Exposure to Bacterial CpG DNA Protects from Airway Allergic Inflammation by Expanding Regulatory Lung Interstitial Macrophages.

Living in a microbe-rich environment reduces the risk of developing asthma. Exposure of humans or mice to unmethylated CpG DNA (CpG) from bacteria reproduces these protective effects, suggesting a major contribution of CpG to microbe-induced asthma resistance. However, how CpG confers protection remains elusive. We found that exposure to CpG expanded regulatory lung interstitial macrophages (IMs) from monocytes infiltrating the lung or mobilized from the spleen. Trafficking of IM precursors to the lung was independent of CCR2, a chemokine receptor required for monocyte mobilization from the bone marrow. Using a mouse model of allergic airway inflammation, we found that adoptive transfer of IMs isolated from CpG-treated mice recapitulated the protective effects of CpG when administered before allergen sensitization or challenge. IM-mediated protection was dependent on IL-10, given that Il10-/- CpG-induced IMs lacked regulatory effects. Thus, the expansion of regulatory lung IMs upon exposure to CpG might underlie the reduced risk of asthma development associated with a microbe-rich environment.

Re-Evaluating the Relevance of the Oxygen-Glucose Deprivation Model in Ischemic Stroke: The Example of Cdk Inhibition.

Previous research has shown that cyclin-dependent kinases (Cdks) that play physiological roles in cell cycle regulation become activated in post-mitotic neurons after ischemic stroke, resulting in apoptotic neuronal death. In this article, we report our results using the widely used oxygen-glucose deprivation (OGD) in vitro model of ischemic stroke on primary mouse cortical neurons to investigate whether Cdk7, as part of the Cdk-activating kinase (CAK) complex that activates cell cycle Cdks, might be a regulator of ischemic neuronal death and may potentially constitute a therapeutic target for neuroprotection. We found no evidence of neuroprotection with either pharmacological or genetic invalidation of Cdk7. Despite the well-established idea that apoptosis contributes to cell death in the ischemic penumbra, we also found no evidence of apoptosis in the OGD model. This could explain the absence of neuroprotection following Cdk7 invalidation in this model. Neurons exposed to OGD seem predisposed to die in an NMDA receptor-dependent manner that could not be prevented further downstream. Given the direct exposure of neurons to anoxia or severe hypoxia, it is questionable how relevant OGD is for modeling the ischemic penumbra. Due to remaining uncertainties about cell death after OGD, caution is warranted when using this in vitro model to identify new stroke therapies.

[Ulcerative Zoon's balanitis]. / Balanite de Zoon ulcérée.

Zoon's balanitis is a rare genital dermatosis of unknown etiology, usually presenting as a red-orange macule or plaque with a «cayenne pepper¼ appearance on the glans and/or foreskin. Unfortunately, atypical presentations are not uncommon, including vegetating or ulcerative lesions. Usually, it affects middle-age to older uncircumcised men. Although it is a benign pathology, Zoon's balanitis may be superimposed on another inflammatory or neoplastic dermatosis. As topical treatments are generally non satisfactory and relapses are usual on treatment with-drawal, circumcision remains an interesting option with usually a rapid and complete remission of the symptoms.

La balanite de Zoon est une dermatose génitale peu fréquente d'étiologie inconnue, se présentant habituellement par une macule ou une plaque de couleur rouge- orange avec un aspect en «poivre de cayenne¼, sur le gland et/ou le prépuce. Néanmoins, des formes atypiques, végétantes ou ulcérées, sont possibles. Elle touche plus souvent l'homme d'âge moyen à avancé et non circoncis. Il s'agit d'une pathologie bénigne, mais qui peut se surajouter à d'autres dermatoses inflammatoires ou néoplasiques. Les traitements topiques ne sont pas très efficaces et entraînent généralement une récidive à l'arrêt, tandis que la circoncision permet souvent une disparition rapide et complète des symptômes.

Effects of Low Intensity Pulsed Ultrasound Stimulation on the Temporal Dynamics of Irradiated Bone Tissue Healing: A Histomorphometric Study in Rabbits.

The present study evaluated the influence of Low-Intensity Pulsed Ultrasound (LIPUS) on the regeneration processes of non-critical-size bone defects in irradiated and non-irradiated rabbit tibias. Bone defects were surgically created on both tibiae of six rabbits. The control group had no additional treatment. In one intervention group, one tibia was irradiated with 15 Gy in a single dose. A second group was treated with LIPUS, and a third with a combination of both treatments. The control samples showed 83.10% ± 17.79% of bone repair after 9 weeks, while the irradiated bone had regenerated significantly less during the same period (66.42% ± 29.36%). The LIPUS treatment on irradiated bones performed a 79.21% ± 21.07% bone fill and could not significantly improve the response compared to the non-treated irradiated specimens. However, LIPUS treatment on non-irradiated bone showed bone formations beyond the size defect (115.91% ± 33.69%), which was a highly significant increase when compared to the control group or any irradiated group. The application of ultrasound to healthy bone produced highly significant and enhanced bone formations with 36.70% more regenerated bone when compared to the same application on irradiated bone. LIPUS vibration stimuli may be considered as a promising complementary treatment approach in non-irradiated bone regeneration procedures to shorten the treatment and enhance bone healing. In irradiated bones, the effect of ultrasound application is less clear, and further studies are needed to refine the dynamics of the present results.

Four-color multiplex real-time PCR assay prototype targeting azithromycin resistance mutations in Mycoplasma genitalium.

BACKGROUND: The worldwide expansion of macrolide-resistant Mycoplasma genitalium (MG) in cases of genital infections has led to an increased recurrence rate of these infections after first-line azithromycin treatment. By detecting the presence of azithromycin-resistant MG, the patient's antibiotic treatment can be targeted and the spread of resistance prevented. With this aim in mind, macrolide-resistance detection kits are helpful tools for the physician. METHODS: Azithromycin resistance mutations in MG are targeted using a four-color multiplex real-time RT-PCR assay. Tested targets include plasmid DNA (as positive controls) as well as macrolide-sensitive and macrolide-resistant genomic DNA from characterized cell lines and clinical samples. RESULTS: The analytical data presented here were generated from plasmid DNA and genomic RNA/DNA and include adaptation to an internal control, specificity between targets, specificity vs non-MG species, limit of detection (LoD) and interference studies (co-infection and endogenous substances). The clinical data were based on the application of the assay to clinical samples characterized by sequencing. CONCLUSIONS: A new NAAT (nucleic acid amplification test) prototype has been developed in collaboration with the Diagenode s.a. company, this prototype targets MG and azithromycin-resistance mutations in that pathogen.

[A Massive Open Oneline Course (MOOC) on pratical histology: A goal, a tool, a large public ! Return on a first experience]. / Un « Massive Open Online Course ¼ (MOOC) sur des travaux pratiques en histologie : un objectif, un outil, un public varié ! Retour sur une première expérience.

A goal ! The MOOC entitled "Introduction to Histology, A Human Tissue Exploration" correspond to our vision of the practice of General Histology, which is based on the ability to diagnose 5 families of biological tissues. Ultimately, participants must be able to recognize the different types of cells and all the surrounding elements in order to understand how they organize themselves to form tissues with specific functions. A tool ! This know-how is based on reasoning from observations of microscopic structures. Learners are therefore invited to manipulate a virtual microscope to explore biological samples on histological slides digitized. Annotations, comments, drawings or photos are associated with landmarks that enrich the study of these histological sections. A target audience ! Two educational paths allow deepening the subject in a different way and thus matching the goals or motivations of each one. After a first year of experience, usage statistics and surveys of our learners show that the MOOC Histo has allowed each of them to find an interest and federate a community of motivated learners.

Validating instructional design and predicting student performance in histology education: Using machine learning via virtual microscopy.

As a part of modern technological environments, virtual microscopy enriches histological learning, with support from large institutional investments. However, existing literature does not supply empirical evidence of its role in improving pedagogy. Virtual microscopy provides fresh opportunities for investigating user behavior during the histology learning process, through digitized histological slides. This study establishes how students' perceptions and user behavior data can be processed and analyzed using machine learning algorithms. These also provide predictive data called learning analytics that enable predicting students' performance and behavior favorable for academic success. This information can be interpreted and used for validating instructional designs. Data on the perceptions, performances, and user behavior of 552 students enrolled in a histology course were collected from the virtual microscope, Cytomine®. These data were analyzed using an ensemble of machine learning algorithms, the extra-tree regression method, and predictive statistics. The predictive algorithms identified the most pertinent histological slides and descriptive tags, alongside 10 types of student behavior conducive to academic success. We used these data to validate our instructional design, and align the educational purpose, learning outcomes, and evaluation methods of digitized histological slides on Cytomine®. This model also predicts students' examination scores, with an error margin of <0.5 out of 20 points. The results empirically demonstrate the value of a digital learning environment for both students and teachers of histology.

Effects of Minocycline Hydrochloride as an Adjuvant Therapy for a Guided Bone Augmentation Procedure in The Rat Calvarium.

This in vivo study reports the influence of minocycline-HCl administration on extra-skeletal bone generation in a Guided Bone Augmentation model, utilizing titanium caps placed on the intact as well as perforated calvaria of rats. The test group was administered 0.5 mg/mL minocycline-HCl with the drinking water, and the amount of bone tissue in the caps was quantified at three time points (4, 8 and 16 weeks). A continuously increased tissue fill was observed in all groups over time. The administration of minocycline-HCl as well as perforation of the calvaria increased this effect, especially with regard to mineralization. The strongest tissue augmentation, with 1.8 times that of the untreated control group, and, at the same time, the most mineralized tissue (2.3× over untreated control), was produced in the combination of both treatments, indicating that systemic administration of minocycline-HCl has an accelerating and enhancing effect on vertical bone augmentation.

Design, Synthesis, and Evaluation of Novel Pyruvate Dehydrogenase Kinase Inhibitors.

AIMS: The present work describes the synthesis and the biological evaluation of novel compounds acting as pyruvate dehydrogenase kinase (PDK) inhibitors. These drugs should become a new therapeutic approach for the treatment of pathologies improved by the control of the blood lactate level. METHODS: Four series of compounds belonging to N-(4-(N-alkyl/aralkylsulfamoyl)phenyl)-2- methylpropanamides and 1,2,4-benzothiadiazine 1,1-dioxides were prepared and evaluated as PDK inhibitors. RESULTS: The newly synthesized N-(4-(N-alkyl/aralkylsulfamoyl)phenyl)-2-methylpropanamides structurally related to previously reported reference compounds 4 and 5 were found to be potent PDK inhibitors (i.e. 10d: IC50 = 41 nM). 1,2,4-Benzothiadiazine 1,1-dioxides carrying a (methyl/ trifluoromethyl)-propanamide moiety at the 6-position were also designed as conformationally restricted ring-closed analogues of N-(4-(N-alkyl/aralkylsulfamoyl)phenyl)-2-hydroxy-2-methylpropanamides. Most of them were found to be less potent than their ring-opened analogues. Interestingly, the best choice of hydrocarbon side chain at the 4-position was the benzyl chain, providing 11c (IC50 = 3.6 µM) belonging to "unsaturated" 1,2,4-benzothiadiazine 1,1-dioxides, and 12c (IC50 = 0.5 µM) belonging to "saturated' 1,2,4-benzothiadiazine 1,1-dioxides. CONCLUSION: This work showed that ring-closed analogues of N-(4-(N-alkyl/aralkylsulfamoyl) phenyl)- 2-hydroxy-2-methylpropanamides were less active as PDK inhibitors than their corresponding ring-opened analogues. However, the introduction of a bulkier substituent at the 4-position of the 1,2,4-benzothiadiazine 1,1-dioxide core structure, such as a benzyl or a phenethyl side chain, was allowed, opening the way to the design of new inhibitors with improved PDK inhibitory activity.

Ustekinumab in psoriasis immunopathology with emphasis on the Th17-IL23 axis: a primer.

Psoriasis is a chronic relapsing immunoinflammatory dermatosis that is commonly associated with systemic comorbidities. The pathogenic importance of interleukin (IL)-12 and IL-23 is beyond doubt, as well as the involvement of T helper cells (Th)1 and Th17 cells. There is upregulation of the p40 subunit shared by IL-12 and IL-23 and of the IL-23 p19 subunit, but not an increased expression of the IL-12 p35 subunit. This indicates that IL-23 appears more involved than IL-12 in the pathogenesis of psoriatic plaques. Ustekinumab is a fully human monoclonal antibody of the immunoglobulin (Ig) G1 class targeting the p40 subunit common to both IL-12 and IL-23, thus inhibiting both IL-12 and IL-23 receptor-mediated signalling. Ustekinumab is part of the recent biologic therapies active in psoriasis, autoimmune arthritides, and inflammatory bowel diseases.

Field melanin mapping of the hairless scalp.

BACKGROUND: Mottled subclinical melanoderma (MSM) is frequently seen on facial skin using the ultraviolet light enhanced visualization (ULEV) method. The corresponding aspect on the hairless scalp remains unknown. OBJECTIVE: To explore the field distribution of melanin on the scalp of fair-skinned Caucasian subjects. METHOD: The scalp was examined in 43 men with androgenic alopecia. The Visioscan(®) camera provided the ULEV pictures. Another optical (Visioface(®) Quick) device was used under white light illumination followed by colour contrast enhancement. This was reached after specific computer filtration of the cyan hue wavelengths. RESULTS: Under white light illumination, the scalp looked normal. MSM patterns were disclosed by both optical procedures as evenly scattered discrete patchy fields of hypermelanosis. The smaller rounded spots were restricted to the lips of the hair infundibula. Larger irregularly shaped spots predominated in the interfollicular areas. A few hypomelanotic spots were scattered over the scalp. CONCLUSION: The present observations based on dual optical methods possibly provide information about a patterned pathobiology of melanocytes on the scalp. The spotty MSM pattern looked similar to the reported aspects on the face. It somewhat resembled the widespread PUVA-induced lentiginosis.

Recent trends in specular light reflectance beyond clinical fluorescence diagnosis.

Under specific light illumination, particularly ultraviolet (UV) and near-UV light stimulation, the skin produces both specular light reflectance and, possibly, specific fluorescent emission. These properties offer diagnostic clues and disclose some peculiar functions of the skin. A series of superficial infections (erythrasma, some tinea capitis types, tinea/pityriasis versicolor, dermatophytoses, etc.) and pilosebaceous follicles enriched in Propionibacterium spp show fluorescence. This latter characteristic is downgraded or lost while on some anti-acne treatments. A quenching effect of fluorescence is observed following the application of sunscreens. The (pre)neoplastic areas prepared for methylaminolevulinate photodynamic therapy (MAL-PDT) show reddish fluorescence following drug metabolisation producing porphyrins by the abnormal activated cells. Of note, when using a recording sensitive CCD camera instead of casual visual observation, skin fluorescence may be superimposed on the specular reflectance of the incident light. With the current technology, these situations are not distinguished with confidence. Any harsh and scaly lesion appears brighter following yellowish specular light reflectance. Stratum corneum samplings collected on clear self-adhesive discs or cyanoacrylate skin surface strippings are conveniently examined ex vivo, taking advantage of the same optical properties.

Photoaging under recreational sunbeds.

BACKGROUND: Artificial sources of restricted light wavelength, particularly tanning beds, are progressively gaining importance in photoaging. OBJECTIVE: To assess the kinetics and the long-term evolution of skin pigmentation and tensile functions in sunbed worshippers over a period of 8 years. METHODS: Photoaging was explored in women who were both sunshine and sunbed worshippers. A series of 65 phototype III women aged 31-46 years completed a 100-month survey. Quarterly assessments were performed on the forearms to measure (a) the skin color individual typology angle (ITA°), (b) the extent in mottled subclinical melanoderma (MSM) using the ultraviolet light-enhanced visualization method and (c) the rheological properties of skin. RESULTS: A progressive increase in both skin extensibility and hysteresis was observed, contrasting with a decrease in biologic elasticity. These rheological changes were correlated with the ITA° changes, but not with the MSM extent. The kinetics of evolution of each test variable were distinct over time. DISCUSSION: This work is the first attempt at evaluating the kinetics of changes in physical parameters during a long period of frequent exposures to tanning sunbeds and sunshine for lifestyle purposes. The alterations were quite important in the color, MSM and rheological functions of the skin.

Facing up to the imperceptible perspiration. Modulatory influences by diabetic neuropathy, physical exercise and antiperspirant.

BACKGROUND: Sweating is variably altered by physical exercise, diabetic neuropathy and antiperspirants. METHODS: Skin temperature, skin surface water loss (SSWL), the Corneometer(®) average capacitance (CMAC) and skin capacitance mapping (SCM) were measured before and after moderate physical exercise in 20 healthy subjects. The effect of 5% aluminium chloride hexahydrate (ACH) in a water solution was similarly tested. The same assessments were performed in 20 diabetic patients at rest. RESULTS: Diabetic neuropathy appeared at rest as an increased (compensatory) SCM on the forearms without obvious modification on the hypohidrotic legs. On ACH sites after exercise, SCM revealed both a lowered number of active sweat glands and a lighter stratum corneum (SC) (dryness). In addition, CMAC and SSWL were decreased on ACH sites at rest and at completion of exercise. CONCLUSION: In diabetic neuropathy, the compensatory hyperhidrosis is more easily disclosed than the hypohidrosis. ACH affects both sweat excretion and the SC hydration.

The skin landscape following nonoptical capacitance imaging.

Skin capacitance is an electrical property measurable at the level of the stratum corneum. Values are related to the moisture content of the tissue. Using the silicon image sensor technology, it has recently become possible to measure capacitance at 50 microm intervals on the skin surface. The resulting nonoptical picture corresponds to skin capacitance imaging. This novel tool for dermatologists highlights with precision the hollow skin surface patterns, including dermatoglyphics, the patterns of the shallow skin lines, and wrinkles. The topographic heterogeneity in skin surface hydration is made visible. In addition, the sweat gland and follicular openings are disclosed. Skin disorders induced by surfactants or presenting as hyperkeratotic areas, including some neoplasms, may show typical aspects. It is concluded that skin capacitance imaging brings added value to the clinical assessment performed by dermatologists.

Angiogenic fast-growing melanomas and their micrometastases.

Malignant melanoma (MM), particularly its fast-growing type, is prone to interstitial, intravascular and extravascular migratory metastases. There is no information linking their growth fraction, the configuration of the MM advancing edge, the extent in vascularity and the propensity to metastatic progression. The objective of this study was to determine the growth fraction, the size of vascularity and the contours of the progression border of 32 fast-growing MM with regard to the presence or absence of a micrometastatic spread inside the skin and overt metastases in the sentinel lymph nodes. In vivo high resolution colorimetry was performed as a clinical estimate of MM vascularity. Euclidean geometry and fractal analysis were used on immunohistochemical sections. The relative microvasculature profile area beneath MM, and the fractal dimension D of the MM frontline were assessed. The MIB/Ki-67 index was determined in MM cells. Value a* of colorimetry was similarly increased in the presence or absence of micro-metastases. No difference in growth fraction was revealed between these neoplasms. Correlations were found between vascularity and angiotropism, and between the micrometastatic process and the sentinel lymph node involvement. By contrast, no correlation was shown between vascularity and the fractal D dimension of the MM advancing edge. In sum, this study establishes a link between the extent of MM growth fraction, vascularity and the presence of dermal and nodal micrometastases in fast-growing MM.

Updating corneofungimetry: a bioassay exploring dermatomycoses and antifungal susceptibility.

Superficial dermatomycoses are frequent conditions in humans and animals. Specific treatment modalities have been designed using a variety of different antifungal compounds. The need for antifungal susceptibility testing (AST) has been growing steadily over the last two decades due to the extending number of newer antifungal agents. Objective inter- and intraindividual comparisons of their respective efficacies are nearly impossible to perform in vivo. Currently, a series of standardized AST methods and interpretative guidelines have been designed. However, their clinical relevance for dermatomycoses is not consistent. The corneofungimetry bioassay was designed to test comparatively a series of antifungals on pathogenic fungi growing on sheets of human stratum corneum. Computerized morphometric assessments bring numerical values allowing statistical comparisons. Variants of corneofungimetry address more specific aspects related to fungal cell adhesion, fungitoxicity and lipid-dependent fungi.

Exploratory Assessment of Oxygen Flow-Assisted Cutaneous Administration of Methotrexate for Superficial Basal Cell Carcinoma, Mycosis Fungoides, and Extramammary Paget Disease.

The molecular weight of methotrexate (MTX) makes cutaneous penetration difficult. Oxygen flow could enhance the skin permeation of MTX diluted in the proprietary LP3 carrier system. This pilot study aims to assess the efficacy, safety, and tolerance of oxygen flow-assisted LP3-MTX3% for treating superficial skin cancers. Patients with superficial basal cell carcinoma (n = 12), extramammary Paget disease (n = 5), classic mycosis fungoides (MF; n = 10), and folliculotropic MF (n = 6) were included in the study and were treated with four weekly applications of oxygen flow-assisted LP3-MTX3%. Photographs and biopsies were performed before and one month after treatment. At one month after treatment, the mean superficial basal cell carcinoma erythema-crusting-thickness clinical score, the extramammary Paget disease erythema-oozing-scaling/hyperkeratosis-pain/pruritus clinical score, and the modified composite assessment of index lesion severity classic MF and folliculotropic MF scores were improved by 77.5% ± 17.1% (P < 0.0001), 66.7% ± 22.9% (P = 0.011), 51.3% ± 32.2% (P = 0.0007), and 27.8% ± 32.0% (P = 0.086), respectively. At one month after treatment, histology revealed partial and total clearances for superficial basal cell carcinoma (1/12, 11/12), extramammary Paget disease (4/5, 1/5), classic MF (8/10, 2/10), and folliculotropic MF (6/6, 0/6). Tolerance was excellent and no pain was observed. MTX was never detectable in serum at baseline and 1, 2, 3, 8, 24, 48, and 72 hours post-treatment. In conclusion, the interesting therapeutic efficacy of oxygen flow-assisted LP3-MTX3% for treating superficial basal cell carcinoma, extramammary Paget disease, and MF lesions prompts further studies on a larger scale.

Cutaneous Breast Cancer Metastases Successfully Treated Using an Oxygen Flow Assisted Topical Administration of Methotrexate (OFAMTX).

INTRODUCTION: Cutaneous metastases of breast cancer remain a therapeutic challenge. Oxygen flow-assisted topical administration of methotrexate 5% (OFAMTX, 5% methotrexate in a carrier solution) has recently been proven to be an efficacious alternative treatment for extramammary Paget's disease, which is considered to be an in situ mammary adenocarcinoma of the epidermis. CASE REPORT: A 51-year-old patient with triple negative breast cancer presenting with biopsy-proven skin metastases on the chest agreed to a treatment with OFAMTX5%. The treatment duration was 2 weeks and consisted of twice-weekly sessions with OFAMTX5% applied to an area of skin of approximately 40 cm2. Skin biopsies were performed before and 2 months after procedure. The tolerance to the treatment was excellent, and no pain sensations were experienced. Two months post-procedure the treated area presented a post-inflammatory hyperpigmentation. No residual metastatic lesions were detectable on the control skin biopsy. Six months post-procedure the patient is still in clinical remission. DISCUSSION: OFAMTX5% represents an alternative skin-directed, painless, patient-friendly and efficacious adjuvant treatment for superficial metastatic lesions of breast cancer. Larger series are required to evaluate the potential of OFAMTX5% for the treatment of superficial metastatic lesions of breast cancer.