Aleksandra Gregorius

Liver sinusoidal endothelial cells (LSECs) function and NAFLD; NO-based therapy targeted to the liver

Liver sinusoidal endothelial cells (LSECs) present unique, highly specialised endothelial cells in the body. Unlike the structure and function of typical, vascular endothelial cells, LSECs are comprised of fenestrations, display high endocytic capacity and play a prominent role in maintaining overall liver homeostasis. LSEC dysfunction has been regarded as a key event in multiple liver disorders; however, its role and diagnostic, prognostic and therapeutic significance in nonalcoholic fatty liver disease (NAFLD) is still neglected. The purpose of this review is to provide an overview of the importance of LSECs in NAFLD. Attention is focused on the LSECs-mediated NO-dependent mechanisms in NAFLD development. We briefly describe the unique, highly specialised phenotype of LSECs and consequences of LSEC dysfunction on function of hepatic stellate cells (HSC) and hepatocytes. The potential efficacy of liver selective NO donors against liver steatosis and novel treatment approaches to modulate LSECs-driven liver pathology including NAFLD are also highlighted.

AFM-based detection of glycocalyx degradation and endothelial stiffening in the db/db mouse model of diabetes

Degradation of the glycocalyx and stiffening of endothelium are important pathophysiological components of endothelial dysfunction. However, to our knowledge, these events have not been investigated in tandem in experimental diabetes. Here, the mechanical properties of the glycocalyx and endothelium in ex vivo mouse aorta were determined simultaneously in indentation experiments with an atomic force microscope (AFM) for diabetic db/db and control db/+ mice at ages of 11–19 weeks. To analyze highly heterogeneous aorta samples, we developed a tailored classification procedure of indentation data based on a bi-layer brush model supplemented with Hertz model for quantification of nanomechanics of endothelial regions with and without the glycocalyx surface. In db/db mice, marked endothelial stiffening and reduced glycocalyx coverage were present already in 11-week-old mice and persisted in older animals. In contrast, reduction of the effective glycocalyx length was progressive and was most pronounced in 19-week-old db/db mice. The reduction of the glycocalyx length correlated with an increasing level of glycated haemoglobin and decreased endothelial NO production. In conclusion, AFM nanoindentation analysis revealed that stiffening of endothelial cells and diminished glycocalyx coverage occurred in early diabetes and were followed by the reduction of the glycocalyx length that correlated with diabetes progression.

A new in vitro protocol for assessing phototoxiceffects of xenobiotics on human keratinocytes

Routine protocols of phototoxicity tests are based on cultured mouse fibroblasts, mainly because these cells are robust and easy to culture in vitro. However, in a real-life situation, phototoxic reactions take place primarily in the epidermis, comprised of keratinocytes – cells which differ substantially from fibroblasts with regard to structure and function. Therefore, keratinocyte cultures seem more appropriate for the phototoxicity testing of xenobiotics, such as cosmetic ingredients or drugs. Aim: To design and implement a test protocol for in vitro assessment of phototoxic properties of xenobiotics in normal human keratinocytes. Material and methods: As a starting point, we applied the EU-approved protocol for testing phototoxicity in mouse fibroblast cultures (3T3 Neutral Red Uptake Phototoxicity Assay, DB-ALM No. 78). The protocol was modified and adjusted in a series of experiments to the specific demands of cultured normal human keratinocytes. After obtaining a stable growth of keratinocytes in microcultures, the cells were exposed for 1 hour to model agents with phototoxic properties known from clinical observations: chlorpromazine, 8-methoxypsoralen, chloroquine, promethazine, etofenamate, ketoprofen, doxycycline, lymecycline, and isotretinoin in a series of concentrations of 0, 1, 3, 11, 33, and 100 μg/ml. Subsequently, the cultures were exposed to the cumulative dose of 5 J/cm2 of artificial sunlight using the EU-recommended solar simulator. The survival of keratinocytes was assessed by their uptake of neutral red (NR) dye. Results: Using the proposed test protocol, we were able to achieve stable growth of normal adult human keratinocytes in vitro. In the absence of phototoxic agents, no effects of light on cell viability were noticeable up to the dose of 10 J/cm2. The proposed system was capable of demonstrating phototoxicity of model phototoxic xenobiotics selected for the tests, which was in line with the clinical experience regarding phototoxic effects of these agents in humans. Conclusions: We have developed an in vitro protocol for assessment of the phototoxic potential of xenobiotics in normal human keratinocytes. Its functionality and reliability has been confirmed by tests results with known phototoxic agents. Although more difficult to culture than mouse fibroblasts, and therefore neglected in routine phototoxicity testing, human keratinocytes seem more appropriate for predicting in vitro phototoxic effects of xenobiotics in human skin, as phototoxic processes predominantly involve the epidermis which consists of keratinocytes.

Optimization of the basophil activation test in the diagnosis and qualification for treatment by means of allergen-specific immunotherapy in children with respiratory allergy to house dust mites

Diagnosis of diseases, especially qualification for treatment should be based on the methods properly optimized in order to minimize the risk of incorrect qualification to unnecessary treatments, or neglecting treatment wherever it is needed. Among possible alternatives for routine allergy diagnostic tests, hopeful mentions can be found of the basophil activation test (BAT), which replicates in vitro reactions that occur in the body during basophil response to allergens, thus giving a chance for forecasting the severity of allergic reaction. Aim The aim of the study was to optimize the basophil activation test in the detection of house dust mite allergy in Polish children with allergic respiratory diseases. Patients and methods The study involved 32 patients, with symptoms of asthma or allergic rhinitis, qualified for sIT with the D pteronyssinus allergen by an experienced pediatric allergist. The control group consisted of an equal number of sex- and agematched children with the same clinical diagnoses, yet sensitized to allergens other than D. pteronyssinus .I n all patients, the BAT test was performed with five dilutions of D. pteronyssinus allergen. In order to detect possible cross-reactivity, BAT was also carried out with one dilution of D. farinae. The results were analyzed by the means of ROC. Results The highest diagnostic efficiency in the analyzed population was yielded by the cut-off of 9.76% of activated basophils after activation with a single allergen concentration of 2.25 ng/ml (sensitivity 90,63%, specificity 100%). The computed value differed significantly from the cut-off value of 15% proposed by the manufacturer of the test. Qualification of the patients with the use of the proposed protocol and cut-off value did not differ from the “gold standard”, i.e. qualification by the physician, while adoption of the parameters proposed by the manufacturer would result in a significant difference in this regard. Conclusions