Skin mast cells protect mice against vaccinia virus by triggering mast cell receptor S1PR2 and releasing antimicrobial peptides

Mast cells (MCs) are well-known effectors of allergic reactions and are considered sentinels in the skin and mucosa. In addition, through their production of cathelicidin, MCs have the capacity to oppose invading pathogens. We therefore hypothesized that MCs could act as sentinels in the skin against viral infections using antimicrobial peptides. In this study, we demonstrate that MCs react to vaccinia virus (VV) and degranulate using a membrane-activated pathway that leads to antimicrobial peptide discharge and virus inactivation. This finding was supported using a mouse model of viral infection. MC-deficient (Kit(wsh-/-)) mice were more susceptible to skin VV infection than the wild type animals, whereas Kit(wsh-/-) mice reconstituted with MCs in the skin showed a normal response to VV. Using MCs derived from mice deficient in cathelicidin antimicrobial peptide, we showed that antimicrobial peptides are one important antiviral granule component in in vivo skin infections. In conclusion, we demonstrate that MC presence protects mice from VV skin infection, MC degranulation is required for protecting mice from VV, neutralizing Ab to the L1 fusion entry protein of VV inhibits degranulation apparently by preventing S1PR2 activation by viral membrane lipids, and antimicrobial peptide release from MC granules is necessary to inactivate VV infectivity.     

Protective effects of hemin and tetrakis(4-benzoic acid)porphyrin on bacterial mutagenesis and mouse skin carcinogenesis induced by 7, 12-dimethylbenz[a]anthracene

Porphyrins which are widespread in nature can interfere with the actions of certain carcinogens and mutagens, and have also been used clinically in photodynamic therapy (PDT) of tumors. Porphyrins such as chlorophyll, chlorophyllin (CHL) and hemin are known to inactivate various mutagens by forming complexes with them. Tetrakis(4-benzoic acid)porphyrin (TBAP) has been developed as a photosensitizer for PDT and its metal complex, MnTBAP has been shown to be efficacious in a variety of in vitro and in vivo oxidative stress models of human diseases. In the present study, we have found that TBAP and hemin exert concentration-related inhibition of his(+) reversion in Salmonella typhimurium TA100 induced by 7, 12-dimethylbenz[a]anthracene (DMBA), and significantly reduced both incidence and multiplicity of skin tumors when topically applied prior to treatment of 12-O-tetradecanoylphorbol-13-acetate in female ICR mice. Covalent DNA binding of DMBA in mouse skin was also significantly inhibited by topical application of TBAP or hemin as well as CHL. These results suggest the chemopreventive potential of compounds containing a porphyrin nucleus.     

美白祛斑药物对强脉冲光作用下的鼠皮黑色素的影响

观察美白祛斑药物对强脉冲光(intensive pulse light, IPL)作用下的鼠皮黑色素的影响,探讨IPL作用下,皮肤的黑色素的增生和美白药物之间的作用关系,以期探寻美白药物在减少光子嫩肤术并发症的作用机制.以小白鼠作为研究对象,用IPL在一定的波长及能量密度照射活体小鼠皮肤, 在照射后15 min涂抹低浓度的维A酸霜,分别在照射后的4 h、5 d,用显微镜观察涂霜剂、没涂霜剂的皮肤黑色素及其周边组织的变化情况;在照射后的1 d用多光子显微镜观察涂霜剂、没涂霜剂皮肤组织的变化.讨论了美白祛斑药物在光子嫩肤过程中抑制黑色素生成的过程,为如何减少光子嫩肤术并发症提供参考和借鉴.     

The cardiac repair benefits of inflammation do not persist: evidence from mast cell implantation

Multiple mechanisms contribute to progressive cardiac dysfunction after myocardial infarction (MI) and inflammation is an important mediator. Mast cells (MCs) trigger inflammation after MI by releasing bio‐active factors that contribute to healing. c‐Kit‐deficient (Kit^W/W‐v) mice have dysfunctional MCs and develop severe ventricular dilatation post‐MI. We explored the role of MCs in post‐MI repair. Mouse wild‐type (WT) and Kit^W/W‐v MCs were obtained from bone marrow (BM). MC effects on fibroblasts were examined in vitro by proliferation and gel contraction assays. MCs were implanted into infarcted mouse hearts and their effects were evaluated using molecular, cellular and cardiac functional analyses. In contrast to WT, Kit^W/W‐v MC transplantation into Kit^W/W‐v mice did not improve cardiac function or scar size post‐MI. Kit^W/W‐v MCs induced significantly reduced fibroblast proliferation and contraction compared to WT MCs. MC influence on fibroblast proliferation was Basic fibroblast growth factor (bFGF)‐dependent and MC‐induced fibroblast contractility functioned through transforming growth factor (TGF)‐β. WT MCs transiently rescue cardiac function early post‐MI, but the benefits of BM cell implantation lasted longer. MCs induced increased inflammation compared to the BM‐injected mice, with increased neutrophil infiltration and infarct tumour necrosis factor‐α (TNF‐α) concentration. This augmented inflammation was followed by increased angiogenesis and myofibroblast formation and reduced scar size at early time‐points. Similar to the functional data, these beneficial effects were transient, largely vanishing by day 28. Dysfunctional Kit^W/W‐v MCs were unable to rescue cardiac function post‐MI. WT MC implantation transiently enhanced angiogenesis and cardiac function. These data suggest that increased inflammation is beneficial to cardiac repair, but these effects are not persistent.     

Rapamycin inhibition of CFA-induced lymphangiogenesis in PLN is independent of mast cells

Elucidation of the events responsible for the interaction between lymphatic endothelial cells (LECs) and mast cells (MCs) may prove to be a valuable source for controlling lymphangiogenesis. In the present study, we compared immunohistochemical and RT-PCR findings of the popliteal lymph node (PLN) and footpad skin in C57BL/6J and WBB6F1 mice, the MC-deficient strain. The results indicated that MCs play certain role in complete Freund’s adjuvant-induced intranodal lymphangiogenesis. VEGF-A, VEGFR-2 and TNF-α were crucial factors in lymphangiogenesis both in the PLN and skin. Moreover, the in vivo administration of the specific mTOR inhibitor, rapamycin inhibited lymphangiogenesis independent of MCs in PLN rather than in the skin. Further study on anti-lymphangiogenic effect will contribute to our understanding of LEC and MC modulation in pathological lymphangiogenesis.     

Combination of PDT and inhibitor treatment affects melanoma cells and spares keratinocytes

Photodynamic therapy (PDT) is a potential tool in cancer treatment. Today this therapy is established among others for the treatment of nonmelanoma skin cancer. However, the more dangerous skin cancer--the melanoma--still has to be removed by surgery. Therefore, we investigated the effects of PDT and additional administration of heme oxygenase I (HO-I) and poly(ADP-ribose) polymerase (PARP) inhibitors on the treatment of melanoma cells in comparison to nonmalignant keratinocytes. Therefore, cocultures were established with WM451LU melanoma cells and HaCaT keratinocytes. In the coculture some 65% melanoma cells and 35% HaCaT cells were present before PDT, whereas after PDT the proportion was 41% melanoma cells and 59% HaCaT cells. Combination of both inhibitors improves these results to only 16% melanoma cells and 84% HaCaT cells. PDT is, therefore, a potent skin cancer treatment, which might also be interesting for melanoma treatment. The cytotoxic effects of PDT are largely mediated by ROS. Addition of HO-I and PARP inhibitors could improve the efficiency of photodynamic treatment.     

Inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultraviolet-induced pigmentation

A pomegranate extract (PE) from the rind containing 90% ellagic acid was tested for its skin-whitening effect. PE showed inhibitory activity against mushroom tyrosinase in vitro, and the inhibition by the extract was comparable to that of arbutin, which is a known whitening agent. PE, when administered orally, also inhibited UV-induced skin pigmentation on the back of brownish guinea pigs. The intensity of the skin-whitening effect was similar between guinea pigs fed with PE and those fed with L-ascorbic acid. PE reduced the number of DOPA-positive melanocytes in the epidermis of UV-irradiated guinea pigs, but L-ascorbic acid did not. These results suggest that the skin-whitening effect of PE was probably due to inhibition of the proliferation of melanocytes and melanin synthesis by tyrosinase in melanocytes. PE, when taken orally, may be used as an effective whitening agent for the skin.     

Mechanism and inhibitory effect of galangin and its flavonoid mixture from Alpinia officinarum on mushroom tyrosinase and B16 murine melanoma cells

The whitening effects of the flavonoid constituents of Alpinia officinarum Hance were investigated on melanin biosynthesis in B 16 mouse melanoma cells, tyrosinase inhibition and UV absorption. The melanin content was reduced to 1.276 microg /10(5) cell for flavonoid mixture and 1.161 microg /10(5) cell for galangin while the melanin control was 1.632 microg/10(5) cell. Both flavonoid mixture and galangin reduced melanin production with an inhibition of 21.81% and 28.86% at a concentration of 26.5 microg/mL and 29 microg/mL (107.4 microM), respectively. Tyrosinase inhibition by the flavonoid mixture and galangin were higher at lower concentrations and galangin showed competitive inhibition at a concentration less than 21.23 microg/mL which was soluble. In addition, the flavonoid mixture and galangin showed a broad absorption band at 270 approximately 290 nm related to the UV-B area. These observations suggest that galangin may be a whitening agent and a promising candidate for prevention of skin cancer. This is the first full scale report on the evaluation of the whitening effect of galangin.     

光动力疗法治疗感染性疾病的动物模型

光动力疗法(photodynamic therapy,PDT)是利用特定波长的激发光照射生物靶标上的光敏剂,从而产生活性氧并有效杀伤多种耐药病原体的新型治疗方式,具有作用广、安全可控、不易耐受等优点。大量体外实验已证实了PDT疗效,但目前动物实验数据较少,且治疗参数不一,一定程度上影响了PDT在临床治疗中的广泛应用。本文综述近年来PDT用于体内抗感染治疗的动物模型构建、治疗方案设计等方面的研究进展,为未来PDT抗感染研究及临床应用提供参考。     

Methadone enhances the effectiveness of 5‐aminolevulinic acid‐based photodynamic therapy for squamous cell carcinoma and glioblastoma in vitro

Although having shown promising clinical outcomes, the effectiveness of 5‐aminolevulinic acid‐based photodynamic therapy (ALA‐PDT) for squamous cell carcinoma (SCC) and glioblastoma remains to be improved. The analgesic drug methadone is able to sensitize various tumors to chemotherapy. In this in vitro study, the influence of methadone to the effectiveness of ALA‐PDT for SCC (FADU) and glioblastoma (A172) was investigated on the protoporphyrin IX (PpIX) fluorescence, survival rates, apoptosis, and cell cycle phase, each with or without the presence of methadone. The production of PpIX was increased by methadone in FADU cells while it was decreased in A172 cells. The survival rates of both cell lines treated by ALA‐PDT were significantly reduced by the combination with methadone (P < .05). Methadone also significantly increased the percentage of apoptotic cells and improved the effect of ALA‐PDT on the cell cycle phase arrest in the G0/G1 phase (P < .05). This study demonstrates the potential of methadone to influence the cytotoxic effect of ALA‐PDT for both SCC and glioblastoma cell lines.      

Comparison of two methods for noninvasive determination of carotenoids in human and animal skin: Raman spectroscopy versus reflection spectroscopy

Based on compelling in vivo and in vitro studies on human skin, carotenoids are thought to be of great interest as powerful antioxidants acting to prevent free‐radical‐induced damages, including premature skin ageing and the development of skin diseases such as cancer. Among the available techniques that are suitable for noninvasive determination of carotenoids in human skin, are resonance Raman spectroscopy (RRS) and reflection spectroscopy (RS). For RS, a LED‐based miniaturized spectroscopic system (MSS) was developed for noninvasive measurement of carotenoids in human skin. The optimization and subsequent calibration of the MSS was performed with the use of RRS. A strong correlation between the carotenoid concentration determined by the RS and for the RRS system was achieved for human skin in vivo (R = 0.88) and for bovine udder skin in vitro (R = 0.81). (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Design,synthesis and photocytotoxicity of upconversion nanoparticles: Potential applications for near‐infrared photodynamic and photothermal therapy

Photodynamic therapy (PDT) and photothermal therapy (PTT) are emerging modalities for the treatment of tumors and nonmalignant conditions, based on the use of photosensitizers to generate singlet oxygen or heat, respectively, upon light (laser) irradiation. They have potential advantages over conventional treatments, being minimally invasive with precise spatial‐temporal selectivity and reduced side effects. However, most clinically employed PDT agents are activated at visible (vis) wavelengths for which the tissue penetration and, hence, effective treatment depth are compromised. In addition, the lipophilicity of near‐infrared (NIR) photothermal agents limits their use and efficiency. To achieve combined PDT/PTT effects, both excitation wavelengths need to be tuned into the NIR spectral window of biological tissues. This paper reports the synthesis of neodymium‐doped upconversion nanoparticles (NaYF₄:Yb,Er,Nd@NaYF₄:Nd) that convert 800 nm light into vis wavelengths, which can then activate conventional photosensitizers on the nanoparticle surface for PDT. Covalently bonded IR‐780 dyes can readily be activated by 800 nm laser irradiation. The PEGylated nanoplatform exhibited a narrow size distribution, good stability and efficient generation of singlet oxygen under laser irradiation. The in vitro photocytotoxicity of this engineered nanoplatform as either a PDT or PTT agent in HeLa cells is demonstrated, while fluorescence microscopy in nanoplatform‐incubated cells highlights its potential for bioimaging.     

Beta‐Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration via a cAMP‐Dependent Mechanism and Wound Angiogenesis

Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar‐free foetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta‐adrenoceptors (β‐AR) are G protein‐coupled receptors (GPCRs) expressed on all skin cell‐types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β‐AR‐mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β‐AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)‐dependent and protein kinase A (PKA)‐independent mechanisms as demonstrated through use of an EPAC agonist that auto‐inhibited the cAMP‐mediated β‐AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that β‐AR activation reduced pro‐angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible β‐AR‐mediated autocrine and paracrine anti‐angiogenic mechanisms. In more complex environments, β‐AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β‐AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β‐AR agonists could be promising anti‐angiogenic modulators in skin. J. Cell. Physiol. 230: 356–365, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Erythrocytes-the 'house elves' of photodynamic therapy.

Photodynamic therapy (PDT) and fluorescence diagnosis (FD) are being developed for a number of clinical applications. Since fluorophores and photosensitising drugs are usually given systemically their effect on blood elements are of significant importance. Photodynamic effects on erythrocytes occur naturally in patients with erythropoietic protoporphyria (EPP). Exposure to small fluences, as obtained by the erythrocytes when they pass capillaries in the skin, leads to transfer of the photosensitiser protoporphyrin IX (PP IX), from EPP erythrocytes to endothelial cells. Thus, the erythrocytes are partly protected while the endothelial cells suffer photodamage. During photodynamic therapy in vivo erythrocytes are regularly photosensitised. This side effect is partly intended but mostly unwanted, and a summary of this topic is given. Furthermore, the effect of UV-A on erythrocytes that is accompanied with the formation of bilirubin is reviewed. Erythrocytes serve as convenient model cells for experimental research. Such use of erythrocytes to screen new photosensitisers may be of limited value. A combination of photohaemolysis and haemoglobin oxygenation may become the basis for an assay for in vitro phototoxicity. Erythrocytes from birds are good model cells for exploration of physiological and molecular mechanisms involved in PDT. A potential mechanism of PDT induced behaviour resembling apoptosis in erythrocytes is provided.PDT for sterilisation of erythrocyte concentrates has a potential for medical use. Photodynamic effects on the erythrocytes themselves should be avoided. This is realised by choosing a virus-selective photosensitiser, low fluences and treatment of the concentrates with agents like dipyridamole and antioxidants. Future aspects of applications of photosensitisation of red blood cells are discussed.     

Unveiling Ga(III) phthalocyanine—a different photosensitizer in neuroblastoma cellular model

Phthalocyanines (Pc) and their metallated derivatives are strongly considered for photodynamic therapy (PDT) possessing unique properties as possible new photosensitizers (PS). We have used toxicological assessments, real‐time monitoring of cellular impedance, and imagistic measurements for assessing the in vitro dark toxicity and PDT efficacy of Ga(III)‐Pc in SHSy5Y neuroblastoma cells. We have established the non‐toxic concentration range of Ga(III)‐Pc, a compound which shows a high intracellular accumulation, with perinuclear distribution in confocal microscopy. By choosing Ga(III)Pc non‐toxic dose, we performed in vitro experimental PDT hampering cellular proliferation. Our proposed Ga(III)‐Pc could complete a future PS panel for neuroblastoma alternate therapy.     

Extracellular vesicle‐enclosed miR‐486‐5p mediates wound healing with adipose‐derived stem cells by promoting angiogenesis

Adipose‐derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC‐secreted extracellular vesicles (EV) carry diverse cargos such as microRNAs (miRNAs) to participate in the ASC‐based therapies. Considering its effects, we aimed to investigate the role of ASC‐EVs in the cutaneous wound healing accompanied with the study on the specific cargo‐medicated effects on wound healing. Two full‐thickness excisional skin wounds were created on mouse dorsum, and wound healing was recorded at the indicated time points followed by histological analysis and immunofluorescence staining for CD31 and α‐SMA. Human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMECs) were co‐cultured with EVs isolated from ASC (ASC‐EVs), respectively, followed by the evaluation of their viability and mobility using CCK‐8, scratch test and transwell migration assays. Matrigel‐based angiogenesis assays were performed to evaluate vessel‐like tube formation by HMECs in vitro. ASC‐EVs accelerated the healing of full‐thickness skin wounds, increased re‐epithelialization and reduced scar thickness whilst enhanced collagen synthesis and angiogenesis in murine models. However, miR‐486‐5p antagomir abrogated the ASC‐EVs‐induced effects. Intriguingly, miR‐486‐5p was found to be highly enriched in ASC‐EVs, exhibiting an increase in viability and mobility of HSFs and HMECs and enhanced the angiogenic activities of HMECs. Notably, we also demonstrated that ASC‐EVs‐secreted miR‐486‐5p achieved the aforesaid effects through its target gene Sp5. Hence, our results suggest that miR‐486‐5p released by ASC‐EVs could be a critical mediator to develop an ASC‐based therapeutic strategy for wound healing.     

In vivo monitoring blood‐brain barrier permeability using spectral imaging through optical clearing skull window

The blood‐brain barrier (BBB) plays a key role in the health of the central nervous system. Opening the BBB is very important for drug delivery to brain tissues to enhance the therapeutic effect on brain diseases. It is necessary to in vivo monitor the BBB permeability for assessing drug release with high resolution; however, an effective method is lacking. In this work, we developed a new method that combined spectral imaging with an optical clearing skull window to in vivo dynamically monitor BBB opening caused by 5‐aminolevulinic acid (5‐ALA)‐mediated photodynamic therapy (PDT), in which the Evans blue dye (EBd) acted as an indicator of the BBB permeability. Using this method, we effectively monitored the cerebrovascular EBd leakage process. Moreover, the analysis of changes in the vascular and extravascular EBd concentrations demonstrated that the PDT‐induced BBB opening exhibited spatiotemporal differences in the cortex. This spectral imaging method based on the optical clearing skull window provides a low‐cost and simply operated tool for in vivo monitoring BBB opening process. This has a high potential for the visualization of drug delivery to the central nervous system. Thus, it is of tremendous significance in brain disease therapy. Monitoring the changes in PDT‐induced BBB permeability by evaluating the EBd concentration using an optical clearing skull window. (A) Entire brains and coronal sections following treatment of PDT with/without an optical clearing skull window after injection of EBd. (B) Typical EBd distribution maps before and after laser irradiation captured by the spectral imaging method. (Colorbar represents the EBd concentration).      

Involvement of Nitric Oxide in UVB‐Induced Pigmentation in Guinea Pig Skin

Ultraviolet (UV) B irradiation evokes erythema and delayed pigmentation in skin, where a variety of toxic and modulating events are known to be involved. Nitric oxide (NO) is generated from l ‐arginine by NO synthases (NOS). Production of NO is enhanced in response to UVB‐stimulation and has an important role in the development of erythema. NO has recently been demonstrated as a melanogen which stimulates melanocytes in vitro, however, no known in vivo data has been reported to support this finding. In this study, we investigated the contribution of NO with UV‐induced pigmentation in an animal model using an NOS inhibitor. UVB‐induced erythema in guinea pig skin was reduced when an NOS inhibitor, l ‐NAME (N‐nitro‐ l ‐arginine methylester hydrochloride), was topically applied to the skin daily, beginning 3 days before UVB‐irradiation. Delayed pigmentation and an increased number of DOPA‐positive melanocytes in the skin were markedly suppressed by sequential daily treatment with l ‐NAME. Furthermore, melanin content 13 days after UVB‐irradiation was significantly lower in skin treated with l ‐NAME than in the controls. In contrast, d ‐NAME (N‐nitro‐ d ‐arginine methylester hydrochloride), an ineffective isomer of l ‐NAME, demonstrated no effect on these UV‐induced skin responses. These results suggest that NO production may contribute to the regulation of UVB‐induced pigmentation.