Biophysikalische Untersuchungen an bakteriellen Modellmembranen bezüglich ihrer lateralen Organisationund der Interaktion mit humanen β-Defensinen

Die Zellhülle Gram-negativer Bakterien besteht aus zwei Membranen. Die äußere Membran steht dabei im Fokus dieser Arbeit. Die Lipidmatrix der äußeren Membran besteht aus einer Phospholipidmischung im Fall der inneren Monoschicht und aus Lipopolysacchariden im Fall der äußeren Monoschicht. Die Zellhülle von Mykobakterien, dem Auslöser der Tuberkolose, ist noch nicht vollständig bekannt. Ausgehend von einem in der Zellhülle vorkommenden Lipid, wurden erste biophysikalische Untersuchungen durchgeführt. Eine Vielzahl biophysikalische Techniken, wie die Rasterkraftmikroskopie, elektrische und optische Messungen an planaren Rekonstituionssystemen der Membran, wurden verwendet, um Untersuchungen der physikalischen Eigenschaften der Membranen und den Einfluss humaner β-Defensine zu untersuchen. Die Ergebnisse dieser Untersuchungen zeigen, dass die beiden Monoschichten der rekonstituierten äußeren Membran Gram-negativer Bakterien sich gegenseitig bezüglich ihrer Phasenseparation ibeeinflussen. Durch die Erstellung von Kraftkarten konnte gezeigt werden, dass die Adhäsion zwischen den Fettsäureketten unterschiedlich in fluiden und rigiden Lipiddomänen ist. Diese Ergebnisse führen zu einer modellhaften Vorstellung der Interaktion zwischen den beiden Monoschichten der äußeren Membran Gram-negativen Bakterien. Das erste Angriffszeil für antimikrobielle Peptide ist die äußere Membran der Gram-negativen Bakterien. In der vorliegenden Arbeit wurde das humane β-Defensin-2 (hBD-2), hBD-3 und zwei Derivate des hBD-3 für Untersuchungen zur Interaktion zwischen humanen β-Defensinen und der äußeren Membran Gram-negativer Bakterien verwendet. Humane β-Defensine verfügen über drei Disulfidbrücken. Um den Einfluss dieser zu untersuchen, wurden zwei humane β-Defensin Derivate ohne und mit einer Disulfidbrücke synthetisiert. Die Ergebnisse der biologischen Messungen zeigten unterschiedliche antimikrobielle Aktivitäten der beiden Derivate im Vergleich zum ursprünglichem hBD-3. Aus biophysikalischen Untersuchungen konnte ein generalisiertes Modell der Wechselwirkungen zwischen humanen β-Defensinen und der äußeren Membran aufgestellt werden. Demnach bilden humane β-Defensine keine Poren definierter Geometrie, sondern führen durch einen auf dem carpet-like Mechanismus basierenden Prozess zu einer Störung der Membranintegrität.The cell envelope of Gram-negative bacteria consists of two membranes. The outer membrane is in the focus of the present study. The lipid matrix of the outer membrane consists of a mixture of phospholipids in the inner leaflet and of lipopolysaccaride in the outer leaftlet. The cell envelope of mycobacteria is not completely investigated. In this study biophysical experiments with one lipid of the cell envelope of mycobacteria were conducted. Several biophysical techniques like atomic force microscopy, electrical and optical measurements on planar reconstitution systems were performed to investigate the physical properties of the outer membrane of Gram-negative bacteria and the influence of human β-defenses on these membranes. The results of this work show that the two leaflets of the bilayer influences eachother by means of the phaseseparation of the lipids. By force mapping measurements it could be shown that the adhesion between the fatty acids differs in fluid and rigid lipid domains. These results leads to a model for the interaction of the two leaflets of the outer membrane of Gram-negative bacteria. The first target for antibacterial peptides is the outer membrane of Gram negative bacteria. In the present work human β-defensine-2 (hBD-2), hBD-3 and two derivates of the human β-defensine-3 were used for the experiments. Humane β-defensines consists of three disulfide-bonds. To investigate the influence of the disulfide-bonds two human β-defensines derivates without and with one disulfide-bond were synthesized. The results from biological measurements showed different antimicrobial activities of the two derivates and hBD-3. From biophysical investigations a generalized model for the interaction between human β-defense and membrans was developed: Human β-defensines does not form pores of a defined geometry, but leads to a disturbance of membrane integrity by a ‘carpet-like’ mechanism

Current NAFLD guidelines for risk stratification in diabetic patients have poor diagnostic discrimination

Patients with type 2 diabetes (T2D) are at risk for non-alcoholic fatty liver disease (NAFLD) and associated complications. This study evaluated the performance of international (EASL-EASD-EASO) and national (DGVS) guidelines for NAFLD risk stratification. Patients with T2D prospectively underwent ultrasound, liver stiffness measurement (LSM) and serum-based fibrosis markers. Guideline-based risk classification and referral rates for different screening approaches were compared and the diagnostic properties of simplified algorithms, genetic markers and a new NASH surrogate (FAST score) were evaluated. NAFLD risk was present in 184 of 204 screened patients (age 64.2 ± 10.7 years; BMI 32.6 ± 7.6 kg/m2). EASL-EASD-EASO recommended specialist referral for 60–77% depending on the fibrosis score used, only 6% were classified as low risk. The DGVS algorithm required LSM for 76%; 25% were referred for specialised care. The sensitivities of the diagnostic pathways were 47–96%. A simplified referral strategy revealed a sensitivity/specificity of 46/88% for fibrosis risk. Application of the FAST score reduced the referral rate to 35%. This study (a) underlines the high prevalence of fibrosis risk in T2D, (b) demonstrates very high referral rates for in-depth hepatological work-up, and (c) indicates that simpler referral algorithms may produce comparably good results and could facilitate NAFLD screening

A new topical panthenol-containing emollient: skin-moisturizing effect following single and prolonged usage in healthy adults, and tolerability in healthy infants

Purpose: Two studies were conducted with a new topical panthenol-containing emollient (NTP-CE) to investigate the skin-moisturizing effect in healthy adults and tolerability in healthy infants. Methods: In Study 1 (N = 44), a single skin application of NTP-CE was performed followed by a 4-week twice-daily application. Skin hydration and stratum corneum (SC) water content change (using Raman spectroscopy) were measured. In the 4-week Study 2 (N = 65, aged 3–25 months), NTP-CE tolerability was assessed using a 5-point scoring system; skin hydration was determined in a subset (N = 21). Results: In Study 1, mean AUC0 − 24 h for skin capacitance change from baseline was 302.03 i.u. with NTP-CE and −15.90 i.u. in control areas (p < .001). With NTP-CE (at 4 h), the water content within the upper SC part was reduced (−45.10 vs. −13.39 g/cm2, p = .013) and the water gradient increased (0.51 vs. 0.11 g/cm4, p = .036), indicating relocation of water into deeper layers. In Study 2, there was no statistically significant change from baseline in mean cutaneous tolerability scores. At days 7, 14, and 28, skin hydration had increased by 42%, 54%, and 49%, respectively (all p < .001). Conclusions: Single and prolonged NTP-CE usage is associated with sustained and deep skin moisturization. NTP-CE is well tolerated by healthy infants

A new topical panthenol-containing emollient: Results from two randomized controlled studies assessing its skin moisturization and barrier restoration potential, and the effect on skin microflora

Purpose: Two randomized, intra-individual comparison studies were performed in healthy subjects to evaluate the skin moisturization and barrier restoration potential of a new topical panthenol-containing emollient (NTP-CE) (Study 1), and its effect on skin microflora (Study 2). Methods: In Study 1 (N = 23), two skin areas, one challenged with 0.5% sodium dodecyl sulfate (SDS) solution and one unchallenged, were treated with NTP-CE for 3 weeks. Transepidermal water loss (TEWL), skin hydration, and intercellular lipid lamellae (ICLL) organization were measured at regular intervals during the study. In Study 2 (N = 20), quantitative bacterial cultures were obtained over 6 h from a skin area undergoing wash stress with 10% SDS with subsequent single application of NTP-CE. Results: In Study 1, mean AUC for TEWL reduction from baseline was more pronounced with NTP-CE compared with control (−168.36 vs. −123.38 g/m2/h, p = 0.023). NTP-CE use was also associated with statistically significant improvements in stratum corneum hydration and an increase in mean ICLL length from baseline (day 22: 120.61 vs. 35.85 nm/1000 nm2, p < 0.001). In Study 2, NTP-CE use had no negative impact on bacterial viability. Conclusions: NTP-CE use has favorable and lasting effects on barrier function and repair as well as skin hydration without negatively influencing bacterial viability