Proteome analysis, bioinformatic prediction and experimental evidence revealed immune response down-regulation function for serum-starved human fibroblasts.

Emerging evidence indicates that fibroblasts play pivotal roles in immunoregulation by producing various proteins under health and disease states. In the present study, for the first time, we compared the proteomes of serum-starved human skin fibroblasts and peripheral blood mononuclear cells (PBMCs) using Nano-LC-ESI-tandem mass spectrometry. This analysis contributes to a better understanding of the underlying molecular mechanisms of chronic inflammation and cancer, which are intrinsically accompanied by growth factor deficiency.The proteomes of starved fibroblasts and PBMCs consisted of 307 and 294 proteins, respectively, which are involved in lymphocyte migration, complement activation, inflammation, acute phase response, and immune regulation. Starved fibroblasts predominantly produced extracellular matrix-related proteins such as collagen/collagenase, while PBMCs produced focal adhesion-related proteins like beta-parvin and vinculin which are involved in lymphocyte migration. PBMCs produced a more diverse set of inflammatory molecules like heat shock proteins, while fibroblasts produced human leukocytes antigen-G and -E that are known as main immunomodulatory molecules. Fifty-four proteins were commonly found in both proteomes, including serum albumin, amyloid-beta, heat shock cognate 71 kDa, and complement C3. GeneMANIA bioinformatic tool predicted 418 functions for PBMCs, including reactive oxygen species metabolic processes and 241 functions for starved fibroblasts such as antigen processing and presentation including non-classical MHC -Ib pathway, and negative regulation of the immune response. Protein-protein interactions network analysis indicated the immunosuppressive function for starved fibroblasts-derived human leucocytes antigen-G and -E. Moreover, in an in vitro model of allogeneic transplantation, the immunosuppressive activity of starved fibroblasts was experimentally documented. Conclusion: Under serum starvation-induced metabolic stress, both PBMCs and fibroblasts produced molecules like heat shock proteins and amyloid-beta, which can have pathogenic roles in auto-inflammatory diseases such as rheumatoid arthritis, type 1 diabetes mellitus, systemic lupus erythematosus, aging, and cancer. However, starved fibroblasts showed immunosuppressive activity in an in vitro model of allogeneic transplantation, suggesting their potential to modify such adverse reactions by down-regulating the immune system.

New Rapid Helicobacter Pylori Blood Test Based on Dual Detection of FliD and CagA Antibodies for On-Site Testing.

Helicobacter pylori is the most prevalent bacterial infection, affecting half of the world's population, with a high morbidity and mortality rate.1,2 Several invasive and noninvasive testing procedures are available, and their selective use serves the specific needs of diverse clinical scenarios. For gastric cancer prevention, mass screening is necessary and requires a noninvasive, rapid, accurate and cost-effective test. For this purpose H pylori serology currently seems to be the preferred noninvasive diagnostic method. Population-based testing and treatment for H pylori is cost effective in high-risk countries, but less effective in low- and medium-risk countries.3,4 Many serologic tests are available on the market, with inconsistent performance often being observed. Therefore, international guidelines recommend considering only serologic tests with high accuracy that have been validated in the respective local populations. To date, no rapid point-of-care test (POCT) has reached a sufficient degree of accuracy.

Ninety-six-hour starved peripheral blood mononuclear cell supernatant inhibited LA7 breast cancer stem cells induced tumor via reduction in angiogenesis and alternations in Gch1 and Spr expressions.

Introduction: The microenvironment of solid tumors such as breast cancer is heterogeneous and complex, containing different types of cell, namely, cancer stem cells and immune cells. We previously reported the immunoregulatory behavior of the human immune cell in a solid tumor microenvironment-like culture under serum starvation stress for 96 h. Here, we examined the effect of this culture-derived solution on breast cancer development in rats. Method: Ninety-six-hour starved PBMCs supernatant (96 h-SPS) was collected after culturing human PBMCs for 96 h under serum starvation condition. Breast cancer stem cells, LA7 cell line, was used for in vitro study by analyzing gene expression status and performing cytotoxicity, proliferation, scratch wound healing assays, followed by in vivo tumor induction in three groups of mature female Sprague Dawley rats. Animals were treated with 96 h-SPS or RPMI and normal saline as control, n = 6 for each group. After biochemical analysis of iron, lactate, and pH levels in the dissected tumors, Ki67 antigen expression, angiogenesis, and necrosis evaluation were carried out. Metabolic-related gene expression was assessed using RT-qPCR. Moreover, 96 h-SPS composition was discovered by Nano-LC-ESI-MS/MS. Results: 96 h-SPS solution reduced the LA7 cell viability, proliferation, and migration and Gch1 and Spr genes expression in vitro (p< 0.05), whereas stemness gene Oct4 was upregulated (p< 0.01). The intracellular lactate was significantly decreased in the 96 h-SPS treated group (p = 0.007). In this group, Gch1 and Spr were significantly downregulated (p< 0.05), whereas the Sox2 and Oct4 expression was not changed significantly. The number of vessels and mitosis (Ki67+ cells) in the 96 h-SPS-treated group was significantly reduced (p = 0.024). The increased rate of necrosis in this group was statistically significant (p = 0.04). Last, proteomics analysis revealed candidate effectors' components of 96 h-SPS solution. Conclusion: 96 h-SPS solution may help to prevent cancer stem cell mediated tumor development. This phenomenon could be mediated through direct cytotoxic effects, inhibition of cell proliferation and migration in association with reduction in Gch1 and Spr genes expression, angiogenesis and mitosis rate, and necrosis augmentation. The preliminary data obtained from the present study need to be investigated on a larger scale and can be used as a pilot for further studies on the biology of cancer development.

Downregulation of Stemness Genes and Induction of Necrosis in Rat LA7 Cancer Stem Cells Induced Tumors Treated with Starved Fibroblasts Culture Supernatant.

BACKGROUND: Stem cell differentiation therapy is a promising strategy in cancer treatment. we show that protein cocktail prepared from serum starved fibroblasts has therapeutic potential based on this strategy. METHODS: The condition medium was prepared from foreskin isolated fibroblasts and analyzed by Liquid chromatography electrospray ionization mass spectrometry-mass spectrometry (LC-ESI-MS/MS). LA7 mammary gland cancer stem cells originated tumors were induced in Sprague Dawley rats. The rats treated subcutaneously with DMEM (group A), condition medium (group B), or normal saline (group C) once daily for 7 days. Then the tumors were removed and divided into the two parts, one part was used to quantify gene expression by stem-loop RT-qPCR assay and the other part was used for Hematoxylin & Eosin (H & E), Giemsa, and immunohistochemistry (IHC) staining. RESULTS: All induced tumors appeared as sarcomatoid carcinoma (SC). Immunohistochemistry staining confirmed this conclusion by recognizing the tumor as Ki67+, cytokeratin+, vimentine+, and estrogen receptor negative SC. RT-qPCR analysis revealed that Oct4-, Sox-2, Nanog- gene expression was much reduced in the condition medium treated tumors versus proper controls (p< 0.05). Tissue necrosis was more prevalent in this group while tumors volume was diminished almost by 40%. The LC-ESI-MS/MS analysis unrevealed the stemness reducing and the cell death inducing proteins such as, pigment epithelium-derived factor (PEDF), insulin like growth factor binding protein-5 (IGFBP-5) and -7 (IGFBP-7) in the condition medium. CONCLUSION: This study showed that the substances released from starved human fibroblasts were able to down-regulate the stemness-related genes and induce necrosis in LA7 derived tumors.

Loss of endogenous RNF43 function enhances proliferation and tumour growth of intestinal and gastric cells.

Ring finger protein 43 (RNF43) is an E3 ubiquitin ligase that has been described to be frequently mutated in gastrointestinal cancers. RNF43 downregulation was associated with distant metastasis, TNM stage and poorer survival in patients with gastric and colorectal cancers. Functional analysis has shown that overexpressed RNF43 negatively regulates Wnt signalling by ubiquitinating Frizzled receptors and targeting them for degradation and by sequestering T-cell factor 4 (TCF4) to the nuclear membrane, thereby inhibiting Wnt-mediated transcription. In the stomach, RNF43 overexpression was shown to impair stem-like properties and to be negatively correlated with expression of Wnt-target genes. In this study, we show that RNF43 knockdown enhances the tumourigenic potential of gastric and colorectal cancer cell lines in vitro and in vivo. Thus, loss of RNF43 leads to increased proliferation and anchorage-independent growth as well as increased invasive capacity. In a xenograft model, RNF43 depletion enhanced tumour growth. Furthermore, we established two mouse models in which mutations in the RING domain of RNF43 were introduced. In the intestine and colon, loss of Rnf43 did not induce changes in epithelial architecture or proliferation. In contrast, in the stomach, thickening of the mucosa, hyperplasia and cellular atypia were observed in these mice. Notably, this was independent of elevated Wnt signalling. Together, our results show that RNF43 plays a tumour suppressive role in gastric and colorectal cancer cells and that the loss of its function alters gastric tissue homeostasis in vivo.

Helicobacter pylori adhesin HopQ engages in a virulence-enhancing interaction with human CEACAMs.

Helicobacter pylori specifically colonizes the human gastric epithelium and is the major causative agent for ulcer disease and gastric cancer development. Here, we identify members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family as receptors of H. pylori and show that HopQ is the surface-exposed adhesin that specifically binds human CEACAM1, CEACAM3, CEACAM5 and CEACAM6. HopQ-CEACAM binding is glycan-independent and targeted to the N-domain. H. pylori binding induces CEACAM1-mediated signalling, and the HopQ-CEACAM1 interaction enables translocation of the virulence factor CagA into host cells and enhances the release of pro-inflammatory mediators such as interleukin-8. Based on the crystal structure of HopQ, we found that a ß-hairpin insertion (HopQ-ID) in HopQ's extracellular 3+4 helix bundle domain is important for CEACAM binding. A peptide derived from this domain competitively inhibits HopQ-mediated activation of the Cag virulence pathway, as genetic or antibody-mediated abrogation of the HopQ function shows. Together, our data suggest the HopQ-CEACAM1 interaction to be a potentially promising novel therapeutic target to combat H. pylori-associated diseases.

The E3 ligase RNF43 inhibits Wnt signaling downstream of mutated ß-catenin by sequestering TCF4 to the nuclear membrane.

Given its fundamental role in development and cancer, the Wnt-ß-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family. We detected endogenous RNF43 in the nucleus of human intestinal crypt and colon cancer cells. We found that RNF43 physically interacted with T cell factor 4 (TCF4) in cells and tethered TCF4 to the nuclear membrane, thus silencing TCF4 transcriptional activity even in the presence of constitutively active mutants of ß-catenin. This inhibitory mechanism was disrupted by the expression of RNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of the Wnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate that RNF43 inhibits the Wnt pathway downstream of oncogenic mutations that activate the pathway. Mimicking or enhancing this inhibitory activity of RNF43 may be useful to treat cancers arising from aberrant activation of the Wnt pathway.

High Frequency of vacA s1m2 Genotypes Among Helicobacter pylori Isolates From Patients With Gastroduodenal Disorders in Kermanshah, Iran.

BACKGROUND: Helicobacter pylori infection and related diseases outcome are mediated by a complex interplay between bacterial, host and environmental factors. Several distinct virulence factors of H. pylori have been shown to be associated with different clinical outcomes. Here we focused on vacA and cagA genotypes of H. pylori strains isolated from patients with gastric disorder. OBJECTIVES: The aim of this study was to determine the frequency of two toxins and genotypes of VacA toxin in patients referred to a central hospital in the west of Iran (Imam Reza hospital, Kermanshah) during 2011 - 2012. PATIENTS AND METHODS: Samples were collected from patients infected with H. pylori. Gastric biopsy specimens from the stomach antrum and corpus were cultured. PCR analysis was performed for genotyping H. pylori vacA and cagA genes. RESULTS: Helicobacter pylori was isolated from 48% (96/200) of patients with gastroduodenal disorders. In 81/96 (84%) cases, the cagA gene was present. Among different genotypes of vacA, two s1m2 and s2m2 genotypes were dominant with frequency of 39.5% and 50%, respectively. The frequency of the s1m1 genotype was 7.2% (7/96), which is much lower than elsewhere. H. pylori isolates with positive results for cagA gene and vacA s1m2 genotypes showed statistically significant correlation with peptic ulcer (s1m2 13/34 [38.2%] P = 0.003). However, isolates of H. pylori infection with cagA gene and vacA s2m2 genotypes were significantly associated with development of gastritis (s2m2 41/42 [97.6%] P = 0.000). CONCLUSIONS: About 90% of H. pylori strains potentially contained vacA s2m2 and s1m2 genotypes. Infection with H. pylori strain containing the cagA gene or the vacA s1m1 and s1m2 genotypes was associated with increased incidence of peptic ulcer disease (PUD).

H. pylori virulence factors: influence on immune system and pathology.

Helicobacter pylori is the most widespread chronic bacterial agent in humans and is well recognized for its association with ulcer disease and gastric cancer, with both representing major global health and socioeconomic issues. Given the high level of adaptation and the coevolution of this bacterium with its human host, a thorough and multidirectional view of the specific microbiological characteristics of this infection as well as the host physiology is needed in order to develop novel means of prevention of therapy. This review aims to pinpoint some of these potentially important angles, which have to be considered mutually when studying H. pylori's pathogenicity. The host's biological changes due to the virulence factors are a valuable pillar of H. pylori research as are the mechanisms by which bacteria provoke these changes. In this context, necessary adhesion molecules and significant virulence factors of H. pylori are discussed. Moreover, metabolism of the bacteria, one of the most important aspects for a better understanding of bacterial physiology and consequently possible therapeutic and prophylactic strategies, is addressed. On the other hand, we discuss the recent experimental proofs of the "hygiene hypothesis" in correlation with Helicobacter's infection, which adds another aspect of complexity to this infection.

The Sox17CreERT2 knock-in mouse line displays spatiotemporal activation of Cre recombinase in distinct Sox17 lineage progenitors.

The HMG-box transcription factor Sox17 is essential for endoderm formation, vascular development, and definitive hematopoiesis. To investigate the fate of distinct Sox17-expressing progenitor cells in a spatiotemporal manner, we generated a hormone-inducible CreERT2 knock-in mouse line. By homologous recombination we fused a codon improved, ligand-dependent estrogen receptor Cre recombinase by an intervening viral T2A sequence for co-translational cleavage to the 3' coding region of Sox17. Induction of Cre activity by administration of tamoxifen at defined time points of early mouse development and subsequent genetic lineage tracing confirmed the inducibility and tissue specificity of Cre recombination. Furthermore, Cre activity could be selectively induced in extra-embryonic and embryonic endoderm lineages, the primitive gut tube, and in endothelial cells of the vascular system as well as in the hemogenic endothelium of the dorsal aorta. The Sox17CreERT2 mouse line therefore represents a new tool for genetic lineage tracing in a tissue-specific manner and in addition enables lineage-restricted functional analysis.

Helicobacter bilis gamma-glutamyltranspeptidase enhances inflammatory stress response via oxidative stress in colon epithelial cells.

Helicobacter bilis (H. bilis) infection is associated with cases of inflammatory bowel Disease, thyphlocolitis, hepatitis and cholecystitis. However, little is known about the bacterial virulence determinants or the molecular mechanisms involved. Recently, H. bilis γ-glutamyltranspeptidase (HBgGT) was shown to be a virulence factor decreasing host cell viability. Bacterial gGTs play a key role in synthesis and degradation of glutathione and enables the bacteria to utilize extracellular glutamine and glutathione as sources of glutamate. gGT-mediated loss of cell viability has so far been linked to DNA damage via oxidative stress, but the signaling cascades involved herein have not been described. In this study, we identified enhanced ROS production induced by HBgGT as a central factor involved in the activation of the oxidative stress response cascades, which finally activate CREB, AP-1 and NF-κB in H. bilis infected colon cancer cells. IL-8, an important pro-inflammatory chemokine that is a common downstream target of these transcription factors, was up-regulated upon H. bilis infection in an HBgGT dependent manner. Moreover, the induction of these signaling responses and inflammatory cytokine production in host cells could be linked to HBgGT-mediated glutamine deprivation. This study implicates for the first time HBgGT as an important regulator of signaling cascades regulating inflammation in H. bilis infected host epithelial cells that could be responsible for induction of inflammatory disorders by the bacterium.

Carcinogenic bacterial pathogen Helicobacter pylori triggers DNA double-strand breaks and a DNA damage response in its host cells.

The bacterial pathogen Helicobacter pylori chronically infects the human gastric mucosa and is the leading risk factor for the development of gastric cancer. The molecular mechanisms of H. pylori-associated gastric carcinogenesis remain ill defined. In this study, we examined the possibility that H. pylori directly compromises the genomic integrity of its host cells. We provide evidence that the infection introduces DNA double-strand breaks (DSBs) in primary and transformed murine and human epithelial and mesenchymal cells. The induction of DSBs depends on the direct contact of live bacteria with mammalian cells. The infection-associated DNA damage is evident upon separation of nuclear DNA by pulse field gel electrophoresis and by high-magnification microscopy of metaphase chromosomes. Bacterial adhesion (e.g., via blood group antigen-binding adhesin) is required to induce DSBs; in contrast, the H. pylori virulence factors vacuolating cytotoxin A, γ-glutamyl transpeptidase, and the cytotoxin-associated gene (Cag) pathogenicity island are dispensable for DSB induction. The DNA discontinuities trigger a damage-signaling and repair response involving the sequential ataxia telangiectasia mutated (ATM)-dependent recruitment of repair factors--p53-binding protein (53BP1) and mediator of DNA damage checkpoint protein 1 (MDC1)--and histone H2A variant X (H2AX) phosphorylation. Although most breaks are repaired efficiently upon termination of the infection, we observe that prolonged active infection leads to saturation of cellular repair capabilities. In summary, we conclude that DNA damage followed by potentially imprecise repair is consistent with the carcinogenic properties of H. pylori and with its mutagenic properties in vitro and in vivo and may contribute to the genetic instability and frequent chromosomal aberrations that are a hallmark of gastric cancer.

A comprehensive analysis of the COL29A1 gene does not support a role in eczema.

BACKGROUND: Based on a recent positional cloning approach, it was claimed that the collagen 29A1 gene (COL29A1), which encodes an epidermal collagen, represents a major risk gene for eczema underlying a previously reported linkage to chromosome 3q21. However, thus far, not a single replication attempt has been published, and no definitive functional data have been provided. OBJECTIVES: We aimed to determine whether COL29A1 polymorphisms contribute to eczema susceptibility and whether COL29A1 expression is altered in eczema. METHODS: We investigated the reported association of COL29A1 variants with eczema, subtypes of eczema, and eczema-related traits in 5 independent and large study populations comprehensively phenotyped for allergic diseases: a set of 1687 German patients with eczema and 2387 population control subjects, a collection of 274 German families with eczema-diseases children, a cross-sectional population of German children (n = 3099), the Swedish population-based birth cohort Children Allergy and Milieu in Stockholm, an Epidemiologic Study (BAMSE) (n = 2033), and the European cross-sectional Prevention of Allergy-Risk Factors for Sensitization Related to Farming and Anthroposophic Lifestyle (PARSIFAL) study (n = 3113). An additional set of 19 COL29A1 coding single nucleotide polymorphisms was analyzed in BAMSE and PARSIFAL. COL29A1 expression was investigated by using in situ hybridization. RESULTS: We found no evidence for a relationship between COL29A1 polymorphisms and eczema. The equivalence test rejected the hypothesis of association even excluding small effects. In situ hybridization carried out on biopsy specimens from lesional and nonlesional skin of patients with eczema and from healthy control subjects did not show any differences in the cellular distribution pattern of COL29A1 expression at the mRNA level. CONCLUSIONS: Our results suggest that COL29A1 is unlikely to contain genetic variants that have a major effect on eczema or atopy susceptibility.

TNF receptor I on human keratinocytes is a binding partner for staphylococcal protein A resulting in the activation of NF kappa B, AP-1, and downstream gene transcription.

Primary human keratinocytes and immortalized HaCaT cells were analysed for their capacity to bind purified staphylococcal protein A (SpA). Co-incubation with FITC-labelled SpA led to a dose-depending attachment. Pull-down experiments with cellular extracts revealed the TNFα receptor I (TNF RI) as binding partner on keratinocytes. Thus, we next looked for expression of this receptor in human epidermis and cultured keratinocytes. TNF RI is strongly expressed on all keratinocytes analysed, both at the mRNA and protein level and activation by SpA at optimal doses of 50-100 µg/ml resulted in the phosphorylation of the TNF RI downstream kinases MEK1/2, JNK1/2, and p38 subsequently leading to translocation of the p65 NF kappa B subunit and AP-1 into the nucleus. This translocation was then followed by increased expression of IL-8 and COX-2, two known NF kappa B-induced pro-inflammatory genes. To further test the relevance of our findings, we analysed in vitro production of over 100 strains isolated from atopic eczema showing that more than 85% of the tested strains produced extracellular SpA in substantial amounts. Thus, besides superantigens, haemolysins, and other cell wall components, Staphylococcus aureus exerts pro-inflammatory stimuli on human keratinocytes through the production of SpA signalling through TNF RI.

Helicobacter pylori induces miR-155 in T cells in a cAMP-Foxp3-dependent manner.

Amongst the most severe clinical outcomes of life-long infections with Helicobacter pylori is the development of peptic ulcers and gastric adenocarcinoma--diseases often associated with an increase of regulatory T cells. Understanding H. pylori-driven regulation of T cells is therefore of crucial clinical importance. Several studies have defined mammalian microRNAs as key regulators of the immune system and of carcinogenic processes. Hence, we aimed here to identify H. pylori-regulated miRNAs, mainly in human T cells. MicroRNA profiling of non-infected and infected human T cells revealed H. pylori infection triggers miR-155 expression in vitro and in vivo. By using single and double H. pylori mutants and the corresponding purified enzymes, the bacterial vacuolating toxin A (VacA) and gamma-glutamyl transpeptidase (GGT) plus lipopolysaccharide (LPS) tested positive for their ability to regulate miR-155 and Foxp3 expression in human lymphocytes; the latter being considered as the master regulator and marker of regulatory T cells. RNAi-mediated knockdown (KD) of the Foxp3 transcription factor in T cells abolished miR-155 expression. Using adenylate cyclase inhibitors, the miR-155 induction cascade was shown to be dependent on the second messenger cyclic adenosine monophosphate (cAMP). Furthermore, we found that miR-155 directly targets the protein kinase A inhibitor alpha (PKIalpha) mRNA in its 3'UTR, indicative of a positive feedback mechanism on the cAMP pathway. Taken together, our study describes, in the context of an H. pylori infection, a direct link between Foxp3 and miR-155 in human T cells and highlights the significance of cAMP in this miR-155 induction cascade.

Double-stranded RNA induces an antiviral defense status in epidermal keratinocytes through TLR3-, PKR-, and MDA5/RIG-I-mediated differential signaling.

Emerging evidence suggests an important role for human epidermal keratinocytes in innate immune mechanisms against bacterial and viral skin infections. The proinflammatory effect of viral infections can be mimicked by double-stranded RNA (dsRNA). Herein, we demonstrate that keratinocytes express all known dsRNA sensing receptors at a constitutive and inducible level, and that they use several downstream signaling pathways leading to a broad pattern of gene expression, not only proinflammatory and immune response genes under the control of NF-kappaB, but also genes under transcriptional control of IRF3. As a consequence, dsRNA, a stimulus for TLR3, protein kinase R (PKR), and the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5, induces a status of antiviral defense in keratinocytes. Using inhibitors for the various dsRNA signaling pathways and specific small interfering RNA for TLR3, RIG-I, and MDA5, we demonstrated that in human keratinocytes, TLR3 seems to be necessary for NF-kappaB but not for IRF3 activation, whereas RIG-I and MDA5 are crucial for IRF3 activation. PKR is essential for the dsRNA response in both signaling pathways and thus represents the central antiviral receptor for dsRNA stimulation. Moreover, human keratinocytes up-regulate TLR7, the receptor for single-stranded RNA, in response to stimulation with dsRNA, which renders keratinocytes functionally responsive to the TLR7 agonist gardiquimod, a member of the imidazoquinoline antiviral immune response modifier family. Thus, in addition to building a physical barrier against infectious pathogens, keratinocytes are specially equipped with a full antiviral defense program that enables them to efficiently target viral infections of the skin.