EAHP European Statements Survey 2018, focusing on Section 1: Introductory Statements and Governance, Section 3: Production and Compounding, and Section 4: Clinical Pharmacy Services.

OBJECTIVES: The 2018 EAHP European Statements Survey focused on sections 1, 3 and 4 of the European Statements of Hospital Pharmacy. Statistical data on the level of implementation and on the main barriers to implementation of the Statements were collected. A further aim was to identify barriers in general, such as lack of awareness. METHODS: An online questionnaire was sent to all hospital pharmacies in EAHP member countries. Data were analysed at Keele University School of Pharmacy, UK. As with previous reports, the survey was divided into three sections: section A, asking general questions about the hospital pharmacy; Section B, addressing questions about the current activity of pharmacists around each statement from Sections 1, 3 and 4; and Section C, focusing on their ability to implement the statements. RESULTS: 719 complete responses were obtained from a sample of 5164 hospital pharmacies, giving a response rate of 14% (719/5164). Section A results indicated that 45% (323/719) of responders worked in teaching hospitals, 79% (568/719) of hospital pharmacies had 10 or fewer pharmacists, and 48% (345/719) of hospital pharmacies served over 500 beds. Section B results found a high percentage of positive responses for activity in section 1 (introductory statements and governance) and section 3 (production and compounding). However, responses to questions in section 4 (clinical pharmacy services) were more variable, with 6 of the 15 questions being answered positively by less than half of respondents. The five questions that revealed the lowest implementation levels were then analysed in greater detail. These questions corresponded to Statements 4.4, 4.5, 4.8, 1.1, and 4.2, which need the greatest effort for implementation. The major identified barriers to implementation were 'lack of capacity' and that 'other health professionals in the hospital fulfil the tasks'. CONCLUSIONS: This survey provides useful information on the implementation status (and the barriers to, and drivers of implementation) of sections 1, 3 and 4 of the Statements. This will allow the EAHP to plan its implementation support programme for its members. To increase the quality of data, as well as the feedback to hospital pharmacies, the EAHP is planning to combine the survey with the self-assessment tool of the European Statements of Hospital Pharmacy.

Results of EAHP's 2018 Survey on Medicines Shortages.

AIMS AND OBJECTIVES: The aim of the 2018 EAHP Survey on Medicines Shortages was to provide a clearer picture on the issue of medicines shortages, including the impact on hospital pharmacists' time, budgets and the effect on patient care. METHODS: A survey was conducted by the EAHP, collecting information from European hospital pharmacists on the shortage situation in their respective countries. The survey ran from 19 March 2018 to 11 June 2018. Keele University, UK analysed and compared the results to those of the 2014 survey. RESULTS: There were 1666 responses to the 2018 survey, which represented a threefold increase from the 2014 survey which received 607 responses. Ninety per cent of respondents answered 'Yes' when asked if shortages of medicines are a current problem in delivering the best care to patients, while only 7% of respondents answered 'No', and 3% 'Unsure'.Problems with shortages of antimicrobials were most commonly reported (77% of respondents reporting this as an issue in 2018 vs 57% in 2014), followed by preventative medicines (43% in 2018 vs 20% in 2014) and anaesthetics (39% in 2018 vs 27% in 2014). Fifty-nine per cent of respondents have seen care delayed as a consequence of medication shortages, with cancellations of care (31% of respondents), medication errors (25% of respondents) and suboptimal treatment for patients (25% of respondents) also being frequently reported.Sixty-three per cent of respondents reported having had to pay a higher price to procure from alternate sources most of the time or always when there was a shortage of a medicine. CONCLUSIONS: Medicines shortages is an increasing problem across Europe and is having an adverse impact on patient care. Medicines shortages are adding to hospital pharmacists' time pressures and have an adverse budgetary impact. More timely information about impending shortages and how long they will last is seen as necessary to help manage the problem.

EAHP European Statements Survey 2017, focusing on sections 2 (Selection, Procurement and Distribution), 5 (Patient Safety and Quality Assurance) and 6 (Education and Research).

OBJECTIVES: The 2017 EAHP European Statements Survey focussed on sections 2, 5 and 6 of the European Statements of Hospital Pharmacy. Statistical data on the level of implementation and on the main barriers to implementation of the Statements were collected. A further aim was to identify barriers in general, such as lack of awareness. METHODS: An online questionnaire was sent to all hospital pharmacies in European Association of Hospital Pharmacists (EAHP) member countries. Data were analysed at Keele University School of Pharmacy, UK by and the EAHP Survey Group. RESULTS: There were 783 complete responses (response rate 17.4%). Some 42% of responders worked in teaching hospitals, 76% of hospital pharmacies had 10 or less pharmacists, and 46% of hospital pharmacies served over 500 beds.Five questions revealing the lowest implementation levels were further analysed in greater detail. Only 30% of respondents reported that their hospital pharmacists routinely publish hospital pharmacy practice research, and only 50% are involved in the development of local or national guidelines. 45% of respondents reported that computerised decision support was used to reduce the risk of medication errors in their hospitals, 69% stated that they had contingency plans for medicines shortages and 60% answered that they had had reason to contact their medicines authority because of drug shortages. 63% reported that the transcription step had been eliminated from the medicines administration process. CONCLUSIONS: The survey has provided the EAHP with an overview of the implementation level as well as the barriers to and drivers of implementation of sections 2, 5 and 6. This is important for informing the plans of EAHP and its members so that implementation can be fully supported.

Ultra-high resolution X-ray structures of two forms of human recombinant insulin at 100 K.

The crystal structure of a commercially available form of human recombinant (HR) insulin, Insugen (I), used in the treatment of diabetes has been determined to 0.92 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16,000 keV (λ = 0.77 Å). Refinement carried out with anisotropic displacement parameters, removal of main-chain stereochemical restraints, inclusion of H atoms in calculated positions, and 220 water molecules, converged to a final value of R = 0.1112 and Rfree = 0.1466. The structure includes what is thought to be an ordered propanol molecule (POL) only in chain D(4) and a solvated acetate molecule (ACT) coordinated to the Zn atom only in chain B(2). Possible origins and consequences of the propanol and acetate molecules are discussed. Three types of amino acid representation in the electron density are examined in detail: (i) sharp with very clearly resolved features; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry; (iii) poor density and difficult or impossible to model. An example of type (ii) is observed for the intra-chain disulphide bridge in chain C(3) between Sγ6-Sγ11 which has two clear conformations with relative refined occupancies of 0.8 and 0.2, respectively. In contrast the corresponding S-S bridge in chain A(1) shows one clearly defined conformation. A molecular dynamics study has provided a rational explanation of this difference between chains A and C. More generally, differences in the electron density features between corresponding residues in chains A and C and chains B and D is a common observation in the Insugen (I) structure and these effects are discussed in detail. The crystal structure, also at 0.92 Å and 100 K, of a second commercially available form of human recombinant insulin, Intergen (II), deposited in the Protein Data Bank as 3W7Y which remains otherwise unpublished is compared here with the Insugen (I) structure. In the Intergen (II) structure there is no solvated propanol or acetate molecule. The electron density of Intergen (II), however, does also exhibit the three types of amino acid representations as in Insugen (I). These effects do not necessarily correspond between chains A and C or chains B and D in Intergen (II), or between corresponding residues in Insugen (I). The results of this comparison are reported. Graphical abstract Conformations of PheB25 and PheD25 in three insulin structures: implications for biological activity? Insulin residues PheB25 and PheD25 are considered to be important for insulin receptor binding and changes in biological activity occur when these residues are modified. In porcine insulin and Intergen (II) PheB25 adopts conformation B and PheD25 conformation D. However, unexpectedly PheB25 in Insugen (I) human recombinant insulin adopts two distinct conformations corresponding to B and D, Figure 1 and PheD25 adopts a single conformation corresponding to B not D, Figure 2. Conformations of this residue in the ultra-high resolution structure of Insugen (I) are therefore unique within this set. Figures were produced with Biovia, Discovery Studio 2016.

EAHP European Statements survey 2015.

OBJECTIVES: The 2015 EAHP European Statements survey was related to sections 2, 5 and 6 of the European Statements of Hospital Pharmacy (Statements). In addition to collection of statistical data about the level of implementation of the Statements, it was also intended to identify important barriers to their implementation. METHODS: The online questionnaire was sent to all hospital pharmacies in EAHP member countries. Data were analysed by researchers from Keele University School of Pharmacy, UK and the EAHP Survey Group. RESULTS: There were a total of 949 responses (response rate 18%). In the first part of the survey, data was collected on hospital pharmacy setting. While almost half of hospital pharmacies served over 500 beds, 80% of hospital pharmacies had 10 or less pharmacists. In section B, we gathered evidence about the degree of implementation of sections 2, 5 and 6 of the Statements and the main barriers to and drivers of implementation. Five questions with the lowest implementation level were then further analysed. Only five countries had 50% or more of hospital pharmacies reporting that the hospital pharmacists routinely publish hospital pharmacy practice research. 67% of participants stated that they had contingency plans for medicines shortages. The majority of countries (n=20) have less than half of respondents using computerised decision support to reduce the risk of medication errors. When asked if an audit had been undertaken in the past 3 years to identify priorities in medicines use processes, the mean percentage of positive responses for a country was 58%. CONCLUSIONS: EAHP has gained an informative overview of the implementation level as well as the barriers to and drivers of implementation in sections 2, 5 and 6. This is essential to inform the plans for EAHP to best support their implementation.

EAHP statements survey 2016: sections 1, 3 and 4 of the European Statements of Hospital Pharmacy.

BACKGROUND: The 2016 European Association of Hospital Pharmacists (EAHP) Statements survey builds on previous surveys and focuses on sections 1, 3 and 4. OBJECTIVE: To collect statistical data about the level of implementation of the Statements, and identify important barriers to their implementation. METHODS: An online questionnaire was sent to all hospital pharmacies in EAHP member countries. Data were analysed by researchers from Keele University School of Pharmacy, UK and the EAHP Survey Group. If an incomplete survey was submitted, the quantitative data were not used, although any free-text responses were incorporated. RESULTS: The overall response rate was 16% (904 out of 5711 requests) with 730 complete responses. In the first part of the survey, data were collected on the hospital pharmacy setting. While almost half (n=335) of hospital pharmacies served over 500 beds, 77% (n=564) of hospital pharmacies had ≤10 pharmacists. In section B, evidence was gathered about the degree of implementation of sections 1, 3 and 4 of the Statements and the main barriers to, and drivers of, implementation. The questions related to production and compounding (section 3) received very positive responses (all questions from this section received at least a 70% positive response rate), indicating that responders are having less difficulty implementing these statements compared with others. The introductory statements and governance questions (section 1) received a more mixed response. Only 343 (47%) responses indicated that the pharmacists worked routinely as part of multidisciplinary team. Many of the questions relating to clinical pharmacy services (section 4) received a more negative response overall, with six questions receiving <50% positive responses. CONCLUSIONS: This iteration of the survey provides the EAHP with further insight into the implementation of the Statements across the member countries as well as the barriers to, and drivers of, implementation in sections 1, 3 and 4. This is essential to inform the plans for EAHP to best support their implementation.

Melatonin, hypnotics and their association with fracture: a matched cohort study.

OBJECTIVES: although melatonin prescribing in England has been increasing in recent years, there have been no large scale studies on the safety of melatonin compared to other medical treatments for insomnia. The primary aim of this study was to examine the association between exposure to melatonin, hypnotic benzodiazepines (temazepam, nitrazepam) or Z-drugs (zolpidem, zopiclone) and fracture risk. DESIGN: retrospective cohort study. SETTING: 309 general practices contributing to The Health Improvement Network (THIN) between 2008 and 2013. PARTICIPANTS: 1,377 patients aged 45 years and older prescribed melatonin; 880 patients prescribed hypnotic benzodiazepines; 1,148 patients prescribed Z-drugs and 2,752 unexposed controls matched by age, gender and practice. MAIN OUTCOME: fracture following prescription of study drugs ascertained from practice records. RESULTS: the unadjusted hazard ratios for fracture during the follow-up period were 1.90 (95% CI 1.41-2.57) for melatonin, 1.70 (95% CI 1.18-2.46) for hypnotic benzodiazepines and 2.03 (95% CI 1.45-2.84) for Z-drugs. After adjustment for 26 covariates, the hazard ratios were 1.44 (95% CI 1.01-2.04) for melatonin, 1.26 (95% CI 0.82-1.92) for hypnotic benzodiazepines and 1.52 (95% CI 1.04-2.23) for Z-drugs. Only patients with three or more melatonin prescriptions had elevated risk. The mean time to fracture was 1.04 years and there was no significant difference in mean time to fracture between the cohorts. CONCLUSIONS: in this large cohort of patients attending UK primary care, prescriptions for melatonin and Z-drugs were associated with a significantly increased risk of fracture. With the use of melatonin increasing steadily overtime, this study adds to the literature on the safety profile of this drug.

EAHP European Statements baseline survey 2015: first steps on the implementation journey.

An implementation plan was developed in conjunction with the publication of the European Statements of Hospital Pharmacy. Subsequently a baseline survey on the status of the Statements was conducted with specific questions on awareness, capability and capacity seen as crucial to informing future implementation plans. The baseline survey showed that, 18 months after agreement at the European Summit of Hospital Pharmacy, hospital pharmacists across Europe have a growing awareness of the Statements. There is a significant variation across Europe with awareness levels ranging from 100% to less than 30%. Capacity within European Healthcare systems to achieve implementation is challenging with less than 10% of respondents in most countries indicating sufficient capacity to implement the services described in the Statements. Staff skills and experience to implement the changes was identified by respondents at less than 20% in many countries. The implementation plan will need to be constantly updated and modified to sustain progress.

EAHP European Statements baseline survey 2015: results.

OBJECTIVES: The European Statements baseline survey was designed to give an insight to how well the European Statements of Hospital Pharmacy (the Statements) are being implemented and to help inform the European Association of Hospital Pharmacists (EAHP) implementation strategy for the Statements. METHODS: The online questionnaire was sent to all hospital pharmacies in EAHP member countries. More than 1000 pharmacists completed the survey, which was analysed by Keele University and presented to EAHP. RESULTS: The survey provided general data for each country about the staffing levels and skill mix within each hospital, how well each hospital was implementing each Statement and finally about what were the barriers to implementation for European hospital pharmacists. The survey identified that, generally, the sections covering more traditional roles of hospital pharmacists (procurement, compounding) were being well implemented-96% of respondents indicated that their hospitals had clear processes in place around the procurement of medicines; 91% indicated "When medicines require manufacture or compounding, we either produce them in our hospital pharmacy or we outsource to an approved provider". Compared with that, clinical pharmacy services, multidisciplinary approaches and active engagement in education and practice research activities are challenging areas in most EAHP member countries. In only 29% of hospitals, the pharmacists enter all medicines used onto the patient's medical record on admission. 49% of responders agreed that the pharmacists in their hospital routinely publish hospital pharmacy practice research. Insufficient capacity and capability in terms of staffing and resources seemed to be main reasons given for low implementation of some of the Statements. CONCLUSIONS: After analysis of data from more than 1000 questionnaires, EAHP gained a necessary overview of Statements implementation level and barriers to implementation, needed for informed and efficient progress of EAHP implementation projects.

EAHP European Statements baseline survey 2015: methodology.

The European Statements of Hospital Pharmacy are a set of hospital pharmacy practice standards published by the European Association of Hospital Pharmacists (EAHP) for European health systems to ensure safe, effective and optimal use of medicines in collaboration with multidisciplinary teams. Keele University was commissioned to conduct a survey among European hospital pharmacists to establish a baseline to assess awareness of the Statements and to identify any barriers to their implementation. The survey was conducted from January 2015 to March 2015, spanning 16 languages and 34 countries. The survey consisted of three sections: ▸ Section A: general questions about the participant's hospital pharmacy, such as workforce skill-mix and number of beds served; ▸ Section B: questions about the current activity of pharmacists around each statement; ▸ Section C: questions about the hospital's readiness and ability to implement the statements. In section B, a value was allocated to each response to rate the degree to which they were able to comply with each statement (where 1=never able to comply, 5=always complied). In section C, they were asked to what degree they agreed with the question (1 for strongly disagree, 5 for strongly agree). A response of 1 or 2 was deemed to indicate some difficulty in complying with that statement-a 'negative response'. Where this was the case, the participant was given the option to provide a free text response explaining the reasons for this difficulty. The full results are discussed in a subsequent paper (see page 69). Overall, the response rate was 18%, but the variation in this was marked. 22 of the 34 countries had a response rate of over 30%. The methods used for this survey results informs the methodology and scope of future EAHP surveys Recommendations for future surveys are as follows: ▸ Keep the survey short and easy to complete (to within 20 min); ▸ Specifically enquire for each question if capacity and capability are the key barriers to implementation; ▸ Construct survey response options for each question to identify barriers other than capacity and capability; ▸ Identify the key drivers for change in countries where implementation has occurred or is occurring; ▸ Compile better intelligence about the number of surveys sent out in the first place (as those countries with a low response rate sent out lots of invites to participate which may be unrealistic); ▸ A named person (country coordinator) to send out invite survey link; ▸ Weekly reminders should be sent out by the named person (country coordinator).

KdPT, a tripeptide derivative of alpha-melanocyte-stimulating hormone, suppresses IL-1 beta-mediated cytokine expression and signaling in human sebocytes.

Acne is the most common inflammatory skin disease in which IL-1 plays a central role. Although alpha-melanocyte-stimulating hormone has immunomodulatory effects, its usefulness as an anti-inflammatory agent in acne is hampered owing to its lipid- and pigment-inducing effects via activation of melanocortin receptors (MC-Rs). We used the immortalized human sebocyte line SZ95 as an in vitro model to investigate the anti-inflammatory potential of KdPT, a tripeptide derivative of the C-terminal end of alpha-melanocyte-stimulating hormone. KdPT potently suppressed IL-1beta-induced IL-6 and IL-8 expression. Mechanistically, KdPT decreased IL-1beta-mediated IkappaBalpha degradation, reduced nuclear accumulation of p65, and attenuated DNA binding of NF-kappaB. Moreover, KdPT reduced IL-1beta-mediated generation of intracellular reactive oxygen species, which contributed to IL-1beta-mediated cytokine induction. KdPT also reduced cell surface binding of fluorochrome-labeled IL-1beta in SZ95 sebocytes. Analysis of the crystal structure of the complex between IL-1beta/IL-1R type I (IL-1RI), followed by computer modeling of KdPT and subsequent modeling of the peptide receptor complex with the crystal structure of IL-1RI via manual docking, further predicted that the tripeptide, through several H-bonds and one hydrophobic bond, interacts with the IL-1RI. Importantly, KdPT did not bind to MC-1Rs, as demonstrated by blocking experiments with a peptide analog of Agouti signaling protein and by binding assays using MC-1R-expressing B16 melanoma cells. Accordingly, KdPT failed to induce melanogenesis. Our data demonstrate a promising anti-inflammatory potential of KdPT and point toward novel future directions in the treatment of acne-as well as of various other IL-1-mediated inflammatory diseases-with this small molecule.

Stretch-tuneable dielectric mirrors and optical microcavities.

We demonstrate how tuneable Distributed Bragg Reflectors (DBRs) and resonant micro-cavities can be built by a scalable layer assembly of the transparent utility rubbers polydimethylsiloxane and polystyrene-polyisoprene. Stretching the devices by more than 60% leads to an affine contraction of the layer thicknesses thereby tuning both DBR and cavity modes across the entire visible spectrum. Such rapidly- and reversibly- stretch-tuneable cavities can be used in tuneable micro-lasers and for quantitative optical strain sensing applications.

Enhanced DNA binding capacity on up-regulated epidermal wild-type p53 in vitiligo by H2O2-mediated oxidation: a possible repair mechanism for DNA damage.

Vitiligo is characterized by a patchy loss of inherited skin color affecting approximately 0.5% of individuals of all races. Despite the absence of the protecting pigment and the overwhelming evidence for hydrogen peroxide (H(2)O(2))-induced oxidative stress in the entire epidermis of these patients, there is neither increased photodamage/skin aging nor a higher incidence for sun-induced nonmelanoma skin cancer. Here we demonstrate for the first time increased DNA damage via 8-oxoguanine in the skin and plasma in association with epidermal up-regulated phosphorylated/acetylated p53 and high levels of the p53 antagonist p76(MDM2). Short-patch base-excision repair via hOgg1, APE1, and polymerasebeta DNA repair is up-regulated. Overexpression of Bcl-2 and low caspase 3 and cytochrome c levels argue against increased apoptosis in this disease. Moreover, we show the presence of high epidermal peroxynitrite (ONOO(-)) levels via nitrotyrosine together with high nitrated p53 levels. We demonstrate by EMSA that nitration of p53 by ONOO(-) (300 x 10(-6) M) abrogates DNA binding, while H(2)O(2)-oxidized p53 (10(-3) M) enhances DNA binding capacity and prevents ONOO(-)-induced abrogation of DNA binding. Taken together, we add a novel reactive oxygen species to the list of oxidative stress inducers in vitiligo. Moreover, we propose up-regulated wild-type p53 together with p76(MDM2) as major players in the control of DNA damage/repair and prevention of photodamage and nonmelanoma skin cancer in vitiligo.

Computer simulation of heterogeneous single nucleotide polymorphisms in the catalase gene indicates structural changes in the enzyme active site, NADPH-binding and tetramerization domains: a genetic predisposition for an altered catalase in patients with vitiligo?

Patients with vitiligo have low levels/activities of catalase in their lesional and non-lesional epidermis as well as in their epidermal melanocytes under in vitro conditions while the levels of catalase mRNA are unaltered. This defect leads to a build-up of hydrogen peroxide (H(2)O(2)) in the 10(-3) m range in the epidermis of these patients. In this context, it was realized that 10(-3) m H(2)O(2) deactivates catalase. Along this line, it was also suspected that catalase in patients with vitiligo possesses a special sensitivity to this reactive oxygen species (ROS), and indeed several heterozygous single nucleotide polymorphisms (SNPs) have been documented in the cat gene of these patients. Based on the 3D structure of human catalase monomer, we have modelled the influence of three selected SNPs on the enzyme active site, on the NADPH- as well as the tetramerization-binding domains. Our results show that these SNPs severely alter catalase structurally, which in turn should make the enzyme more susceptible to ROS compared with wild-type enzyme. Taken together, the work presented herein together with the earlier results on SNPs in the cat gene suggests a genetic predisposition for an altered catalase in patients with vitiligo.

Presence of epidermal allantoin further supports oxidative stress in vitiligo.

Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the hydroxylation of hypoxanthine to xanthine and finally to uric acid in purine degradation. These reactions generate H(2)O(2) yielding allantoin from uric acid when reactive oxygen species accumulates. The presence of XO in the human epidermis has not been shown so far. As patients with vitiligo accumulate H(2)O(2) up to mm levels in their epidermis, it was tempting to examine whether this enzyme and consequently allantoin contribute to the oxidative stress theory in this disease. To address this question, reverse transcription-polymerase chain reaction, immunoreactivity, western blot, enzyme kinetics, computer modelling and high performance liquid chromatography/mass spectrometry analysis were carried out. Our results identified the presence of XDH/XO in epidermal keratinocytes and melanocytes. The enzyme is regulated by H(2)O(2) in a concentration-dependent manner, where concentrations of 10(-6 )m upregulates the activity. Moreover, we demonstrate the presence of epidermal allantoin in acute vitiligo, while this metabolite is absent in healthy controls. H(2)O(2)-mediated oxidation of Trp and Met in XO yields only subtle alterations in the enzyme active site, which is in agreement with the enzyme kinetics in the presence of 10(-3 )m H(2)O(2). Systemic XO activities are not affected. Taken together, our results provide evidence that epidermal XO contributes to H(2)O(2)-mediated oxidative stress in vitiligo via H(2)O(2)-production and allantoin formation in the epidermal compartment.

Methionine sulfoxide reductases A and B are deactivated by hydrogen peroxide (H2O2) in the epidermis of patients with vitiligo.

Patients with the depigmentation disorder vitiligo have low catalase expression/activities and constantly accumulate 10(-3) M hydrogen peroxide (H(2)O(2)) in their skin. Such high concentrations of H(2)O(2) oxidize L-methionine residues in proteins and peptides to (R and S)-methionine sulfoxide diasteriomers. In vivo FT-Raman Spectroscopy revealed the presence of methionine sulfoxide in the depigmented skin of patients with active vitiligo. In normal healthy human skin, methionine sulfoxide reductases A and B specifically reduce methionine sulfoxides (S) and (R), respectively, back to L-methionine consequently repairing oxidatively damaged proteins and peptides. In this report, we show that the expression/activities of MSRA and MSRB are significantly decreased in the epidermis of patients with vitiligo compared to healthy controls. Also, we used recombinant human MSRA and MSRB1 to show that both enzymes are deactivated by 10(-3) M H(2)O(2) by 85 and 40%, respectively. Structural modelling based on the crystal structure of human MSRA revealed that the active site of this enzyme is significantly altered after H(2)O(2)-mediated oxidation of L-methionine, L-tryptophan, and L-cysteine residues in its active site. Taken together, our results confirm that very important anti-oxidant enzymes are seriously affected in acute vitiligo.

Structural and functional alterations in the beta2-adrenoceptor are caused by a point mutation in patients with atopic eczema.

The density of beta2-adrenoceptors is significantly decreased in both keratinocytes and peripheral blood lymphocytes from patients with atopic eczema. Furthermore both cell types showed a sixfold increase in the K(D) for the specific binding of the non-specific antagonists (-)-[(3)H]CGP 12177 and [(125) I]CYP to keratinocytes and lymphocytes respectively compared with healthy controls. Based on these results polymorphism in the beta2-adrenoceptor gene was suspected. Consequently the entire intronless beta2-adrenoceptor gene was isolated from whole blood and by RT-PCR from keratinocyte extracts of nine patients with atopic eczema and four healthy controls. DNA sequence analysis of nine atopic eczema patients confirmed a substitution in codon (1618) GCC (Ala(119)) to GAC (Asp(119)). This point mutation is expressed on the third transmembrane helix only 13A away from the established agonist/antagonist binding site at Asp(113). Computer modelling of this third transmembrane helix revealed substantial structural changes in the mutant compared with the wild type. Epidermal keratinocytes were established from one patient with atopic eczema (homozygote), the mother (heterozygote) and one age-matched healthy control. Cells were grown in media containing different concentrations of l-phenylalanine and receptor densities were determined. The results showed that cells with atopic eczema showed an increased sensitivity to l-phenylalanine concentrations with a narrow homeostasis compared with healthy controls. The heterozygous mother was only 50% as sensitive as the child. In summary, the results indicate that atopic eczema is associated with a single point mutation in the beta2-adrenoceptor gene leading to an impaired adrenergic response in the epidermis of these patients.

Calcium-activated butyrylcholinesterase in human skin protects acetylcholinesterase against suicide inhibition by neurotoxic organophosphates.

The human epidermis holds an autocrine acetylcholine production and degradation including functioning membrane integrated and cytosolic butyrylcholinesterase (BuchE). Here we show that BuchE activities increase 9-fold in the presence of calcium (0.5x10(-3)M) via a specific EF-hand calcium binding site, whereas acetylcholinesterase (AchE) is not affected. (45)Calcium labelling and computer simulation confirmed the presence of one EF-hand binding site per subunit which is disrupted by H(2)O(2)-mediated oxidation. Moreover, we confirmed the faster hydrolysis by calcium-activated BuchE using the neurotoxic organophosphate O-ethyl-O-(4-nitrophenyl)-phenylphosphonothioate (EPN). Considering the large size of the human skin with 1.8m(2) surface area with its calcium gradient in the 10(-3)M range, our results implicate calcium-activated BuchE as a major protective mechanism against suicide inhibition of AchE by organophosphates in this non-neuronal tissue.

Oxidative stress via hydrogen peroxide affects proopiomelanocortin peptides directly in the epidermis of patients with vitiligo.

The human skin holds the capacity for autocrine processing of the proopiomelanocortin (POMC)-derived peptides. Recent data demonstrated the presence and functionality of ACTH, alpha- and beta-melanocyte-stimulating hormone (MSH), and beta-endorphin in the regulation of skin pigmentation, and a role has been put forward for alpha-MSH as an effective antioxidant. In patients with vitiligo, decreased epidermal POMC processing and low alpha-MSH levels were documented previously. These patients accumulate hydrogen peroxide (H2O2) in the 10(-3) M range in their epidermis. Therefore, we examined the involvement of H2O2 on POMC-derived peptides as possible targets for oxidation by this reactive oxygen species. To address this, we employed immunofluorescence labelling, dot blot analysis, Fourier transform Raman spectroscopy, functionality studies, and computer simulation of the peptide structures. We demonstrate H2O2-mediated oxidation of epidermal ACTH, alpha-MSH, and beta-endorphin in vitiligo owing to oxidation of methionine residues in the sequences of these peptides. Moreover, we show that oxidized beta-endorphin loses its function in the promotion of pigmentation in melanocytes. These changes are reversible upon the reduction of H2O2 levels by a pseudocatalase PC-KUS. Moreover, oxidation of alpha-MSH can be prevented by the formation of a 1:1 complex with the abundant cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin. Thus, using vitiligo, we demonstrate that H2O2 can affect pigmentation via epidermal POMC peptide redox homeostasis.

Computer simulation of native epidermal enzyme structures in the presence and absence of hydrogen peroxide (H2O2): potential and pitfalls.

The human epidermis is especially vulnerable to oxidative stress, which in turn leads to oxidation of important antioxidant enzymes, other proteins, and peptides. Molecular dynamic computer modelling is a new powerful tool to predict or confirm oxidative stress-mediated structural changes consequently altering the function of enzymes/proteins/peptides. Here we used examples of important epidermal antioxidant enzymes before and after hydrogen peroxide (H(2)O(2))-mediated oxidation of susceptible amino-acid residues (i.e. tryptophan, methionine, cysteine, and selenocysteine), which can affect enzyme active sites, cofactor binding, or dimerization/tetramerization domains. Computer modelling predicts that enzyme active sites are altered by H(2)O(2)-mediated oxidation in thioredoxin reductase (TR) and acetylcholinesterase (AchE), whereas cofactor nicotinamide adenine dinucleotide phosphate (reduced form) binding is affected in both catalase and TR but not in glutathione peroxidase. Dimerization is prevented in catalase. These structural changes lead to impaired functionality. Fourier transform-Raman- and Fluorescence spectroscopy together with enzyme kinetics support the results. There are limitations of modelling as demonstrated on the AchE substrate-binding domain, where the computer predicted deactivation, which could not be confirmed by enzyme kinetics. Computer modelling coupled with classical biochemical techniques offers a new powerful tool in cutaneous biology to explore oxidative stress-mediated metabolic changes in the skin.