BACKGROUND:Although a role for ultraviolet radiation (UVR) in cutaneous malignant melanoma (CMM) development is accepted, there is debate over the magnitude and mechanisms given its association with intermittent but not chronic exposure. OBJECTIVES:To assess new ideas and data on the subject, review some debated topics, bringing a molecular view to epidemiological observations. METHODS:We reviewed some recent advances in the field of epidemiology and genetics, including phenome-wide association studies, evolutionary genetics related to skin cancer, and mechanisms of UVR-induced DNA adduct formation. RESULTS:High rates of CMM are strongly correlated with light colored skin across the globe. CMM shares risk factors associated with UVR sensitivity with keratinocyte cancer (KC). CMM risk is dominated by MC1R, a gene regulating the proportions of black and red melanin produced. An emerging mutagenic mechanism involves reactive melanin, particularly red pheomelanin, that can itself induce DNA adducts. CONCLUSION:Demographically, epidemiologically, and mechanistically, pigmentation status is central to CMM risk and a shared genetic susceptibility, comprising several pigmentation genes, between CMM and KCs. In the general population, CMM risk is associated with pale skin and poor tanning ability, mechanistically due to a relative lack of protection against UVR adduct formation, or perhaps via an alternate manner in individuals with abundant pheomelanin. Overall, evidence suggests that UVR exposure impacts CMM risk.

Supplementary Figures 1-2, Table 1 from Spontaneous and UV Radiation–Induced Multiple Metastatic Melanomas in <i>Cdk4^R24C/R24C/TPras</i> Mice

Mast cells contain chemicals needed for the body’s response to extrinsic challenges in various tissues, including the skin. Little is known about the genetic control of mast cell numbers in unchallenged skin. Overabundance of mast cells in mastocytosis, resulting from their excess production in the bone marrow, is characterized by somatic KIT alteration, although such variants are not always found in cutaneous mastocytosis. It appears that many germline variants can predispose to this condition, including those near OTX2, ABCA2, CYP2B5, RPTN (Nedoszytko et al., 2020), CEBPA, LICO1412, and TBLXR1 (Galatà et al., 2021).

Animal microbiota have complex interactions with hosts and environment that determines its composition. Yet the ability of hosts to determine their microbiota composition is less well studied. In this study, to investigate the role host genetics in determining skin microbiota, we used 30 different mouse strains from the recombinant inbred panel, the Collaborative Cross. Murine skin microbiota composition was strongly dependent on murine strain with > 50% of the variation explained by murine strain. In particular, a quantitative trait locus on chromosome 4 associates both with Staphylococcus abundance and principal-component multi-trait analyses. Additionally, excisional wound associated changes in microbiota composition were not uniform across mouse strains and were host-specific, the genetic background accounting for about 40% of the variation in microbiota. Genetic background also had the highest effect on the healing speed of wounds accounting for over 50% of the variation while mouse age and microbiota composition change accounted only for 20% and 5% of the healing speed despite reaching statistical significance. In conclusion, host genetics has a significant impact on the skin microbiota composition during both homeostasis and wound healing. These findings have long reaching implications in our understanding of associations between microbiota dysbiosis and disease.### Competing Interest StatementThe authors have declared no competing interest.

Animal microbiota are shaped and maintained not only through microbiota-environmental interactions but also through host-microbiota interactions. The effects of the microbiota on the host has been the source of intense research in recent years, indicating a role for resident microbes in a range of conditions from obesity and mood disorders to atopic dermatitis and chronic wounds. Yet the ability of hosts to determine their microbiota composition is less well studied. In this study, we investigated the role host genetics plays in determining skin microbiota. We used 30 different mouse strains from the advanced recombinant inbred mouse panel, the Collaborative Cross, with PERMANOVA, GWAS and PCA-based GWAS analyses to demonstrate that murine skin microbiota composition is strongly dependent on murine strain. In particular, a quantitative trait locus on chromosome 4 associates both with Staphylococcus abundance and principal-component multi-trait analyses. Additionally, we used a full thickness excisional wound healing model to investigate the relative contributions from the skin microbiota and/or host genetics on wound healing speed. Wound associated changes in skin microbiota composition were observed and were in many instances host-specific. Despite reaching statistical significance, the wound-associated changes in skin microbiota accounted for only a small amount of the variance in wound healing speeds, with the majority attributable to mouse genotype (strain) and age. Host genetics has a significant impact on the skin microbiota composition during both homeostasis and wound healing. These findings have long reaching implications in our understanding of associations between microbiota dysbiosis and disease.

Assisted vaginal birth can be lifesaving but also has implications for both mother and baby.This presentation will look at the current evidence available to maximising outcomes for maternal and neonatal considerations but also those skills required for the next generation of obstetricians

Maintenance of genomic stability is critical to prevent diseases such as cancer. As such, eukaryotic cells have multiple pathways to efficiently detect, signal and repair DNA damage. One common form of exogenous DNA damage comes from ultraviolet B (UVB) radiation. UVB generates cyclobutane pyrimidine dimers (CPD) that must be rapidly detected and repaired to maintain the genetic code. The nucleotide excision repair (NER) pathway is the main repair system for this type of DNA damage. Here, we determined the role of the human Single-Stranded DNA Binding protein 2, hSSB2, in the response to UVB exposure. We demonstrate that hSSB2 levels increase in vitro and in vivo after UVB irradiation and that hSSB2 rapidly binds to chromatin. Depletion of hSSB2 results in significantly decreased Replication Protein A (RPA32) phosphorylation and impaired RPA32 localisation to the site of UV-induced DNA damage. Delayed recruitment of NER protein Xeroderma Pigmentosum group C (XPC) was also observed, leading to increased cellular sensitivity to UVB. Finally, hSSB2 was shown to have affinity for single-strand DNA containing a single CPD and for duplex DNA with a two-base mismatch mimicking a CPD moiety. Altogether our data demonstrate that hSSB2 is involved in the cellular response to UV exposure.

OBJECTIVE:The aim of the study was to obtain an in-depth understanding of the experience of women who received non-surgical treatment for endometrial adenocarcinoma (EAC) or endometrial hyperplasia with atypia (EHA). Enhanced understanding of women's experiences of non-surgical treatment is essential to inform counselling of the growing number of patients in this field. METHODS:Individual semi-structured interviews were conducted with 21 women who received conservative (non-surgical hormonal) treatment for early stage EAC or EHA using the levonorgestrel intrauterine device (LNG-IUD) as part of the feMMe trial (NCT01686126). All interviews were audiotaped and transcribed verbatim prior to content analysis. RESULTS:Of the 21 women interviewed, ten received conservative treatment for early stage EAC and 11 received conservative treatment for EHA. Five overarching themes were identified: i) extensive information and support needs (e.g. understanding of how the LNG-IUD treatment worked); ii) gratitude for treatment choice and non-surgical options (e.g. avoidance of potential risks associated with surgery); iii) onco-fertility (e.g. desire to maintain reproductive potential); iv) patient experience of overweight and obesity related to EAC development (e.g. history of trauma and disordered eating, multiple unsuccessful weight loss attempts); and v) patient experience of treatment options and actual non-surgical treatment (e.g. desire for early referral to counselling services). CONCLUSIONS:This qualitative investigation enabled novel insights into the treatment preferences and decision-making process of women with newly diagnosed EHA and EAC when offered non-surgical treatment options. These insights facilitate the development of pragmatic guidance and decision support tools that could be tested in future clinical trials.

To date little research has investigated the genetic determinants of cutaneous microbiota composition and how microbiota composition can effect acute wound healing. Here we used 114 mice totalling 30 different mouse strains from an advanced cross-breeding program, The Collaborative Cross, and performed large, 1.5x1.5cm, full excisional wounds of mouse dorsal skin. 16sRNA sequencing immediately before wounding and at days 3 and 10 post-wounding show microbiota compositional changes are largely strain dependent with different mouse strains showing different changes in microbiota composition as a result of wounding. Principle component regression of centred-log ratio abundances and wound healing speed, time-to-wound closure, show an adjusted R-squared <5%. PERMANOVA analysis suggests <4% of variance in microbiota compositional changes during healing are explained by wounding alone with >40% variance explained by mouse strain and mouse strain specific responses to wounding. Murine microbiota composition is largely mouse strain dependent with microbiota changes during wound healing largely determined by mouse strain specific responses.

Mouse dorsal coat hair types, guard, awl, auchene and zigzag, develop in three consecutive waves. To date, it is unclear if these hair types are determined genetically through expression of specific factors or can change based on their mesenchymal environment. We undertook a novel approach to this question by studying individual hair type in 67 Collaborative Cross (CC) mouse lines and found significant variation in the proportion of each type between strains. Variation in the proportion of zigzag, awl and auchene, but not guard hair, was largely due to germline genetic variation. We utilised this variation to map a quantitative trait locus (QTL) on chromosome 12 that appears to influence a decision point switch controlling the propensity for either second (awl and auchene) or third wave (zigzag) hairs to develop. This locus contains two strong candidates, Sostdc1 and Twist1, each of which carry several ENCODE regulatory variants, specific to the causal allele, that can influence gene expression, are expressed in the developing hair follicle, and have been previously reported to be involved in regulating human and murine hair behaviour, but not hair subtype determination. Both of these genes are likely to play a part in hair type determination via regulation of BMP and/or WNT signalling.

Malignant melanoma is the most aggressive skin cancer. A majority of cancer-associated deaths are due to the invasion of cancer cells to distant tissues. Thus, it is of great importance to understand the mechanisms of metastatic spread. A potential marker for this is BRN2, encoded by the POU3F2 gene, that interacts with SOX transcription factors (Cook and Sturm, 2008Cook A.L. Sturm R.A. POU domain transcription factors: BRN2 as a regulator of melanocytic growth and tumourigenesis.Pigment Cell Melanoma Res. 2008; 21: 611-626Crossref PubMed Scopus (50) Google Scholar, Malik et al., 2018Malik V. Zimmer D. Jauch R. Diversity among POU transcription factors in chromatin recognition and cell fate reprogramming.Cell Mol Life Sci. 2018; 75: 1587-1612Crossref PubMed Scopus (30) Google Scholar). While some studies consider BRN2 as a positive regulator of microphthalmia-associated transcription factor (MITF), which favors proliferation (Goodall et al., 2004aGoodall J. Martinozzi S. Dexter T.J. Champeval D. Carreira S. Larue L. et al.Brn-2 expression controls melanoma proliferation and is directly regulated by beta-catenin.Mol Cell Biol. 2004; 24: 2915-2922Crossref PubMed Scopus (100) Google Scholar, Wellbrock et al., 2008Wellbrock C. Rana S. Paterson H. Pickersgill H. Brummelkamp T. Marais R. Oncogenic BRAF regulates melanoma proliferation through the lineage specific factor MITF.PLOS ONE. 2008; 3e2734Crossref PubMed Scopus (198) Google Scholar), other studies propose a dual function for BRN2 in both tumor proliferation and invasion, which could be controlled via the upregulation or downregulation of MITF, respectively (Simmons et al., 2017Simmons J.L. Pierce C.J. Al-Ejeh F. Boyle G.M. MITF and BRN2 contribute to metastatic growth after dissemination of melanoma.Sci Rep. 2017; 7: 10909Crossref PubMed Scopus (27) Google Scholar, Wellbrock and Arozarena, 2015Wellbrock C. Arozarena I. Microphthalmia-associated transcription factor in melanoma development and MAP-kinase pathway targeted therapy.Pigment Cell Melanoma Res. 2015; 28: 390-406Crossref PubMed Scopus (133) Google Scholar). BRN2 and MITF are present in two distinct sub-populations of cells in 3-dimensional culture (Thurber et al., 2011Thurber A.E. Douglas G. Sturm E.C. Zabierowski S.E. Smit D.J. Ramakrishnan S.N. et al.Inverse expression states of the BRN2 and MITF transcription factors in melanoma spheres and tumour xenografts regulate the NOTCH pathway.Oncogene. 2011; 30: 3036-3048Crossref PubMed Scopus (66) Google Scholar) and melanoma patient biopsies (Goodall et al., 2008Goodall J. Carreira S. Denat L. Kobi D. Davidson I. Nuciforo P. et al.Brn-2 represses microphthalmia-associated transcription factor expression and marks a distinct subpopulation of microphthalmia-associated transcription factor-negative melanoma cells.Cancer Res. 2008; 68: 7788-7794Crossref PubMed Scopus (134) Google Scholar, Pinner et al., 2009Pinner S. Jordan P. Sharrock K. Bazley L. Collinson L. Marais R. et al.Intravital imaging reveals transient changes in pigment production and Brn2 expression during metastatic melanoma dissemination.Cancer Res. 2009; 69: 7969-7977Crossref PubMed Scopus (151) Google Scholar) in which the expression of each transcription factor is mutually exclusive. The relationship between MITF and BRN2 has also been further characterized by showing a role for MITF in the reduction of BRN2 protein levels (Boyle et al., 2011Boyle G.M. Woods S.L. Bonazzi V.F. Stark M.S. Hacker E. Aoude L.G. et al.Melanoma cell invasiveness is regulated by miR-211 suppression of the BRN2 transcription factor.Pigment Cell Melanoma Res. 2011; 24: 525-537Crossref PubMed Scopus (128) Google Scholar). However, recent single cell gene expression efforts on human primary melanoma cells suggests that the situation may not always be "black and white" with some melanoma cell-expressing genes associated with both high and low levels of BRN2-associated genes in vivo (Ennen et al., 2017Ennen M. Keime C. Gambi G. Kieny A. Coassolo S. Thibault-Carpentier C. et al.MITF-high and MITF-low cells and a novel subpopulation expressing genes of both cell states contribute to intra- and intertumoral heterogeneity of primary melanoma.Clin Cancer Res. 2017; 23: 7097-7107Crossref PubMed Scopus (34) Google Scholar). Here we have assessed the expression levels of BRN2 in various types of melanocytic lesions using extremely sensitive immunohistochemistry with Tyramide signal amplification. We approached this by looking for melanocytic cells positive for both SOX10 and BRN2 (SOX10+/BRN2+). While BRN2 is not detectable in normal melanocytes, strong expression of the nuclear BRN2 protein was seen in dermal nevi and congenital dermal nevi (Figure 1a). In epidermal melanocytic lesions, we observed much less BRN2 positivity (Figure 1a and Supplementary Figure S1). The proportion of SOX10+/BRN2+ cells was quantified and stratified based on their location in skin using InForm Cell Analysis software (Perkin Elmer, Waltham, MA). The results demonstrate that most dermal nevus cells, particularly in dermal congenital nevi, express BRN2. In epidermal lesions, the proportion of BRN2-positive cells is much lower than that in the dermal lesions, although it increases during progression (Figure 1b). Few or no BRN2-positive cells were observed in benign epidermal nevi, with slightly higher levels in superficial spreading melanomas and invasive epidermal malignant melanomas, although not approaching the very high levels seen in dermal congenital nevi (Supplementary Figures S1 and S2). A schematic description of the BRN2 positivity of melanocytic lesions is presented in Figure 1c. Our findings are in agreement with recent studies in which benign epidermal nevi were found to not express significant BRN2 protein, while the expression increases during disease progression (Zeng et al., 2018Zeng H. Jorapur A. Shain A.H. Lang U.E. Torres R. Zhang Y. et al.Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation.Cancer Cell. 2018; 34: 56-68.e9Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Hence, we set out to further investigate the control of BRN2 expression in the congenital dermal nevi. Previous studies in melanoma have found that activation of the mitogen-activated protein kinase pathway in melanoma, in particular, the mutation of BRAF (Goodall et al., 2004bGoodall J. Wellbrock C. Dexter T.J. Roberts K. Marais R. Goding C.R. The Brn-2 transcription factor links activated BRAF to melanoma proliferation.Mol Cell Biol. 2004; 24: 2923-2931Crossref PubMed Scopus (101) Google Scholar), nuclear accumulation of β-catenin (Goodall et al., 2004aGoodall J. Martinozzi S. Dexter T.J. Champeval D. Carreira S. Larue L. et al.Brn-2 expression controls melanoma proliferation and is directly regulated by beta-catenin.Mol Cell Biol. 2004; 24: 2915-2922Crossref PubMed Scopus (100) Google Scholar), PI3K activation (Bonvin et al., 2012Bonvin E. Falletta P. Shaw H. Delmas V. Goding C.R. A phosphatidylinositol 3-kinase-Pax3 axis regulates Brn-2 expression in melanoma.Mol Cell Biol. 2012; 32: 4674-4683Crossref PubMed Scopus (34) Google Scholar), or E2F1 (Zeng et al., 2018Zeng H. Jorapur A. Shain A.H. Lang U.E. Torres R. Zhang Y. et al.Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation.Cancer Cell. 2018; 34: 56-68.e9Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) activate the promoter of BRN2. BRN2 has recently been shown to suppress apoptosis and reprogram DNA repair, suggesting that BRN2 contributes to the generation of melanoma with a high somatic mutation burden (Herbert et al., 2019Herbert K. Binet R. Lambert J.P. Louphrasitthiphol P. Kalkavan H. Sesma-Sanz L. et al.BRN2 suppresses apoptosis, reprograms DNA damage repair, and is associated with a high somatic mutation burden in melanoma.Genes Dev. 2019; 33: 310-332Crossref PubMed Scopus (15) Google Scholar). We therefore examined the expression of BRN2 in relation to the activation of either the mitogen-activated protein kinase (BRAF V600E mutation and extracellular signal–regulated kinase 1/2 phosphorylation) or PI3K (PTEN) pathway in 10 congenital dermal nevi (Figure 2 and Supplementary Table S1). All the congenital dermal nevi expressed high levels of PTEN, therefore suggesting that the PI3K/protein kinase B pathway is not activated in these lesions. In addition, five of the 10 were positive for the BRAF V600E mutation as assessed using a specific antibody for staining. Interestingly, BRAF V600E staining did not correlate with BRN2 expression or phosphorylated extracellular signal–regulated kinase 1/2 staining (Supplementary Table S1). This data may suggest a different form of control of BRN2 expression in congenital dermal nevi compared with cutaneous melanoma cells. While we observed strong BRN2 staining in congenital dermal nevi, there was also some weaker BRN2 staining in the invasive epidermal malignant melanomas. To investigate whether BRN2 expression is associated with melanoma survival in a large cohort of melanoma patients, we analyzed the testicular germ cell tumor skin melanoma cohort, which restricts us to assessing the POU3F2 RNA but not the protein levels. Across this cohort, POU3F2 levels just reach the level of significance (P = 0.048) (Supplementary Figure S3) for correlation with overall survival but not for disease-free survival (P = 0.18), suggesting a somewhat complex relationship between the two variables. Notwithstanding, it is possible that the propensity of certain tumor cells to metastasize may not depend on the overall level of gene expression but rather may be dependent on the presence of just a few positive cells that may initiate metastatic spread. In conclusion, the levels of BRN2 expression in melanocytic cells throughout melanoma progression in vivo is becoming clearer. It does not appear to be involved in the day-to-day function of normal melanocytes. In primary epidermal lesions, the proportion of melanocytic cells positive for BRN2 increases only mildly during progression. Strikingly, the expression of BRN2 protein is overwhelmingly the highest in dermal congenital nevi, and to a lesser extent, in non-congenital benign dermal nevi, suggesting that high BRN2 expression may be in some way associated with the localization of melanocytic cells in the dermal component, away from the familiar cues from associated keratinocytes. However, this begs the question as to why these lesions carry such high levels of BRN2 expression, because only very large giant congenital nevi (which we did not study) are at increased risk for transformation. BRN2 may possibly function in non-metastatic melanocytic cells to some degree regulating the expression of "neural" type genes, as congenital lesions probably emanate from the neural crest-derived melanocytes trapped in the dermis during development. It was previously shown that such lesions harbor a more neural gene expression profile than other epidermal melanocytic lesions (Hamid et al., 1989Hamid Q.A. Bishop A.E. Springall D.R. Adams C. Polak J.M. Quaba A.A. et al.Neuron-specific enolase and its mRNA are highly expressed in large congenital nevi: a study using immunocytochemistry, biochemical assay, and in situ hybridization.J Mol Neurosci. 1989; 1: 85-91Crossref PubMed Scopus (2) Google Scholar). Not applicable. Experiments were undertaken with the University of Queensland Human Research Ethics Committee A & B approval (approval number 2011001201/HREC/11/QPAH/363). Patients gave written, informed consent to participate. Arash Chitsazan: https://orcid.org/0000-0002-8449-8154 Blake Ferguson: https://orcid.org/0000-0002-5643-6976 Duncan Lambie: https://orcid.org/0000-0002-0102-6638 Herlina Y. Handoko: https://orcid.org/0000-0003-3153-2248 Brian Gabrielli: https://orcid.org/0000-0003-3933-1651 Graeme J. Walker: https://orcid.org/0000-0002-9392-8769 Glen M Boyle: https://orcid.org/0000-0002-1385-529X. The authors state no conflict of interest. This work was supported by the Melanoma Research Alliance , Washington, DC, USA, and the National Health and Medical Research Council (NHMRC) of Australia. Conceptualization: AC, GW, GB; Formal Analysis: AC, GW, DL; Funding Acquisition: GW, GB; Investigation: AC, BF, HYH; Project Administration: GW, GB; Resources: DL, BG; Supervision: GW, GB; Writing: AC, GW, GB. De-identified patient samples were subjected to a histopathological diagnosis by an experienced pathologist (DL). Experiments were undertaken with the University of Queensland Human Research Ethics Committee A & B approval (approval number 2011001201/HREC/11/QPAH/363). Patients gave written, informed consent to participate. Sections from paraffin-embedded lesions were dewaxed and treated with Dako low pH antigen retrieval solution at 100 °C for 15 minutes (Carpinteria, CA). Endogenous peroxidase activity was quenched in 3% hydrogen peroxide and sections blocked with 5% bovine serum albumin. Primary BRN2 (D2C1L Rabbit mAb #12137, Cell Signaling Technology, Danvers, MA) and Sox10 (sc-17342, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were applied, followed by the appropriate secondary antibodies. After washing, the signal was amplified using a Perkin Elmer Tyramide amplification kit (FITC and CY5, respectively). The slides were washed, counterstained with DAPI, and coverslipped using Dako mounting medium. Slides were scanned using the Vectra spectral imaging system (PerkinElmer, Waltham, MA). Images were scanned at ×4 magnification. Multispectral images were captured using Phenochart software and scanned from 420 nm to 720 nm using Vectra 3.1 Automated quantitative pathology imaging system. Three multispectral images per sample were analyzed using InForm analysis software. Positive cells were segmented and counted per ×20 magnification field. Sections (5 μm) were incubated with primary antibodies (BRAF V600E was detected using the Ventana system; BRN2 - D2C1L Rabbit mAb #12137, Cell Signaling; PTEN – 138G6 Rabbit mAb #9559, Cell Signaling; phosphorylated extracellular signal–regulated kinase 1/2 – 20G11 Rabbit mAb #4376, Cell Signaling) overnight at 4 °C before incubation with the appropriate biotinylated secondary antibodies and before incubation with the streptavidin horseradish peroxidase conjugate for 20 minutes (1:600 dilution in phosphate-buffered saline; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and subsequently probed with the chromagen substrate 3-amino-9-ethylcarbazole for 5 minutes (DakoCytomation, Glostrup, Denmark). All the slides were counterstained with routine hematoxylin. To calculate the melanoma survival measures associated with gene expression, we used the Gene Expression Profiling Interactive Analysis database (http://gepia.cancer-pku.cn).Supplementary Figure S2BRN2 staining in nevi and melanomas. Comparison of three tumors of each subtypes. Images of 4-plex fluorescent immunohistochemistry with Tyramide signal amplification staining of BRN2 in (a) benign dermal and congenital nevi, (b) epidermal lesions from benign nevi to invasive MM. Red arrows point to the major location of tumor within either the epidermis or dermis. Yellow Bars = 600 μm; Green Bars = 400 μm; Red Bars = 200 μm. MM, malignant melanoma.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S3POU3F2 RNA expression and melanoma survival across the SKCM cohort. (a) Significant association between high or low levels of POU3F2 gene expression and overall survival in SKCM patients across the testicular germ cell tumors cohort (P = 0.048). (b) Association between high or low levels of POU3F2 and disease-free survival. Dotted lines indicate the 95% confidence intervals. HR, Cox PH hazard ratio; SKCM, skin cutaneous melanoma; TPM, transcripts per million.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Table S1Immunohistochemical Staining of Human Congenital Dermal NeviSampleBRN2PTENBRAF (V600E)ppERK1/2Score (1–3)CytoplasmicNuclearScore (1–3)CytoplasmicNuclearScore (1–3)CytoplasmicNuclearScore (1–3)CytoplasmicNuclear12050%2260%00000023060%2210%33000030003310%00000042020%2230%1103110%53080%3350%000215%6101%3310%3300007000335%33000083060%2250%33000091010%3280%0002030%10201%3270%3302120%Abbreviation: ERK, extracellular signal–regulated kinase. Open table in a new tab Abbreviation: ERK, extracellular signal–regulated kinase.

Skin and wound microbiome have been identified as key modulators of health and disease. Although the general composition of this microbiome follows large trends there are large inter-individual variation. Wound healing is a complex multistep process that involves an inflammatory phase that conditions many of the subsequent stages of healing. Skin barrier rupture results in exposure to bacterial compounds suggesting a key role of the wound microbiome in the healing process. We here aimed to systematically study the skin bacterial composition in normal and wounded animals in 32 different strains of mice from the collaborative cross. The healing speed of excisional wounds varied significantly across strains (P<0.0001) allowing their separation into fast and slow groups. 16S sequencing was performed on faeces and skin swabs prior to wounding and at D3 and D10 post-wounding in 115 mice from all strains (n=3-5 per strain). Although all collaborative cross strains originated from 8 common founders and although they were maintained in the same animal facility, we observed significant changes in alpha diversity between strains (p=0.00024, ANOVA) and at different time points (p=0.00096) during healing. Strain and therefore genetic background was the most important factor determining bacterial composition. Genome wide association study of abundance of individual commensal bacteria at genus level identified loci strongly predictive of high abudance. Within these loci, genes regulating the activity of NLRP3 or other members of the innate immunity carried significant polymorphisms. In conclusions, skin and wound microbial composition affects the healing of wounds across multiple murine strains. However this should be considered in the context of the background genetic of murine strains.

Genetic variation conferring resistance and susceptibility to carcinogen-induced tumorigenesis is frequently studied in mice. We have now turned this idea to melanoma using the collaborative cross (CC), a resource of mouse strains designed to discover genes for complex diseases. We studied melanoma-prone transgenic progeny across seventy CC genetic backgrounds. We mapped a strong quantitative trait locus for rapid onset spontaneous melanoma onset to Prkdc, a gene involved in detection and repair of DNA damage. In contrast, rapid onset UVR-induced melanoma was linked to the ribosomal subunit gene Rrp15. Ribosome biogenesis was upregulated in skin shortly after UVR exposure. Mechanistically, variation in the 'usual suspects' by which UVR may exacerbate melanoma, defective DNA repair, melanocyte proliferation, or inflammatory cell infiltration, did not explain melanoma susceptibility or resistance across the CC. Instead, events occurring soon after exposure, such as dysregulation of ribosome function, which alters many aspects of cellular metabolism, may be important.

Melanocytes can group together in nevi, commonly thought to form because of intrinsic somatic mutations involving MAPK pathway activation. However, the role of the microenvironment, in particular keratinocytes, in nevogenesis is rarely studied. Melanocytes proliferate during the hair follicle growth phase and in some basal cell carcinomas, allowing us to construct keratinocyte gene expression clusters correlated with melanocyte activation. We asked whether such correlations are evident in the more subtle context of regulation of melanocyte behavior in normal skin. We considered genes which, when mutated in keratinocytes in mice, lead to nevogenesis. Across the human GTEx normal skin database, their expression was correlated with that of keratinocyte cytokines KITLG, HGF, FGF2, EDN1, and melanocyte markers. These cytokines have pleiotropic effects on melanocyte-specific and pigmentation genes and also influence mast cell gene expression. We show five classes of keratinocyte genes that, via germline genetic variation, influence melanocyte activity. These include genes involved in SHH signaling, structural keratins, ribosomal biogenesis, and stem cell governance. In agreement with the finding of KITLG linked to nevogenesis in human genome-wide association studies, we provide evidence that specific keratinocyte cytokines are components of networks that may drive or exacerbate nevus development.

Wound healing is a complex multistep process that involves an inflammatory phase that conditions many of the subsequent stages of healing. Skin barrier rupture results in exposure to bacterial compounds suggesting a key role of the wound microbiome in the healing process. We here aimed to systematically study the healing of skin excisional wounds in 72 different strains of mice from the collaborative cross to evaluate wound healing. In 30 strains we also evaluated associations between bacterial composition in normal and wounded skin at different time points and healing speed. The healing speed of excisional wounds varied significantly across the 72 strains (P<0.0001) allowing their separation into fast, slow or intermediate groups. 16S sequencing was performed on faeces and skin swabs prior to wounding and at D3 and D10 post-wounding in 150 mice from 30 strains. Although all collaborative cross strains originated from 8 common founders and although they were maintained in the same animal facility, we observed significant changes in alpha diversity between strains (p=0.00024, ANOVA) and at different time points (p=0.00096) during healing. Using multivariate partial least regression models integrating microbiome and wound healing data at various time points we further showed that skin microbial composition at D3 but not D10 tended to predict healing classification of strains. The gut microbiome composition at baseline allowed a better classification of the fastest healing strains. In conclusions, wound microbial composition affects the healing of wounds across multiple murine strains. However this should be considered in the context of the background genetic of murine strains.

Susceptibility to the development of allergic contact dermatitis (ACD) is variable amongst individuals and has been attributed to polymorphisms in genes associated with the immune response. Previous studies sought to determine the genetic factors underlying hapten sensitization, yet few have examined the subsequent stages of ACD resulting in dermatitis. In this study our aim was to determine the underlying genetic factors that contribute to the development of contact hypersensitivity (CHS), the murine model of ACD. 38 different strains of mice (n=5 per strain) from the Collaborative Cross (CC), a murine resource developed for investigation of complex genetic traits, were exposed in a reproducible manner to a strong sensitizer, oxazolone, and later challenged to determine intensity of the dermatitis at various time points and the recovery from the inflammation. The CHS response showed remarkable variation in both the initial inflammatory phase and in their subsequent recovery across the 38 strains. In genome wide association studies we identified major effect quantitative trait loci associated with these phenotypes and identified Bach2 and Gilz (Lod score of 10.1 and 15.4 respectively, pBach2 displayed reduced levels of regulatory T cells at baseline and higher proportions of CD8+IFNg+ T cells upon challenge. These candidates point to an essential role of the MAPK/ERK pathway in the intensity and the recovery of the dermatitis associated with CHS and open new possibilities for targeted therapeutic intervention.

Allergic contact dermatitis (ACD) is a T-cell–mediated delayed hypersensitivity reaction characterized by the occurrence of inflammatory skin lesions following contact with a particular allergen.1El Beidaq A. Link C.W. Hofmann K. Frehse B. Hartmann K. Bieber K. et al.In vivo expansion of endogenous regulatory T cell populations induces long-term suppression of contact hypersensitivity.J Immunol. 2016; 197: 1567-1576Crossref PubMed Scopus (12) Google Scholar Nonprotein allergens, or haptens, capable of eliciting contact dermatitis include a wide range of substances encountered within occupational or domestic settings.2Vocanson M. Hennino A. Cluzel-Tailhardat M. Saint-Mezard P. Benetiere J. Chavagnac C. et al.CD8+ T cells are effector cells of contact dermatitis to common skin allergens in mice.J Invest Dermatol. 2006; 126: 815-820Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar ACD has a reported median prevalence of approximately 20% across Europe and North America and is continuing to increase with limited therapeutic options addressing its burden.3Peiser M. Tralau T. Heidler J. Api A.M. Arts J.H. Basketter D.A. et al.Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects: current knowledge assembled at an international workshop at BfR, Germany.Cell Mol Life Sci. 2012; 69: 763-781Crossref PubMed Scopus (0) Google Scholar The development of ACD requires an initial sensitization event where cutaneous exposure to a hapten results in an innate immune response. The initial inflammatory response is suppressed by CD4+ regulatory T cells; however, upon a subsequent immune challenge by the same hapten, an inflammatory reaction mediated by CD8+ T cells results in the typical pathology of ACD.2Vocanson M. Hennino A. Cluzel-Tailhardat M. Saint-Mezard P. Benetiere J. Chavagnac C. et al.CD8+ T cells are effector cells of contact dermatitis to common skin allergens in mice.J Invest Dermatol. 2006; 126: 815-820Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Repeated exposure to haptens has been identified as an occupational risk that can lead to the development of contact dermatitis.3Peiser M. Tralau T. Heidler J. Api A.M. Arts J.H. Basketter D.A. et al.Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects: current knowledge assembled at an international workshop at BfR, Germany.Cell Mol Life Sci. 2012; 69: 763-781Crossref PubMed Scopus (0) Google Scholar However, it is clear that there is variation in individual susceptibility to develop clinically significant allergic reactions. More precisely, susceptibility to the development of ACD is related to the presence of genetic variations such as single nucleotide polymorphisms in genes relevant to this process.3Peiser M. Tralau T. Heidler J. Api A.M. Arts J.H. Basketter D.A. et al.Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects: current knowledge assembled at an international workshop at BfR, Germany.Cell Mol Life Sci. 2012; 69: 763-781Crossref PubMed Scopus (0) Google Scholar Our current knowledge regarding the impact of genetic polymorphisms on contact dermatitis is largely a result of multiple candidate gene–specific studies, with limited whole-genome expression analyses and genome-wide association study approaches.3Peiser M. Tralau T. Heidler J. Api A.M. Arts J.H. Basketter D.A. et al.Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects: current knowledge assembled at an international workshop at BfR, Germany.Cell Mol Life Sci. 2012; 69: 763-781Crossref PubMed Scopus (0) Google Scholar, 4Kim D.S. Kim D.H. Lee H. Jee H. Lee Y. Chang M.Y. et al.A genome-wide association study in Koreans identifies susceptibility loci for allergic nickel dermatitis.Int Arch Allergy Immunol. 2013; 162: 184-186Crossref PubMed Scopus (3) Google Scholar Importantly, most previous studies have addressed the genetic susceptibility in sensitization, but the ensuing development of dermatitis has seldom been interrogated. Determining the underlying genetics of the subsequent inflammatory response is of particular relevance, because this is directly responsible for patients' symptoms. Recently, the development of a large panel of recombinant inbred mouse lines known as the Collaborative Cross (CC) has provided a new model to study complex genetic traits. The genome of each CC strain has been comprehensively characterized and therefore we hypothesized that quantitative trait loci (QTL) can be determined for contact hypersensitivity (CHS) by investigating this particular trait across multiple strains.5Ram R. Mehta M. Balmer L. Gatti D.M. Morahan G. Rapid identification of major-effect genes using the collaborative cross.Genetics. 2014; 198: 75-86Crossref PubMed Scopus (14) Google Scholar In this study, we used the CC to investigate the genetic factors underlying CHS, the murine model of ACD in which hapten sensitization and subsequent immune challenge can be fully controlled and standardized.1El Beidaq A. Link C.W. Hofmann K. Frehse B. Hartmann K. Bieber K. et al.In vivo expansion of endogenous regulatory T cell populations induces long-term suppression of contact hypersensitivity.J Immunol. 2016; 197: 1567-1576Crossref PubMed Scopus (12) Google Scholar, 2Vocanson M. Hennino A. Cluzel-Tailhardat M. Saint-Mezard P. Benetiere J. Chavagnac C. et al.CD8+ T cells are effector cells of contact dermatitis to common skin allergens in mice.J Invest Dermatol. 2006; 126: 815-820Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar We assessed CHS in 38 strains of CC mice by measuring the increase in ear thickness at various time points after a single standardized oxazalone challenge (see this article's Methods section in the Online Repository at www.jacionline.org). At each time point, there were marked differences in response between CC strains in both the acute response to immune challenge and postchallenge recovery (see Results and Discussion sections and Fig E1 in this article's Online Repository at www.jacionline.org). To determine QTL that control the acute CHS response, we first performed genome-wide linkage analysis on the basis of increase in ear thickness at D1 postchallenge (Fig 1, A; see the Methods section in the Online Repository). We identified a major effect QTL on mouse chromosome 4 (−Log10(P) = 10.1) in the interval from 19.7 to 33.1 megabases (Mb) (Fig 1, B). Examination of the founder haplotype coefficients through the linked interval on chromosome 4 showed that the causal variant was derived from the 129SlvJ founder strain (hereafter termed 129S) (Fig 1, C and D). To determine candidate genes within this interval, DNA variants between susceptible and resistant strains were identified from the Sanger Mouse Genomes database.6Keane T.M. Goodstadt L. Danecek P. White M.A. Wong K. Yalcin B. et al.Mouse genomic variation and its effect on phenotypes and gene regulation.Nature. 2011; 477: 289-294Crossref PubMed Scopus (1058) Google Scholar No 129S-specific missense variants were identified in any gene within this locus; however, a search of the ENCODE database revealed 34 possible 129S-specific regulatory variants affecting 9 coding regions. Of these, Bach2 was the most plausible biological candidate associated with the observed phenotype (for further discussion, see Results and Discussion sections in the Online Repository). Indeed, we found this causal candidate to be carried by strains within which the most rapid ear-swelling responses were observed. When comparing the SAT strain carrying the 129S haplotype and mounting rapid and intense CHS at D1 to NOD that carried a different haplotype at this locus, we observed a tendency toward reduced regulatory T cells at baseline (P = .06) (Fig 1, G) and significantly increased IFN-γ–secreting CD8+ T cells in the former (Fig 1, H). The strains did not differ in total levels of CD4 or CD8 (Fig 1, I). This shows the importance of the CD8+ T-cell response in the phenotype. Of importance, the strains also differed in their macrophage but not neutrophil response, with SAT mice increasing massively the proportion of macrophages (see Fig E2 in this article's Online Repository at www.jacionline.org). We also mapped a second QTL to chromosome 15 (−Log10(P) = 7.8), in the interval between 32.2 and 55.4Mb, and determined the causal variant to be derived from the CAST founder strain (Fig 1, E and F). Within this interval we identified Tnfrsf11b, Ebag9, and Klf10 as candidate genes, with the CAST haplotype displaying unique noncoding variants of Klf10 and Ebag9 within this locus (for further discussion, see Results and Discussion sections in the Online Repository). We next analyzed the genetic control over the rate of recovery of ear swelling following immune challenge (Fig 2, A). Recovery from dermatitis is an important clinical phenomenon because in its absence ACD lesions tend to spread locally and systemically beyond the initial area of contact with the allergen. Remarkably, in several strains the inflammation following immune challenge was maintained over 7 days or worsened despite limiting the challenge to a single application of oxazolone (see Fig E1). We again calculated the genome-wide significance threshold and identified a bifurcated peak on chromosome X that was highly significant (−Log10(P) = 17.0) (Fig 2, B) with a 2-logarithm of the odds to the base 10 (LOD) drop between 137.2 and 141.8 Mb. Examination of the founder haplotype coefficients through the linked interval on chromosome X identified that the causal variant was derived from the CAST founder strain (Fig 2, C). There were only 7 genes with CAST-specific missense variants within this region, as well as 18 genes carrying regulatory variants in the ENCODE database (see Table E1 in this article's Online Repository at www.jacionline.org). Among these genes, we identified Gilz (also known as Tsc22d3) as a biological candidate associated with the recovery following immune challenge given its role in the response to glucocorticoids and IL-10 (Fig 2, D; see the Discussion section in the Online Repository). When considering recovery as an outcome, we observed several other loci with suggestive linkage, all of which returned LOD scores greater than 10 (Fig 2, B and D; see Table E1). These intervals include chromosome 1, 188.1 to 191.0 Mb; chromosome 14, 19.2 to 21.7 Mb; chromosome 15, 71.6 to 76.1 and 83.3 to 93.2 Mb; and chromosome 19, 50.1 to 53.9 Mb. When examining these intervals and the variants corresponding to the founder haplotype most strongly associated with the phenotype, we identified several biologically relevant candidates that may modify CHS recovery. These included Tgfb2, Kcnk5, Lrrk2, Ptk2, Cyp11b1, Shoc2, and Dusp5 (for further discussion, see Results and Discussion sections in the Online Repository). Together with the previously identified candidates Gilz and Bach2, many of these genes were identified as members of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway (Fig 2, E). Finally, we performed further genome-wide scans for linkage at each day after immune challenge up to D4, also comparing the linkage peaks detected here with those associated with nonrecovery at D7. This allowed the confirmation of previously identified loci that failed to reach statistical significance across multiple time points and identification of additional candidate genes (for further discussion, see Fig E2 and Results and Discussion sections in this article's Online Repository at www.jacionline.org). In this study, we have demonstrated that genetic variability underlies substantial differences in CHS responses and we have identified genetic loci strongly associated with the response to CHS immune challenge in the mouse. The most important associations were with the level of inflammation (acute response to immune challenge), and level of recovery (postchallenge recovery), which both revealed strong gene candidates explaining the observed phenotype. These were identified as Bach2 and Gilz, respectively, and are of significant interest because both are known modifiers of CHS and immune responses in general.7Vahedi G. Kanno Y. Furumoto Y. Jiang K. Parker S.C. Erdos M.R. et al.Super-enhancers delineate disease-associated regulatory nodes in T cells.Nature. 2015; 520: 558-562Crossref PubMed Scopus (118) Google Scholar, 8Beaulieu E. Morand E.F. Role of GILZ in immune regulation, glucocorticoid actions and rheumatoid arthritis.Nat Rev Rheumatol. 2011; 7: 340-348Crossref PubMed Scopus (66) Google Scholar Beyond these 2 genes, we have also identified a large number of additional loci and candidate genes possibly associated with the CHS response in multiple independent analyses over time. Altogether, these findings point to the possibility that the MAPK/ERK signaling pathway is a key regulator of the development and recovery of CHS. In conclusion, our data overwhelmingly support a strong genetic basis for the development of ACD. The intensity of the dermatitis as well as the recovery from the dermatitis were regulated by key loci where candidate genes regulate the MAPK/ERK pathway. These findings have far-reaching implications in the management of ACD and point toward new therapeutic targets. ACD is a T-cell–mediated delayed hypersensitivity reaction within the skin and has a high prevalence worldwide.E1Peiser M. Tralau T. Heidler J. Api A.M. Arts J.H. Basketter D.A. et al.Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects: current knowledge assembled at an international workshop at BfR.Germany. Cell Mol Life Sci. 2012; 69: 763-781Crossref PubMed Scopus (256) Google Scholar ACD occurs following hapten sensitization upon contact with the skin, with subsequent exposures resulting in inflammatory skin lesions, namely, dermatitis or eczema.E2Vocanson M. Hennino A. Cluzel-Tailhardat M. Saint-Mezard P. Benetiere J. Chavagnac C. et al.CD8+ T cells are effector cells of contact dermatitis to common skin allergens in mice.J Invest Dermatol. 2006; 126: 815-820Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Susceptibility to the development of ACD is clearly variable among individuals and is attributed to polymorphisms in genes associated with the immune response.E1Peiser M. Tralau T. Heidler J. Api A.M. Arts J.H. Basketter D.A. et al.Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects: current knowledge assembled at an international workshop at BfR.Germany. Cell Mol Life Sci. 2012; 69: 763-781Crossref PubMed Scopus (256) Google Scholar Previous studies have sought to determine the genetic factors underlying hapten sensitization, yet few have examined the genetic determinants driving the different stages of subsequent dermatitis in this context.E1Peiser M. Tralau T. Heidler J. Api A.M. Arts J.H. Basketter D.A. et al.Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects: current knowledge assembled at an international workshop at BfR.Germany. Cell Mol Life Sci. 2012; 69: 763-781Crossref PubMed Scopus (256) Google Scholar In this study, we used strains from the CC, a murine resource developed for investigation of complex genetic traits, to evaluate ACD responses using a standard CHS system. The CC was developed from 8 founder strains of mice (A/J, C57BL/6J, 129S1SvImJ, NOD/LtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) whose genomes have been sequenced.E3Churchill G.A. Airey D.C. Allayee H. Angel J.M. Attie A.D. Beatty J. et al.The Collaborative Cross, a community resource for the genetic analysis of complex traits.Nat Genet. 2004; 36: 1133-1137Crossref PubMed Scopus (818) Google Scholar The CC mice were developed using a strategy that ensures that each strain has genetic contribution from each of the founder strains, resulting in a population with large phenotypic and genetic diversity that models the amount of variation present in the human population.E3Churchill G.A. Airey D.C. Allayee H. Angel J.M. Attie A.D. Beatty J. et al.The Collaborative Cross, a community resource for the genetic analysis of complex traits.Nat Genet. 2004; 36: 1133-1137Crossref PubMed Scopus (818) Google Scholar Because the genome of each CC strain has been comprehensively characterized, investigation of a particular trait across multiple strains can determine the underlying QTL associated with the observed phenotype.E4Ram R. Mehta M. Balmer L. Gatti D.M. Morahan G. Rapid identification of major-effect genes using the collaborative cross.Genetics. 2014; 198: 75-86Crossref PubMed Scopus (31) Google Scholar To date, the CC has provided insights into the underlying genetic factors important for a number of conditions, including inflammatory bowel disease,E5Rogala A.R. Morgan A.P. Christensen A.M. Gooch T.J. Bell T.A. Miller D.R. et al.The Collaborative Cross as a resource for modeling human disease: CC011/Unc, a new mouse model for spontaneous colitis.Mamm Genome. 2014; 25: 95-108Crossref PubMed Scopus (58) Google Scholar melanoma susceptibility,E6Ferguson B. Ram R. Handoko H.Y. Mukhopadhyay P. Muller H.K. Soyer H.P. et al.Melanoma susceptibility as a complex trait: genetic variation controls all stages of tumor progression.Oncogene. 2015; 34: 2879-2886Crossref PubMed Scopus (24) Google Scholar, E7Chitsazan A. Ferguson B. Ram R. Mukhopadhyay P. Handoko H.Y. 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All work in this study was conducted according to the Australian Code for the Care and Use of Animals for Scientific Purposes, 8th Edition, 2013, and institutional ethical requirements. A minimum of 3 female mice at age 8 weeks for each of 38 strains of the CC were used for CHS experiments. Mice were sensitized at age 8 weeks by treating with 150 μL of 5% oxazolone (4-ethoxymethylene-2-phenyloxazol-5-one) (Sigma-Aldrich, St Louis, Mo), a strong sensitizer, in an 80% acetone/20% corn oil vehicle solution on their abdomen that had been shaved previously using electric clippers. Seven days later, ear-thickness measurements were taken from both ears using a digital micrometer (Mitutoyo, Sakado, Japan). Following initial measurements, mice were challenged with a single application of 20 μL of 2% oxazolone in vehicle to both surfaces of their left ear, and vehicle-only control to their right ear. This time point was taken as day (D) 0, and subsequent ear-thickness measurements were taken at D1, D2, D3, D4, and D7 postchallenge. Ear swelling is a widely used measure to assess T-cell–mediated delayed-type hypersensitivity reactions including ACD.E10Christensen A.D. Skov S. Haase C. Local and systemic effects of co-stimulatory blockade using cytotoxic T lymphocyte antigen-4-immunoglobulin in dinitrofluorobenzene- and oxazolone-induced contact hypersensitivity in mice.Clin Exp Immunol. 2013; 171: 220-230Crossref PubMed Scopus (4) Google Scholar, E11Zhu Z. Luo Y. Yu J. Gao J. Zhang Y. Xiao C. et al.Sema4D is required in both the adaptive and innate immune responses of contact hypersensitivity.Mol Immunol. 2016; 78: 98-104Crossref PubMed Scopus (10) Google Scholar, E12Jung S.H. Sun X. Ryu W.S. Yang B.S. 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Lee H.G. et al.Protein tyrosine phosphatase conjugated with a novel transdermal delivery peptide, astrotactin 1-derived peptide recombinant protein tyrosine phosphatase (AP-rPTP), alleviates both atopic dermatitis-like and psoriasis-like dermatitis.J Allergy Clin Immunol. 2018; 141: 137-151Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Ears and lymph nodes were harvested. Cells were stained for 20 minutes at 4°C with the fluorophore-conjugated cell surface markers CD3 PB, CD4 PETR, CD8 PerCPCy5.5, CD25 PE, IL10 PE, Ly6c APCCy7, Ly6g PE, and IFNg FITC. After washing, cells were fixed and then permeabilized for Foxp3 staining according to the manufacturer's protocol (Biolegend, San Diego, Calif). Isotype controls were used to exclude nonspecific fluorescence. Analysis was performed using a Gallios cytometer (Beckman Coulter, Brea, Calif). The data were analyzed using Kaluza software (Beckman Coulter). Regulatory T cells were analyzed on the basis of CD25 and FoxP3 expression, gating on CD3+ population. CD8+IFNg+ cells and T cells based on CD4 and CD8 expression were analyzed gating on CD3+ population. At each time point, ear-thickness measurements for the challenged ear were calculated as a percentage of the vehicle-control–treated ear thickness. These data were plotted over time according to strain. For each strain, the acute response to immune challenge was determined as the percentage increase in ear thickness of the challenged ear compared to the vehicle-control ear thickness at D1. Postchallenge recovery was calculated as the difference between D1 and D7 measurements, which were calculated as a percentage difference between challenged and vehicle-treated ears. Negative values indicated overall recovery, and positive values indicated worsening of inflammation. The means calculated for each strain across each of the investigated phenotypes were used for subsequent QTL mapping. The construction of the CC founder haplotypes is based on the method described in Ram et al.E4Ram R. Mehta M. Balmer L. Gatti D.M. Morahan G. Rapid identification of major-effect genes using the collaborative cross.Genetics. 2014; 198: 75-86Crossref PubMed Scopus (31) Google Scholar For mapping, we used a logistic regression matrix model over the reconstructed haplotypes matrix to produce genome-wide distribution of P values (ANOVA chi-squared). For QTL mapping, we calculated significance values on the basis of 10,000 permutations, to limit the possibility of type I error. Striking differences in response for both the acute reaction to immune challenge and postchallenge recovery were observed across the 38 CC strains we analyzed. In the acute response, mean percentage increase in ear thickness was observed to vary between 55% (VIT strain) and 233% (SAT strain) (Fig E1, A). Regarding postchallenge recovery, when comparing ear thickness between D1 and D7, 33 of the 38 strains analyzed were observed to have a negative value for percentage change in ear thickness, demonstrating an overall recovery from the initial inflammatory response (Fig E1, A). However, the remaining 5 strains failed to recover and displayed a worsening response, as demonstrated by an increased ear thickness at D7 compared with D1. In particular, the BEM strain exhibited the worst recovery, with a mean increase of 290% in ear thickness from D1. The variation in response among strains is best demonstrated visually with a heat map of percentage change in ear thickness over time (Fig E1, B). Notably, this illustrates that at each time point the extremes of responses are exhibited by different strains, suggesting that particular genes are likely to be influencing the immune response at different times postchallenge. Genome-wide linkage analysis based on the acute CHS response (defined by increase in ear thickness at D1 postchallenge) identified a major effect QTL on chromosome 4 between 19.7 and 33.1 Mb, with the causal variant found to be derived from the 129S founder strain. Within this interval, we identified Bach2 (BTB Domain and CNC Homolog 2) as a candidate gene associated with the acute CHS response. BACH2 is a transcription factor involved in B-cell development and differentiation into plasma cells.E16Zhou Y. Wu H. Zhao M. Chang C. Lu Q. The Bach family of transcription factors: a comprehensive review.Clin Rev Allergy Immunol. 2016; 50: 345-356Crossref PubMed Scopus (69) Google Scholar In addition, it has been described as an essential factor regulating T-cell function. It is expressed in CD4+ and CD8+ memory T cells and acts as a repressor of T-cell superenhancers, mediating many of their effector functions during terminal differentiation.E17Vahedi G. Kanno Y. Furumoto Y. Jiang K. 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Schwab S.R. et al.Increased generation of Foxp3(+) regulatory T cells by manipulating antigen presentation in the thymus.Nat Commun. 2016; 7: 10562Crossref PubMed Scopus (42) Google Scholar However, sustained overexpression of this gene has been shown not to alter CHS.E26Stolina M. Dwyer D. Ominsky M.S. Corbin T. Van G. Bolon B. et al.Continuous RANKL inhibition in osteoprotegerin transgenic mice and rats suppresses bone resorption without impairing lymphorganogenesis or functional immune responses.J Immunol. 2007; 179: 7497-7505Crossref PubMed Scopus (57) Google Scholar Notably, the CAST haplotype displayed unique noncoding variants of Klf10 and Ebag9 within this locus. Ebag9 is a negative regulator of T-cell cytotoxicity and its loss has been associated with increased CD8+ gra

Importance:Standard treatment for endometrial cancer involves removal of the uterus, tubes, ovaries, and lymph nodes. Few randomized trials have compared disease-free survival outcomes for surgical approaches. Objective:To investigate whether total laparoscopic hysterectomy (TLH) is equivalent to total abdominal hysterectomy (TAH) in women with treatment-naive endometrial cancer. Design, Setting, and Participants:The Laparoscopic Approach to Cancer of the Endometrium (LACE) trial was a multinational, randomized equivalence trial conducted between October 7, 2005, and June 30, 2010, in which 27 surgeons from 20 tertiary gynecological cancer centers in Australia, New Zealand, and Hong Kong randomized 760 women with stage I endometrioid endometrial cancer to either TLH or TAH. Follow-up ended on March 3, 2016. Interventions:Patients were randomly assigned to undergo TAH (n = 353) or TLH (n = 407). Main Outcomes and Measures:The primary outcome was disease-free survival, which was measured as the interval between surgery and the date of first recurrence, including disease progression or the development of a new primary cancer or death assessed at 4.5 years after randomization. The prespecified equivalence margin was 7% or less. Secondary outcomes included recurrence of endometrial cancer and overall survival. Results:Patients were followed up for a median of 4.5 years. Of 760 patients who were randomized (mean age, 63 years), 679 (89%) completed the trial. At 4.5 years of follow-up, disease-free survival was 81.3% in the TAH group and 81.6% in the TLH group. The disease-free survival rate difference was 0.3% (favoring TLH; 95% CI, -5.5% to 6.1%; P = .007), meeting criteria for equivalence. There was no statistically significant between-group difference in recurrence of endometrial cancer (28/353 in TAH group [7.9%] vs 33/407 in TLH group [8.1%]; risk difference, 0.2% [95% CI, -3.7% to 4.0%]; P = .93) or in overall survival (24/353 in TAH group [6.8%] vs 30/407 in TLH group [7.4%]; risk difference, 0.6% [95% CI, -3.0% to 4.2%]; P = .76). Conclusions and Relevance:Among women with stage I endometrial cancer, the use of total abdominal hysterectomy compared with total laparoscopic hysterectomy resulted in equivalent disease-free survival at 4.5 years and no difference in overall survival. These findings support the use of laparoscopic hysterectomy for women with stage I endometrial cancer. Trial Registration:clinicaltrials.gov Identifier: NCT00096408; Australian New Zealand Clinical Trials Registry: CTRN12606000261516.

Giant congenital nevi are associated with clinical complications such as neurocutaneous melanosis and melanoma. Virtually nothing is known about why some individuals develop these lesions. We previously identified the sonic hedgehog (Shh) pathway regulator Cdon as a candidate nevus modifier gene. Here we validate this by studying Cdon knockout mice, and go on to establishing the mechanism by which Shh exacerbates nevogenesis. Cdon knockout mice develop blue nevi without the need for somatic melanocyte oncogenic mutation. In a mouse model carrying melanocyte NRASQ61K, we found that strain backgrounds that carry genetic variants that cause increased keratinocyte Shh pathway activity, as measured by Gli1 and Gli2 expression, develop giant congenital nevi. Shh components are also active adjacent to human congenital nevi. Mechanistically, this exacerbation of nevogenesis is driven via the release of the melanocyte mitogen endothelin-1 from keratinocytes. We then suppressed nevus development in mice using Shh and endothelin antagonists. Our work suggests an aspect of nevus development whereby keratinocyte cytokines such as endothelin-1 can exacerbate nevogenesis, and provides potential therapeutic approaches for giant congenital nevi. Furthermore, it highlights the notion that germline genetic variation, in addition to somatic melanocyte mutation, can strongly influence the histopathological features of melanocytic nevi.