Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response.

Cancer immunotherapy approaches target signaling pathways that are highly synonymous between CD4 and CD8 T-cell subsets and, therefore, often stimulate nonspecific lymphocyte activation, resulting in cytotoxicity to otherwise healthy tissue. The goal of our study was to identify intrinsic modulators of basic T lymphocyte activation pathways that could discriminately bolster CD8 anti-tumor effector responses. Using a Tbc1d10c null mouse, we observed marked resistance to a range of tumor types conferred by Tbc1d10c deficiency. Moreover, tumor-bearing Tbc1d10c null mice receiving PD-1 or CTLA-4 monotherapy exhibited a 33% or 90% cure rate, respectively. While Tbc1d10c was not expressed in solid tumor cells, Tbc1d10c disruption selectively augmented CD8 T-cell activation and cytotoxic effector responses and adoptive transfer of CD8 T cells alone was sufficient to recapitulate Tbc1d10c null tumor resistance. Mechanistically, Tbc1d10c suppressed CD8 T-cell activation and anti-tumor function by intersecting canonical NF-κB pathway activation via regulation of Map3k3-mediated IKKß phosphorylation. Strikingly, none of these cellular or molecular perturbations in the NF-κB pathway were featured in Tbc1d10c null CD4 T cells. Our findings identify a Tbc1d10c-Map3k3-NF-κB signaling axis as a viable therapeutic target to promote CD8 T-cell anti-tumor immunity while circumventing CD4 T cell-associated cytotoxicity and NF-κB activation in tumor cells.

Pre-validation of an in vitro skin irritation test for medical devices using the reconstructed human tissue model EpiDerm™.

Assessment of dermal irritation is an essential component of the safety evaluation of medical devices. Reconstructed human epidermis (RhE) models have replaced rabbit skin irritation testing for neat chemicals and their mixtures (OECD Test Guideline 439). However, this guideline cannot be directly applied to the area of medical devices (MD) since their non-toxicity assessment is largely based on the testing of MD extracts that may have very low irritation potential. Therefore, the RhE-methods previously validated with neat chemicals needed to be modified to reflect the needs for detection of low levels of potential irritants. A protocol employing RhE EpiDerm was optimized in 2013 using known irritants and spiked polymers (Casas et al., 2013, TIV). In 2014 and 2015 MatTek In Vitro Life Science Laboratories (IVLSL) and RIVM assessed the transferability of the assay. After the successful transfer and standardization of the protocol, 17 laboratories were trained in the use of the protocol in the preparation for the validation. Laboratories produced data with 98% agreement of predictions for the selected references and controls. We conclude that a modified RhE skin irritation test has the potential to address the skin irritation potential of the medical devices. Standardization and focus on the technical issues is essential for accurate prediction.

Upregulation of Extracellular Matrix Genes Corroborates Clinical Efcacy of Human Fibroblast-Derived Growth Factors in Skin Rejuvenation.

Skin care products may use various active ingredients to support skin rejuvenation including growth factors and other molecules that help to regenerate extracellular matrix (ECM) and promote skin repair. The biological effect of skin care products with a strong anti-aging claim was assessed in gene expression analyses using an in vitro human skin model. Application of products containing human fibroblast-derived growth factors resulted in signifcant upregulation of genes encoding ECM components including collagens and elastin. Human fibroblasts cultured under hypoxic conditions show increased gene expression of stem cell markers, and their conditioned media could possibly further support skin rejuvenation. Furthermore, a double-blind, randomized, placebo-controlled study was con-ducted in subjects with moderate to severe facial photodamage to assess the cosmetic clinical efficacy of a product containing human fibroblast-derived growth factors. The test product group demonstrated significantly greater reductions in the appearance of fne lines/wrinkles, coarse line/wrinkles, and overall photodamage, compared to the placebo group. Altogether, the results suggest that human fibroblast-derived growth factors support skin rejuvenation by stimulating dermal fibroblasts to generate ECM.

Transcriptional profiling of epidermal barrier formation in vitro.

BACKGROUND: Barrier function is integral to the health of epithelial tissues. Currently, there is a broad need to develop and improve our knowledge with regard to barrier function for reversal of mild skin irritation and dryness. However, there are few in vitro models that incorporate modulations of both lipids and epidermal differentiation programs for pre-clinical testing to aid in the understanding of barrier health. OBJECTIVE: We have generated a reconstituted epidermis on a decellularized dermis (DED) and characterized its barrier properties relative to human epidermis in order to determine its utility for modeling barrier formation and repair. METHODS: We followed the process of epidermal differentiation and barrier formation through immunocytochemistry and transcriptional profiling. We examined barrier functionality through measurements of surface pH, lipid composition, stratum corneum water content, and the ability to demonstrate topical dose-dependent exclusion of surfactant. RESULTS: Transcriptional profiling of the epidermal model during its formation reveals temporal patterns of gene expression associated with processes regulating barrier function. The profiling is supported by gradual formation and maturation of a stratum corneum and expression of appropriate markers of epidermis development. The model displays a functional barrier and a water gradient between the stratum corneum and viable layers, as determined by confocal Raman spectroscopy. The stratum corneum layer displays a normal acidic pH and an appropriate composition of barrier lipids. CONCLUSION: The epidermal model demonstrates its utility as an investigative tool for barrier health and provides a window into the transcriptional regulation of multiple aspects of barrier formation.

Adolescent school-based vaccination in Australia.

Adolescents have become an increasingly prominent target group for vaccination in Australia and other developed countries. Over the past decade, voluntary school-based vaccination programs have evolved to become the primary method of delivering adolescent vaccines funded under Australia's National Immunisation Program (NIP). These programs operate at a state and territory level and offer NIP vaccines to adolescents in specific school grades using local teams of trained vaccine providers. This paper summarises the current operation of voluntary school-based vaccination programs in Australia. Information was obtained through a literature review, semi-structured interviews with those managing and implementing school-based vaccination programs in each jurisdiction and a review of program resources. Available coverage data was obtained from each state or territory. Vaccines are delivered at the school, during school hours, and typically target late primary or early secondary school grades. Written parental consent is required for any vaccine to be administered. Operation of the programs is influenced by various factors at the school and provider level. Despite variability in program implementation, collection and analysis of coverage data, comparable coverage has been achieved across all states and territories. Coverage is higher than that reported by other countries where adolescent vaccines are mandated for school entry or available only through community vaccination providers. Voluntary school-based vaccination programs are an established mechanism for the delivery of adolescent vaccines in Australia and vaccines offered will continue to evolve in light of national recommendations. Current gaps in evidence include a detailed understanding of the influence of procedural factors on uptake, the best ways to maximise consent form return and, standardisation of coverage data reporting.

Hairless and NFκB form a positive feedback loop after UVB and TNFα stimulation.

Hairless (HR) is a nuclear protein with corepressor activity whose exact function in the skin remains to be determined. Mutations in both human and mouse Hairless lead to hair loss accompanied by the appearance of papules, a disorder called atrichia with papular lesions. Furthermore, mice with mutations in HR are known to have a higher susceptibility to ultraviolet radiation-induced tumorigenesis, suggesting that HR plays a crucial role in the epidermal UVB response. Using normal human keratinocytes (NHKs) and keratinocytes containing a mutation in HR, we found that HR is an early UVB response gene that negatively regulates NFκB mRNA expression. HR mutant keratinocytes have a dysregulated UVB response that includes increased proliferation and the aberrant activation of NFκB effector genes. Additionally, we show that another UVB response gene, TNFα, negatively regulates HR mRNA expression. TNFα-induced negative regulation of HR occurs through a direct interaction of the p65 subunit with a single NFκB-binding domain located in the HR promoter region. Therefore, we show for the first time that HR and NFκB participate in a positive feedback loop that can be initiated either by UVB or TNFα.

L-3-Phosphoserine phosphatase (PSPH) regulates cutaneous squamous cell carcinoma proliferation independent of L-serine biosynthesis.

BACKGROUND: L-3-Phosphoserine phosphatase (PSPH) is a highly conserved and widely expressed member of the haloacid dehalogenase superfamily and the rate-limiting enzyme in l-serine biosynthesis. We previously found Psph expression to be uniquely upregulated in a α6ß4 integrin transgenic mouse model that is predisposed to epidermal hyperproliferation and squamous cell carcinoma (SCC) formation implicating a role for Psph in epidermal homeostasis. OBJECTIVE: We examined the status of PSPH in normal skin epidermis and skin tumors along with its sub-cellular localization in epidermal keratinocytes and its requirement for squamous cell carcinoma (SCC) proliferation. METHODS: First, an immunohistochemical study was performed for PSPH in normal skin and skin cancer specimens and in cultured keratinocytes. Next, biochemical analyses were performed to confirm localization of PSPH and to identify candidate binding proteins. Finally, proliferation and apoptosis studies were performed in human SCC and normal keratinocytes, respectively, transduced with vectors encoding small hairpin RNAs targeting PSPH or overexpressing a phosphatase-deficient PSPH mutant. RESULTS: PSPH is expressed throughout the proliferative layer of the epidermis and hair follicles in rodent and human skin and is highly induced in SCC. In keratinocytes, PSPH is a cytoplasmic protein that primarily localizes to endosomes and is present primarily as a homodimer. Knock down of PSPH dramatically diminished SCC cell proliferation and cyclin D1 levels in the presence of exogenous of l-serine production suggesting a non-canonical role for PSPH in epithelial carcinogenesis. CONCLUSIONS: Psph is highly induced in proliferative normal keratinocytes and in skin tumors. PSPH appears to be critical for the proliferation of SCC cells; however, this phenomenon may not involve the phosphoserine metabolic pathway.

mda-7/IL-24: multifunctional cancer-specific apoptosis-inducing cytokine.

"Differentiation therapy" provides a unique and potentially effective, less toxic treatment paradigm for cancer. Moreover, combining "differentiation therapy" with molecular approaches presents an unparalleled opportunity to identify and clone genes mediating cancer growth control, differentiation, senescence, and programmed cell death (apoptosis). Subtraction hybridization applied to human melanoma cells induced to terminally differentiate by treatment with fibroblast interferon (IFN-beta) plus mezerein (MEZ) permitted cloning of melanoma differentiation associated (mda) genes. Founded on its novel properties, one particular mda gene, mda-7, now classified as a member of the interleukin (IL)-10 gene family (IL-24) because of conserved structure, chromosomal location, and cytokine-like properties has become the focus of attention of multiple laboratories. When administered by transfection or adenovirus-transduction into a spectrum of tumor cell types, melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) induces apoptosis, whereas no toxicity is apparent in normal cells. mda-7/IL-24 displays potent "bystander antitumor" activity and also has the capacity to enhance radiation lethality, to induce immune-regulatory activities, and to inhibit tumor angiogenesis. Based on these remarkable attributes and effective antitumor therapy in animal models, this cytokine has taken the important step of entering the clinic. In a Phase I clinical trial, intratumoral injections of adenovirus-administered mda-7/IL-24 (Ad.mda-7) was safe, elicited tumor-regulatory and immune-activating processes, and provided clinically significant activity. This review highlights our current understanding of the diverse activities and properties of this novel cytokine, with potential to become a prominent gene therapy for cancer.

Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase decreases UVB-induced activator protein-1 and cyclooxygenase-2 in a SKH-1 hairless mouse model.

Activation of activator protein-1 (AP-1) and increased expression of cyclooxygenase-2 (COX-2) have been clearly shown to play a functional role in UVB-induced skin tumor promotion. In this study, we examined UVB-induced signal transduction pathways in SKH-1 mouse epidermis leading to increases in COX-2 expression and AP-1 activity. We observed rapid increases in p38 mitogen-activated protein kinase (MAPK) signaling through activation of p38 MAPK and its downstream target, MAPK activated protein kinase-2. UVB also increased phosphatidylinositol 3-kinase (PI3K) signaling as observed through increases in AKT and GSK-3beta phosphorylation. Activation of the p38 MAPK and PI3K pathways results in the phosphorylation of cyclic AMP-responsive element binding protein, which was also observed in UVB-irradiated SKH-1 mice. Topical treatment with SB202190 (a specific inhibitor of p38 MAPK) or LY294002 (a specific inhibitor of PI3K) significantly decreased UVB-induced AP-1 activation by 84% and 68%, respectively, as well as COX-2 expression. Our data show that in mouse epidermis, UVB activation of the p38 MAPK and PI3K pathways leads to AP-1 activation and COX-2 expression.

Ultraviolet A-induced modulation of Bcl-XL by p38 MAPK in human keratinocytes: post-transcriptional regulation through the 3'-untranslated region.

We examined the effect of inhibiting p38 MAPK on UVA-irradiated HaCaT cells, a spontaneously immortalized human keratinocyte cell line. Recent work from our laboratory has shown that UVA (250 kJ/m2) induces a rapid phosphorylation of p38 MAPK in the HaCaT cell line. Inhibition of p38 MAPK activity through the use of a specific inhibitor, SB202190, in combination with UVA treatment induced a rapid cleavage of caspase-9, caspase-8, and caspase-3, whereas UVA irradiation alone had no effect. Similarly, cleavage of the caspase substrate poly(ADP-ribose) polymerase was observed in UVA-irradiated HaCaT cells treated with SB202190 or in cells expressing a dominant-negative p38 MAPK. No effect of p38 MAPK inhibition upon caspase cleavage was observed in mock-irradiated HaCaT cells. In addition, increases in apoptosis were observed in UVA-irradiated cells treated with SB202190 by morphological analysis with no significant apoptosis occurring from UVA irradiation alone. Similar results were obtained by using normal human epidermal keratinocytes. UVA induced expression of the anti-apoptotic Bcl-2 family member, Bcl-XL, with abrogation of expression by using the p38 MAPK inhibitor SB202190. Overexpression of Bcl-XL prevented poly(ADP-ribose) polymerase cleavage induced by the combination of UVA and p38 MAPK inhibition. UVA enhanced the stability of Bcl-XL mRNA through increases in p38 MAPK activity. We determined that increases in UVA-induced expression of Bcl-XL occur through a posttranscriptional mechanism mediated by the 3'-untranslated region (UTR). We used Bcl-XL 3'-UTR luciferase constructs to determine the mechanism by which UVA increased Bcl-XL mRNA stability. Additionally, RNA binding studies indicate that UVA increases the binding of RNA-binding proteins to Bcl-XL 3'-UTR mRNA, which can be decreased by using SB202190. In conclusion, p38 MAPK and Bcl-XL expression play critical roles in the survival of UVA-irradiated HaCaT cells.

UVA-mediated activation of signaling pathways involved in skin tumor promotion and progression.

Each year more than 1,000,000 cases of non-melanoma skin cancer (NMSC) are diagnosed in the Unites States. Solar radiation has been described as an important etiological factor in the development of NMSC. UVA comprises the largest portion of solar radiation reaching the surface of the earth (90-99%) and has been described to lead to benign tumor formation as well as malignant cancers, squamous cell carcinomas (SCCs). While much research has focused upon the effects of UVB radiation, little is known about UVA-induced signaling pathways and their role in tumor promotion. Here we focus on UVA-mediated activation of mitogen-activated protein kinase (MAPK) pathways and their role in activator protein-1 (AP-1) mediated transcription and cyclooxygenase-2 (COX-2) expression. AP-1 and COX-2 have been found to play a role in angiogenesis in other tissues. We propose UVA-mediated increases in AP-1 and COX-2 may play a role in tumor promotion through increases in interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF). Since MAPKs, specifically p38 and JNK, appear to play a major role in the expression of UVA-induced AP-1 and COX-2, pharmacological inhibitors may be of benefit in the chemoprevention of non-melanoma skin cancer.

The role of JNK and p38 MAPK activities in UVA-induced signaling pathways leading to AP-1 activation and c-Fos expression.

To further delineate ultraviolet A (UVA) signaling pathways in the human keratinocyte cell line HaCaT, we examined the potential role of mitogen-activated protein kinases (MAPKs) in UVA-induced activator protein-1 (AP-1) transactivation and c-Fos expression. UVA-induced phosphorylation of p38 and c-Jun N-terminal kinase (JNK) proteins was detected immediately after irradiation and disappeared after approximately 2 hours. Conversely, phosphorylation of extracellular signal-regulated kinase was significantly inhibited for up to 1 hour post-UVA irradiation. To examine the role of p38 and JNK MAPKs in UVA-induced AP-1 and c-fos transactivations, the selective pharmacologic MAPK inhibitors, SB202190 (p38 inhibitor) and SP600125 (JNK inhibitor), were used to independently treat stably transfected HaCaT cells in luciferase reporter assays. Both SB202190 and SP600125 dose-dependently inhibited UVA-induced AP-1 and c-fos transactivations. SB202190 (0.25-0.5 microM) and SP600125 (62-125 nM) treatments also primarily inhibited UVA-induced c-Fos expression. These results demonstrated that activation of both JNK and p38 play critical role in UVA-mediated AP-1 transactivation and c-Fos expression in these human keratinocyte cells. Targeted inhibition of these MAPKs with their selective pharmacologic inhibitors may be effective chemopreventive strategies for UVA-induced nonmelanoma skin cancer.

The role of p38 in UVA-induced cyclooxygenase-2 expression in the human keratinocyte cell line, HaCaT.

We examined the expression of cycloxygenase-2, the rate-limiting enzyme in the production of prostaglandins, in the UVA-irradiated human keratinocyte cell line, HaCaT. UVA induced a dose-dependent increase in COX-2 at the protein level at 2 and 4 h post-irradiation and at the mRNA level at 1 and 2 h post-irradiation. Experiments using semi-quantitative RT-PCR demonstrate that UVA increased the half-life of the COX-2 message by more than fourfold in the presence of Actinomycin D (with a half life between 4 and 8 h post-irradiation), suggesting that UVA induction of COX-2 is post-transcriptionally regulated. Through the use of the specific p38 inhibitor, SB202190, increases in COX-2 message and protein levels were abrogated in UVA-irradiated cells. In UVA-irradiated cells treated with SB202190, the half-life of the COX-2 message was decreased to basal levels (between 1 and 2 h post-irradiation), indicating that p38 was responsible for the stabilization of the message. Luciferase activity was increased in UVA-irradiated cells transfected with reporter constructs containing the 3' UTR of COX-2, a region containing AU-rich elements (AREs). These regulatory sequences of AUUUA have been proposed as one mechanism of post-transcriptional regulation. Increases observed in luciferase activity could be decreased using a p38 dominant-negative construct. We report for the first that UVA can induce COX-2 expression in the human keratinocyte cell line, HaCaT. Additionally, p38 appears to play a critical role in the UVA-induced expression of COX-2 in these keratinocytes and may serve as a potential drug target in the chemoprevention of skin cancer.