). Echocardiography showed a significant reduction in infarction size and improvement in post AMI remodelling as evidenced by an attenuated loss of ejection fraction., Conclusion: These data indicate that infusion of IA-PBMC cell suspension in experimental AMI circumvented inflammation, caused preferential homing of regenerative EPC and replaced infarcted myocardium.

Epidermal keratinocytes (KCs) express antimicrobial peptides as a part of the innate immune response. It has recently been shown that the culture supernatant of Escherichia coli induces the expression of S100A7c (psoriasin) in KCs and that S100A7c efficiently kills E. coli. Here we have investigated which of the microbial components triggers the up-regulation of S100A7c expression. Exposure of human primary KCs to ligands of the human Toll-like receptors (TLRs) revealed that only the TLR5 ligand flagellin strongly induced the expression of S100A7c mRNA and protein, whereas all other TLR ligands had no significant effect. In contrast to the supernatant from flagellated wild-type (WT) E. coli, the supernatant of a flagellin-deficient E. coli strain (DeltaFliC) did not induce S100A7c expression. Small interfering RNA-mediated knockdown of TLR5 expression suppressed the ability of KCs to up-regulate S100A7c expression in response to both flagellin and WT E. coli supernatant. Taken together, our data demonstrate that bacterial flagellin is essential and sufficient for the induction of S100A7c expression in KCs by E. coli.

Caspase-14, a protease involved in skin barrier formation, is specifically expressed in epidermal keratinocytes (KCs). Here, we mapped three start sites of transcription of the human caspase-14 gene and analyzed the upstream chromosomal region for promoter activity. Reporter gene assays identified a core promoter region proximal to the first exon and a distal regulatory region which differentially suppressed promoter activity in KC and other cells. Sequence elements in the proximal promoter were bound by the transcription factors AP-1 (JunB, c-Jun, JunD, Fra-1 and Fra-2) and NFkappaB (p50 and RelB). Our data reveal the basic organization of the human caspase-14 promoter and suggest an important role of AP-1 and NFkappaB in the transcriptional control of caspase-14.

Background: Histidase (histidine ammonia lyase) converts histidine into urocanic acid, the main ultraviolet (UV) light absorption factor of the stratum corneum. It is unknown if and how histidase is regulated in the epidermis., Objective: We have investigated the transcriptional regulation of histidase expression in epidermal keratinocytes., Methods: Human epidermal keratinocytes were cultured in vitro and exposed to UV irradiation, a number of cytokines and all-trans retinoic acid (ATRA) (1 microM). Keratinocyte differentiation was triggered by maintaining confluent cells in monolayer culture and by establishing three-dimensional skin equivalents. The mRNA expression level of histidase in keratinoytes as well as in the epidermis and other tissues was determined by quantitative real-time PCR. Protein expression was determined by Western blot analysis., Results: Human epidermis contained higher levels of histidase transcripts than all other tissues investigated. Expression of histidase strongly increased at the mRNA and protein levels during differentiation of primary keratinocytes in vitro. Treatment of keratinocytes with UVA and UVB did not significantly change the expression level of histidase. By contrast, ATRA suppressed histidase expression almost completely., Conclusions: Our results show that histidase is upregulated during keratinocyte differentiation and that ATRA but not UV irradiation modulates the expression level of histidase. Suppression of histidase-mediated production of urocanic acid may contribute to the increase in UV sensitivity that is caused by treatment with retinoids.

To investigate the role of the angiogenic cytokine vascular endothelial growth factor (VEGF) during pregnancy and lactation, we used mice in which VEGF had been inactivated in mammary gland epithelial cells. Pups born to mutant mothers failed to thrive, displaying little milk in their stomachs. However, when they were transferred to control mothers they developed normally. Investigation of the mammary gland morphology revealed that lobulo-alveolar expansion into the fat pad was not complete in lactating mutant glands, and an accumulation of fat globules was evident in their secretory epithelium. In contrast to control glands, lactating mutant glands failed to up-regulate mRNAs for genes involved in milk secretion. Blood vessel density was comparable in pregnant mice of both groups but was only half that of controls in lactating mutant mice. FITC-labeled albumin injected intravenously (i.v.) into lactating mice extravasated rapidly and accumulated in the mammary gland epithelial cells in control animals, but was almost completely retained within the vessels in the mutants. Injection of recombinant VEGF i.v. reversed this effect. These findings demonstrate that mammary epithelium-derived VEGF is partially dispensable for angiogenesis during pregnancy and lactation, and by regulating vascular function during lactation, this factor is crucial to mammary gland differentiation and milk production.