A human 3D immune competent full-thickness skin model mimicking dermal dendritic cell activation.

We have integrated dermal dendritic cell surrogates originally generated from the cell line THP-1 as central mediators of the immune reaction in a human full-thickness skin model. Accordingly, sensitizer treatment of THP-1-derived CD14-, CD11c+ immature dendritic cells (iDCs) resulted in the phosphorylation of p38 MAPK in the presence of 1-chloro-2,4-dinitrobenzene (DNCB) (2.6-fold) as well as in degradation of the inhibitor protein kappa B alpha (IκBα) upon incubation with NiSO4 (1.6-fold). Furthermore, NiSO4 led to an increase in mRNA levels of IL-6 (2.4-fold), TNF-α (2-fold) and of IL-8 (15-fold). These results were confirmed on the protein level, with even stronger effects on cytokine release in the presence of NiSO4: Cytokine secretion was significantly increased for IL-8 (147-fold), IL-6 (11.8-fold) and IL-1ß (28.8-fold). Notably, DNCB treatment revealed an increase for IL-8 (28.6-fold) and IL-1ß (5.6-fold). Importantly, NiSO4 treatment of isolated iDCs as well as of iDCs integrated as dermal dendritic cell surrogates into our full-thickness skin model (SM) induced the upregulation of the adhesion molecule clusters of differentiation (CD)54 (iDCs: 1.2-fold; SM: 1.3-fold) and the co-stimulatory molecule and DC maturation marker CD86 (iDCs ~1.4-fold; SM:~1.5-fold) surface marker expression. Noteworthy, the expression of CD54 and CD86 could be suppressed by dexamethasone treatment on isolated iDCs (CD54: 1.3-fold; CD86: 2.1-fold) as well as on the tissue-integrated iDCs (CD54: 1.4-fold; CD86: 1.6-fold). In conclusion, we were able to integrate THP-1-derived iDCs as functional dermal dendritic cell surrogates allowing the qualitative identification of potential sensitizers on the one hand, and drug candidates that potentially suppress sensitization on the other hand in a 3D human skin model corresponding to the 3R principles ("replace", "reduce" and "refine").

Identification of pathological transcription in autosomal dominant polycystic kidney disease epithelia.

Autosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways. Here we performed transcriptomic analyses of Pkd1- and Pkd2-deficient mIMCD3 kidney epithelial cells followed by a meta-analysis to integrate all published ADPKD transcriptomic data sets. Based on the hypothesis that Pkd1 and Pkd2 operate in a common pathway, we first determined transcripts that are differentially regulated by both genes. RNA sequencing of genome-edited ADPKD kidney epithelial cells identified 178 genes that are concordantly regulated by Pkd1 and Pkd2. Subsequent integration of existing transcriptomic studies confirmed 31 previously described genes and identified 61 novel genes regulated by Pkd1 and Pkd2. Cluster analyses then linked Pkd1 and Pkd2 to mRNA splicing, specific factors of epithelial mesenchymal transition, post-translational protein modification and epithelial cell differentiation, including CD34, CDH2, CSF2RA, DLX5, HOXC9, PIK3R1, PLCB1 and TLR6. Taken together, this model-based integrative analysis of transcriptomic alterations in ADPKD annotated a conserved core transcriptomic profile and identified novel candidate genes for further experimental studies.

Quantifying snowfall from orographic cloud seeding.

Climate change and population growth have increased demand for water in arid regions. For over half a century, cloud seeding has been evaluated as a technology to increase water supply; statistical approaches have compared seeded to nonseeded events through precipitation gauge analyses. Here, a physically based approach to quantify snowfall from cloud seeding in mountain cloud systems is presented. Areas of precipitation unambiguously attributed to cloud seeding are isolated from natural precipitation (<1 mm h-1). Spatial and temporal evolution of precipitation generated by cloud seeding is then quantified using radar observations and snow gauge measurements. This study uses the approach of combining radar technology and precipitation gauge measurements to quantify the spatial and temporal evolution of snowfall generated from glaciogenic cloud seeding of winter mountain cloud systems and its spatial and temporal evolution. The results represent a critical step toward quantifying cloud seeding impact. For the cases presented, precipitation gauges measured increases between 0.05 and 0.3 mm as precipitation generated by cloud seeding passed over the instruments. The total amount of water generated by cloud seeding ranged from 1.2 × 105 m3 (100 ac ft) for 20 min of cloud seeding, 2.4 × 105 m3 (196 ac ft) for 86 min of seeding to 3.4 x 105 m3 (275 ac ft) for 24 min of cloud seeding.

Filtration based assessment of CTCs and CellSearch® based assessment are both powerful predictors of prognosis for metastatic breast cancer patients.

BACKGROUND: The assessment of circulating tumor cells (CTCs) has been shown to enable monitoring of treatment response and early detection of metastatic breast cancer (MBC) recurrence. The aim of this study was to compare a well-established CTC detection method based on immunomagnetic isolation with a new, filtration-based platform. METHODS: In this prospective study, two 7.5 ml blood draws were obtained from 60 MBC patients and CTC enumeration was assessed using both the CellSearch® and the newly developed filtration-based platform. We analyzed the correlation of CTC-positivity between both methods and their ability to predict prognosis. Overall survival (OS) was calculated and Kaplan-Meier curves were estimated with thresholds of ≥1 and ≥5 detected CTCs. RESULTS: The CTC positivity rate of the CellSearch® system was 56.7% and of the filtration-based platform 66.7%. There was a high correlation of CTC enumeration obtained with both methods. The OS for patients without detected CTCs, regardless of the method used, was significantly higher compared to patients with one or more CTCs (p < 0.001). The median OS of patients with no CTCs vs. ≥ 1 CTC assessed by CellSearch® was 1.83 years (95% CI: 1.63-2.02) vs. 0.74 years (95% CI: 0.51-1.52). If CTCs were detected by the filtration-based method the median OS times were 1.88 years (95% CI: 1.74-2.03) vs. 0.59 years (95% CI: 0.38-0.80). CONCLUSIONS: The newly established EpCAM independently filtration-based system is a suitable method to determine CTC counts for MBC patients. Our study confirms CTCs as being strong predictors of prognosis in our population of MBC patients.

Precipitation formation from orographic cloud seeding.

Throughout the western United States and other semiarid mountainous regions across the globe, water supplies are fed primarily through the melting of snowpack. Growing populations place higher demands on water, while warmer winters and earlier springs reduce its supply. Water managers are tantalized by the prospect of cloud seeding as a way to increase winter snowfall, thereby shifting the balance between water supply and demand. Little direct scientific evidence exists that confirms even the basic physical hypothesis upon which cloud seeding relies. The intent of glaciogenic seeding of orographic clouds is to introduce aerosol into a cloud to alter the natural development of cloud particles and enhance wintertime precipitation in a targeted region. The hypothesized chain of events begins with the introduction of silver iodide aerosol into cloud regions containing supercooled liquid water, leading to the nucleation of ice crystals, followed by ice particle growth to sizes sufficiently large such that snow falls to the ground. Despite numerous experiments spanning several decades, no direct observations of this process exist. Here, measurements from radars and aircraft-mounted cloud physics probes are presented that together show the initiation, growth, and fallout to the mountain surface of ice crystals resulting from glaciogenic seeding. These data, by themselves, do not address the question of cloud seeding efficacy, but rather form a critical set of observations necessary for such investigations. These observations are unambiguous and provide details of the physical chain of events following the introduction of glaciogenic cloud seeding aerosol into supercooled liquid orographic clouds.