Practical approach to the use of daylight photodynamic therapy with topical methyl aminolevulinate for actinic keratosis: a European consensus

INTRODUCTION Daylight-mediated photodynamic therapy has been shown to be an effective therapy for actinic keratoses (AKs) and a simple and tolerable treatment procedure in three randomized Scandinavian studies and two recent Phase III randomized controlled studies in Australia and Europe. OBJECTIVES To establish consensus recommendations for the use of daylight photodynamic therapy (DL-PDT) using topical methyl aminolaevulinate (MAL) in European patients with AKs. METHODS The DL-PDT consensus recommendations were developed on behalf of the European Society for Photodynamic Therapy in Dermatology and comprised of 10 dermatologists from different European countries with experience in how to treat AK patients with PDT. Consensus was developed based on literature review and experience of the experts in the treatment of AK using DL-PDT. RESULTS The recommendations arising from this panel of experts provide general guidance on the use of DL-PDT as a dermatological procedure with specific guidance regarding patient selection, therapeutic indications, when to treat, pre-treatment skin preparation, MAL application and daylight exposure for patients with AK in different countries of Europe. CONCLUSIONS This consensus recommendation provides a framework for physicians to perform DL-PDT with MAL cream while ensuring efficiency and safety in the treatment of patients with AK in different European countries.

Efficacy of topical tacrolimus for oral lichen planus: real-life experience in a retrospective cohort of patients with a review of the literature

BACKGROUND AND OBJECTIVE Management of oral lichen planus (OLP) is challenging and therapeutic options are limited. The use of topical tacrolimus has shown promising results. We reviewed our daily life experience with topical tacrolimus in OLP patients. METHODS This retrospective unicentre study included all 21 patients with OLP, which were evaluated over a 53-month period and treated with topical tacrolimus. Patients were initially given a topical preparation of 0.1% tacrolimus twice daily. The response to treatment was assessed using a 4-point scale at month 2 and 6: complete response of affected area (CR), major remission (>50%, MR), partial remission (25-50%, PR) and either no response (<25%) or worsening. The pain score was also assessed using a 3-point scale. RESULTS Four of 21 patients (19%) showed a CR at month 2, whereas at month 6, 7 (33%) had a CR. For patients who reported MR (n = 2) and PR (n = 8) at month 2, the therapy was continued. Of those, at 6 months, three patients showed a CR, while four maintained a PR. The pain score improved during treatment. After 2 months of therapy, eight of 10 patients with an initial high pain score achieved a significant improvement. In patients starting with moderate pain an improvement was observed in one of seven patients. Overall, for three patients there was a complete loss of pain, while in nine there was a reduction. Except for transitory burning sensation and altered taste sensation, no relevant side-effects were reported. CONCLUSION This retrospective analysis confirms that topical tacrolimus is a valuable therapeutic option in severe or treatment-resistant OLP. Our findings in daily practice suggested nevertheless that the efficacy of topical tacrolimus is overestimated with regard to both complete response and pain reduction.

Does Full Wound Rupture following Median Pilonidal Closure Alter Long-Term Recurrence Rate?

OBJECTIVE The purpose of this study was to examine the recurrence rate of wound rupture in primary pilonidal sinus disease (PSD) after median closure. SUBJECTS AND METHODS A total of 583 patients from the German military cohort were interviewed. We compared the choice of surgical therapy, wound dehiscence (if present) and long-term recurrence-free survival for patients with primary open treatment, marsupialization and primary median treatment (closed vs. secondary open, respectively). Actuarial recurrence rate was determined using the Kaplan-Meier calculation with a follow-up of up to 20 years after primary PSD surgery. RESULTS Patients with excision followed by primary open wound treatment showed a significantly lower 5- than 10-year recurrence rate (8.3 vs. 11.2%) compared to the patients with primary midline closure (17.4 vs. 20.5%, p = 0.03). The 20-year recurrence rate was 28% in primary open wound treatment versus 44% in primary midline closure without wound rupture. In contrast to these findings, long-term recurrence rates following secondary open wound treatment (12.2% at 5 years vs. 17.1% at 10 years) tended to be higher (although not significantly, p = 0.57) compared to primary open treatment (8.3% at 5 years vs. 11.2% at 10 years). There was no statistical difference in long-term recurrence rates between secondary open and primary midline closure (p = 0.7). Hence, despite only a short wound closure time experienced before wound rupture, the patient does not fully benefit from an open wound treatment in terms of recurrence rate. CONCLUSION The postoperative pilonidal sinus wound rupture of primary midline closures did not significantly increase the 5- and 10-year long-term recurrence rates compared to uneventfully healing primary midline closures.

Multiple functions of gingival and mucoperiosteal fibroblasts in oral wound healing and repair

Fibroblasts are cells of mesenchymal origin. They are responsible for the production of most extracellular matrix in connective tissues and are essential for wound healing and repair. In recent years, it has become clear that fibroblasts from different tissues have various distinct traits. Moreover, wounds in the oral cavity heal under very special environmental conditions compared with skin wounds. Here, we reviewed the current literature on the various interconnected functions of gingival and mucoperiosteal fibroblasts during the repair of oral wounds. The MEDLINE database was searched with the following terms: (gingival OR mucoperiosteal) AND fibroblast AND (wound healing OR repair). The data gathered were used to compare oral fibroblasts with fibroblasts from other tissues in terms of their regulation and function during wound healing. Specifically, we sought answers to the following questions: (i) what is the role of oral fibroblasts in the inflammatory response in acute wounds; (ii) how do growth factors control the function of oral fibroblasts during wound healing; (iii) how do oral fibroblasts produce, remodel and interact with extracellular matrix in healing wounds; (iv) how do oral fibroblasts respond to mechanical stress; and (v) how does aging affect the fetal-like responses and functions of oral fibroblasts? The current state of research indicates that oral fibroblasts possess unique characteristics and tightly controlled specific functions in wound healing and repair. This information is essential for developing new strategies to control the intraoral wound-healing processes of the individual patient.

Local Heat Application for the Treatment of Buruli Ulcer: Results of a Phase II Open Label Single Center Non Comparative Clinical Trial.

BACKGROUND Buruli ulcer (BU) is a necrotizing skin disease most prevalent among West African children. The causative organism, Mycobacterium ulcerans, is sensitive to temperatures above 37°C. We investigated the safety and efficacy of a local heat application device based on phase change material. METHODS In a phase II open label single center noncomparative clinical trial (ISRCTN 72102977) under GCP standards in Cameroon, laboratory confirmed BU patients received up to 8 weeks of heat treatment. We assessed efficacy based on the endpoints 'absence of clinical BU specific features' or 'wound closure' within 6 months ("primary cure"), and 'absence of clinical recurrence within 24 month' ("definite cure"). RESULTS Of 53 patients 51 (96%) had ulcerative disease. 62% were classified as World Health Organization category II, 19% each as category I and III. The average lesion size was 45 cm(2). Within 6 months after completion of heat treatment 92.4% (49 of 53, 95% confidence interval [CI], 81.8% to 98.0%) achieved cure of their primary lesion. At 24 months follow-up 83.7% (41 of 49, 95% CI, 70.3% to 92.7%) of patients with primary cure remained free of recurrence. Heat treatment was well tolerated; adverse effects were occasional mild local skin reactions. CONCLUSIONS Local thermotherapy is a highly effective, simple, cheap and safe treatment for M. ulcerans disease. It has in particular potential as home-based remedy for BU suspicious lesions at community level where laboratory confirmation is not available. CLINICAL TRIALS REGISTRATION ISRCT 72102977.

First Staphylococcal Cassette Chromosome mec Containing a mecB-Carrying Gene Complex Independent of Transposon Tn6045 in a Macrococcus caseolyticus Isolate from a Canine Infection.

A methicillin-resistant mecB-positive Macrococcus caseolyticus (strain KM45013) was isolated from the nares of a dog with rhinitis. It contained a novel 39-kb transposon-defective complete mecB-carrying staphylococcal cassette chromosome mec element (SCCmecKM45013). SCCmecKM45013 contained 49 coding sequences (CDSs), was integrated at the 3' end of the chromosomal orfX gene, and was delimited at both ends by imperfect direct repeats functioning as integration site sequences (ISSs). SCCmecKM45013 presented two discontinuous regions of homology (SCCmec coverage of 35%) to the chromosomal and transposon Tn6045-associated SCCmec-like element of M. caseolyticus JCSC7096: (i) the mec gene complex (98.8% identity) and (ii) the ccr-carrying segment (91.8% identity). The mec gene complex, located at the right junction of the cassette, also carried the β-lactamase gene blaZm (mecRm-mecIm-mecB-blaZm). SCCmecKM45013 contained two cassette chromosome recombinase genes, ccrAm2 and ccrBm2, which shared 94.3% and 96.6% DNA identity with those of the SCCmec-like element of JCSC7096 but shared less than 52% DNA identity with the staphylococcal ccrAB and ccrC genes. Three distinct extrachromosomal circularized elements (the entire SCCmecKM45013, ΨSCCmecKM45013 lacking the ccr genes, and SCCKM45013 lacking mecB) flanked by one ISS copy, as well as the chromosomal regions remaining after excision, were detected. An unconventional circularized structure carrying the mecB gene complex was associated with two extensive direct repeat regions, which enclosed two open reading frames (ORFs) (ORF46 and ORF51) flanking the chromosomal mecB-carrying gene complex. This study revealed M. caseolyticus as a potential disease-associated bacterium in dogs and also unveiled an SCCmec element carrying mecB not associated with Tn6045 in the genus Macrococcus.

Severe postoperative wound healing disturbance in a patient with alpha-1-antitrypsin deficiency: the impact of augmentation therapy

Wound healing disturbance is a common complication following surgery, but the underlying cause sometimes remains elusive. A 50-year-old Caucasian male developed an initially misunderstood severe wound healing disturbance following colon and abdominal wall surgery. An untreated alpha-1-antitrypsin (AAT) deficiency in the patient's medical history, known since 20 years and clinically apparent as a mild to moderate chronic obstructive pulmonary disease, was eventually found to be at its origin. Further clinical work-up showed AAT serum levels below 30% of the lower reference value; phenotype testing showed a ZZ phenotype and a biopsy taken from the wound area showed the characteristic, disease-related histological pattern of necrotising panniculitits. Augmentation therapy with plasma AAT was initiated and within a few weeks, rapid and adequate would healing was observed. AAT deficiency is an uncommon but clinically significant, possible cause of wound healing disturbances. An augmentation therapy ought to be considered in affected patients during the perioperative period.

Rapid temperature-dependent wound closure following adipose fin clipping of Atlantic salmon Salmo salar L.

Three groups of Atlantic salmon were kept at a constant temperature of 4, 10 and 14 °C. The adipose fins were removed; six fish/group were sampled at 11 subsequent time points post-clipping. Samples were prepared for histopathological examination to study the course of re-epithelization. A score sheet was developed to assess the regeneration of epidermal and dermal cell types. Wounds were covered by a thin epidermal layer between 4 and 6 h post-clipping at 10 and 14 °C. In contrast, wound closure was completed between 6 and 12 h in fish held at a constant temperature of 4 °C. By 18 h post-clipping, superficial cells, cuboidal cells, prismatic basal cells and mucous cells were discernible in all temperature groups, rapidly progressing towards normal epidermal structure and thickness. Within the observation period, only minor regeneration was found in the dermal layers. A positive correlation between water temperature and healing rates was established for the epidermis. The rapid wound closure rate, epidermal normalization and the absence of inflammatory reaction signs suggest that adipose fin clipping under anaesthesia constitutes a minimally invasive method that may be used to mark large numbers of salmon presmolts without compromising fish welfare.

Spatial Distribution of Stem Cell-Like Keratinocytes in Dissected Compound Hair Follicles of the Dog

Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i) compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii) the lower isthmus (comprising the bulge) harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii) unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv) the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients.

Development of a Novel Rabies Simulation Model for Application in a Non-endemic Environment.

Domestic dog rabies is an endemic disease in large parts of the developing world and also epidemic in previously free regions. For example, it continues to spread in eastern Indonesia and currently threatens adjacent rabies-free regions with high densities of free-roaming dogs, including remote northern Australia. Mathematical and simulation disease models are useful tools to provide insights on the most effective control strategies and to inform policy decisions. Existing rabies models typically focus on long-term control programs in endemic countries. However, simulation models describing the dog rabies incursion scenario in regions where rabies is still exotic are lacking. We here describe such a stochastic, spatially explicit rabies simulation model that is based on individual dog information collected in two remote regions in northern Australia. Illustrative simulations produced plausible results with epidemic characteristics expected for rabies outbreaks in disease free regions (mean R0 1.7, epidemic peak 97 days post-incursion, vaccination as the most effective response strategy). Systematic sensitivity analysis identified that model outcomes were most sensitive to seven of the 30 model parameters tested. This model is suitable for exploring rabies spread and control before an incursion in populations of largely free-roaming dogs that live close together with their owners. It can be used for ad-hoc contingency or response planning prior to and shortly after incursion of dog rabies in previously free regions. One challenge that remains is model parameterisation, particularly how dogs' roaming and contacts and biting behaviours change following a rabies incursion in a previously rabies free population.

Gene expression profile in newborn rat lungs after two days of recovery of mechanical ventilation.

BACKGROUND Preterm infants having immature lungs often require respiratory support, potentially leading to bronchopulmonary dysplasia (BPD). Conventional BPD rodent models based on mechanical ventilation (MV) present outcome measured at the end of the ventilation period. A reversible intubation and ventilation model in newborn rats recently allowed discovering that different sets of genes modified their expression related to time after MV. In a newborn rat model, the expression profile 48 h after MV was analyzed with gene arrays to detect potentially interesting candidates with an impact on BPD development. METHODS Rat pups were injected P4-5 with 2 mg/kg lipopolysaccharide (LPS). One day later, MV with 21 or 60% oxygen was applied during 6 h. Animals were sacrified 48 h after end of ventilation. Affymetrix gene arrays assessed the total gene expression profile in lung tissue. RESULTS In fully treated animals (LPS + MV + 60% O(2)) vs. controls, 271 genes changed expression significantly. All modified genes could be classified in six pathways: tissue remodeling/wound repair, immune system and inflammatory response, hematopoiesis, vasodilatation, and oxidative stress. Major alterations were found in the MMP and complement system. CONCLUSION MMPs and complement factors play a central role in several of the pathways identified and may represent interesting targets for BPD treatment/prevention.Bronchopulmonary dysplasia (BPD) is a chronic lung disease occurring in ~30% of preterm infants born less than 30 wk of gestation (1). Its main risk factors include lung immaturity due to preterm delivery, mechanical ventilation (MV), oxygen toxicity, chorioamnionitis, and sepsis. The main feature is an arrest of alveolar and capillary formation (2). Models trying to decipher genes involved in the pathophysiology of BPD are mainly based on MV and oxygen application to young mammals with immature lungs of different species (3). In newborn rodent models, analyses of lung structure and gene and protein expression are performed for practical reasons directly at the end of MV (4,5,6). However, later appearing changes of gene expression might also have an impact on lung development and the evolution towards BPD and cannot be discovered by such models. Recently, we developed a newborn rat model of MV using an atraumatic (orotracheal) intubation technique that allows the weaning of the newborn animal off anesthesia and MV, the extubation to spontaneous breathing, and therefore allows the evaluation of effects of MV after a ventilation-free period of recovery (7). Indeed, applying this concept of atraumatic intubation by direct laryngoscopy, we recently were able to show significant differences between gene expression changes appearing directly after MV compared to those measured after a ventilation-free interval of 48 h. Immediately after MV, inflammation-related genes showed a transitory modified expression, while another set of more structurally related genes changed their expression only after a delay of 2 d (7). Lung structure, analyzed by conventional 2D histology and also by 3D reconstruction using synchrotron x-ray tomographic microscopy revealed, 48 h after end of MV, a reduced complexity of lung architecture compared to the nonventilated rat lungs, similar to the typical findings in BPD. To extend these observations about late gene expression modifications, we performed with a similar model a full gene expression profile of lung tissue 48 h after the end of MV with either room air or 60% oxygen. Essentially, we measured changes in the expression of genes related to the MMPs and complement system which played a role in many of the six identified mostly affected pathways.

Prevalence estimation of tick-borne encephalitis virus (TBEV) antibodies in dogs from Finland using novel dog anti-TBEV IgG MAb-capture and IgG immunofluorescence assay based on recombinant TBEV subviral particles

Tick-borne encephalitis (TBE) is one of the most dangerous human neurological infections occurring in Europe and Northern parts of Asia with thousands of cases and millions vaccinated against it. The risk of TBE might be assessed through analyses of the samples taken from wildlife or from animals which are in close contact with humans. Dogs have been shown to be a good sentinel species for these studies. Serological assays for diagnosis of TBE in dogs are mainly based on purified and inactivated TBEV antigens. Here we describe novel dog anti-TBEV IgG monoclonal antibody (MAb)-capture assay which is based on TBEV prME subviral particles expressed in mammalian cells from Semliki Forest virus (SFV) replicon as well as IgG immunofluorescence assay (IFA) which is based on Vero E6 cells transfected with the same SFV replicon. We further demonstrate their use in a small-scale TBEV seroprevalence study of dogs representing different regions of Finland. Altogether, 148 dog serum samples were tested by novel assays and results were compared to those obtained with a commercial IgG enzyme immunoassay (EIA), hemagglutination inhibition test and IgG IFA with TBEV infected cells. Compared to reference tests, the sensitivities of the developed assays were 90-100% and the specificities of the two assays were 100%. Analysis of the dog serum samples showed a seroprevalence of 40% on Åland Islands and 6% on Southwestern archipelago of Finland. In conclusion, a specific and sensitive EIA and IFA for the detection of IgG antibodies in canine sera were developed. Based on these assays the seroprevalence of IgG antibodies in dogs from different regions of Finland was assessed and was shown to parallel the known human disease burden as the Southwestern archipelago and Åland Islands in particular had considerable dog TBEV antibody prevalence and represent areas with high risk of TBE for humans.

The Honey Bee Pathosphere of Mongolia: European Viruses in Central Asia

Parasites and pathogens are apparent key factors for the detrimental health of managed European honey bee subspecies, Apis mellifera. Apicultural trade is arguably the main factor for the almost global distribution of most honey bee diseases, thereby increasing chances for multiple infestations/infections of regions, apiaries, colonies and even individual bees. This imposes difficulties to evaluate the effects of pathogens in isolation, thereby creating demand to survey remote areas. Here, we conducted the first comprehensive survey for 14 honey bee pathogens in Mongolia (N = 3 regions, N = 9 locations, N = 151 colonies), where honey bee colonies depend on humans to overwinter. In Mongolia, honey bees, Apis spp., are not native and colonies of European A. mellifera subspecies have been introduced ~60 years ago. Despite the high detection power and large sample size across Mongolian regions with beekeeping, the mite Acarapis woodi, the bacteria Melissococcus plutonius and Paenibacillus larvae, the microsporidian Nosema apis, Acute bee paralysis virus, Kashmir bee virus, Israeli acute paralysis virus and Lake Sinai virus strain 2 were not detected, suggesting that they are either very rare or absent. The mite Varroa destructor, Nosema ceranae and four viruses (Sacbrood virus, Black queen cell virus, Deformed wing virus (DWV) and Chronic bee paralysis virus) were found with different prevalence. Despite the positive correlation between the prevalence of V. destructor mites and DWV, some areas had only mites, but not DWV, which is most likely due to the exceptional isolation of apiaries (up to 600 km). Phylogenetic analyses of the detected viruses reveal their clustering and European origin, thereby supporting the role of trade for pathogen spread and the isolation of Mongolia from South-Asian countries. In conclusion, this survey reveals the distinctive honey bee pathosphere of Mongolia, which offers opportunities for exciting future research.

Neonicotinoid insecticides severely affect honey bee queens

Queen health is crucial to colony survival of social bees. Recently, queen failure has been proposed to be a major driver of managed honey bee colony losses, yet few data exist concerning effects of environmental stressors on queens. Here we demonstrate for the first time that exposure to field realistic concentrations of neonicotinoid pesticides during development can severely affect queens of western honey bees (Apis mellifera). In pesticide-exposed queens, reproductive anatomy (ovaries) and physiology (spermathecal-stored sperm quality and quantity), rather than flight behaviour, were compromised and likely corresponded to reduced queen success (alive and producing worker offspring). This study highlights the detriments of neonicotinoids to queens of environmentally and economically important social bees, and further strengthens the need for stringent risk assessments to safeguard biodiversity and ecosystem services that are vulnerable to these substances.

Differential diagnosis of the honey bee trypanosomatids Crithidia mellificae and Lotmaria passim

Trypanosomatids infecting honey bees have been poorly studied with molecular methods until recently. After the description of Crithidia mellificae (Langridge and McGhee, 1967) it took about forty years until molecular data for honey bee trypanosomatids became available and were used to identify and describe a new trypanosomatid species from honey bees, Lotmaria passim (Evans and Schwarz, 2014). However, an easy method to distinguish them without sequencing is not yet available. Research on the related bumble bee parasites Crithidia bombi and Crithidia expoeki revealed a fragment length polymorphism in the internal transcribed spacer 1 (ITS1), which enabled species discrimination. In search of fragment length polymorphisms for differential diagnostics in honey bee trypanosomatids, we studied honey bee trypanosomatid cell cultures of C. mellificae and L. passim. This research resulted in the identification of fragment length polymorphisms in ITS1 and ITS1-2 markers, which enabled us to develop a diagnostic method to differentiate both honey bee trypanosomatid species without the need for sequencing. However, the amplification success of the ITS1 marker depends probably on the trypanosomatid infection level. Further investigation confirmed that L. passim is the dominant species in Belgium, Japan and Switzerland. We found C. mellificae only rarely in Belgian honey bee samples, but not in honey bee samples from other countries. C. mellificae was also detected in mason bees (Osmia bicornis and Osmia cornuta) besides in honey bees. Further, the characterization and comparison of additional markers from L. passim strain SF (published as C. mellificae strain SF) and a Belgian honey bee sample revealed very low divergence in the 18S rRNA, ITS1-2, 28S rRNA and cytochrome b sequences. Nevertheless, a variable stretch was observed in the gp63 virulence factor.