Response Of Mouse Skin Research Articles

Double-negative T cells ameliorate psoriasis by selectively inhibiting IL-17A-producing γδlow T cells

BackgroundPsoriasis is a chronic immune-mediated skin condition. Although biologic treatments are effective in controlling psoriasis, some patients do not respond or lose response to these therapies. Thus, new strategies for psoriasis treatment are still urgently needed. Double-negative T cells (DNT) play a significant immunoregulatory role in autoimmune diseases. In this study, we aimed to evaluate the protective effect of DNT in psoriasis and explore the underlying mechanism.MethodsWe conducted a single adoptive transfer of DNT into an imiquimod (IMQ)-induced psoriasis mouse model through tail vein injection. The skin inflammation and IL-17A producing γδ T cells were evaluated.ResultsDNT administration significantly reduced the inflammatory response in mouse skin, characterized by decreased skin folds, scales, and red patches. After DNT treatment, the secretion of IL-17A by RORc+ γδlow T cells in the skin was selectively suppressed, resulting in an amelioration of skin inflammation. Transcriptomic data suggested heightened expression of NKG2D ligands in γδlow T cells within the mouse model of psoriasis induced by IMQ. When blocking the NKG2D ligand and NKG2D (expressed by DNT) interaction, the cytotoxic efficacy of DNT against RORc+IL17A+ γδlow T cells was attenuated. Using Ccr5−/− DNT for treatment yielded evidence that DNT migrates into inflamed skin tissue and fails to protect IMQ-induced skin lesions.ConclusionsDNT could migrate to inflamed skin tissue through CCR5, selectively inhibit IL-17-producing γδlow T cells and finally ameliorate mouse psoriasis. Our study provides feasibility for using immune cell therapy for the prevention and treatment of psoriasis in the clinic.

Critical roles of irradiance in the regulation of UVB-induced inflammasome activation and skin inflammation in human skin keratinocytes.

UVB dosage is generally regarded as the most critical factor that determines the severity of UVB-induced skin erythema. However, recent studies have demonstrated that different UV irradiances induce varying biological responses in mouse skin even at constant UV doses. UVB-induced inflammasome activation is particularly observed in human skin keratinocytes, which are classified as immunocompetent cells, but not in mouse skin keratinocytes, which do not express sufficient inflammasome complex components. In human skin UVB-induced sunburn reactions, NLRP1 inflammasome activation critically mediates the inflammatory responses. Here, we employed primary human skin keratinocytes to explore the impact of different irradiances of a constant UVB dosage on inflammasome activation and related inflammatory responses. Our findings indicated that low-irradiance UVB induced relatively stronger NLRP1 inflammasome activation, which manifested as more active IL-1β, IL-18 release, and enhanced procaspase-1 cleavage compared to high-irradiance UVB at the same dose. Irradiance did not influence cell lysis or the expression of inflammasome complex proteins including NLRP1, proIL-1β, proIL-18, procaspase-1, and ASC. The UVB-induced TNF-α and cyclooxygenase-2 expression was also relatively higher in keratinocytes exposed to low-irradiance UVB. Low-irradiance UVB also increased reactive oxygen species production. UVB-triggered signaling analysis revealed that low-irradiance UVB resulted in more prominent p38 and JNK activation. Therefore, our findings indicated that, in addition to the role of total dosage, irradiance crucially modulates UVB-elicited inflammation in human skin keratinocytes, thus providing novel insights into human skin photobiology.

Wwox Deficiency Causes Downregulation of Prosurvival ERK Signaling and Abnormal Homeostatic Responses in Mouse Skin.

Deficiency of tumor suppressor WW domain-containing oxidoreductase (WWOX) in humans and animals leads to growth retardation and premature death during postnatal developmental stages. Skin integrity is essential for organism survival due to its protection against dehydration and hypothermia. Our previous report demonstrated that human epidermal suprabasal cells express WWOX protein, and the expression is gradually increased toward the superficial differentiated cells prior to cornification. Here, we investigated whether abnormal skin development and homeostasis occur under Wwox deficiency that may correlate with early death. We determined that keratinocyte proliferation and differentiation were decreased, while apoptosis was increased in Wwox–/– mouse epidermis and primary keratinocyte cultures and WWOX-knockdown human HaCaT cells. Without WWOX, progenitor cells in hair follicle junctional zone underwent massive proliferation in early postnatal developmental stages and the stem/progenitor cell pools were depleted at postnatal day 21. These events lead to significantly decreased epidermal thickness, dehydration state, and delayed hair development in Wwox–/– mouse skin, which is associated with downregulation of prosurvival MEK/ERK signaling in Wwox–/– keratinocytes. Moreover, Wwox depletion results in substantial downregulation of dermal collagen contents in mice. Notably, Wwox–/– mice exhibit severe loss of subcutaneous adipose tissue and significant hypothermia. Collectively, our knockout mouse model supports the validity of WWOX in assisting epidermal and adipose homeostasis, and the involvement of prosurvival ERK pathway in the homeostatic responses regulated by WWOX.

Topical Vitamin D Receptor Antagonist/Partial-Agonist Treatment Induces Epidermal Hyperproliferation via RARγ Signaling Pathways

Vitamin D and A derivatives are well-known endogenous substances responsible for skin homeostasis. In this study we topically treated shaved mouse skin with a vitamin D agonist (MC903) or vitamin D antagonist/partial agonist (ZK159222) and compared the changes with acetone (control treatment) treatment for 14 days. Topical treatment with ZK159222 resulted in increased expression of genes involved in retinoic acid synthesis, increased retinoic acid concentrations and increased expression of retinoid target genes. Clustering the altered genes revealed that heparin-binding epidermal growth factor-like growth factor, the main driver of epidermal hyperproliferation, was increased via RARγ-mediated pathways, while other clusters of genes were mainly decreased which were comparable to the changes seen upon activation of the RARα-mediated pathways. In summary, we conclude that epidermal hyperproliferation of mouse skin in response to a topically administered vitamin D receptor antagonist/partial agonist (ZK159222) is induced via increased retinoic acid synthesis, retinoic acid levels and increased RARγ-mediated pathways.

Bucillamine Inhibits UVB-Induced MAPK Activation and Apoptosis in Human HaCaT Keratinocytes and SKH-1 Hairless Mouse Skin.

Ultraviolet B (UVB) radiation is known as a culprit in skin carcinogenesis. We have previously reported that bucillamine (N-[2-mercapto-2-methylpropionyl]-L-cysteine), a cysteine derivative with antioxidant and anti-inflammatory capacity, protects against UVB-induced p53 activation and inflammatory responses in mouse skin. Since MAPK signaling pathways regulate p53 expression and activation, here we determined bucillamine effect on UVB-mediated MAPK activation in vitro using human skin keratinocyte cell line HaCaT and in vivo using SKH-1 hairless mouse skin. A single low dose of UVB (30mJcm-2 ) resulted in increased JNK/MAPK phosphorylation and caspase-3 cleavage in HaCaT cells. However, JNK activation and casaspe-3 cleavage were inhibited by pretreatment of HaCaT cells with physiological doses of bucillamine (25 and 100µm). Consistent with these results, bucillamine pretreatment in mice (20mgkg-1 ) inhibited JNK/MAPK and ERK/MAPK activation in skin epidermal cells at 6-12 and 24h, respectively, after UVB exposure. Moreover, bucillamine attenuated UVB-induced Ki-67-positive cells and cleaved caspase-3-positive cells in mouse skin. These findings demonstrate that bucillamine inhibits UVB-induced MAPK signaling, cell proliferation and apoptosis. Together with our previous report, we provide evidence that bucillamine has a photoprotective effect against UV exposure.

Inhibitory effects of 7,7′-bromo-curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation

This study aimed to determine the capacity of 7,7′-bromo-curcumin (CUR-Br), a curcumin analogue with higher chemical stability than curcumin (CUR), in the suppression of mouse ear edema. Male CD-1 mice were topically pre-treated with either CUR or CUR-Br for 30 min prior to an application of 12-O-tetradecanoylphorbol-13-acetate. After 6 h, mice were killed, and ear punches were measured for their weight and thickness as a marker of edema and inflammation. CUR-Br demonstrated a higher anti-inflammatory efficacy compared to CUR. CUR and CUR-Br at 1.0 μmol suppressed the TPA-induced increase in the ear weight by 26.0% and 57.2%, and decreased TPA-induced increase in the ear thickness by 22.2% and 84.7%, respectively. The inhibitory effects of Cur-Br were associated with decreased levels of inflammatory cytokines (IL-1β, IL-2, IL-6, KC/GRO, IL-10, IL-17, and IL-23). In addition, CUR-Br significantly downregulated expression of pro-inflammatory signaling proteins such as p-STAT3, STAT3, PI3K, AKT, p-p65, and COX-2. Overall, our results demonstrated that the curcumin analogue, CUR-Br, showed stronger anti-inflammatory properties than CUR in inhibiting TPA-induced inflammatory response in mouse skin.

The different roles of 5-HT1A/2A receptors in fluoxetine ameliorated pigmentation of C57BL/6 mouse skin in response to stress

Background5-HT1A receptor was participated in fluoxetine induced melanogenesis in melanocytes and in normal C57BL/6 mice, but we know little about whether other 5-HT receptors are involved in regulation of fluoxetine promotes pigmentation. ObjectiveTo investigate the role of 5-HT receptors in regulation of fluoxetine ameliorates chronic unpredictable mild stress (CUMS) and chronic restraint stress (CRS) induce hypopigmentation in C57BL/6 mice. MethodsCUMS and CRS were used to induce depigmentation in mice and evaluate the effect of fluoxetine. Western blot, immunohistochemistry and Q-PCR assay were used to determine the levels of protein and mRNA. Masson Fontana staining was used for melanin staining and FITC-Phalloidin staining was used to detect the expression of F-actin. Zebrafish and B16F10 cells were used for the mechanism research. ResultsFluoxetine (2.6 mg/kg, ig) ameliorated hypopigmentation induced by CUMS and CRS in mice, significantly increased the mRNA and protein levels of 5-HT1 A and 5-HT2 A receptors in mice and B16F10 cells. The effect of fluoxetine on melanogenesis in B16F10 cells and zebrafish were inhibited by WAY100635 (a selective 5-HT1 A receptor antagonist) and ketanserin (a 5-HT2 A receptor antagonist), respectively. Activation of p38 MAPK signaling pathways was contributed to fluoxetine induced melanogenesis and inhibited by WAY100635, but not ketanserin. However, ketanserin selectively weakened the action of fluoxetine promoted migration and up-regulated Rab27a protein expression in B16F10 cells. Conclusions5-HT1 A and 2 A receptors contribute to melanogenesis and migration property of fluoxetine. The newly revealed mechanism indicates that fluoxetine and its analogues may be a potential drug for treatment of depigmentation disorders.

1162 BMAL1 and CLOCK proteins in regulating the sensitivity of human keratinocytes to UVB-induced apoptosis

A diverse array of biological processes is under circadian controls. It has been reported that UV-induced apoptosis and DNA damage responses in mouse skin are time-of-day dependent and regulated by core clock proteins, including BMAL1, CLOCK, PERs, and CRYs. The aim of this study is to investigate the circadian controls of UV responses in human keratinocytes, using the siRNA-mediated loss-of-function approach. We found that low-dose (5 mJ/cm2) of UVB triggered an oscillation of BMAL1 and CLOCK mRNA expression in the immortal HaCat human keratinocyte cell line, as well as primary human keratinocytes. In HaCat cells, depletion of BMAL1 or CLOCK reduced the percentage of UVB-induced apoptotic cells from 43% (in controls) to 29% (p < 0.001) and 31% (P < 0.05), respectively. Moreover, knockdown of either clock gene strongly blocked UVB-induced expression of tumor necrosis factor α (TNFα) and phosphorylation of NF-κB. These results suggest that BMAL1-CLOCK proteins may promote cell death by regulating the TNFα-NF-κB mediated extrinsic apoptotic pathway in UVB-irradiated HaCat keratinocytes. Given the critical impact of UVB on photo-aging and photo-carcinogenesis, mechanistic elucidation and understanding of circadian controls on UVB effects in human skin will be beneficial for prevention and treatment of skin-related diseases including cancers.

P44 Does the slow-releasing hydrogen sulfide donor GYY4137 controls inflammation and pruritogen responses in mouse skin?

A growing body of evidence suggests that hydrogen sulphide (H2S) plays a crucial role in both physiological and pathophysiological process, including heart failure, inflammatory lung and articular diseases, erectile dysfunction, diabetes, among others. Poor diabetes control in women (mean glycosylated hemoglobin level The aim of this study is to determine the effectiveness of the slow H2S-releasing donor GYY4137 in reducing itch behaviour and cutaneous inflammation in mouse dorsal skin. The animal protocols were approved by the local ICB-USP Ethics Committee. In order to produce itching behaviour, male BALB/c mice (25–30 g) were anaesthetised with isoflurano and i.d. injected with histamine (1 μmol site−1) alone or co-injected with GYY4137 (0.3–30 nmol site−1; 0.05 ml). Itching behaviour was estimated by measuring the number of scratching bouts over a 40 min period. In a separate set of experiments, animals were pre-treated (−40 min) with the ATP-sensitive K+ (KATP) channel blocker glibenclamide, and then i.d. injected with histamine and Na2S (3 nmol site−1). In addition, we investigated the presence of mRNA for the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST) in the mouse naive skin via qualitative RT-PCR. Data are presented as mean ± SEM and were analyzed by ANOVA followed by the Dunnett test. Values of P The i.d. injection of histamine significantly increased the itching frequency as compared to its vehicle Tyrode. Increasing doses of GYY4137 (1–10 nmol site−1) dose-dependently reduced histamine-induced pruritus but failed to inhibit plasma extravasation evoked by histamine. Suppression of histamine-induced pruritus by the H2S donor was not reversed by glibenclamide. A marked mRNA expression for CBS and 3-MST, but not for CSE, was seen in the mouse naive skin. These findings indicate that low doses of GYY4137 in the skin decrease histamine-induced pruritus without affecting the associated local inflammation. This protective effect does not appear to be mediated by KATP channels activation. Financial support: FAPESP, CAPES and CNPq.

High-power femtosecond-terahertz pulse induces a wound response in mouse skin

Terahertz (THz) technology has emerged for biomedical applications such as scanning, molecular spectroscopy, and medical imaging. Although a thorough assessment to predict potential concerns has to precede before practical utilization of THz source, the biological effect of THz radiation is not yet fully understood with scant related investigations. Here, we applied a femtosecond-terahertz (fs-THz) pulse to mouse skin to evaluate non-thermal effects of THz radiation. Analysis of the genome-wide expression profile in fs-THz-irradiated skin indicated that wound responses were predominantly mediated by transforming growth factor-beta (TGF-β) signaling pathways. We validated NFκB1- and Smad3/4-mediated transcriptional activation in fs-THz-irradiated skin by chromatin immunoprecipitation assay. Repeated fs-THz radiation delayed the closure of mouse skin punch wounds due to up-regulation of TGF-β. These findings suggest that fs-THz radiation initiate a wound-like signal in skin with increased expression of TGF-β and activation of its downstream target genes, which perturbs the wound healing process in vivo.

Phenethyl Isothiocyanate Inhibits 12-O-Tetradecanoylphorbol-13-Acetate-Induced Inflammatory Responses in Mouse Skin

Phenethyl isothiocyanate (PITC) is the hydrolysis product of the glucosinolate gluconasturtiin in cruciferous vegetables. This study was conducted to determine whether PITC inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in the mouse ear. Topical application of 5 nmol of TPA to mouse ears markedly increased the ear weight, expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 protein, and phosphorylation of the inhibitor of κB (IκB) α, AKT, and extracellular signal-regulated protein kinase (ERK) 1/2 and reduced IκBα protein levels. Pretreatment with PITC (150-450 nmol) significantly suppressed these TPA-induced inflammatory responses. We also determined whether low concentrations of PITC (0.5-5 μmol/L) inhibited lipopolysaccharide (LPS)-stimulated inflammatory responses in Raw264.7 cells. PITC dose-dependently reduced the LPS-induced secretion of nitric oxide, prostaglandin E(2), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, as well as COX-2 and iNOS protein expression. PITC also attenuated LPS-induced increases in iNOS, COX-2, IL- 6, IL-1β, and TNF-α mRNA levels, as well as the promoter-dependent transcriptional activation of the genes for iNOS and COX-2. PITC inhibited LPS-induced IκBα phosphorylation and degradation and subsequently reduced LPS-induced p65 nuclear translocation and the transcriptional activity of nuclear factor-κB (NF-κB), which was accompanied by a reduction in ERK1/2 and AKT phosphorylation. The results of this study demonstrated that PITC effectively inhibits inflammatory responses in vivo and in vitro, which may be mediated via the inhibition of AKT and ERK1/2 activation, leading to subsequent inhibition of the transcriptional activity of NF-κB.

Nordihydroguaiaretic Acid from Creosote Bush (Larrea tridentata) Mitigates 12‐O‐Tetradecanoylphorbol‐13‐Acetate‐Induced Inflammatory and Oxidative Stress Responses of Tumor Promotion Cascade in Mouse Skin

Nordihydroguaiaretic acid (NDGA) is a phenolic antioxidant found in the leaves and twigs of the evergreen desert shrub, Larrea tridentata (Sesse and Moc. ex DC) Coville (creosote bush). It has a long history of traditional medicinal use by the Native Americans and Mexicans. The modulatory effects of topically applied NDGA was studied on acute inflammatory and oxidative stress responses in mouse skin induced by stage I tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). Double TPA treatment adversely altered many of the marker responses of stage I skin tumor promotion cascade. Pretreatment of NDGA in TPA-treated mice mitigated cutaneous lipid peroxidation and inhibited production of hydrogen peroxide. NDGA treatment also restored reduced glutathione level and activities of antioxidant enzymes. Elevated activities of myeloperoxidase, xanthine oxidase and skin edema formation in TPA-treated mice were also lowered by NDGA indicating a restrained inflammatory response. Furthermore, results of histological study demonstrated inhibitory effect of NDGA on cellular inflammatory responses. This study provides a direct evidence of antioxidative and anti-inflammatory properties of NDGA against TPA-induced cutaneous inflammation and oxidative stress corroborating its chemopreventive potential against skin cancer.

Mice deficient in epidermal PPARγ exhibit loss of sebaceous glands as well as augmented apoptosis and inflammation in response to ultraviolet B

Previous pharmacological studies have provided conflicting results regarding the relevance of PPARγ in photobiology. In these current studies, we utilize a cytokeratin 14 promoter driven CRE‐LoxP strategy to examine how loss of epidermal PPARγ (KO mice) alters ultraviolet B (UVB) responses in mouse skin. We first demonstrate an increase in PPARγ ligand production in lipid extracts of mouse epidermis irradiated with 1500 J/m2 of UVB. We next show that after 24 hrs, KO mice exhibit a marked increase (&gt; 4‐fold) in UVB‐induced apoptosis as measured by epidermal caspase 3 activity. We next assessed UVB‐induced inflammatory responses. At 24 and 72 hrs post‐irradiation, KO mice exhibited increased inflammation as determined by significant increases in skin thickness, myeloperoxidase activity and inflammatory cell infiltrates. Given that inflammatory infiltrates typically occur at 36–48 hrs post‐UVB, these studies suggest an earlier onset of inflammation in KO mice. Finally, in data unrelated to UVB irradiation, adult KO mice also exhibited almost complete loss of sebaceous glands. This data provides compelling evidence that epidermal PPARγ serves an important role in regulating the acute epidermal photoresponse and is necessary for sebaceous gland development and/or maintenance. Supported by funding by the National Institutes of Health and the Prevent Cancer Foundation.

UVB Radiation Induces Apoptosis in Keratinocytes by Activating a Pathway Linked to “BLT2-Reactive Oxygen Species”

The role of reactive oxygen species (ROS) in UVB-induced apoptosis has been established, but the molecular mechanisms of their production in response to UVB irradiation in keratinocytes are not well understood. In this study, we demonstrate that levels of BLT2, a low-affinity leukotriene B(4) receptor, and its ligands (LTB(4) and 12(S)-HETE) are greatly increased by UVB irradiation and are responsible for the UVB-induced ROS generation in human keratinocytes. Blockade of BLT2 with a BLT2-specific antagonist, LY255283, or with siBLT2 attenuated ROS production and apoptotic cell death detected by a number of criteria. Moreover, we found that the NADPH oxidase family protein Nox1 lies downstream of BLT2 and mediates UVB-induced ROS production and apoptosis. Topical treatment of mouse epidermal skin with LY255283 gave significant protection against UVB-induced sunburn-associated apoptotic damage. Finally, when BLT2-overexpressing transgenic mice were irradiated with UVB, we observed more extensive skin apoptosis. Taken together, our results demonstrate that a "BLT2-Nox1"-linked pathway has a crucial role in UVB-induced ROS generation and mediates apoptosis in human keratinocytes.

Leukotriene B4 Mediates Sphingosylphosphorylcholine-Induced Itch-Associated Responses in Mouse Skin

In atopic dermatitis, the concentration in the skin of sphingosylphosphorylcholine (SPC), which is produced from sphingomyelin by sphingomyelin deacylase, is increased. In the present study, we investigated the itch-eliciting activity of SPC and related substances and the mechanisms of SPC action in mice. An intradermal injection of SPC, but not sphingomyelin and sphingosine, induced scratching, an itch-associated response, which was not suppressed by a deficiency in mast cells or the H(1) histamine receptor antagonist terfenadine. The action of SPC was inhibited by the mu-opioid receptor antagonist naltrexone. SPC action also was inhibited by the 5-lipoxygenase inhibitor zileuton and the leukotriene B(4) antagonist ONO-4057, but not by the cyclooxygenase inhibitor indomethacin. Moreover, SPC action was inhibited by the antiallergic agent azelastine, which suppresses the action and production of leukotriene B(4). Administration of SPC to the skin and to primary cultures of keratinocytes increased leukotriene B(4) production. SPC increased intracellular Ca(2+) ion concentration in primary cultures of dorsal root ganglion neurons and keratinocytes. These results suggest that SPC induces itching through a direct action on primary afferents and leukotriene B(4) production of keratinocytes. Sphingomyelin deacylase and SPC receptors may be previously unreported targets for antipruritic drugs.

Acute allergic skin response as a new tool to evaluate the allergenicity of whey hydrolysates in a mouse model of orally induced cow’s milk allergy

Hypoallergenic milk formulae are used for cow's milk allergic infants and may be a good option for infants at risk. Clinical studies have shown that the protein source or the hydrolysis methodology used may influence the effectiveness in infants stressing the importance of adequate pre-clinical testing of hypoallergenic formulae in an in vivo model of orally induced cow's milk allergy. This study was undertaken to introduce a new read-out system to measure the residual allergenicity of whey hydrolysates on both the sensitization and challenge phase of orally induced cow's milk allergy in mice. Mice were sensitized orally to whey or a partial whey hydrolysate (pWH) to measure the residual sensitizing capacity. To predict the residual allergenicity of hydrolysates, whey allergic mice were challenged in the ear with pWH, extensive whey hydrolysate or an amino acid-based formula. An acute allergic skin response (ear swelling at 1 h), whey-specific serum antibodies, and local MCP-1 concentrations were measured. In contrast to whey, oral sensitization with pWH did not result in the induction of whey-specific antibodies, although a minor residual skin response to whey was observed after challenge. Skin exposure to whey hydrolysates showed a hydrolysation dependent reduction of the acute allergic skin response in whey allergic mice. In contrast to whey, skin exposure to pWH did not enhance tissue MCP-1 levels. The acute allergic skin response in mice orally sensitized to cow's milk proteins reveals a new pre-clinical tool which might provide information about the residual sensitizing capacity of hydrolysates supporting the discussion on the use of hypoallergenic formulae in high risk children. This mouse model might be a relevant model for the screening of new hypoallergenic formulae aimed to prevent or treat cow's milk allergy.

In Vitro and In Vivo Anti-Allergic Effects of Arctium lappa L.

The discovery of drugs that can be used for the treatment of allergic disease is important in human health. Arctium lappa Linne (Compositae) (AL) has been used as a traditional medicine in Brazil and throughout Asia and is known to have an anti-inflammatory effect. In this study, the inhibitory effects of AL on degranulation and the release of mediators as well as on inhibition of cys-leukotriene biosynthesis by basophils were investigated. AL was selected out of 10,000 herbal extracts in a set-up for high throughput screening in which the degree of degranulation was monitored by the release of beta-hexosaminidase from rat basophil leukemia (RBL-2H3) cells. The AL extract significantly reduced degranulation and biosynthesis of cys-leukotrienes of human basophils in peripheral blood mono-nuclear cells (PBMCs) (50% inhibitory concentration [IC(50)] = 8.3 and 11.4 microg/ml, respectively). Viability and metabolic activity of the PBMCs were not affected. Although arctiin, the active component of AL that has been described in the literature, was not able to reduce degranulation in RBL-2H3 cells, a single high-performance liquid chromatography (HPLC) fraction from the AL extract inhibited beta-hexosaminidase release (IC(50) = 22.2 microg/ml). Topical administration of an aqueous extract of AL (5 mg/ear) on the ear of whey-sensitized mice 4 hrs before challenge with whey in the ear inhibited acute ear swelling by 50% in an in vivo cow's milk allergic model. The extract had no effect in this model when administered orally. In conclusion, the active component present in the active HPLC fraction of the AL extract was able to significantly reduce the release of inflammatory mediators through inhibition of degranulation and cys-leukotriene release in vitro. In addition, this active component was able to inhibit acute skin response in mice in vivo, indicating that AL is a very promising natural component for use in anti-allergic treatment.

Curcumin decreases 12- O -tetradecanoylphorbol-13-acetate-induced protein kinase C translocation to modulate downstream targets in mouse skin

Curcumin has been shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumour promotion and some of the TPA-responsive markers in mouse skin. However, its mechanism of action is not fully elucidated. The present study focuses on understanding the role of protein kinase C (PKC), the major cellular receptor of TPA, in mediating TPA-induced biological responses in mouse skin and subsequently, elucidating the effects of curcumin on PKC and its downstream target molecules. As compared with controls, single topical application of TPA (5 nmol) to skin increased the translocation of PKC from cytosolic to particulate fraction, determined in terms of activity and protein levels. Ro-31- 8220 (PKC inhibitor, 1 nmol) when applied topically, alone or prior to TPA, inhibited PKC activity in both the compartments but did not affect the TPA-induced protein translocation. In contrast, though curcumin (10 mumol) alone did not alter the basal activity/levels, its pre-treatment decreased the TPA-induced translocation of PKC isozymes (alpha, beta, gamma, epsilon, eta), resulting in appropriate alterations in activity. Despite differences in modes of action of Ro-31-8220 (activity inhibition) and curcumin (decreasing translocation) in modulating PKC, their pre-treatment blunted the TPA-induced levels of mitogen-activated protein kinases and transcription factors (c-jun, c-fos and nuclear factor-kappa B) and downstream target proteins associated with cell proliferation (cyclin D1 and ornithine decarboxylase), cell death (Bax and Bcl2), inflammation (cyclooxygenase-2 and prostaglandin E2) and oxidative stress (8-hydroxy-2'-deoxyguanosine) in skin. These results demonstrate the crucial role of PKC in TPA-mediated cellular responses in skin and that curcumin modulates transmembrane signal transduction via PKC to affect TPA-induced biochemical and molecular alterations in mouse skin.

Deficient inflammatory response to UV radiation in neonatal mice

Mechanisms of juvenile susceptibility to cancer are not well understood. The immune response in neonates favors nonresponsiveness or T(H)2-dominant responses, raising the question of a role for neonatal immunity in this susceptibility. We have investigated the postulate that the inflammatory response differs in neonatal and adult skin. We found no inflammatory infiltrate into neonatal mouse skin in response to UV irradiation as a function of time, dose, or wavelength, although UV-induced DNA damage was readily detected. In contrast, UV irradiation of adult mice initiated a dose- and time-dependent influx of inflammatory cells, chiefly CD11b(+)Ly6G(+) neutrophils, into the skin, detected by immunohistochemistry and quantitated by FACS analysis. This inflammatory response was initiated by UVB (290-320 nm) but not by UVA (320-400 nm). Further, in neonates, in contrast to adults, neither topical trinitrochlorobenzene (TNCB) nor i.p. thioglycollate initiated an inflammatory infiltrate. Conversely, topical TNCB applied to neonates was tolerogenic, resulting in a subsequent antigen-specific decrease of the contact-hypersensitivity response in adults. Neonatal blood contained abundant neutrophils, which exhibited impaired chemotaxis to the chemokine growth-related oncogene-alpha but efficient chemotaxis to the bacterial product fMLP, concomitant with decreased expression of CXCR2 but normal levels of CD11b. We propose this neonatal deficiency in the inflammatory response is a significant, previously unrecognized factor in neonatal immune tolerance and may contribute to neonatal susceptibility to cancer, including melanoma and other UV-induced cancers.

Preclinical pharmacodynamic studies of Aurora A inhibition by MLN8054

13059 Background: The mitotic kinase Aurora A is implicated in the development of multiple tumor types. MLN8054 is an oral, potent and selective small-molecule inhibitor of Aurora A with broad efficacy in preclinical models of cancer. Inhibition of Aurora A by MLN8054 induces accumulation of mitotic cells, followed by apoptosis. This study explores relationships between Aurora A inhibition, mitotic index, and tumor growth inhibition for xenograft models with different sensitivity to MLN8054. The marker response in mouse skin was also studied. Methods: Mice bearing subcutaneous xenografts were dosed orally qd or bid with MLN8054 for 21 days. Pharmacodynamic markers were studied after 1–2 doses. Formalin-fixed xenograft tissues were stained with the mitotic markers pHisH3 and MPM2, or with an antibody to the T288 autophosphorylation site on Aurora A. Tumor growth inhibition (TGI) was calculated using the formula 100 - [ΔT/ΔC * 100], where ΔT is the volume change for treated tumors, and ΔC is the volume change for control tumors. Results: HCT116 human colon xenografts were sensitive to MLN8054 on a qd or bid schedule (84% and 96% TGI respectively for 30mg/kg dose). The T288 autophosphorylation site was used to directly demonstrate inhibition of Aurora A, which resulted in dose-dependent duration of the elevation in mitotic index. Efficacy was similar for qd vs bid dosing of 30mg/kg MLN8054, and accordingly we found that a single dose was sufficient to elevate the mitotic index for about 20–24h in this model. SW480 human colon xenografts have MLN8054 sensitivity similar to that of HCT116, but more modest effects on mitotic index were observed. The mitotic index profile of SW480 is similar to that of MDA-MB-231 xenografts, the most insensitive model studied. Elevated mitotic index was also observed in mouse skin. Conclusions: We found that mitotic index measurements coupled with the T288 autophosphorylation site as a direct marker of Aurora A activity are useful for monitoring inhibition of Aurora A by MLN8054 in tumor and/or skin biopsies. In a sensitive model, greater duration of mitotic index elevation results in greater efficacy. Our continuing work aims to better understand the differences in marker and efficacy responses between xenograft lines, incorporating the pT288 antibody as a direct marker of Aurora A inhibition. [Table: see text]