Bortezomib in relapsed/refractory immune thrombotic thrombocytopenic purpura: A single-centre retrospective cohort and systematic literature review.

Immune thrombotic thrombocytopenic purpura (iTTP) is a rare and life-threatening haematological condition. Initial treatment involves plasma exchange (PLEX), corticosteroids, caplacizumab and rituximab. In relapsed and refractory cases despite initial treatments, further immune-modulating therapy includes the proteasome inhibitor, bortezomib. Evidence for bortezomib in this setting is limited to case reports and case series. We report our experience and perform a systematic review of the literature. We identified 21 publications with 28 unique patients in addition to our cohort of eight patients treated with bortezomib. The median age of patients was 44 years (IQR: 27-53) and 69% female. They were usually in an initial, refractory presentation of iTTP where they had received PLEX, corticosteroids, rituximab and another line of therapy. After bortezomib administration, 72% of patients had a complete response, with 85% maintaining a durable response without relapse at the last follow-up.

Immature Platelet Fraction as a Biomarker for Disease Severity in Pediatric Respiratory Coronavirus Disease 2019.

In this retrospective single-institution cohort study of 113 hospitalized pediatric patients with respiratory coronavirus disease 2019, those admitted to the intensive care unit or requiring mechanical ventilation had significantly higher immature platelet fractions than those who did not require intensive care unit-level care or ventilation. Immature platelet fraction may be an accessible biomarker for disease severity in pediatric respiratory coronavirus disease 2019.

Evaluation of head and neck soft tissue sarcoma 8th edition pathologic staging system and proposal of a novel stage grouping system.

BACKGROUND: The AJCC 8th edition issued a dedicated staging system for head and neck soft tissue sarcomas (HN-STS) with 2 and 4 cm tumor cut-off points, as well as a T4 classification based on invasion of adjacent structures. Stage groupings were not provided due to a paucity of data. METHODS: We identified HN-STS patients undergoing primary surgery without neoadjuvant therapy patients in the Surveillance, Epidemiology, and End Results (SEER) database. We used multivariable analysis to examine adverse prognosticators. Then, using, recursive partitioning analysis (RPA), we established a stage grouping system that was externally validated in the National Cancer Database (NCDB). RESULTS: Multivariable analysis in the SEER cohort (N = 546) demonstrated worsened survival with tumors invading adjacent structures (P < 0.001) and increasing de-differentiation (P < 0.001). There was no prognostic difference based on size for T1-3 tumors; however, when assessed as a continuous variable, a 5 cm tumor size cut-off point was predictive of outcome. RPA generated a stage grouping system with the following five-year overall survival: RPA Stage I (pT1-3N0-1G1-2M0) 71.2%, RPA Stage II (pT4abN0-1G1-2M0/pT1-3N0-1G3-4M0) 53.4%, and RPA Stage III (pT4abN0-1G3-4M0) 17.5%. This was successfully externally validated in the NCDB cohort (P < 0.001). CONCLUSIONS: We confirm the importance of structural invasion and grade and demonstrate that the currently used size cut-off points are not prognostic. We propose a novel stage grouping system. A 5 cm tumor size cut-off point for tumor stage should be further evaluated.

Pathologic staging changes in oral cavity squamous cell carcinoma: Stage migration and implications for adjuvant treatment.

BACKGROUND: The eighth edition of the AJCC Cancer Staging Manual (AJCC 8) incorporates depth of invasion (DOI) into the pathologic tumor (pT) classification and pathologic extranodal extension (pENE) into the pathologic nodal (pN) classification for oral cavity squamous cell carcinoma (OCSCC). This study evaluated the incidence and prognostic importance of stage migration as a result of these changes in the AJCC 8 staging system. METHODS: From the National Cancer Database, cohorts were identified from patients with OCSCC undergoing definitive surgery between 2004 and 2013 for pT (n = 7184), pN (n = 13,627), and pathologic stage (pStage) analysis (n = 5580). RESULTS: DOI and pENE were prognostic in all groups except for pN3 according to the seventh edition of the AJCC Cancer Staging Manual (AJCC 7). Upstaging was seen in 12.4% of patients for the pT classification, in 13.3% for the pN classification, and in 24.8% for the overall pStage grouping. Notably, upstaging led to similar or improved 5-year overall survival (OS) for every AJCC 8 pT/N classification except pStage IVB. Patients with AJCC 7 pT1 tumors that were upstaged to AJCC 8 pT3 tumors had improved OS in comparison with the remainder of the pT3 group (71.7% vs 43.7%; P < .0001). A multivariable analysis of upstaged pT3N0 patients demonstrated a reduced risk of death with the receipt of postoperative radiotherapy (PORT; hazard ratio, 0.56; 95% confidence interval, 0.33-0.95; P = .03). CONCLUSIONS: Upstaging is common in AJCC 8, and patients with upstaged tumors demonstrate improved survival; these factors should be kept in mind when one is interpreting data with the new staging system. PORT may reduce deaths among newly upstaged pT3N0 patients, and further study is needed in this area.

Radiation therapy treatment facility and overall survival in the adjuvant setting for locally advanced head and neck squamous cell carcinoma.

BACKGROUND: Treatment at high-volume surgical facilities (HVSFs) provides a survival benefit for patients with head and neck squamous cell carcinomas (HNSCCs); however, it is unknown what role postoperative radiation therapy (PORT) plays in achieving the improved outcomes. METHODS: From the National Cancer Database, 6844 patients with locally advanced invasive HNSCCs of the oral cavity, oropharynx, larynx, and hypopharynx who underwent definitive surgery with PORT between 2004 and 2013 were identified. HVSFs were those in the top percentile for annual case volume during this period. RESULTS: The median follow-up was 54 months. Compared with a lower volume surgical facility (LVSF), an HVSF improved 5-year overall survival (OS; 57.7% at HVSFs vs 52.5% at LVSFs; P = .0003). Overall, 31.6% of the patients changed their radiation therapy (RT) facility after surgery, with this being more common at HVSFs (39.1% vs 28.9% at LVSFs; P < .001). Among those patients undergoing surgery at an HVSF, remaining at the same facility for RT improved 5-year OS (63.1% vs 49.3% with a facility change; P < .0001). A propensity score-matched cohort of patients treated at HVSFs confirmed the improved 5-year OS when patients remained at the treating HVSF for RT (59.2% vs 50.7% with a facility change; P = .005). In a multivariate analysis, treatment at an HVSF and remaining there for RT resulted in a reduced hazard of death (hazard ratio, 0.81; 95% confidence interval, 0.69-0.94; P = .006). CONCLUSIONS: The survival benefit associated with HVSFs persists only when patients remain at the facility for RT, and this suggests that facility specialization and/or high-volume PORT may assist in driving the OS improvement.

Patterns of failure in high-metastatic node number human papillomavirus-positive oropharyngeal carcinoma.

BACKGROUND: The 8th edition American Joint Committee on Cancer staging system for resected HPV-positive oropharynx carcinoma (HPV+ OPC) highlights high node number as a critical determinant of survival. We sought to characterize outcomes and patterns of failure in patients with high pathologically involved node number oropharynx cancer. METHODS: We retrospectively identified 116 HPV+ OPC patients sequentially treated with neck dissection and either resection or intraoperative brachytherapy of the primary tumor between 2010 and 2016. External beam radiation was given based on the pathologic findings. Cox proportional hazards regression was used for multivariate analysis. RESULTS: With a median follow-up of 27 months, the 3-year overall survival and progression free survival (PFS) were 89% and 81%, respectively. On multivariate analysis, ≥5 involved lymph nodes was significantly associated with worse PFS (hazard ratio 4.3, 95% confidence interval (CI) 1.5-12.0, P = 0.001). Rates of 3-year locoregional recurrence (LRR) in patients with ≤4 vs ≥5 were 6% and 22% (log-rank P = 0.12). Rates of 3-year distant metastases (DM) were 12% and 53% between ≤4 and ≥5 (log-rank P < 0.001). CONCLUSION: Our findings confirm that patients with 5 or more involved lymph nodes appear to have substantially worsened rates of disease recurrence. While these patients appear to be at high risk of both LRR and DM, the predominant mechanism of failure is distant, and the rate of DM in this group was over 50%. Dedicated clinical trials in this patient population are warranted with a focus on mitigating the high DM rate.

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.

The risk of level IB nodal involvement in oropharynx cancer: Guidance for submandibular gland sparing irradiation.

PURPOSE: Xerostomia remains a common side effect of head and neck irradiation. Conflicting data exist regarding the likelihood of level IB involvement for patients with oropharyngeal squamous cell cancer (OPSCC), and data are limited on this risk in patients with human papillomavirus-positive disease. This study examined surgically treated OPSCC to determine the risk of pathologic level IB nodal involvement and to identify a cohort of patients in whom ipsilateral level IB radiation therapy may be safely omitted. METHODS AND MATERIALS: A total of 102 submandibular nodal dissections were identified (92 ipsilateral and 10 contralateral) in 92 patients from 2010 to 2016 in those undergoing primary surgical treatment and dissection of ipsilateral level IB lymph nodes. Radiographically positive cases were excluded. Retrospective chart review was used for data collection, and the rate of pathologic level IB involvement was determined. RESULTS: The ipsilateral level IB nodal station had negative imaging and pathologically positive nodes at rates of 4.3% in OPSCC and 5.3% in human papillomavirus-positive OPSCC. Positive node burden in the ipsilateral neck at stations other than IB appeared to correlate with the risk of pathologic positive IB (pIB+) nodes: 50% of pathologically IB-negative patients had 2 or more positive nodes versus 75% of pIB+ patients who had 4 or more positive nodes. CONCLUSIONS: Our data indicate a low risk of pathologic level IB involvement in early-stage OPSCC. High positive node burden in stations near level IB may be associated with a higher chance of pathologic level IB involvement.

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.

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.

Butyrylcholinesterase is present in the human epidermis and is regulated by H2O2: more evidence for oxidative stress in vitiligo.

The human epidermis holds the capacity for autocrine cholinergic signal transduction, but the presence of butyrylcholinesterase (BchE) has not been shown so far. Our results demonstrate that this compartment transcribes a functional BchE. Its activity is even higher compared to acetylcholinesterase (AchE). Moreover, we show that BchE is subject to regulation by H(2)O(2) in a concentration-dependent manner as it was recently described for AchE. Epidermal BchE protein expression and enzyme activities are severely affected by H(2)O(2) in vitiligo as previously demonstrated for AchE. Removal/reduction of H(2)O(2) by a pseudocatalase PC-KUS yields normal/increased protein expression and activities. H(2)O(2)-mediated oxidation of methionine residues in BchE was confirmed by FT-Raman spectroscopy. Computer simulation supported major alteration of the enzyme active site and its tetramerisation domain suggesting deactivation of the enzyme due to H(2)O(2)-mediated oxidation. Based on our results we conclude that H(2)O(2) is a major player in the regulation of the cholinergic signal via both AchE and BchE and this signal is severely affected in the epidermis of patients with active vitiligo.