Metabolic activation and toxicological evaluation of polychlorinated biphenyls in Drosophila melanogaster.

Degradation of polychlorinated biphenyls (PCBs) is initiated by cytochrome P450 (CYP) enzymes and includes PCB oxidation to OH-metabolites, which often display a higher toxicity than their parental compounds. In search of an animal model reflecting PCB metabolism and toxicity, we tested Drosophila melanogaster, a well-known model system for genetics and human disease. Feeding Drosophila with lower chlorinated (LC) PCB congeners 28, 52 or 101 resulted in the detection of a human-like pattern of respective OH-metabolites in fly lysates. Feeding flies high PCB 28 concentrations caused lethality. Thus we silenced selected CYPs via RNA interference and analyzed the effect on PCB 28-derived metabolite formation by assaying 3-OH-2',4,4'-trichlorobiphenyl (3-OHCB 28) and 3'-OH-4',4,6'-trichlorobiphenyl (3'-OHCB 28) in fly lysates. We identified several drosophila CYPs (dCYPs) whose knockdown reduced PCB 28-derived OH-metabolites and suppressed PCB 28 induced lethality including dCYP1A2. Following in vitro analysis using a liver-like CYP-cocktail, containing human orthologues of dCYP1A2, we confirm human CYP1A2 as a PCB 28 metabolizing enzyme. PCB 28-induced mortality in flies was accompanied by locomotor impairment, a common phenotype of neurodegenerative disorders. Along this line, we show PCB 28-initiated caspase activation in differentiated fly neurons. This suggested the loss of neurons through apoptosis. Our findings in flies are congruent with observation in human exposed to high PCB levels. In plasma samples of PCB exposed humans, levels of the neurofilament light chain increase after LC-PCB exposure, indicating neuronal damage. In summary our findings demonstrate parallels between Drosophila and the human systems with respect to CYP mediated metabolism and PCB mediated neurotoxicity.

Four-year follow-up of two patients on maintenance therapy with fondaparinux and mycophenolate mofetil for microthrombotic antiphospholipid syndrome.

OBJECTIVES: We discuss two patients with antiphospholipid syndrome (APS) who presented with critical ischemia of both lower extremities due to arterial microthrombi. They received multimodality therapy emergently: anticoagulation, immunosuppression, and therapeutic plasma exchange (TPE). Then they were maintained on anticoagulation with fondaparinux and immunosuppression with mycophenolate mofetil (MMF), and were followed for 4 years. METHODS: Two patients with APS with ischemia and necrosis of their distal lower extremities were treated emergently with anticoagulation (intravenous heparin), immunosuppression (prednisone), and TPE. They were maintained on anticoagulation with fondaparinux and immunosuppression with MMF. RESULTS: Neither patient had recurrent microthrombotic disease during a 4-year follow-up. CONCLUSIONS: As described in our small cohort, patients with APS who suffer from microthrombotic arterial disease may benefit from maintenance therapy of anticoagulation with fondaparinux and immunosuppression with MMF, an approach which may be worthy of further trial. Fondaparinux does not require attention to diet, monitoring, and cumbersome bridging that is typical of warfarin therapy. MMF provides immunosuppression while sparing the side effects of steroid treatment.

Direct biological effects of fractional ultrapulsed CO2 laser irradiation on keratinocytes and fibroblasts in human organotypic full-thickness 3D skin models.

Molecular effects of various ablative and non-ablative laser treatments on human skin cells-especially primary effects on epidermal keratinocytes and dermal fibroblasts-are not yet fully understood. We present the first study addressing molecular effects of fractional non-sequential ultrapulsed CO2 laser treatment using a 3D skin model that allows standardized investigations of time-dependent molecular changes ex vivo. While histological examination was performed to assess morphological changes, we utilized gene expression profiling using microarray and qRT-PCR analyses to identify molecular effects of laser treatment. Irradiated models exhibited dose-dependent morphological changes resulting in an almost complete recovery of the epidermis 5 days after irradiation. On day 5 after laser injury with a laser fluence of 100 mJ/cm2, gene array analysis identified an upregulation of genes associated with tissue remodeling and wound healing (e.g., COL12A1 and FGF7), genes that are involved in the immune response (e.g., CXCL12 and CCL8) as well as members of the heat shock protein family (e.g., HSPB3). On the other hand, we detected a downregulation of matrix metalloproteinases (e.g., MMP3), differentiation markers (e.g., LOR and S100A7), and the pro-inflammatory cytokine IL1α.Overall, our findings substantiate the understanding of time-dependent molecular changes after CO2 laser treatment. The utilized 3D skin model system proved to be a reliable, accurate, and reproducible tool to explore the effects of various laser settings both on skin morphology and gene expression during wound healing.

[Double sensitization to PR10 and PR-14 proteins]. / Doppelsensibilisierung auf PR-10- und PR-14-Proteine.

A patient with mild oral allergy syndrome presented with a history of anaphylaxis induced by both hazelnuts and peaches. The ensuing work-up showed a double sensitization to proteins in both pathogenesis-related group 10 (e.g. Bet v1, Cor a1, Pru p1) and 14 (e.g. Pru p3, Cor a8). Such double sensitization profiles are increasingly being recognized in Europe.

[Retinoids in dermatopharmacology]. / Retinoide in der Dermatopharmakologie.

BACKGROUND: Retinoids are important in regulating cell proliferation and differentiation and play an important role in the body, including the skin. OBJECTIVES: Our objective is to review the current medical literature regarding use, effects and side-effects of topical and systemic retinoids used for therapy. METHODS: Pubmed/Medline electronic database was searched for relevant German and English literature. RESULTS: The group of retinoids used for therapeutic purposes includes both naturally occurring and chemically synthesized vitamin A derivates. Because of their influence on keratinization and epithelial differentiation, as well on the proliferation of benign and malignant keratinocytes, retinoids have found a wide application in the field of dermatopharmacology. CONCLUSION: Retinoids are among the most efficacious drugs used in the treatment of dermatological disorders and have a wide range of biological effects. Thorough knowledge about side-effects and comprehensive information for the patient are essential for safe treatment with retinoids.

Molecular microarray analysis reveals allergen- and exotoxin-specific IgE repertoires in children with atopic dermatitis.

BACKGROUND: Children suffering from atopic dermatitis frequently show allergen-specific sensitization. However, the corresponding IgE-recognition patterns have not yet been extensively characterized using multiallergen microarrays. OBJECTIVE: To provide comprehensive, molecular IgE repertoires in paediatric patients with atopic dermatitis using microarray technology. METHODS: Sera of 140 affected children were screened with a protein microarray containing a panel of 95 inhalant, food and staphylococcal antigen components. In addition, total serum IgE levels and further clinical parameters were recorded. RESULTS: At a mean total IgE level of 1528 kU/L, the number of sensitizations varied from 0 to 32 per patient, and regression analysis revealed a significant association between total IgE and the quantity of recognized antigens. A total of 78 single allergen and microbial components elicited at least one IgE response, while 11 plant and 13 non-plant molecules were recognized by more than 10% of patients. Specific IgE against Staphylococcus aureus could be detected in 14% of children. Sensitization rates against the studied allergen molecules differed significantly when stratified by age. Whereas reactivity against inhalant allergens and SEC was lowest in the youngest group (<24 months) reaching highest values in children ≥ 72 months, IgE responses against food allergen components peaked in younger age groups (0-48 months) and clearly declined in patients of higher age. The large amount of microarray data could be aggregated by centroid cluster analysis revealing valid allergen clusters possibly linked with higher disease severity as determined by multivariate analysis of covariance. CONCLUSION: Allergenic molecule microarray analysis can be regarded as a suitable research tool for large-scale IgE screening in infants and children with atopic dermatitis (AD). Still, further studies in well-defined populations are needed to exactly identify its tangible benefits in the diagnostic and therapeutic management of affected patients in daily clinical practice.

[Anaphylaxis to PR-10 proteins (Bet v1 homologues)]. / Anaphylaxie auf PR-10-Proteine (Bet v1-Homologe).

Secondary food allergies to PR-10 proteins (Bet v1 homologues) are the most common food allergies in Germany. Clinically they present with an oral allergy syndrome (intraoral pruritus and perhaps swelling). When drinks containing PR-10 proteins are rapidly consumed, for example after sporting activities, large concentrations of allergen can be reached without any intraoral symptoms and then lead to anaphylaxis. This phenomenon has often been described for soja milk and occurred in our case with an apple drink with 60% fruit concentration. It seems likely that such cases of anaphylaxis are not adequately represented in the anaphylaxis registry.

[Morbihan disease as a special form of rosacea: review of pathogenesis and new therapeutic options]. / Morbus Morbihan als Sonderform der Rosazea: Einblick in die Pathogenese und neue Therapieoptionen.

Morbihan disease is classified as a special form of rosacea, which presents with persistent facial erythema and solid edema because of marked involvement of the lymphatic vessels. The cheeks, nose and forehead are particularly affected. Currently, the treatment options of this cosmetically very disturbing disease are limited. However, every attempt should be made to provide treatment because of the great emotional suffering of the patients. We review some new currently available therapeutic options for Morbihan disease. In our patient, we were able to achieve a significant improvement with systemic isotretinoin 30 mg/day over a period of 12 months.

Dexpanthenol modulates gene expression in skin wound healing in vivo.

Topical application of dexpanthenol is widely used in clinical practice for the improvement of wound healing. Previous in vitro experiments identified a stimulatory effect of pantothenate on migration, proliferation and gene regulation in cultured human dermal fibroblasts. To correlate these in vitro findings with the more complex in vivo situation of wound healing, a clinical trial was performed in which the dexpanthenol-induced gene expression profile in punch biopsies of previously injured and dexpanthenol-treated skin in comparison to placebo-treated skin was analyzed at the molecular level by Affymetrix® GeneChip analysis. Upregulation of IL-6, IL-1ß, CYP1B1, CXCL1, CCL18 and KAP 4-2 gene expression and downregulation of psorasin mRNA and protein expression were identified in samples treated topically with dexpanthenol. This in vivo study might provide new insight into the molecular mechanisms responsible for the effect of dexpanthenol in wound healing and shows strong correlations to previous in vitro data using cultured dermal fibroblasts.

Evaluation of the i-STAT point-of-care capillary whole blood prothrombin time and international normalized ratio: comparison to the Tcoag MDAII coagulation analyzer in the central laboratory.

BACKGROUND: Point-of-care devices for performing a prothrombin time/international normalized ratio (PT/INR) using capillary blood samples are being increasingly used to monitor patients receiving anticoagulation therapy. However, the performance of some devices has been shown to be suboptimal and there are only limited published data comparing specific devices to various central laboratory coagulation analyzers. We report an evaluation of the iSTAT PT/INR with a comparison to the Tcoag MDA II analyzer. METHODS: We obtained simultaneous capillary/venous samples on 20 healthy volunteers for a normal range study and on 50 anticoagulated patients for a clinical evaluation. Testing was performed by phlebotomists. We also obtained 68 near simultaneous capillary/venous test results for assessment of performance by non-laboratory personnel. The criteria for determining clinical equivalence of the iSTAT to the MDA II were (1) same clinical category (subtherapeutic INR<2, therapeutic INR 2-3, and supratherapeutic INR>3) or (2) paired values within ≤ 0.4 INR. RESULTS: Forty nine of 50 patient sample pairs collected by phlebotomists showed acceptable clinical agreement. Sixty one (61) of 68 patient sample pairs collected by nurses showed acceptable agreement. In all discordant cases the differences were minor and would have resulted in either no or minimal change in therapy. CONCLUSIONS: The iSTAT PT/INR compares well to the MDA II when performed by phlebotomists or nurses.

Ultraviolet B radiation and reactive oxygen species modulate interleukin-31 expression in T lymphocytes, monocytes and dendritic cells.

BACKGROUND: Interleukin (IL)-31 is a novel Th2 T-cell cytokine that induces pruritus and dermatitis in transgenic mice. While enhanced mRNA expression of this cytokine is detected in skin samples of inflammatory skin diseases, the regulation of IL-31 expression is poorly understood. OBJECTIVES: To assess the effects of ultraviolet (UV) B radiation and H2O2 on IL-31 mRNA and protein expression in skin and different peripheral blood mononuclear cells (PBMCs). METHODS: The effects of UVB radiation and H2O2, as a prototypic reactive oxygen species, on IL-31 mRNA and protein expression were analysed in various inflammation-related cells and murine skin tissue. RESULTSTreatment of cells with UVB radiation and H2 O2 strongly induced IL-31 mRNA and protein expression in human PBMCs and in the skin of SKH-1 mice. Following exposure to UVB or H2O2, we observed increased expression of IL-31 mRNA in T cells, monocytes, macrophages, and immature and especially mature dendritic cells. H2O2 treatment but not UVB radiation led to a moderate upregulation of IL-31 mRNA expression in epidermal keratinocytes and dermal fibroblasts. Pretreatment of T lymphocytes with the MAPK p38 inhibitor SB203580 or the MEK1 inhibitor U0126 reduced the stimulatory effect of H2O2. These experiments suggest that p38 is involved in the regulation of IL-31 expression in human skin. CONCLUSIONS: Our studies reveal that UVB and reactive oxygen species stimulate the expression of IL-31 in PBMCs and skin, especially in T cells, monocytes and monocyte-derived dendritic cells.

Microarrays of recombinant Hevea brasiliensis proteins: a novel tool for the component-resolved diagnosis of natural rubber latex allergy.

BACKGROUND: Component-resolved diagnosis using microarray technology has recently been introduced in clinical allergology, but its applicability in patients with natural rubber latex (NRL) allergy has not been investigated. OBJECTIVES: To evaluate the utility of microarray-based immunoglobulin (Ig) E detection in the diagnostic workup of NRL allergy and to compare this new diagnostic tool with established methods of NRL-specific IgE detection. METHODS: We investigated 52 adults with immediate-type NRL allergy and 50 control patients. Determination of specific serum IgE against 8 recombinant Hevea brasiliensis allergen components was performed using a customized allergen microarray and a conventional fluorescence enzyme immunoassay (FEIA). RESULTS: The panel of microarrayed allergen components was shown to represent a comprehensive repertoire of clinically relevant NRL proteins. NRL-specific IgE recognition patterns and sensitization rates determined by microarray analysis were similar to those obtained by conventional FEIA. The diagnostic sensitivity rates of combined single-component data were not significantly different for the respective recombinant test system, whereas the sensitivity level of extract-based FEIA analysis was markedly higher. CONCLUSION: The current study provides evidence that microarrays of recombinant NRL allergen components are a suitable new tool for the diagnosis of NRL-specific sensitization.They show performance characteristics comparable to those of current diagnostic tests and could be indicated in small children in whom only limited blood volumes are obtainable. Further large-scale studies in unselected patient populations and in high-risk groups are warranted before the microarray can be introduced into routine management of patients with NRL allergy.

High-resolution transcriptional profiling of chemical-stimulated dendritic cells identifies immunogenic contact allergens, but not prohaptens.

Allergic contact dermatitis is a complex syndrome and knowledge about the in vitro detection of small-molecular-weight compounds, particularly prohaptens, is limited. Therefore, we investigated chemical-induced gene expression changes in human antigen-presenting cells upon stimulation with immunogenic contact allergens, prohaptens and irritants. Monocyte-derived dendritic cells (moDCs) and THP-1 cells were stimulated with the prohapten cinnamic alcohol (CAlc), the hapten cinnamic aldehyde (CAld), an irritant and an obligatory sensitizer in vitro. Whole-genome screening and consecutive PCR analysis of differential gene expression in moDCs stimulated with either CAld or the obligatory sensitizer revealed coregulation of 11 marker genes which were related to immunological reactions (IL-8, CD1e, CD200R1, PLA2G5, TNFRSF11A), oxidative or metabolic stress responses (AKR1C3, SLC7A11, GCLM) or other processes (DPYLS3, TFPI, TRIM16). In contrast, the prohapten CAlc and the irritant did not change marker gene expression. In THP-1 cells, CAld and the positive control elicited similar expression changes in only 4 of the previously identified genes (IL-8, TRIM16, CD200R1, GCLM). In conclusion, we provide important insights into the pathophysiological basis of allergic contact dermatitis, identify marker genes suitable for skin hazard assessment and demonstrate that contact-allergenic prohaptens escape in in vitro detection if their skin metabolism is not taken into account.

Expression and induction of cytochrome p450 isoenzymes in human skin equivalents.

Organotypic skin models are frequently used for a wide range of applications and latterly also for dermatotoxicological studies. To evaluate their practicability for the investigation of xenobiotic metabolism in human skin we compared three types of organotypic skin models, acquired by purchase from different manufacturers, to a self-constructed in-house model with regard to cytochrome P450 (CYP) isoenzyme expression on mRNA and protein level and the inducibility of these enzymes by aryl hydrocarbon receptor ligands. To induce enzyme activity, models were treated with benzanthracene, liquor carbonis detergens, pix lithanthracis or dimethyl sulfoxide as a solvent control. RNA was isolated by phenol-chloroform extraction and purified. Gene expression patterns were studied by cDNA microarray analysis. Microarray data were confirmed by real-time PCR. For quality control of the models and to detect and localize enzyme expression, immunofluorescence staining was performed with antibodies against CYPs and structure proteins. The immunofluorescence staining demonstrated the regular structure of our models. We could provide evidence for the expression of CYP types 1A1, 1B1, 2E1, 2C and 3A5 in organotypic skin models. The expression of CYP1A1 and CYP1B1 was highly inducible by treatment with liquor carbonis detergens. The proof of the expression and inducibility of CYP enzymes in organotypic skin models suggests that skin equivalents are a valuable tool that can emulate CYP-dependent metabolism of drugs and other xenobiotics in human skin.

Tacrolimus modulates dendritic cell activation in the sensitization phase of allergic contact dermatitis.

BACKGROUND: Knowledge of the effect of topically applied calcineurin antagonists such as tacrolimus on the sensitization phase of allergic contact dermatitis is currently limited. OBJECTIVE: To investigate tacrolimus-dependent immunomodulation on gene expression alterations in human antigen-presenting cells which are stimulated with small-molecular-weight contact allergens. METHODS: Monocyte-derived dendritic cells (moDC) and THP-1 cells were stimulated with the contact sensitizer cinnamic aldehyde (CAld) and compared with the very strong experimental sensitizer 2,4,6-trinitrobenzene sulfonic acid (TNBS) in vitro. Quantitative PCR analysis was used to detect gene expression changes, particularly of interleukin (IL) 8, as an indicator of differential dendritic cell (DC) gene expression after sensitizer stimulation in the absence or presence of tacrolimus and betamethasone at two different concentrations. RESULTS: DC activation was clearly demonstrated by a significant IL-8 upregulation after 24 h, whereas tacrolimus or betamethasone alone did not affect IL-8 baseline expression. Betamethasone and, to a lesser extent, tacrolimus led to a marked reduction of chemical-induced IL-8 expression by TNBS and CAld. CONCLUSION: The results of the present study support the hypothesis that the calcineurin inhibitor tacrolimus has modulatory effects on human antigen-presenting cells during the sensitization phase of allergic contact dermatitis. In addition, moDC as well as THP-1 cells may serve as a system to study immune-modulating effects of drugs such as glucocorticoids or calcineurin antagonists.

In vitro detection and characterization of drug hypersensitivity using flow cytometry.

BACKGROUND: The lymphocyte transformation test (LTT) is the only in vitro test for detecting drug sensitization at the cellular level irrespective of the reaction's phenotype. However, the LTT includes working with radioactive substances and is considered impracticable for routine laboratory investigation. OBJECTIVE: The aim of this study was to assess drug-specific cytokine production by means of flow cytometry as an alternative nonradioactive approach which may be more appropriate for routine testing and may provide in addition more information about the pathophysiology of the reaction than proliferation-based assays, like the LTT. METHOD: Peripheral blood mononuclear cells of 19 patients were incubated with culprit drugs (n = 28) or irrelevant antigens (n = 10). Ten healthy persons served as controls for all different drugs (n = 15). Intracellular interleukin (IL)-5, interferon (IFN)-gamma and IL-10 production was investigated using flow cytometry. Accuracy of the flow cytometry test system was confirmed using different statistical tests, i.e. receiver operating characteristic curve and Mann-Whitney rank test. In addition, drug-specific secretion of IL-5, IL-2 and IFN-gamma were analysed using enzyme-linked immunosorbent assay (ELISA). RESULTS: Drug-specific cytokine production could be demonstrated in 75% of the patients using flow cytometry and in 79% using ELISA respectively. Combining ELISA and flow cytometry increased the sensitivity to 100%. Analysis of involved T-cell subsets [e.g. CD4(+) or CD8(+); T helper (TH) 1 or TH 2] allowed characterization of the in vitro lymphocyte reactivity pattern. CONCLUSIONS: Analysis of drug-specific cytokine production by means of flow cytometry proved a useful and reliable approach for the in vitro detection and characterization of drug hypersensitivities.