Cutaneous Keratinocytes Research Articles

Reprogramming of epidermal keratinocytes by PITX1 transforms the cutaneous cellular landscape and promotes wound healing.

Cutaneous wound healing is a slow process that often terminates with permanent scarring while oral wounds, in contrast, regenerate damage faster. Unique molecular networks in epidermal and oral epithelial keratinocytes contribute to the tissue-specific response to wounding, but key factors that establish those networks and how the keratinocytes interact with their cellular environment remain to be elucidated. The transcription factor PITX1 is highly expressed in the oral epithelium but is undetectable in cutaneous keratinocytes. To delineate if PITX1 contributes to oral keratinocyte identity, cell-cell interactions, and the improved wound healing capabilities, we ectopically expressed PITX1 in the epidermis of murine skin. Using comparative analysis of murine skin and oral (buccal) mucosa with scRNA-seq and spatial transcriptomics, we found that PITX1 expression enhances epidermal keratinocyte migration, proliferation, and alters differentiation to a quasi-oral keratinocyte state. PITX1+ keratinocytes reprogram intercellular communication between skin-resident cells to mirror buccal tissue while also stimulating the influx of neutrophils that establish a pro-inflammatory environment. Furthermore, PITX1+ skin heals significantly faster than control skin via increased keratinocyte activation and migration and a tunable inflammatory environment. These results illustrate that PITX1 programs oral keratinocyte identity and cellular interactions while also revealing critical downstream networks that promote wound closure.

3D Porous Polymer Scaffold-Conjugated KGF-Mimetic Peptide Promotes Functional Skin Regeneration in Chronic Diabetic Wounds.

The re-epithelialization process gets severely dysregulated in chronic nonhealing diabetic foot ulcers/wounds. Keratinocyte growth factor (KGF or FGF-7) is the major modulator of the re-epithelialization process, which regulates the physiological phenotypes of cutaneous keratinocytes. The existing therapeutic strategies of growth factor administration have several limitations. To overcome these, we have designed a KGF-mimetic peptide (KGFp, 13mer) based on the receptor interaction sites in murine KGF. KGFp enhanced migration and transdifferentiation of mouse bone marrow-derived MSCs toward keratinocyte-like cells (KLCs). A significant increase in the expression of skin-specific markers Bnc1 (28.5-fold), Ck5 (14.6-fold), Ck14 (26.1-fold), Ck10 (187.7-fold), and epithelial markers EpCam (23.3-fold) and Cdh1 (64.2-fold) was associated with the activation of ERK1/2 and STAT3 molecular signaling in the KLCs. Further, to enhance the stability of KGFp in the wound microenvironment, it was conjugated to biocompatible 3D porous polymer scaffolds without compromising its active binding sites followed by chemical characterization using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, dynamic mechanical analysis, and thermogravimetry. In vitro evaluation of the KGFp-conjugated 3D polymer scaffolds revealed its potential for transdifferentiation of MSCs into KLCs. Transplantation of allogeneic MSCGFP using KGFp-conjugated 3D polymer scaffolds in chronic nonhealing type 2 diabetic wounds (db/db transgenic, 50-52 weeks old male mice) significantly enhanced re-epithelialization-mediated wound closure rate (79.3%) as compared to the control groups (Untransplanted -22.4%, MSCGFP-3D polymer scaffold -38.5%). Thus, KGFp-conjugated 3D porous polymer scaffolds drive the fate of the MSCs toward keratinocytes that may serve as potential stem cell delivery platform technology for tissue engineering and transplantation.

Superior Drug Delivery Performance of Multifunctional Bilosomes: Innovative Strategy to Kill Skin Cancer Cells for Nanomedicine Application.

Numerous failures in melanoma treatment as a highly aggressive form of skin cancer with an unfavorable prognosis and excessive resistance to conventional therapies are prompting an urgent search for more effective therapeutic tools. Consequently, to increase the treatment efficiency and to reduce the side effects of traditional administration ways, herein, it has become crucial to combine photodynamic therapy as a promising therapeutic approach with the selectivity and biocompatibility of a novel colloidal transdermal nanoplatform for effective delivery of hybrid cargo with synergistic effects on melanoma cells. The self-assembled bilosomes, co-stabilized with L-α-phosphatidylcholine, sodium cholate, Pluronic® P123, and cholesterol, were designated, and the stability of colloidal vesicles was studied using dynamic and electrophoretic light scattering, also provided in cell culture medium (Dulbecco's Modified Eagle's Medium). The hybrid compounds - a classical photosensitizer (Methylene Blue) along with a complementary natural polyphenolic agent (curcumin), were successfully co-loaded, as confirmed by UV-Vis, ATR-FTIR, and fluorescent spectroscopies. The biocompatibility and usefulness of the polymer functionalized bilosome with loaded double cargo were demonstrated in vitro cyto- and phototoxicity experiments using normal keratinocytes and melanoma cancer cells. Thein vitrobioimaging and immunofluorescence study upon human skin epithelial (A375) and malignant (Me45) melanoma cell lines established the protective effect of the PEGylated bilosome surface. This effect was confirmed in cytotoxicity experiments, also determined on human cutaneous (HaCaT) keratinocytes. The flow cytometry experiments indicated the enhanced uptake of the encapsulated hybrid cargo compared to the non-loaded MB and CUR molecules, as well as a selectivity of the obtained nanocarriers upon tumor cell lines. The phyto-photodynamic action provided 24h-post irradiation revealed a more significant influence of the nanoplatform on Me45 cells in contrast to the A375 cell line, causing the cell viability rate below 20% of the control. As a result, we established an innovative and effective strategy for potential metastatic melanoma treatment through the synergism of phyto-photodynamic therapy and novel bilosomal-origin nanophotosensitizers.

1135 Identification of novel derivatives of fisetin with anti-inflammatory and antioxidant activities in cutaneous keratinocytes and macrophage

1135 Identification of novel derivatives of fisetin with anti-inflammatory and antioxidant activities in cutaneous keratinocytes and macrophage

770 Reprogramming of diabetic wounds by SOX2 triggers a pro-healing transcriptional signature

Diabetic foot ulcers (DFUs) are a severe complication of diabetes and a common cause of lower limb amputations and are associated with a high mortality rate. We previously demonstrated that the transcription factor SOX2 establishes a transcriptional network that primes the oral epithelium for rapid wound repair and reprogrammed cutaneous keratinocytes to present accelerated wound resolution both in vitro and in vivo. However, the mechanisms by which SOX2 reverses effects of diabetes during wound healing are not known.

Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes-In Vitro Studies.

Pig-to-human xenotransplantation seems to be the response to the contemporary shortage of tissue/organ donors. Unfortunately, the phylogenetic distance between pig and human implies hyperacute xenograft rejection. In this study, we tested the hypothesis that combining expression of human α1,2-fucosyltransferase (hFUT2) and α-galactosidase A (hGLA) genes would allow for removal of this obstacle in porcine transgenic epidermal keratinocytes (PEKs). We sought to determine not only the expression profiles of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) proteins, but also the relative abundance (RA) of Galα1→3Gal epitopes in the PEKs stemming from not only hFUT2 or hGLA single-transgenic and hFUT2×hGLA double-transgenic pigs. Our confocal microscopy and Western blotting analyses revealed that both rhα1,2-FT and rhα-Gal A enzymes were overabundantly expressed in respective transgenic PEK lines. Moreover, the semiquantitative levels of Galα1→3Gal epitope that were assessed by lectin fluorescence and lectin blotting were found to be significantly diminished in each variant of genetically modified PEK line as compared to those observed in the control nontransgenic PEKs. Notably, the bi-transgenic PEKs were characterized by significantly lessened (but still detectable) RAs of Galα1→3Gal epitopes as compared to those identified for both types of mono-transgenic PEK lines. Additionally, our current investigation showed that the coexpression of two protective transgenes gave rise to enhanced abrogation of Galα→3Gal epitopes in hFUT2×hGLA double-transgenic PEKs. To summarize, detailed estimation of semiquantitative profiles for human α-1,2-FT and α-Gal A proteins followed by identification of the extent of abrogating the abundance of Galα1→3Gal epitopes in the ex vivo expanded PEKs stemming from mono- and bi-transgenic pigs were found to be a sine qua non condition for efficiently ex situ protecting stable lines of skin-derived somatic cells inevitable in further studies. The latter is due to be focused on determining epigenomic reprogrammability of single- or double-transgenic cell nuclei inherited from adult cutaneous keratinocytes in porcine nuclear-transferred oocytes and corresponding cloned embryos. To our knowledge, this concept was shown to represent a completely new approach designed to generate and multiply genetically transformed pigs by somatic cell cloning for the needs of reconstructive medicine and dermoplasty-mediated tissue engineering of human integumentary system.

Alpha 1 adrenoceptor expression in skin, nerves and blood vessels of patients with painful diabetic neuropathy.

Diabetic neuropathy (dNP) patients often suffer from severe neuropathic pain. It was suggested that alpha-1 adrenoceptor (α1-AR) hyperresponsiveness contributes to pain in dNP. The aim of our study was to quantify α1-AR expression using immunohistochemistry in skin biopsies of nine patients with painful diabetic neuropathy compared to 10 healthy controls. Additionally, the association between α1-AR expression and activation with spontaneous and sympathetically maintained pain (SMP) induced by intradermal injection of the α1-agonist phenylephrine was investigated. For control purposes the α2-agonist clonidine was injected in a different session. We found that dermal nerve density was significantly lower in dNP than in controls. However, α1-AR expression was significantly greater on cutaneous blood vessels and keratinocytes of dNP patients than controls. A similar trend, which failed to reach significance, was observed for dermal nerves. Intradermal injection of phenylephrine induced only minor pain, which resolved after a few minutes. Adrenergically evoked pain persisted for more than 15min in only one patient, but none of the patients fulfilled the criteria for SMP (pain increase after injection of phenylephrine and decrease after clonidine). In conclusion, our results imply that SMP does not occur in dNP. However, elevated expression of α1-AR on keratinocytes and dermal blood vessels is an important finding, since this could contribute to dNP progression and supports the theory of receptor up-regulation of denervated structures. The implications of this α1-upregulation should be examined in further studies.

Beta-Genus Human Papillomavirus 8 E6 Destabilizes the Host Genome by Promoting p300 Degradation.

The beta genus of human papillomaviruses infects cutaneous keratinocytes. Their replication depends on actively proliferating cells and, thus, they conflict with the cellular response to the DNA damage frequently encountered by these cells. This review focus on one of these viruses (HPV8) that counters the cellular response to damaged DNA and mitotic errors by expressing a protein (HPV8 E6) that destabilizes a histone acetyltransferase, p300. The loss of p300 results in broad dysregulation of cell signaling that decreases genome stability. In addition to discussing phenotypes caused by p300 destabilization, the review contains a discussion of the extent to which E6 from other β-HPVs destabilizes p300, and provides a discussion on dissecting HPV8 E6 biology using mutants.

Potentiating cutaneous wound healing in young and aged skin with nutraceutical collagen peptides.

Chronic wounds continue to be a burden to healthcare systems, with ageing linked to increased prevalence of chronic wound development. Nutraceutical collagen peptides have been shown to reduce signs of skin ageing, but their therapeutic potential for cutaneous wound healing remains undefined. To determine the potential for nutraceutical collagen peptides to promote cutaneous wound healing in vitro in the context of age. The potential for bovine- or porcine-derived nutraceutical collagen peptides to promote wound healing of primary cutaneous fibroblasts and keratinocytes derived from young and aged individuals in vitro was assessed by two-dimensional scratch and cell-viability assays and by immunofluorescence for the cell proliferation marker, Ki67. The achievement of peptide concentrations in vivo, equivalent to those exerting a beneficial effect on wound healing in vitro, was confirmed by pharmacokinetic studies of hydroxyproline, a biomarker for collagen peptide absorption, following peptide ingestion by healthy individuals over a wide age range. Results demonstrated significant nutraceutical collagen peptide-induced wound closure of both young and aged fibroblasts and keratinocytes, mediated by enhanced cellular proliferation and migration. Analysis of blood levels of hydroxyproline in young and aged individuals following porcine collagen peptide ingestion revealed peak serum/plasma levels after 2h at similar concentrations to those exerting beneficial effects on wound healing in vitro. These data demonstrate the capacity for nutraceutical collagen peptides to promote cutaneous wound closure in vitro, at pharmacologically achievable concentrations in vivo, thereby offering a potential novel therapeutic strategy for the management of cutaneous wounds in young and aged individuals.

Correction: IL-35 Decelerates the Inflammatory Process by Regulating Inflammatory Cytokine Secretion and M1/M2 Macrophage Ratio in Psoriasis.

Zhang, J., Y. Lin, C. Li, X. Zhang, L. Cheng, L. Dai, Y. Wang, F. Wang, G. Shi, Y. Li, Q. Yang, X. Cui, Y. Liu, H. Wang, S. Zhang, Y. Yang, R. Xiang, J. Li, D. Yu, Y. Wei, and H. Deng. 2016. IL-35 decelerates the inflammatory process by regulating inflammatory cytokine secretion and M1/M2 macrophage

Prognostic evaluation of human papillomavirus and p16 in esophageal squamous cell carcinoma.

To the Editor: Esophageal cancer is the sixth most common cancer globally according to the World Health Organization.[1] Due to rapid clinical progression and extremely poor prognosis, the 5-year survival rate of esophageal squamous cell carcinomas (ESCC) remains less than 20%;[2] therefore, further developments in this field are needed. Human papillomavirus (HPV) is a small circular non-enveloped double-stranded DNA virus that primarily infects mucosa and cutaneous keratinocytes[3] and its infection rates in ESCC range from 11.7% to 38.9% worldwide.[4] P16 which is encoded by the CDKN2A gene and known as cyclin-dependent kinase inhibitor 2A, is a widely used immunohistochemical marker in squamous cell carcinomas and associated with high-risk HPV.[5] The present study included 228 cases from Henan Cancer Hospital, immunostaining for p53 and retinoblastoma protein (Rb) were previously performed for 147 samples, and then 73 ESCC cases were randomly selected for detecting HPV infection and p16 expression. A total of 20 cases were selected through healthy gastroscopy physical examination. This study was approved by the Institutional Ethics Committee of Henan Cancer Hospital (No. 201401017). Sections were incubated with HPV (ZM-0217, CAMVIR-1), p16 (ZM-0205, 1C1), p53 (ZM-0405, BP53.12), or Rb (ZM-0223, 13A10) antibodies from Beijing Zhongshan Jinqiao Biotechnology Co. Ltd, China as the primary antibodies and biotinylated goat anti-rabbit IgG as the secondary antibody. The RNA of HPV 16/18 was detected by the RNAscope 2.5 Assay Reagent Kit (Advanced Cell Diagnostics, Inc., Hayward, CA, USA). All statistical analysis were performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Fisher exact test was used to identify predictors for HPV-positive tumors. The analysis of the 5-year survival rate between different groups was performed using the Kaplan-Meier method and log-rank test. Differences with a value of P < 0.05 were considered statistically significant. All statistical tests were two-sided. The characteristics of 73 ESCC patients were analyzed and revealed that there were no significant differences between the groups with respect to age (P = 0.708), stage (P = 0.788), T category (P = 0.417), N category (P = 0.788), differentiation grade (P = 0.141), smoking (P = 0.445), and alcohol consumption (P = 0.940). The positive cases of HPV, p53, p16, and Rb were 33 (45.2%), 40 (54.8%), 5 (6.8%), and 32 (43.8%), respectively. The mRNA expression of HPV 16/18 detected through RNAscope 2.5 Assay was negative. The HPV infection in the esophageal mucosa of 20 normal people was all negative. The cases of co-positive HPV and p16, p53, and Rb were 3 (4.1%), 22 (30.1%), and 12 (16.4%), respectively. Among the 73 ESCC patients (for five cases, survival information could not be confirmed), the total number of deaths was 24, and the 5-year survival was evaluated to compare the prognosis from the different groups. The number of deaths of patients with positive HPV, p16, p53, and Rb were 9, 1, 14, and 12; and the number of deaths of patients with negative HPV, p16, p53, and Rb were 15, 23, 10, and 12, respectively. As shown in Figure 1, there was no significant difference in 5-year survival rate between patients with positive or negative HPV, p16, p53, and Rb (χ2 = 0.664, 0.500, 0.008, and 0.262, P = 0.415, 0.479, 0.929, and 0.609, respectively, all P > 0.05).Figure 1: The Kaplan-Meier curve analysis of 5-year survival rate between patients with positive or negative HPV, p16, p53, and Rb, respectively. Number 1 and 2 indicates positive and negative HPV, p16, p53, or Rb, respectively. HPV: Human papilloma virus; Rb: Retinoblastoma protein.It is well-known that the p53 and p16 tumor suppressor genes are closely related to the cell cycle. Mutations in the p53 and p16 genes can disrupt the cell cycle regulatory mechanism and induce cell hyperplasia and tumors growth. The rate of HPV infection in 73 ESCC patients was 45.3%; however, statistical analysis showed that the HPV, p16, p53, and Rb were not correlated with the 5-year survival rate of ESCC (all P > 0.05). HPV infection may be related to the occurrence of ESCC, but it is not related to the 5-year survival rate of ESCC patients. The relationship between HPV and ESCC should to be further explored based on a large sample size. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Funding This work was supported by grants from the Henan Medical Science and Technology Project (general) (No. 201401017) and the National Natural Science Foundation of China (No. 81602637). Conflicts of interest None.

Potential Noncutaneous Sites of Chelonid Herpesvirus 5 Persistence and Shedding in Green Sea Turtles Chelonia mydas.

Chelonid herpesvirus 5 (ChHV5), the likely etiologic agent of sea turtle fibropapillomatosis (FP), is predicted to be unevenly distributed within an infected turtle, in which productive virus replication and virion shedding occurs in cutaneous tumor keratinocytes. In this study, we measured and compared ChHV5 DNA quantities in tumors, skin, urine, major organs, and nervous tissue samples from green turtles Chelonia mydas. These samples were taken from the carcasses of 10 juvenile green turtles with and without clinical signs of FP that stranded in Florida during 2014. Quantitative PCR for ChHV5 UL30 was used to identify ChHV5 DNA in tumors, skin, heart, kidney, nerves, and urine sampled from five out of five FP-positive and three out of five FP-free turtles. The most frequently co-occurring sites were cutaneous tumor and kidney (n = 4). Novel data presented here include the identification of ChHV5 DNA in kidney, heart, and nerve samples from three FP-free turtles. These data support candidate nontumored anatomic sites of ChHV5 DNA localization and mobilization during two different disease states that may be involved in the ChHV5 infection cycle. Received September 8, 2016; accepted April 17, 2017.

IL-35 Decelerates the Inflammatory Process by Regulating Inflammatory Cytokine Secretion and M1/M2 Macrophage Ratio in Psoriasis.

IL-35 downregulates Th17 cell development and suppresses certain types of autoimmune inflammation such as collagen-induced arthritis and experimental autoimmune uveitis. Psoriasis is thought to be initiated by abnormal interactions between cutaneous keratinocytes and systemic immune cells. However, the role of IL-35 in psoriasis remains unclear. In this study, we assessed IL-35 in three well-known psoriasis models: a human keratinocyte cell line (HaCaT), a keratin 14 (K14)-vascular endothelial growth factor A (VEGF-A)-transgenic (Tg) mouse model, and an imiquimod-induced psoriasis mouse model. First, we found that IL-35 suppressed the expression of IL-6, CXCL8, and S100A7, which are highly upregulated by a mixture of five proinflammatory cytokines in HaCaT. Second, a plasmid coding for the human IL-35 sequence coated with cationic liposomes showed potent immunosuppressive effects on K14-VEGF-A-Tg and imiquimod-induced psoriasis mouse models. In the K14-VEGF-A-Tg model, our results showed that several types of proinflammatory cytokines were significantly reduced, whereas IL-10 was remarkably induced by IL-35. Compared with pcDNA3.1, there was a small number of CD4(+)IL-17(+) T cells and a large number of CD4(+)IL-10(+) and CD4(+)CD25(+)Foxp3(+) T cells in the IL-35 group. Most importantly, we found that IL-35 decreased the total number of macrophages and ratio of M1/M2 macrophages, which has not been reported previously. In addition, compared with dexamethasone, IL-35 showed long-term therapeutic efficacy. In summary, our results strongly indicate that IL-35 plays a potent immunosuppressive role in psoriasis. Thus, IL-35 has potential for development as a new therapeutic strategy for patients with chronic psoriasis and other cutaneous inflammatory diseases.

ID: 21: Ganoderma lucidum-derived triterpenes inhibit PAR2-mediated IL8 production by human keratinocytes

Crude extracts from Ganoderma lucidum, a medical mushroom, are popularly used for treatment of allergic diseases such as atopic dermatitis (AD). Triterpenes are the major active constituents of Ganoderma lucidum. Stimulation of protease-activated receptor-2 (PAR2) on cutaneous keratinocytes induces secretion of various immune and inflammatory cytokines including interleukin 8 (IL8), and thereby plays a role in the development of allergic skin inflammation. Thus, PAR2-mediated signaling has been suggested to be a therapeutic target for the treatment of allergic diseases. This study aimed to evaluate the inhibitory effects of Ganoderma triterpenes on PAR2-mediated IL8 secretion from human keratinocytes. Normal human epidermal keratinocytes (NHEK) were stimulated with PAR2-activating peptide (AP) in the presence or absence of Ganoderma triterpenes in vitro. IL8 secretion from NHEK cells was measured by enzyme-linked immunosorbent assay (ELISA). Chemical structures of triterpenes were determined based on spectroscopic evidence and mass spectral data. Among of Ganoderma triterpenes, ganolucidate F exhibited most potent inhibitory activity on PAR2-mediated IL8 secretion from keratinocytes (IC50 = 12.5 μ M). Ganolucidic acid γ a showed strong inhibitory activity with IC50 value of 29.1 μ M. In addition, ganoderic acid η and ganoderic acid θ showed remarkable inhibitory effects with IC50 values of 64.9 μ M and 109.5 μ M, respectively. Elucidation of relationships between chemical structure of triterpenes and inhibitory activity implied that substitution of hydroxy group at C-23, instead of a carbonyl group, may be a very important structural feature for inhibitory activity. Ganoderma lucidum-derived ganoderic acids could be a candidate drug for treatment of allergic skin diseases.

Prevalence of High risk Human Papillomavirus in cervical dysplasia and cancer samples from twin cities in Pakistan

Human Papilloma Virus (HPV) is small DNA virus mostly infecting mucosa and cutaneous keratinocytes. So far, more than 200 Human papillomaviruses are known. HPV have been divided into high- and low-risk on the basis of their oncogenic potential. High risk HPV is considered to be the main etiological cause for cervical cancer. The current study was designed to screen the local cervical cancer patients from the twin cities of Pakistan for the occurance of high risk HPV. A total of 67 formalin fixed paraffin-embedded samples of cervical cancer biopsies were obtained from the government hospitals in Islamabad and Rawalpindi. Cervical cancer biopsies were examined for the presence of HPV DNA. Polymerase chain reaction (PCR) was used for the amplification of a region in the HPV-L1 gene for the general detection of the Papilloma virus and for the genotype specific detection of high risk HPV 16 and 18 using the GP5/GP6 primers and genotype specific primers, respectively. HPV DNA was detected in 59 out of 67 samples analyzed. 30 samples showed the presence of HPV16 while 22 samples were positive for HPV18. HPV subtype could not be determined in 7 samples. Our results show a strong association between HPV infection and cervical cancer among women in twin cities of Pakistan. One way to minimize the disease burden in relation to HPV infection in Pakistani population is the use of prophylactic vaccines and routine screening. An early diagnosis of HPV infection will allow better health management to reduce the risk of developing cervical cancer.

Increased bilateral expression of α1-adrenoceptors on peripheral nerves, blood vessels and keratinocytes does not account for pain or neuroinflammatory changes after distal tibia fracture in rats

In certain forms of nerve injury and inflammation, noradrenaline augments pain via actions on up-regulated α1-adrenoceptors (α1-ARs). The aim of this study was to use immunohistochemistry to examine α1-AR expression on peripheral neurons, cutaneous blood vessels and keratinocytes after distal tibia fracture and cast immobilization, a model of complex regional pain syndrome type 1. We hypothesized that there would be increased α1-AR expression on neurons and keratinocytes in the injured limb in comparison to the contralateral unaffected limb after distal tibia fracture, in association with inflammatory changes and pain. α1-AR expression was increased on plantar keratinocytes, dermal blood vessels and peripheral nerve fibers at 16weeks after injury both in the fractured and contralateral uninjured limb. Similar changes were seen in controls whose limb had been immobilized in a cast for 4weeks but not fractured. Neurofilament 200 (NF200), a marker of myelinated neurons, and calcitonin gene-related peptide (CGRP), a neuropeptide involved in neuro-inflammatory signaling, decreased 4weeks after fracture and casting but then increased at the 16-week time point. As some of these changes were also detected in the contralateral hind limb, they probably were triggered by a systemic response to fracture and casting. Soon after the cast was removed, intraplantar injections of the α1-AR antagonist prazosin released local vasoconstrictor tone but had no effect on pain behaviors. However, systemic injection of prazosin inhibited behavioral signs of pain, suggesting that fracture and/or casting triggered an up-regulation of α1-ARs in central nociceptive pathways that augmented pain. Together, these findings indicate that α1-AR expression increases in the hind limbs after distal tibia fracture and cast immobilization. However, these peripheral increases do not contribute directly to residual pain.

Brm inhibits the proliferative response of keratinocytes and corneal epithelial cells to ultraviolet radiation-induced damage.

Ultraviolet radiation (UV) from sunlight is the primary cause of skin and ocular neoplasia. Brahma (BRM) is part of the SWI/SNF chromatin remodeling complex. It provides energy for rearrangement of chromatin structure. Previously we have found that human skin tumours have a hotspot mutation in BRM and that protein levels are substantially reduced. Brm−/− mice have enhanced susceptibility to photocarcinogenesis. In these experiments, Brm−/− mice, with both or a single Trp53 allele were exposed to UV for 2 or 25 weeks. In wild type mice the central cornea and stroma became atrophic with increasing time of exposure while the peripheral regions became hyperplastic, presumably as a reparative process. Brm−/−, Trp53+/−, and particularly the Brm−/− Trp53+/− mice had an exaggerated hyperplastic regeneration response in the corneal epithelium and stroma so that the central epithelial atrophy or stromal loss was reduced. UV induced hyperplasia of the epidermis and corneal epithelium, with an increase in the number of dividing cells as determined by Ki-67 expression. This response was considerably greater in both the Brm−/− Trp53+/+ and Brm−/− Trp53+/− mice indicating that Brm protects from UV-induced enhancement of cell division, even with loss of one Trp53 allele. Cell division was disorganized in Brm−/− mice. Rather than being restricted to the basement membrane region, dividing cells were also present in the suprabasal regions of both tissues. Brm appears to be a tumour suppressor gene that protects from skin and ocular photocarcinogenesis. These studies indicate that Brm protects from UV-induced hyperplastic growth in both cutaneous and corneal keratinocytes, which may contribute to the ability of Brm to protect from photocarcinogenesis.

The potent immunosuppressive role of Interleukin (IL)-37 in the pathogenesis of psoriasis (THER6P.846)

Abstract IL-37 has emerged as a fundamental inhibitor of innate immune response. Psoriasis is thought to be initiated by abnormal interactions between cutaneous keratinocytes and systemic immune cells, triggering epidermal hyperplasia, increased inflammatory cells infiltration and high inflammatory mediator production. To research the presumed unique “cooling fires of inflammation” properties of IL-37 in psoriasis, we firstly found that IL-37 was abundantly distributed in human psoriatic plaques (mainly localized in effector memory T cells and macrophages not regulatory T cells). Further, we explored the possible immunomodulatory mechanism of IL-37 in psoriasis models in vitro and in vivo. IL-37 was effective in suppressing the production of chemokines, proinflammatory cytokines and antimicrobial peptides in human keratinocytes which recapitulated numerous features of psoriasis in vitro. IL-37 relieved the psoriatic symptoms of keratin14-VEGF Tg mice (widely-used psoriasis model) including thinner epidermis and diminished lymphocyte infiltration, and greatly dampened Th1 type cytokines production and mast cells recruitment in psoriatic lesions. Our results support the idea that the presence of IL-37 attenuates the inflammatory process in psoriasis by disrupting the linkage between innate immunity and adaptive immune response, indicating a hopeful therapeutic approach for immune-mediated disorders.

IL-37 Ameliorates the Inflammatory Process in Psoriasis by Suppressing Proinflammatory Cytokine Production

IL-37 is a potent inhibitor of innate immunity by shifting the cytokine equilibrium away from excessive inflammation. Psoriasis is thought to be initiated by abnormal interactions between the cutaneous keratinocytes and systemic immune cells, triggering keratinocyte hyperproliferation. In the current study, we assessed IL-37 in two well-known psoriasis models: a human keratinocyte cell line (HaCaT) and the keratin 14 VEGF-A-transgenic mouse model. First, we used the HaCaT cell line, which was transiently transfected with an overexpressing IL-37 vector, and tested the effect of IL-37 on these cells using a mixture of five proinflammatory cytokines. IL-37 was effective in suppressing the production of CXCL8, IL-6, and S100A7, which were highly upregulated by the mixture of five proinflammatory cytokines. Keratin 14 VEGF-A-transgenic mice were treated with plasmid coding human IL-37 sequence-formulated cationic liposomes, and we observed potent immunosuppressive effects over the 18-d period. In this model, we observed reduced systemic IL-10 levels, local IFN-γ gene transcripts, as well as mild mast cell infiltration into the psoriatic lesions of the mice. Immunohistochemical analysis indicated that IL-37 was expressed by effector memory T cells, as well as macrophages, in human psoriatic plaques. In conclusion, our studies strongly indicate that IL-37 plays a potent immunosuppressive role in the pathogenesis of both experimental psoriasis models in vitro and in vivo by downregulating proinflammatory cytokines. Importantly, our findings highlight new therapeutic strategies that can be designed to use this immunosuppressive anti-inflammatory cytokine in psoriasis and other inflammatory cutaneous diseases.

Dicephalic ionic surfactants in fabrication of biocompatible nanoemulsions: Factors influencing droplet size and stability

The present work has been carried out to explore the potential of two novel ionic dicephalic-type surfactants, i.e., positively charged N,N-bis[3,3′-(trimethylammonio)propyl]-dodecanamide dimethylsulphate, C12(TAPAMS)2 and anionic disodium N-dodecyliminodiacetate, C12(COONa)2 for spontaneous formation of biocompatible and stable oil-in-water (o/w) nanoemulsions. Initially, we examined ternary phase diagrams of surfactant–oil–water (SOW) systems containing different ratios of C12(TAPAMS)2 or C12(COONa)2 as the surfactant entity (S), oleic acid, isopropyl palmitate or isopropyl myristate as the oil phase (O) and the water phase (W) by the titration method. Then, all obtained nanoemulsions were characterized by visual and microscopic observations, as well as dynamic light scattering (DLS) measurements of particle sizes and size distributions. Results of cryo-TEM and DLS showed that the studied droplets making up the nanoemulsions have a nearly monodisperse size distribution (DH<100nm, PdI<0.2). Backscattered profiles obtained by the turbidimetric technique proved high kinetic stability of the obtained systems whereas Doppler electrophoresis revealed their large positive or negative ζ-potential. In order to test the nanoemulsions biocompatibility under the surfactants concentration, cytotoxicity of the optimized formulations was evaluated in vitro upon two normal human skin cell lines, i.e., cutaneous keratinocytes (HaCaT) and gingival fibroblasts (HGF). The analyzed parameters – oil composition, surfactant type, and surfactant-to-oil ratio – were all found to influence the droplet size, charge and stability of the systems produced. Our results prove that the studied nanoemulsions, stabilized by dicephalic ionic surfactants, may provide new biocompatible colloidal systems, designated mainly for cosmetics, personal and household care products.