Perioperative elafin for ischaemia-reperfusion injury during coronary artery bypass graft surgery: a randomised-controlled trial.

BACKGROUND: Elafin is a potent endogenous neutrophil elastase inhibitor that protects against myocardial inflammation and injury in preclinical models of ischaemic-reperfusion injury. We investigated whether elafin could inhibit myocardial ischaemia-reperfusion injury induced during coronary artery bypass graft (CABG) surgery. METHODS AND RESULTS: In a randomised double-blind placebo-controlled parallel group clinical trial, 87 patients undergoing CABG surgery were randomised 1:1 to intravenous elafin 200 mg or saline placebo administered after induction of anaesthesia and prior to sternotomy. Myocardial injury was measured as cardiac troponin I release over 48 h (area under the curve (AUC)) and myocardial infarction identified with MRI. Postischaemic inflammation was measured by plasma markers including AUC high-sensitive C reactive protein (hs-CRP) and myeloperoxidase (MPO). Elafin infusion was safe and resulted in >3000-fold increase in plasma elafin concentrations and >50% inhibition of elastase activity in the first 24 h. This did not reduce myocardial injury over 48 h (ratio of geometric means (elafin/placebo) of AUC troponin I 0.74 (95% CI 0.47 to 1.15, p=0.18)) although post hoc analysis of the high-sensitive assay revealed lower troponin I concentrations at 6 h in elafin-treated patients (median 2.4 vs 4.1 µg/L, p=0.035). Elafin had no effect on myocardial infarction (elafin, 7/34 vs placebo, 5/35 patients) or on markers of inflammation: mean differences for AUC hs-CRP of 499 mg/L/48 h (95% CI -207 to 1205, p=0.16), and AUC MPO of 238 ng/mL/48 h (95% CI -235 to 711, p=0.320). CONCLUSIONS: There was no strong evidence that neutrophil elastase inhibition with a single-dose elafin treatment reduced myocardial injury and inflammation following CABG-induced ischaemia-reperfusion injury. TRIAL REGISTRATION NUMBER: (EudraCT 2010-019527-58, ISRCTN82061264).

Human gammadelta T cells produce the protease inhibitor and antimicrobial peptide elafin.

Human gammadelta T cells rapidly secrete pro-inflammatory cytokines in response to T cell receptor-dependent recognition of pyrophosphates produced by many bacteria and parasites. In further support of an important role of gammadelta T cells in the immune defence against infection, human gammadelta T cells have been shown to produce the antimicrobial peptide LL37/cathelicidin. In this study, we have investigated whether gammadelta T cells can produce additional antimicrobial peptides. To this end, we have screened human gammadelta T cell clones by RT-PCR for mRNA expression of a broad range of antimicrobial peptides. While alpha-defensins were absent and beta-defensins (HBD1) present only in rare gammadelta T cell clones, elafin mRNA was induced by supernatant of Pseudomonas aeruginosa grown under static conditions. Elafin is a protease inhibitor that also displays antimicrobial activity. Constitutive intracellular expression of elafin was demonstrated by flow cytometry and Western blot analysis. Furthermore, trappin-2 (pre-elafin) could be immunoprecipitated in cell lysates but also in the supernatant of gammadelta T cells stimulated by Ps. aeruginosa supernatant. Taken together, our studies reveal a novel effector function of gammadelta T cells which might be important for local immune defence.

Treatment of photoaged skin with a cream containing 0.05% isotretinoin and sunscreens.

AIMS: To determine the safety and efficacy of a cream formulation of 0.05% isotretinoin with sunscreens (SPF 15) (I+S) in the treatment of photoaged skin. DESIGN AND SUBJECTS: A 6-month, multicentre, randomized, double-blind, parallel-group, vehicle-controlled study of 346 subjects with photoaged skin, as defined by the presence of fine lines in the periorbital region. The main outcome measure was profilometry measurements of replicas of the periorbital region, taken at 0 and 6 months. Subsidiary outcome measures were clinical scoring of wrinkles/fine lines at 0, 1, 3, 6 and 9 months and histopathology at 0 and 6 months. METHODS AND RESULTS: A total of 172 subjects were randomized to 0.05% I+S and 174 subjects to vehicle. Subjects used the study medication topically, once-daily for 6 months, followed by a 3-month washout period. Profilometry measurements of the distance between the highest peak and lowest valley (Rz) and the distance between all valleys and peaks from mid-line (Ra) showed that subjects using I+S had statistically significant improvement (p<0.05) compared with the vehicle group. Additionally, at all visits, VAS clinical scoring of wrinkles/fine lines showed a statistically significant difference between the groups in favour of I+S. Tolerance assessments showed that more subjects in the I+S group experienced local side effects at the start of the study; however, reports of side effects decreased over time in both groups. CONCLUSION: This study confirms that I+S improves the appearance of fine wrinkles associated with photoaged skin.

Neutrophil serine proteases: potential key regulators of cell signalling during inflammation.

The serine proteases cathepsin G, human leucocyte elastase and proteinase 3 are major contents of neutrophils and are released at sites of inflammation. The common picture of their function was that they do not degrade extracellular proteins specifically. Recent studies provided evidence that these proteases are able to activate specifically pro-inflammatory cytokines and lead to the activation of different receptors. Neutrophil serine proteases might therefore be important regulators of inflammatory processes and are interesting targets for new therapeutic approaches against inflammatory disorders. This review summarizes the current knowledge on the regulation of cell signalling by neutrophil serine proteases.

Mycosis fungoides of the larynx: case report and review of the literature.

Mycosis fungoides is a rare cutaneous lymphoma. Dissemination to extra-cutaneous sites occurs at advanced stages of disease. Laryngeal manifestations of mycosis fungoides have been reported in only 13 cases in the available literature. We present a further calla of mycosis fungoides of the larynx at the terminal stage of disease with an additional manifestation in the left maxillary sinus and review the cases published to date. To our knowledge this is the first reported case of mycosis fungoides with laryngeal and paranasal sinus manifestation. Concluding from the present case and from literature therapy should be palliative to improve quality of life.

Psoriasis scales contain C5a as the predominant chemotaxin for monocyte-derived dendritic cells.

Dendritic cells seem to be of major importance for the pathogenesis of psoriasis. They are increased in number in lesional psoriatic skin which is thought to be due to an increased influx from the peripheral blood regulated by chemotaxins. Using a biological/biochemical approach we have addressed the question whether psoriasis scale extracts contain proteinaceous chemotaxins for dendritic cells. Human monocytes differentiated into dendritic cells by culture with GM-CSF and IL-4 (MoDC) served as responder cells. Chemotactic activity for MoDC was purified by several HPLC-steps. The results of our study show that C5a/C5adesarg is the major chemotactic peptide for MoDC in psoriasis scale extracts. In comparison to other stimuli such as fMLP or monocyte chemotactic peptide 1 (MCP-1) C5a proved to be a most potent and efficient chemotaxin for MoDC. C5a co-eluted with MRP14/calgranulin B which is present in large amounts in psoriasis scale extracts as identified by amino acid sequencing. However, MRP14/calgranulin B did not possess any chemotactic activity for MoDC. Our results provide evidence that C5a/C5adesarg although not specific for dendritic cells seems to be the major chemoattractant for these cells in lesional psoriasis skin.

The trappin gene family: proteins defined by an N-terminal transglutaminase substrate domain and a C-terminal four-disulphide core.

Recently, several new genes have been discovered in various species which are homologous to the well-characterized human epithelial proteinase inhibitor elafin/SKALP (skin-derived anti-leukoproteinase). Because of the high degree of conservation and the similarities in genomic organization, we propose that these genes belong to a novel gene family. At the protein level, the family members are defined by: (1) an N-terminal domain consisting of a variable number of repeats with the consensus sequence Gly-Gln-Asp-Pro-Val-Lys that can act as an anchoring motif by transglutaminase cross-linking, and (2) a C-terminal four-disulphide core or whey acidic protein (WAP) domain, which harbours a functional motif involved in binding of proteinases and possibly other proteins. We have proposed the name trappin gene family as a unifying nomenclature for this group of proteins (trappin is an acronym for TRansglutaminase substrate and wAP domain containing ProteIN, and refers to its functional property of 'getting trapped' in tissues by covalent cross-linking). Analysis of the trappin family members shows extensive diversification in bovidae and suidae, whereas the number of primate trappins is probably limited. Recent biochemical and cell biological data on the human trappin family member elafin/SKALP suggest that this molecule is induced in epidermis by cellular stress. We hypothesize that trappins play an important role in the regulation of inflammation and in protection against tissue damage in stratified epithelia.

Structural changes of human epidermis induced by human leukocyte-derived proteases.

During the process of inflammation human neutrophils release potent serine proteases, such as human leukocyte elastase and cathepsin G. In psoriasis these enzymes are released within the epidermis. To investigate the destructive potential of neutrophil-derived serine proteases these were applied on viable human epidermis as well as full thickness human skin in vitro. Human leukocyte elastase and cathepsin G were found to dissociate keratinocytes from epidermal sheets in a time- and dose-dependent fashion. Significant keratinocyte dissociation was observed 4 h after application of 3 nM human leukocyte elastase. By electron microscopy of elastase- or cathepsin G-treated full thickness human skin, widening of the extracellular space followed by complete separation of keratinocytes without intradesmosomal cleavage was observed. In addition, cathepsin G induced membrane damage as well as destruction of intracellular organelles. Thus, neutrophil-derived serine proteases exert pronounced destructive potential in human epidermis in concentrations likely to appear in lesional psoriatic skin.

Antileukoprotease in human skin: an antibiotic peptide constitutively produced by keratinocytes.

Antileukoprotease (ALP), also known as mucous protease inhibitor or secretory leukoprotease inhibitor, resembles one of the major antiproteases present in human body fluids. It is capable of preventing proteolytic degradation of extracellular matrix proteins by neutrophil-derived serine proteases. ALP was isolated from human callus and detected in supernatants of cultured human primary keratinocytes. ALP mRNA was constitutively expressed in keratinocytes and the expression was not significantly affected by TNF alpha or Interferon gamma stimulation. In microbicidal assays recombinant ALP exhibited antimicrobial activity against several human skin associated microorganisms like P. aeruginosa, S. aureus, S. epidermidis, and C. albicans, indicating that ALP may actively participate in mechanisms allowing homeostasis of bacterial and yeast colonization on human skin. Thus, ALP represents a major soluble serine protease inhibitor and antimicrobial agent expressed in human skin and seems to contribute to the high resistance of the epidermis against proteolysis and infections.

The effect of tamol on human mast cell chymase and plasmin.

Tamol is widely used in the therapy of inflammatory dermatoses. It has pronounced astringent properties and is able to inactivate the neutrophil-derived elastase. Since plasminogen activation and release of mast cell chymase may occur in acute dermatitis, we investigated the inhibitory properties of tamol for these enzymes. Tamol proved to be a potent inhibitor of plasmin and mast cell chymase in concentrations relevant for use in dermatotherapy. The inhibition of mast cell chymase and plasmin by tamol was linear and non-competitive. The inactivation of proteolytic enzymes with the capacity to degrade extracellular-matrix proteins may be one of the major clinical effects of tamol in the treatment of acute inflammatory dermatoses.

[Oral exposure testing in non-aspirin-induced analgesic intolerance]. / Orale Expositionstestungen bei nicht Aspirin-bedingten Analgetikaintoleranzen.

Although intolerance reaction to analgesics are not uncommon, there is still a lack of standardized procedures to diagnose the problem. We retrospectively analyzed results of scratch tests as well as oral challenges with analgesics in order to evaluate risk and diagnostic relevance of these procedures. In 1987-1992 a total of 650 patients with supposed intolerance to drugs were tested by oral challenge. Among them were 98 patients with a positive history of intolerance to non-aspirin analgesics. In 56 patients the intolerance could be verified by oral challenge. In order of decreasing frequency, the most likely agents were propyphenazone, diclofenac, metamizole, ibuprofen, carbamazepine, indomethacin, phenazone (antipyrine), and paracetamol (acteaminophen). Oral provocation showed clear dose-response relationships. For propyphenazone, the half-effective provocation dose was the same for all symptoms (cutaneous, nasal, bronchial, anaphylactoid). Scratch testing was not of diagnostic significance. Standardized test protocols starting with low dose oral challenges are suitable and helpful in minimizing the risk of severe side effects.

Antileukoprotease inhibits stratum corneum chymotryptic enzyme. Evidence for a regulative function in desquamation.

The stratum corneum chymotryptic enzyme (SCCE) has been previously purified from human stratum corneum and resembles a chymotryptic serine endopeptidase involved in physiological detachment of corneocytes from human stratum corneum. From human stratum corneum two inhibitory activities of SCCE could be extracted. These were due to serine protease inhibitors already known to be present in human epidermis, antileukoprotease (secretory leukocyte protease inhibitor) and elafin (skin-derived antileukoprotease). The Inhibition of SCCE by antileukoprotease shows a hyperbolic, mixed type inhibition with an equilibrium dissociation constant of 63 n. Antileukoprotease also inhibits detachment of corneocytes from human plantar callus in vitro almost completely (>96%). In addition, elafin was shown to be a weak inhibitor for SCCE activity, and elafin significantly reduces the shedding of corneocytes. Thus, antileukoprotease, which is known to be produced by human keratinocytes, is likely to be the major physiological inhibitor of SCCE in the epidermis. It seems to be involved in the regulation of desquamation under physiological and pathophysiological conditions.

Treatment of psoriasis with polyethylene sheet bath PUVA.

BACKGROUND: Bath PUVA has been shown to be an effective alternative treatment for psoriasis with fewer systemic side effects than oral methoxsalen (8-MOP). The cost of 8-MOP and the need for a bath unit have prevented wider use of this treatment. OBJECTIVE: We investigated the safety and efficacy of sheet bath PUVA by restricting the volume of the psoralen/bath water solution to 10 L with the aid of a polyethylene sheet. METHODS: Fifty-eight patients with chronic plaque-type psoriasis were treated with bath PUVA in a concentration of 0.5 mg of 8-MOP per liter of water. RESULTS: The group required a median of 17 baths (95% confidence interval [CI], 14-20) for clearance. Total UVA dose for the entire group was 26 J/cm2(95% CI, 18-47). CONCLUSION: Sheet bath PUVA is safe, efficient, and easy. This regimen can significantly reduce the amount of 8-MOP required, thereby resulting in a favorable cost/benefit ratio.

Foil bath PUVA in the treatment of prurigo simplex subacuta.

Prurigo simplex subacuta is a chronic pruritic condition of unknown aetiology. The skin lesions respond to topical corticosteroids, UV-A and UV-B therapy only to a limited degree. Ten patients suffering from prurigo simplex subacuta were treated with foil bath PUVA at a concentration of 0.5 mg 8-methoxypsoralen/l. Using the foil bath method the volume of the psoralen/bath-water solution is restricted to 10 l with the aid of polyethylene foil. The group required a median of 13 (95% CI: 9-19) baths for clearance. The total UV-A dose for the whole group was 19 (95% CI:5-30) J/cm2. Bath PUVA is a safe and well-tolerated therapy in the treatment of prurigo simplex subacuta.

Human eosinophils lack human leukocyte elastase.

Recently, human leukocyte elastase has been detected in human eosinophils. Reinvestigating these findings, 2.5 pg active human leukocyte elastase (E.C. 3.4.21.37) were found per neutrophil isolated from peripheral blood, whereas the elastase activity of eosinophil preparations was linearly correlated with the content of contaminating neutrophils. Also spontaneous or stimulated release of active elastase was absent in eosinophils. By immunohistochemistry no elastase immunoreactivity could be demonstrated in human eosinophils. Therefore, we conclude that human eosinophils do not contain considerable amounts of human leukocyte elastase.

Antiprotease activity in urine of patients with inflammatory skin disorders.

Polymorphonuclear leukocytes contain well-defined proteolytic enzymes in their azurophilic granules that can be released into tissues during inflammation, producing a localized excess of proteases that causes a protease-antiprotease imbalance with subsequent tissue destruction. The antiproteolytic compounds of the epidermis, such as the protease inhibitors elafin and antileukoprotease, are thought to counteract the proteolytic tissue damage. We investigated the urine of patients suffering from inflammatory skin conditions (e.g., erysipelas, psoriasis) for the presence of urinary antiprotease activities. Purification of elastase-inhibitory activities from pooled urine samples by cation exchange high-performance liquid chromatography and preparative and analytical reverse-phase high-performance liquid chromatography yielded two different types of inhibitors. One was a cationic, acid-stable, and elastase-specific inhibitor of M(r) 6,000 by size-exclusion high-performance liquid chromatography. N-terminal amino acid sequence analysis of the first 28 residues showed identity with elafin, an elastase-specific inhibitor recently isolated from psoriatic scales. The second anti-protease activity was due to two forms of urinary bikunin, the inhibitory subunit of inter-alpha-inhibitor. Both bikunin fragments, with M(r) 4,000 and 16,000, were identified by N-terminal amino acid sequence analysis of the first 10 residues and were characterized by an antiproteolytic profile against human leukocyte elastase, cathepsin G, and trypsin. Urinary protease inhibitors may serve as diagnostic markers of inflammatory diseases.

Lesional elastase activity in psoriasis. Diagnostic and prognostic significance.

Human leukocyte elastase, a neutrophil-derived serine protease, is present in psoriatic lesions in an enzymatically active form. Our purpose was to assess the significance of human leukocyte elastase determinations in estimating the inflammatory activity of psoriatic lesions. A standardized method was used to analyse lesional elastase activity. Elastase activities were correlated with erythema, induration and hyperkeratosis of psoriatic lesions in 54 patients. Lesional elastase activities were also determined during treatment with salt-water bathing and UVB irradiation. Lesional elastase activity correlated with skin induration and was inversely correlated with hyperkeratosis of the lesions. Psoriatic lesions with high elastase activity responded well to therapy, whereas lesions with low elastase activity appeared to be comparatively resistant. This study shows that by quantitative determination lesional elastase activities it is possible to distinguish predominantly inflammatory from predominantly hyperproliferative psoriasis. The latter shows delayed responsiveness to topical therapy with salt-water bathing plus UVB irradiation.

[Innovative balneotherapy with reduced bath volume: foil baths]. / Innovative Balneotherapie mit reduzierten Badevolumina: Folienbäder.

Balneo-phototherapy, in which salt baths are followed by UV-B irradiation, has proved successful in the treatment of psoriasis. Because of practical problems, the major one of which is the large turnover of bath solution volumes, balneotherapy has so far been limited to specialized treatment centres. With the aid of a polyethylene foil the volume of bathing solutions needed can be reduced to a total of 41 per bath. Balneotherapy using small bath solution volumes for total body treatment can now be applied as an outpatient regimen for salt bath and bath-PUVA therapy. The methods for establishing balneotherapy with restricted water volumes and the advantages of bath-PUVA over conventional oral psoralen therapy are described.

Antileukoprotease in psoriatic scales.

Antileukoprotease is known to be an antiproteolytic compound of mucous secretions in humans. While searching for peptide-like inhibitors of neutrophil-derived serine proteases in horny layers of human skin, we isolated a potent inhibitor of human leukocyte elastase (EC 3.4.21.37) and cathepsin G (EC 3.4.21.20) from psoriatic scales. This inhibitor showed inhibitory constants for human leukocyte elastase of approximately 0.5-2 x 10(-10) M and for cathepsin G of 2-4 x 10(-9) N. The N-terminal amino acid sequence of the purified peptide matched the sequence of antileukoprotease and both peptides showed the same M(r) on SDS-PAGE. Therefore, antileukoprotease may not only regulate serine protease activities in mucous secretions, but also in skin.

Inhibition of proteinase 3 activity by peptides derived from human epidermis.

Elafin and antileukoprotease are potent peptide-like inhibitors of human leukocyte elastase and have been isolated from human skin and bronchial mucus. Elafin proved to be a potent inhibitor of proteinase 3, whereas other inhibitors of human leukocyte elastase such as antileukoprotease and eglin C proved to be much less effective.