Pigment epithelium derived factor drives melanocyte proliferation and migration in neurofibromatosis café au lait macules.

Background: RASopathies, which include neurofibromatosis type 1 (NF1), are defined by Ras/mitogen-activated protein kinase (Ras/MAPK) pathway activation. They represent a group of clinically related disorders often characterised by multiple Café au Lait Macules (CALMs). Objectives: To determine, using in depth transcriptomic analysis of NF1 melanocytes from CALM and unaffected skin, (1) the gene(s) responsible for melanocyte proliferation and migration, and (2) the activated signalling pathway(s) in NF1 melanoma. Methods: Classical NF1 (n = 2, who develop tumours) and 3bp deletion NF1 (p. Met992del, who do not develop tumours) (n = 3) patients underwent skin biopsies from CALM and unaffected skin. Melanocytes were isolated and propagated, with five replicates from each tissue sample. DNA and RNA were extracted for mutational analysis and transcriptomic profiling with six replicates per sample. Mechanistic determination was undertaken using melanocyte and melanoma cell lines. Results: All CALMs in NF1 were associated with biallelic NF1 loss, resulting in amplification of Ras/MAPK and Wnt pathway signalling. CALMs were also associated with reduced SERPINF1 gene expression (and pigment epithelium-derived factor (PEDF) levels, the reciprocal protein), a known downstream target of the master regulator of melanocyte differentiation microphthalmia-associated transcription factor (MITF), leading to increased melanocyte proliferation, migration and invasion. In classical NF1 and melanoma, but not 3bp deletion NF1, there was also activation of the PI3K/AKT pathway. Pigment epithelium-derived factor was found to reduce cell proliferation and invasion of NF1 melanoma. Conclusions: Melanocyte proliferation and migration leading to CALMs in NF1 arises from biallelic NF1 loss, resulting in RAS/MAPK pathway activation, and reduced expression of the tumour suppressor PEDF. Activation of the PI3K/AKT pathway in classical NF1 and NF1 melanoma may facilitate tumour growth.

HPV8-induced STAT3 activation led keratinocyte stem cell expansion in human actinic keratoses.

Despite epidermal turnover, the skin is host to a complex array of microbes, including viruses, such as HPV, which must infect and manipulate skin keratinocyte stem cells (KSCs) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induced ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses. Together, these results define the "hit-and-run" mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lack melanosome protection and are thus susceptible to sun light-induced malignant transformation.

Targeting CD20-expressing malignant melanoma cells augments BRAF inhibitor killing.

BACKGROUND: Mutant BRAF targeted therapies remain a standard of care for the treatment of metastatic malignant melanoma (MM); however, high initial response rates are tempered by the persistence of residual MM cells that eventually lead to disease recurrence and mortality. As MM recurrence during targeted therapy can present with the simultaneous occurrence of multiple tumour nodules at the original body sites, we hypothesized the presence of an intrinsically resistant MM cell subpopulation. OBJECTIVES: To identify an MM cell subpopulation that is intrinsically resistant to targeted therapy and possibly responsible for MM recurrence. METHODS: Using melanoma cell lines, we defined culture conditions for the reproducible three-dimensional growth of melanospheres to investigate putative cancer stem cell populations. We undertook RNA sequencing and bioinformatic analysis to characterize cell populations between adherent and nonadherent culture, and cells expressing or not expressing CD20. Furthermore, we defined an in vitro assay to evaluate the killing of melanoma cancer stem cells as a therapeutic test using combination therapies targeting driver mutation and CD20. RESULTS: We described the culture conditions that promote MM cells to form melanospheres with a reproducible colony-forming efficiency rate of 0.3-1.3%. RNA sequencing of melanosphere vs. conventional MM cell cultures (n = 6), irrespective of the BRAF mutation status, showed that melanosphere formation was associated with growth and differentiation transcriptional signatures resembling MM tumours. Importantly, melanosphere formation also led to the emergence of a CD20+ MM cell subpopulation, similar to that observed in primary human MM tumours. CD20+ MM cells were resistant to BRAF inhibitor therapy and, consistent with this finding, demonstrated a Forkhead box protein M1 transcriptomic profile (n = 6). Combining BRAF inhibitor and anti-CD20 antibody treatment led to the additional killing of previously resistant CD20+  BRAF mutant MM cells. CONCLUSIONS: In patients with MM that harbour a CD20+ subpopulation, combined therapy with BRAF inhibitor and anti-CD20 antibody could potentially kill residual MM cells and prevent disease recurrence.

CAR T cells and time-limited ibrutinib as treatment for relapsed/refractory mantle cell lymphoma: the phase 2 TARMAC study.

ABSTRACT: CD19-directed chimeric antigen receptor T cells (CAR-T) achieve high response rates in patients with relapsed/refractory mantle cell lymphoma (MCL). However, their use is associated with significant toxicity, relapse concern, and unclear broad tractability. Preclinical and clinical data support a beneficial synergistic effect of ibrutinib on apheresis product fitness, CAR-T expansion, and toxicity. We evaluated the combination of time-limited ibrutinib and CTL019 CAR-T in 20 patients with MCL in the phase 2 TARMAC study. Ibrutinib commenced before leukapheresis and continued through CAR-T manufacture for a minimum of 6 months after CAR-T administration. The median prior lines of therapy was 2; 50% of patients were previously exposed to a Bruton tyrosine kinase inhibitor (BTKi). The primary end point was 4-month postinfusion complete response (CR) rate, and secondary end points included safety and subgroup analysis based on TP53 aberrancy. The primary end point was met; 80% of patients demonstrated CR, with 70% and 40% demonstrating measurable residual disease negativity by flow cytometry and molecular methods, respectively. At 13-month median follow-up, the estimated 12-month progression-free survival was 75% and overall survival 100%. Fifteen patients (75%) developed cytokine release syndrome; 12 (55%) with grade 1 to 2 and 3 (20%) with grade 3. Reversible grade 1 to 2 neurotoxicity was observed in 2 patients (10%). Efficacy was preserved irrespective of prior BTKi exposure or TP53 mutation. Deep responses correlated with robust CAR-T expansion and a less exhausted baseline T-cell phenotype. Overall, the safety and efficacy of the combination of BTKi and T-cell redirecting immunotherapy appears promising and merits further exploration. This trial was registered at www.ClinicalTrials.gov as #NCT04234061.

Spatial proteomics identifies a spectrum of immune dysregulation in acquired bone marrow failure syndromes.

Poor graft function (PGF), manifested by multilineage cytopenias and complete donor chimerism post-allogeneic stem cell transplantation (alloSCT), and acquired aplastic anaemia (AA) are immune-mediated acquired bone marrow (BM) failure syndromes with a similar clinical presentation. In this study, we used spatial proteomics to compare the immunobiology of the BM microenvironment and identify common mechanisms of immune dysregulation under these conditions. Archival BM trephines from patients exhibited downregulation of the immunoregulatory protein VISTA and the M2 macrophage marker and suppressor of T-cell activation ARG1 with increased expression of the immune checkpoint B7-H3 compared to normal controls. Increased CD163 and CD14 expression suggested monocyte/macrophage skewing, which, combined with dysregulation of STING and VISTA, is indicative of an environment of reduced immunoregulation resulting in the profound suppression of hematopoiesis in these two conditions. There were no changes in the immune microenvironment between paired diagnostic AA and secondary MDS/AML samples suggesting that leukaemic clones develop in the impaired immune microenvironment of AA without the need for further alterations. Of the eight proteins with dysregulated expression shared by diagnostic AA and PGF, the diagnostic AA samples had a greater fold change in expression than PGF, suggesting that these diseases represent a spectrum of immune dysregulation. Unexpectedly, analysis of samples from patients with good graft function post-alloSCT demonstrated significant changes in the immune microenvironment compared to normal controls, with downregulation of CD44, STING, VISTA, and ARG1, suggesting that recovery of multilineage haematopoiesis post-alloSCT does not reflect recovery of immune function and may prime patients for the development of PGF upon further inflammatory insult. The demonstrable similarities in the immunopathology of AA and PGF will allow the design of clinical interventions that include both patient cohorts to accelerate therapeutic discovery and translation.

CD200 ectodomain shedding into the tumor microenvironment leads to NK cell dysfunction and apoptosis.

The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor-initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells to suppress MAPK pathway signaling that in turn blocked indirect (IFN-γ release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ-regulated gene transcription and led to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL, which resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell-specific immune checkpoint, which is responsible for NK cell-associated poor outcomes in many cancers.

Regulation of dendritic cell function by Fc-γ-receptors and the neonatal Fc receptor.

Dendritic cells (DCs) express receptors to sense pathogens and/or tissue damage and to communicate with other immune cells. Among those receptors, Fc receptors (FcRs) are triggered by the Fc region of antibodies produced during adaptive immunity. In this review, the role of FcγR and neonatal Fc receptor (FcRn) in DC immunity will be discussed. Their expression in DC subsets and impact on antigen uptake and presentation, DC maturation and polarisation of T cell responses will be described. Lastly, we will discuss the importance of FcR-mediated DC function in the context of immunity during viral infection, inflammatory disease, cancer and immunotherapy.

Identification of Human Cutaneous Basal Cell Carcinoma Cancer Stem Cells.

The cancer stem cell model states that a subset of tumor cells, called "cancer stem cells," can initiate and propagate tumor growth through self-renewal, high proliferative capacity, and their ability to recreate tumor heterogeneity. In basal cell carcinoma (BCC), we have shown that tumor cells that express the cell surface protein CD200 fulfill the cancer stem cell hypothesis. CD200+ CD45- BCC cells represent 0.05-3.96% of all BCC cells and reside in small clusters at the tumor periphery. Using a novel, reproducible in vivo xenograft growth assay, we determined that tumor-initiating cell (TIC) frequencies are approximately 1 per 1.5 million unsorted BCC cells. The CD200+ CD45- BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200+ CD45- cells, representing ~1500-fold enrichment. The methods used to identify and purify CD200+ CD45- BCC cells, as well as characterize gene expression, are described herein.

Identification of Human Cutaneous Squamous Cell Carcinoma Cancer Stem Cells.

Epithelia are under constant threat from environmental carcinogens and none more so than squamous epithelia, which form the outermost linings of our bodies. Hence malignancies of squamous epithelia are collectively the most common cancer type and with the highest mortality, despite a constant cell turnover and only relatively rare long-lived adult tissue stem cells. Genetic analysis from SCC whole genome sequencing reveals commonality in mutated genes, despite various etiological factors. Most SCC types have been shown to exhibit hierarchical growth, in which a high frequency of cancer stem cells is associated with poor prognosis. For human cutaneous SCC (cSCC), we have shown that cancer stem cells express CD133 and that this population can recreate tumor heterogeneity in a novel in vivo model. CD133+ cSCC cells is small subset of tumor cells (~1%) in the outer layer of cSCC that are highly enriched for tumor-initiating capacity (TIC) (~1/400) compared to unsorted cSCC cells (~1/106). Xenografts of CD133+ cSCC recreated the original cSCC tumor histology and organizational hierarchy, while CD133- cells did not. Only CD133+ cells demonstrated the capacity for self-renewal in serial transplantation studies. Hence, cSCC has the potential to be the ideal model in which to study SCC biology.

Within-day variability in microbial concentrations at a UK designated bathing water: Implications for regulatory monitoring and the application of predictive modelling based on historical compliance data.

Prediction of bathing water quality is recommended by the World Health Organization (WHO), the European Union (EU) and the United States Environmental Protection Agency (USEPA) and is an established element in bathing water management designed to protect public health. Most commonly, historical regulatory compliance data are used for model calibration and provide the dependent variable for modelling. Independent (or predictor) variables (e.g. rainfall, river flow and received irradiance) measured over some antecedent period are used to deliver prediction of the faecal indicator concentration measured on the day of the regulatory sample collection. The implied linked assumptions of this approach are, therefore, that; (i) the independent variables accurately predict the bathing-day water quality; which is (ii) accurately characterized by the single regulatory sample. Assumption (ii) will not be the case where significant within-day variability in water quality is evident. This study built a detailed record of water quality change through 60 days at a UK coastal bathing water in 2011 using half-hourly samples each subjected to triplicate filtration designed to enhance enumeration precision. On average, the mean daily variation in FIO concentrations exceeded 1 log10 order, with the largest daily variations exceeding 2 log10 orders. Significant diurnality was observed at this bathing water, which would determine its EU Directive compliance category if the regulatory samples were collected at the same time each day. A sampling programme of this intensity has not been reported elsewhere to date and, if this pattern is proven to be characteristic of other bathing waters world-wide, it has significance for: (a) the design of regulatory sampling programmes; (b) the use of historical data to assess compliance, which often comprises a single sample taken at the compliance point on a regular, often weekly, basis; and (c) the use of regulatory compliance data to build predictive models of water quality.

Global Non-Potential Magnetic Models of the Solar Corona During the March 2015 Eclipse.

Seven different models are applied to the same problem of simulating the Sun's coronal magnetic field during the solar eclipse on 2015 March 20. All of the models are non-potential, allowing for free magnetic energy, but the associated electric currents are developed in significantly different ways. This is not a direct comparison of the coronal modelling techniques, in that the different models also use different photospheric boundary conditions, reflecting the range of approaches currently used in the community. Despite the significant differences, the results show broad agreement in the overall magnetic topology. Among those models with significant volume currents in much of the corona, there is general agreement that the ratio of total to potential magnetic energy should be approximately 1.4. However, there are significant differences in the electric current distributions; while static extrapolations are best able to reproduce active regions, they are unable to recover sheared magnetic fields in filament channels using currently available vector magnetogram data. By contrast, time-evolving simulations can recover the filament channel fields at the expense of not matching the observed vector magnetic fields within active regions. We suggest that, at present, the best approach may be a hybrid model using static extrapolations but with additional energization informed by simplified evolution models. This is demonstrated by one of the models.

Global conditions in the solar corona from 2010 to 2017.

Through reduction of a huge data set spanning 2010-2017, we compare mean global changes in temperature, emission measure (EM), and underlying photospheric magnetic field of the solar corona over most of the last activity cycle. The quiet coronal mean temperature rises from 1.4 to 1.8 MK, whereas EM increases by almost a factor of 50% from solar minimum to maximum. An increased high-temperature component near 3 MK at solar maximum drives the increase in quiet coronal mean temperature, whereas the bulk of the plasma remains near 1.6 MK throughout the cycle. The mean, spatially smoothed magnitude of the quiet Sun magnetic field rises from 1.6 G in 2011 to peak at 2.0 G in 2015. Active region conditions are highly variable, but their mean remains approximately constant over the cycle, although there is a consistent decrease in active region high-temperature emission (near 3 MK) between the peak of solar maximum and present. Active region mean temperature, EM, and magnetic field magnitude are highly correlated. Correlation between sunspot/active region area and quiet coronal conditions shows the important influence of decaying sunspots in driving global changes, although we find no appreciable delay between changes in active region area and quiet Sun magnetic field strength. The hot coronal contribution to extreme ultraviolet (EUV) irradiance is dominated by the quiet corona throughout most of the cycle, whereas the high variability is driven by active regions. Solar EUV irradiance cannot be predicted accurately by sunspot index alone, highlighting the need for continued measurements.

Multi-Scale Gaussian Normalization for Solar Image Processing.

Extreme ultra-violet images of the corona contain information over a wide range of spatial scales, and different structures such as active regions, quiet Sun, and filament channels contain information at very different brightness regimes. Processing of these images is important to reveal information, often hidden within the data, without introducing artefacts or bias. It is also important that any process be computationally efficient, particularly given the fine spatial and temporal resolution of Atmospheric Imaging Assembly on the Solar Dynamics Observatory (AIA/SDO), and consideration of future higher resolution observations. A very efficient process is described here, which is based on localised normalising of the data at many different spatial scales. The method reveals information at the finest scales whilst maintaining enough of the larger-scale information to provide context. It also intrinsically flattens noisy regions and can reveal structure in off-limb regions out to the edge of the field of view. We also applied the method successfully to a white-light coronagraph observation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11207-014-0523-9) contains supplementary material, which is available to authorized users.

Gene and protein responses of human lung tissue explants exposed to ambient particulate matter of different sizes.

CONTEXT: Exposure to ambient particulate air pollution is associated with increased cardiovascular and respiratory morbidity and mortality. It is necessary to understand causal pathways driving the observed health effects, particularly if they are differentially associated with particle size. OBJECTIVES: To investigate the effect of different size ranges of ambient particulate matter (PM) on gene and protein expression in an in vitro model. MATERIALS AND METHODS: Normal human tracheobronchial epithelium (NHTBE) three-dimensional cell constructs were exposed for 24 h to washed ambient PM of different sizes (size 1: 7-615 nm; size 2: 616 nm-2.39 µm; size 3: 2.4-10 µm) collected from a residential street. A human stress and toxicity PCR array was used to investigate gene expression and iTRAQ was used to perform quantitative proteomics. RESULTS: Eighteen different genes of the 84 on the PCR array were significantly dysregulated. Treatment with size 2 PM resulted in the greatest number of genes with altered expression, followed by size 1 and lastly size 3. ITRAQ identified 317 proteins, revealing 20 that were differentially expressed. Enrichment for gene ontology classification revealed potential changes to various pathways. DISCUSSION AND CONCLUSIONS: Different size fractions of ambient PM are associated with dysregulatory effects on the cellular proteome and on stress and toxicity genes of NHTBE cells. This approach not only provides an investigative tool to identify possible causal pathways but also permits the relationship between particle size and responses to be explored.

Medical humanities in Nepal--snakes and ladders.

In the last decade there has been a quantitative growth in medical schools in Nepal, a developing country in South Asia. Medical Humanities (MH) uses disciplines traditionally termed as the humanities in the pursuit of medical educational goals. The subject is slowly developing in Nepal. Sessions have been conducted at Manipal College of Medical Sciences, Pokhara and KIST Medical College, Lalitpur. In this article the authors examine inhibitory factors (snakes) and facilitating factors (ladders) for the development of the subject in Nepal.

Effectiveness of aeration and mixing in the remediation of a saline stratified river.

This study examines the use of an aeration scheme to remediate low oxygen conditions in a saline stratified system. The Tawe estuary was impounded in 1992 and quickly developed saline stratification during the summer months which led to an anoxic hypolimnon. In 1998 trials began in which a suite of aerators was applied to remediate the water quality; the trial was later extended to a full aeration scheme. This study examines pre-aeration conditions in order to delineate conditions under which poor water quality would develop, and would therefore be the conditions when aeration would be necessary. Furthermore, the study compared identical periods within the impoundment during which the following conditions existed: no aeration; and aeration with first 44, then 88, aerators. The study shows that (i) destratification occurred naturally under flows of >10 m3/s, and no low dissolved oxygen conditions were observed at higher flows; (ii) the presence of all levels of aeration had a statistically significant effect upon dissolved oxygen (DO) levels; the effect of increasing the number of aerators was approximately linear; (iii) the average effect of aeration was an increase of up to 3 mg/L DO in the deepest water; (iv) the frequency of low DO conditions decreased from 19% to 3% with the operation of aerators; and (v) aeration is most effective during periods of no tidal incursion and further from the saline water source. This study is the first to demonstrate the effectiveness of aeration in a saline stratified system.

The relationship between nuclear DNA content and leaf strategy in seed plants.

BACKGROUND AND AIMS: Species' 2C-values (mass of DNA in G(1) phase 2n nuclei) vary by at least four orders of magnitude among seed plants. The 2C-value has been shown to be co-ordinated with a number of other species traits, and with environmental variables. A prediction that species 2C-values are negatively related to leaf life span (LL) and leaf mass per area (LMA) is tested. These leaf traits are components of a major dimension of ecological variation among plant species. METHODS: Flow cytometry was used to measure the 2C-values for 41 Australian seed plant species, 40 of which were new to the literature. Where possible, LL and LMA data from the global literature were combined with 2C-values from our data set and online C-value databases. KEY RESULTS: Across all species, weak positive relationships were found between 2C-values and both LL and LMA; however, these did not reflect the relationships within either angiosperms or gymnosperms. Across 59 angiosperm species, there were weak negative relationships between 2C-values and both LL (r2 = 0.13, P = 0.005) and LMA (r2 = 0.15, P = 0.002). These relationships were the result of shifts to longer LL and greater LMA in woody compared with herbaceous growth forms, with no relationships present within growth forms. It was not possible to explain a positive relationship between 2C-values and LMA (r2 = 0.30, P = 0.024) across 17 gymnosperm species. The 2C-value was not related to LL or LMA either across species within orders (except for LMA among Pinales), or as radiation divergences in a model phylogeny. CONCLUSIONS: Gymnosperms appear to vary along a spectrum different from angiosperms. Among angiosperms, weak negative cross-species relationships were associated with growth form differences, and traced to a few divergences deep in the model phylogeny. These results suggest that among angiosperms, nuclear DNA content and leaf strategy are unrelated.