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We have developed a new in vitro skin irritation test based on an open source reconstructed epidermis (OS-REp) with openly accessible protocols for tissue production and test performance. Due to structural, mechanistic and procedural similarity, a blinded catch-up validation study for skin irritation according to OECD Performance Standards (PS) was conducted in three laboratories to promote regulatory acceptance, with OS-REp models produced at a single production site only. While overall sensitivity and predictive capacity met the PS requirements, overall specificity was only 57%. A thorough analysis of the test results led to the assumption that some of the false-positive classifications could have been evoked by volatile skin-irritating chemicals tested in the same culture plate as the non-irritants falsely predicted as irritants. With GC/MS and biological approaches the cross-contamination effect was confirmed and the experimental set-up adapted accordingly. Retesting of the affected chemicals with the improved experimental set-up and otherwise identical protocol resulted in correct classifications as non-irritants. Taking these re-test results into account, 93% overall sensitivity, 70% specificity and 82% accuracy was achieved, which is in accordance with the OECD PS. A sufficient reliability of the method was indicated by a within-laboratory-reproducibility of 85–95% and a between-laboratory-reproducibility of 90%.
hallmark of ageing is the redistribution of body fat. Particularly, subcutaneous fat decreases paralleled by a decrease of skin collagen I are typical for age-related skin atrophy. In this paper, we hypothesize that collagen I may be a relevant molecule stimulating the differentiation of adipose-derived stem cells (ASCs) into adipocytes augmenting subcutaneous fat. In this context lipogenesis, adiponectin, and collagen I receptor expression were determined. Freshly isolated ASCs were characterized by stemness-associated surface markers by FACS analysis and then transdifferentiated into adipocytes by specific medium supplements. Lipogenesis was evaluated using Nile Red staining and documented by fluorescence microscopy or quantitatively measured by using a multiwell spectrofluorometer. Expression of adiponectin was measured by real-time RT-PCR and in cell-free supernatants by ELISA, and expression of collagen I receptors was observed by western blot analysis. It was found that supports coated with collagen I promote cell adhesion and lipogenesis of ASCs. Interestingly, a reverse correlation to adiponectin expression was observed. Moreover, we found upregulation of the collagen receptor, discoidin domain-containing receptor 2; receptors of the integrin family were absent or downregulated. These findings indicate that collagen I is able to modulate lipogenesis and adiponectin expression and therefore may contribute to metabolic dysfunctions associated with ageing.
Recent studies have proven that Dimethylfumarate (DMF) has a marked anti-proliferative impact on diverse cancer entities e.g., on malignant melanoma. To explore its anti-tumorigenic potential, we examined the effects of DMF on human colon carcinoma cell lines and the underlying mechanisms of action. Human colon cancer cell line HT-29 and human colorectal carcinoma cell line T84 were treated with or without DMF. Effects of DMF on proliferation, cell cycle progression, and apoptosis were analyzed mainly by Bromodeoxyuridine (BrdU)- and Lactatdehydrogenase (LDH)-assays, caspase activation, flowcytometry, immunofluorescence, and immunoblotting. In addition, combinational treatments with radiation and chemotherapy were performed. DMF inhibits cell proliferation in both cell lines. It was shown that DMF induces a cell cycle arrest in G0/G1 phase, which is accompanied by upregulation of p21 and downregulation of cyclin D1 and Cyclin dependent kinase (CDK)4. Furthermore, upregulation of autophagy associated proteins suggests that autophagy is involved. In addition, the activation of apoptotic markers provides evidence that apoptosis is involved. Our results show that DMF supports the action of oxaliplatin in a synergetic manner and failed synergy with radiation. We demonstrated that DMF has distinct anti-tumorigenic, cell dependent effects on colon cancer cells by arresting cell cycle in G0/G1 phase as well as activating both the autophagic and apoptotic pathways and synergizes with chemotherapy.
Background: Inflammation, angiogenesis and oxidative stress have been implicated in the pathogenesis of various vascular diseases. Recent evidence suggests that dimethylfumarate (DMF), an antiposriatic and anti-multiple sclerosis agent, possesses anti-inflammatory, anti-oxidative and anti-angiogenic properties. Here, we analyze the influence of DMF on TNF-α-induced expression of the important pro-inflammatory and pro-atherogenic chemokine MCP-1 and investigate the underlying mechanisms of this expression.
Findings: We analyzed constitutive and TNF-α-induced expression of MCP-1 in human umbilical vascular endothelial cells (HUVEC) +/− DMF treatment via enzyme-linkes immunosorbent assay (ELISA). DMF significantly inhibited the protein expression levels in a time- and concentration-dependent manner. Furthermore, MCP-1 mRNA expression was also reduced in response to DMF, as demonstrated by RT-PCR. Thus, the regulation occurs at the transcriptional level. Interestingly, DMF prolonged the TNF-α-induced p38 and JNK phosphorylation in HUVEC, as demonstrated by Western blot analysis; however, the p38 and JNK inhibitor SB203580 did not affect the DMF-conveyed suppression of TNF-α-induced MCP-1 expression. DMF suppressed the TNF-α-induced nuclear translocation and phosphorylation (Serine 536) of p65 in these cells. These results were additionally approved by p65 luciferase promoter assays. Furthermore, we found that DMF slightly inhibited the early degradation of IκBα. In addition, we verified our results using other important inflammatory cytokines such as CCL-5, PDGF-BB, GM-CSF and IL-6.
Conclusion: DMF suppresses various TNF-α-induced pro-inflammatory and pro-atherogenic cytokines/chemokines in human endothelial cells. This action is regulated by reduced p65 activity and nuclear translocation, which can be explained in part by the reduced early degradation of IκBα and more important the reduced phosphorylation of p65 at Serine 536. These effects were independent of the p38, PI3K and p42/44 signaling pathways. As a result, DMF might be suitable for treating patients with vascular diseases.
Meeting Abstract Es wurde eine zellbasierte Wundauflage mit Keratinozyten und Fibroblasten auf Basis einer kommerziellen Wundauflage (Matriderm, Collagen/Elastin-Matrix) generiert, um damit großflächige Verbrennungswunden behandeln zu können. Zunächst wurde die Expansion der Keratinozyten optimiert und die Zeit für die Anzüchtung minimiert. Ausgangsmaterial waren 1–2 cm2 Spalthaut vom Patienten. Epidermis und Dermis wurden nach einer enzymatischen Behandlung mit Thermolysin voneinander getrennt. Aus den beiden Hautkompartimenten wurden durch Trypsin- und Kollagenase I-Behandlung Keratinozyten und Fibroblasten isoliert, welche in Kollagen I-beschichteten Zellkulturflaschen expandiert wurden. Nach 10 Tagen wurden die Fibroblasten auf 100 cm2 Matriderm aufgebracht. Nach einwöchiger submerser Kultivierung wurden die Keratinozyten ausgesät. Eine Woche später wurde die Matrix an die Luft-Flüssigkeitsgrenze angehoben, um die epidermale Differenzierung einzuleiten. Nach 16 Tagen wurde das Hautäquivalent fixiert und immunhistologisch sowie elektronen-mikroskopisch begutachtet. Die Histologie zeigte eine regelgerechte Stratifizierung des epidermalen Anteils. Immunhistologisch ließ sich eine Basalmembran mit Collagen IV und Laminin 5 nachweisen. Proliferative Zellen, nachgewiesen mit KI-67 befanden sich lediglich in der basalen Region der Epidermis. Desmoglein, sowie die Differenzierungsmarker Involucrin und CK 10 wurden suprabasal nachgewiesen. Elektronenmikroskopisch waren die Basalmembran sowie die Zell-Zell-Verbindungen in Form von Desmosmen zu erkennen. Späte Differenzierungsmerkmale, wie granuläre Strukturen und verdickte Zellmembranen, fanden sich im Str. granulosum und Str. corneum. Die Studie zeigt, dass man aus Matriderm eine zellbasierte Wundauflage herstellen kann, die verglichen mit dem Ausgangsmaterial um den Faktor 50–100 vergrößert ist und deren Aufbau normaler Haut weitgehend entspricht.
Evaluation of 2‑methoxyestradiol serum levels as a potential prognostic marker in malignant melanoma
(2021)
Experimental findings indicated that 2‑methoxyestradiol (2‑ME), an endogenous metabolite of 17β‑estradiol, may exhibit anti‑tumorigenic properties in various types of tumour, such as melanoma and endometrial carcinoma. In patients with endometrial cancer, the serum levels of 2‑ME are decreased compared with those in healthy controls, and this finding has been associated with a poor outcome. The aim of the present study was to examine whether the serum levels of 2‑ME are decreased in patients with melanoma, and whether this decrease may be correlated with disease stage and, therefore, serve as a prognostic indicator. ELISA was used to detect serum levels of 2‑ME in patients with stage I‑IV malignant melanoma (MM). A cohort of 78 patients with MM was analysed, along with 25 healthy controls, among whom 15 were women in the second trimester of pregnancy (positive control). As expected, significantly elevated levels of serum 2‑ME were observed in pregnant control patients compared with those in patients with MM and healthy controls. There was no observed correlation between 2‑ME serum levels in patients with MM and disease stage, tumour thickness, lactate dehydrogenase or S100 calcium‑binding protein B levels. In addition, the 2‑ME levels of patients with MM did not differ significantly from those of normal healthy controls. Overall, the findings of the present study indicated that the 2‑ME serum levels in patients with MM were not decreased, and there was no correlation with early‑ or advanced‑stage disease. Therefore, in contrast to published results on endometrial cancer, endogenous serum 2‑ME levels in MM were not found to be correlated with tumour stage and did not appear to be a suitable prognostic factor in MM.
Curcumin—a rhizomal phytochemical from the plant Curcuma longa—is well known to inhibit cell proliferation and to induce apoptosis in a broad range of cell lines. In previous studies we showed that combining low curcumin concentrations and subsequent ultraviolet A radiation (UVA) or VIS irradiation induced anti-proliferative and pro-apoptotic effects. There is still debate whether curcumin induces apoptosis via the extrinsic or the intrinsic pathway. To address this question, we investigated in three epithelial cell lines (HaCaT, A431, A549) whether the death receptors CD95, tumor necrosis factor (TNF)-receptor I and II are involved in apoptosis induced by light and curcumin. Cells were incubated with 0.25–0.5 µg/mL curcumin followed by irradiation with 1 J/cm2 UVA. This treatment was combined with inhibitors specific for distinct membrane-bound death receptors. After 24 h apoptosis induction was monitored by quantitative determination of cytoplasmic histone-associated-DNA-fragments. Validation of our test system showed that apoptosis induced by CH11 and TNF-α could be completely inhibited by their respective antagonists. Interestingly, apoptosis induced by curcumin/light treatment was reversed by none of the herein examined death receptor antagonists. These results indicate a mechanism of action independent from classical death receptors speaking for intrinsic activation of apoptosis. It could be speculated that a shift in cellular redox balance might prompt the pro-apoptotic processes
The anti-cancer properties of curcumin in vitro have been documented. However, its clinical use is limited due to rapid metabolization. Since irradiation of curcumin has been found to increase its anti-cancer effect on several tumor types, this investigation was designed to determine whether irradiation with visible light may enhance the anti-tumor effects of low-dosed curcumin on renal cell carcinoma (RCC) cell growth and proliferation. A498, Caki1, and KTCTL-26 cells were incubated with curcumin (0.1–0.4 µg/mL) and irradiated with 1.65 J/cm2 visible light for 5 min. Controls were exposed to curcumin or light alone or remained untreated. Curcumin plus light, but not curcumin or light exposure alone altered growth, proliferation, and apoptosis of all three RCC tumor cell lines. Cells were arrested in the G0/G1 phase of the cell cycle. Phosphorylated (p) CDK1 and pCDK2, along with their counter-receptors Cyclin B and A decreased, whereas p27 increased. Akt-mTOR-signaling was suppressed, the pro-apoptotic protein Bcl-2 became elevated, and the anti-apoptotic protein Bax diminished. H3 acetylation was elevated when cells were treated with curcumin plus light, pointing to an epigenetic mechanism. The present findings substantiate the potential of combining low curcumin concentrations and light as a new therapeutic concept to increase the efficacy of curcumin in RCC.
Although anti-cancer properties of the natural compound curcumin have been reported, low absorption and rapid metabolisation limit clinical use. The present study investigated whether irradiation with visible light may enhance the inhibitory effects of low-dosed curcumin on prostate cancer cell growth, proliferation, and metastasis in vitro. DU145 and PC3 cells were incubated with low-dosed curcumin (0.1–0.4 µg/mL) and subsequently irradiated with 1.65 J/cm2 visible light for 5 min. Controls remained untreated and/or non-irradiated. Cell growth, proliferation, apoptosis, adhesion, and chemotaxis were evaluated, as was cell cycle regulating protein expression (CDK, Cyclins), and integrins of the α- and β-family. Curcumin or light alone did not cause any significant effects on tumor growth, proliferation, or metastasis. However, curcumin combined with light irradiation significantly suppressed tumor growth, adhesion, and migration. Phosphorylation of CDK1 decreased and expression of the counter-receptors cyclin A and B was diminished. Integrin α and β subtypes were also reduced, compared to controls. Irradiation distinctly enhances the anti-tumor potential of curcumin in vitro and may hold promise in treating prostate cancer.
Hypersensitivity reactions to non‐steroidal anti‐inflammatory drugs (NSAIDs) – a retrospective study
(2020)
Background: The aim of this study was to verify the validity of clinical history and oral provocation challenges of patients with NSAID hypersensitivity and to identify safe alternatives. The COX‐2 inhibitor etoricoxib, in particular, was studied.
Patients and methods: In all, 104 patients with confirmed diagnoses of NSAID hypersensitivity treated at the Department of Dermatology, Frankfurt University Hospital, Germany between 2004 and 2012 were retrospectively studied.
Results: The medical history and hypersensitivity symptoms during oral provocation testing (OPT) largely coincided and were mostly mild to moderate. Acetylsalicylic acid (ASA) was the most frequent trigger both anamnestically (27.9 %) and during OPT (47.8 %). Etoricoxib caused the fewest reactions during OPT (4.2 %). Acetaminophen led to reactions in only 6.7 % of the cases studied although it was named more often in clinical histories (14 %).
Conclusions: OPT should be the aim whenever possible as most symptoms are mild to moderate. To distinguish between selective and cross‐hypersensitivity reactions, ASA should be part of the test protocol. Furthermore, the findings of this study indicate that etoricoxib and acetaminophen are safe treatment alternatives in case of NSAID hypersensitivity. However, these drugs should not be administered without prior OPT in an inpatient setting, as severe symptoms can occur.