The use of tissue-engineered skin to demonstrate the negative effect of CXCL5 on epidermal ultraviolet radiation-induced cyclobutane pyrimidine dimer repair efficiency.

BACKGROUND: Ultraviolet radiation (UVR) is responsible for keratinocyte cancers through the induction of mutagenic cyclobutane pyrimidine dimers (CPDs). Many factors influence CPD repair in epidermal keratinocytes, and a better understanding of those factors might lead to prevention strategies against skin cancer. OBJECTIVES: To evaluate the impact of dermal components on epidermal CPD repair efficiency and to investigate potential factors responsible for the dermal-epidermal crosstalk modulating UVR-induced DNA damage repair in keratinocytes. METHODS: A model of self-assembled tissue-engineered skin containing human primary keratinocytes and fibroblasts was used in this study. RESULTS: We showed that CPD repair in keratinocytes is positively influenced by the presence of a dermis. We investigated the secretome and found that the cytokine CXCL5 is virtually absent from the culture medium of reconstructed skin, compared with media from fibroblasts and keratinocytes alone. By modulating CXCL5 levels in culture media of keratinocytes, we have shown that CXCL5 is an inhibitor of CPD repair. CONCLUSIONS: This work outlines the impact of the secreted dermal components on epidermal UVR-induced DNA damage repair and sheds light on a novel role of CXCL5 in CPD repair.

[Dialogue between general practitioner and patient regarding tobacco and alcohol consumption, from the patient's standpoint]. / Dialogue entre médecin généraliste et patient : les consommations de tabac et d'alcool en question, du point de vue du patient.

BACKGROUND: General practitioners (GP) are key players in screening and counselling for smokers and alcohol drinkers exceeding French guidelines for low-risk consumption thresholds. Tackling the subject from the patients' perspective, the authors aim at estimating the proportion of the population having discussed their smoking and alcohol consumption with their GPs, and to pinpoint the factors associated with their having done so. METHODS: The data (n=6346) are derived from nationwide representative phone survey in mainland France conducted by the French public health monitoring center Baromètre de Santé publique France. RESULTS: Among the persons aged 18-75 having consulted a GP over the last 12 months (82.5%), 36.7% stated that smoking had been discussed in consultation while 16.8% had talked about alcohol use. For both substances, being a man, a smoker, an excessive alcohol user and having a chronic illness were not only independently associated with being questioned by one's doctor, but also with more frequent patient initiative. About 87% considered it normal to be asked by their GP about their alcohol intake, this proportion being higher among men and people with high incomes. CONCLUSION: Even though a large portion of the population would deem it normal to discuss smoking and alcohol intake in consultation with a GP, the subjects are rarely broached. Our results underline the need to bolster efforts at systematic screening for substance use by GPs.

[Smoking and vaping in France]. / Consommation de tabac et usage de cigarette électronique en France.

INTRODUCTION: The aim of the article is to present a summary of current knowledge on tobacco smoking in France available through representative population surveys. STATE OF ART: Smoking is very common in France as more than a quarter (28.7%) of 15-75years old individuals reported daily smoking in 2016. The rate is also high among 15-year-old (14.6%) and half of them (51.8%) have already smoked one cigarette. Since the 1970s, sex differences in smoking behaviour are narrowing. More recently, since 2000, smoking prevalence has tended to decrease among the most advantaged individuals but to increase among less advantaged ones. These social inequalities, which are becoming more and more pronounced, are being observed from smoking initiation, often before the age of 18. Daily users of E-cigarettes represent 2.5% of individuals from 15-75 years in France. They are also tobacco smokers (58.8%) or former smokers (41.2%). PERSPECTIVES: Taking social inequalities in health into consideration, notably regarding smoking initiation, is a key challenge in the fight against smoking. CONCLUSIONS: It is essential to strengthen efforts to decrease the rate of smoking in France. The 2017 Health Barometer and Escapad surveys will allow evaluation of the public policies implemented in 2016 and determine whether they have had an impact on the image of smoking and on smoking rates.

Gambling Type, Substance Abuse, Health and Psychosocial Correlates of Male and Female Problem Gamblers in a Nationally Representative French Sample.

Many studies carried out on treatment-seeking problem gamblers (PG) have reported high levels of comorbid substance use disorders, and mental and physical health problems. Nevertheless, general population studies are still sparse, most of them have been carried out in the United States or Canada, and gender differences have not always been considered. Thus, the aim of this study was to describe the type of games, and psychological and physical correlates in male and female PG in a nationally representative French sample. The total sample studied involved 25,647 subjects aged 15-85 years, including 333 PG and 25,314 non-problem gamblers (NPG). Data were extracted from a large survey of a representative sample of the French general population. They were evaluated for sociodemographic variables, gambling behavior, type of gambling activity, substance use, psychological distress, body mass index, chronic disease, and lack of sleep. Overall, there were significant differences between PG and NPG in gender, age, education, employment and marital status, substance use disorders (alcohol, tobacco, cannabis, cocaine and heroin), psychological distress, obesity, lack of sleep and type of gambling activity. Although male and female PG had different profiles, the gambling type, especially strategic games, appeared as an important variable in the relationship between gender and problem gambling. This research underlines the importance of considering gender differences and gambling type in the study of gambling disorders. Identifying specific factors in the relationship between gender, gambling type and gambling problems may help improve clinical interventions and health promotion strategies.

Educational differences in associations of noticing anti-tobacco information with smoking-related attitudes and quit intentions: findings from the International Tobacco Control Europe Surveys.

This study examined educational differences in associations of noticing anti-tobacco information with smoking-related attitudes and quit intentions among adult smokers. Longitudinal data (N = 7571) from two waves of six countries of the International Tobacco Control (ITC) Europe Surveys were included. Generalized estimating equation analyses and multiple linear and logistic regression analyses were conducted. Higher educated smokers noticed anti-tobacco information slightly more often than lower educated smokers (F(2) = 25.78, P < 0.001). Noticing anti-tobacco information was associated with more negative smoking-related attitudes (ß = 0.05, P < 0.001) and more quit intentions (OR = 1.08, P < 0.001). Among smokers without a quit intention at baseline, a positive association was found for noticing anti-tobacco information at baseline with follow-up quit intention (OR = 1.14, P = 0.003). No other longitudinal associations were found. No educational differences were found in the association of noticing anti-tobacco information with smoking-related attitudes but associations with quit intentions were found only among low (OR = 1.12, P = 0.001) and high educated respondents (OR = 1.11, P < 0.001) and not among moderate educated respondents (OR = 1.02, P = 0.43). Noticing anti-tobacco information may positively influence quit intentions and possibly smoking-related attitudes. Lower educated smokers were as likely to be influenced by anti-tobacco information as higher educated smokers but noticed anti-tobacco information less often; increasing reach of anti-tobacco information may increase impact in this group.

[Increasing trends in screening for addictives behaviors among general practitioners in France]. / Le développement du repérage des pratiques addictives en médecine générale en France.

BACKGROUND: Make an inventory of screening of addictive behaviours among general practice since the end of the 1990s. METHODS: A representative sample of 2,083 general practitioners was surveyed in 2008/2009. They were asked about their prevention practices. RESULTS: Two thirds of the general practitioners (GPs) reported discussing tobacco consumption at least once with each patient. This assessment was less systematic for alcohol (23% of GPs) and cannabis (8% of GPs). Approximatively 70% reported addressing cannabis or alcohol use issues only with patients at risk. One third reported using tobacco smoking screening questionnaires, while there were only 6% in 2003. Only 13% of GPs used alcohol standardized questionnaires, a clear rise since 1998 (1.4% in 1998, 2.0% in 2003). Using alcohol standardized screening tests was more frequent among GPs belonging to a drug addiction network, but no significant link was found with gender, age or area. Only 2% of GPs used cannabis use screening tests. The care for cannabis users seemed particularly linked to the practitioners' inclination to discuss this issue without waiting for a demand arising from the patient. The proportion of practitioners reporting helping patients kick their nicotine addiction in the last seven days proved stable since 2003, after a very significant increase between 1998 and 2003. The proportion of GPs reporting seeing a patient for an alcoholic weaning (52%) was stable since 1998. CONCLUSION: This study illustrates the development of addiction care practices integrating smoking cessation help and, to a more limited extent, screening of alcohol and cannabis abuse, evidenced by the expanded application of standardized questionnaires in general practice. Consideration given to cannabis and alcohol use assessment appeared correlated to GPs feeling at ease with addressing drug use issues, this feeling being linked to their ability to rely on institutional directives or validated screening tools. It seems thus important to encourage the implementation of a health educational approach including early screening and brief intervention during consultation.

The mixed lineage kinase leucine-zipper protein kinase exhibits a differentiation-associated localization in normal human skin and induces keratinocyte differentiation upon overexpression.

Leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase is a recently described protein serine/threonine kinase which belongs to the mixed lineage kinase family. The overall pattern of expression of the leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase gene in embryonic and adult mouse tissues suggested that this kinase could be involved in the regulation of epithelial cell proliferation and differentiation. In order to get more insights into the potential role of leucine-zipper protein kinase in these cellular processes, we characterized its expression in normal human skin, both at the mRNA and protein levels. In situ hybridization, western blotting, and indirect immunofluorescence studies were conducted to localize leucine-zipper protein kinase on various human skin tissues. This is one of the first reports that leucine-zipper protein kinase has a very precise pattern of expression in human skin epithelia, as both mRNA and protein are restricted to the granular layer of the epidermis and inner root sheath of hair follicles. Detection of leucine-zipper protein kinase protein on skin from various body sites, donors of different ages as well as on reconstructed human skin always reveals that leucine-zipper protein kinase is present only in the very differentiated keratinocytes of epidermis and hair follicles. To determine directly whether leucine-zipper protein kinase exhibits any effect on cell growth and differentiation, keratinocytes were transfected with an expression vector harboring the leucine-zipper protein kinase cDNA. The presence of this construct in keratinocytes results in growth arrest together with a concomitant increase in filaggrin expression. Collectively, our results indicate that leucine-zipper protein kinase plays an active part in cellular processes related to terminal differentiation of epidermal keratinocytes.

Multistep production of bioengineered skin substitutes: sequential modulation of culture conditions.

Many studies are being conducted to define the role of growth factors in cutaneous physiology in order to add cytokines in a timely fashion for optimal tissue engineering of skin. This study is aimed at developing a multistep approach for the production of bioengineered skin substitutes, taking into account the effects of various growth factors according to the culture time. The use of a serum-supplemented medium throughout the whole culture period of skin substitutes was compared to the sequential use of specific additives at defined culture steps. Histological analysis revealed that serum was necessary for keratinocyte proliferation and migration on dermal substitutes during the first 2 d after their seeding. However, the serum-free medium presented some advantages when supplemented with different additives at specific culture steps. Interestingly, ascorbic acid added to the dermal substitutes before and after keratinocyte seeding maintained their cuboidal morphology in the basal epidermal layer. In the absence of serum, collagen matrix degradation slowed down, and a better multilayered epidermal organization was obtained, notably with retinoic acid. Stratum corneum formation was also enhanced by fatty acids. Thus, sequential addition of exogenous factors to the medium used to produce skin substitutes can improve their structural features and functional properties in vitro.

Reconstructed human cornea produced in vitro by tissue engineering.

The aim of the present study was to produce a reconstructed human cornea in vitro by tissue engineering and to characterize the expression of integrins and basement membrane proteins in this reconstructed cornea. Epithelial cells and fibroblasts were isolated from human corneas (limbus or centre) and cultured on plastic substrates in vitro. Reconstructed human corneas were obtained by culturing epithelial cells on collagen gels containing fibroblasts. Histological (Masson's trichrome staining) and immunohistological (laminin, type VII collagen, fibronectin as well as beta1, alpha3, alpha4, alpha5, and alpha6 integrin subunits) studies were performed. Human corneal epithelial cells from the limbus yielded colonies of small fast-growing cells when cultured on plastic substrates. They could be subcultured for several passages in contrast to central corneal cells. In reconstructed cornea, the epithelium had 4-5 cell layers by the third day of culture; basal cells were cuboidal. The basement membrane components were already detected after 3 days of culture. Integrin stainings, except for the alpha4 integrin, were also positive after 3 days. They were mostly detected at the epithelium-stroma junction. Such in vitro tissue-engineered human cornea, which shows appropriate histology and expression of basement membrane components and integrins, provides tools for further physiological, toxicological and pharmacological studies as well as being an attractive model for gene expression studies.

Differential expression of collagens XII and XIV in human skin and in reconstructed skin.

Collagens XII and XIV localize near the surface of collagen fibrils and may be involved in epithelial-mesenchymal interactions as well as in the modulation of tissue biomechanical properties. Moreover, human skin fibroblasts cultured in monolayer are known to lose their ability to produce collagen XIV and to switch the transcription of collagen XII from the small splice variant (220 kDa) to the large (320 kDa), whereas the small form is the main form found in human skin. We have investigated the expression patterns of these two molecules in human skin as a function of donor age and anatomic site, by using immunohistology with specific monoclonal antibodies. We demonstrated changes in the expression patterns of collagens XII and XIV in human skin after birth. Moreover, in adult scalp skin, very strong staining of collagen XII fibril bundles was observed around hair follicles, in association with very low expression of collagen XIV. We also investigated the expression of collagens XII and XIV by fibroblasts and keratinocytes cultured in a reconstructed skin. In these culture conditions, fibroblasts recovered their ability to produce collagen XIV and re-expressed the small splice variant of collagen XII. These results could be explained by the deposition of large amounts of collagen fibrils by fibroblasts in this culture system. Thus, the re-expression of these collagens suggests that the deposition of banded collagen fibrils is a pre-requisite for the expression of collagen XIV and small variant of collagen XII.

Grafting on nude mice of living skin equivalents produced using human collagens.

Autologous epidermal transplantation for human burn management is an example of a significant breakthrough in tissue engineering. However, the main drawback with this treatment remains the fragility of these grafts during and after surgery. A new human bilayered skin equivalent (hSE) was produced in our laboratory to overcome this problem. The aim of the present work was to study skin regeneration after hSE grafting onto nude mice. A comparative study was carried out over a period of 90 days, between anchored bovine skin equivalent, hSE and hSE+, the latter containing additional matrix components included at concentrations similar to those in human skin in vivo. The addition of a dermal layer to the epidermal sheet led to successful graft take, enhanced healing, and provided mechanical resistance to the grafts after transplantation. In situ analysis of the grafts showed good ultrastructural organization, including the deposition of a continuous basement membrane 1 week after surgery.

Skin equivalent produced with human collagen.

Several studies have recently been conducted on cultured skin equivalent (SE), prepared using human keratinocytes seeded on various types of dermal equivalents (DE). We previously showed the advantages of our anchorage method in preventing the severe surface reduction of DE due to fibroblast contractile properties in vitro. A new anchored human SE was established in our laboratory in order to obtain a bioengineered tissue that would possess the appropriate histological and biological properties. In order to compare the effects of different collagen origins on the evolution of SE in vitro, human keratinocytes were seeded on three types of anchored DE. A comparative study was carried out between bovine SE (bSE), human SE (hSE), and human skin equivalent containing additional dermal matrix components (hSE+). Immunohistological analysis showed that hSE and hSE+ presented good structural organization, including the deposition of several basement membrane constituents. Higher amounts of transglutaminase, ceramides, and keratin 1 were detected in the epidermal layers of all SE when cultured at the air-liquid interface. However, a 92 kDa gelatinase activity was higher in bovine skin equivalent (bSE) compared to hSE cultures. The use of human collagens comparatively to bovine collagen as SE matricial component delayed the degradation of the dermal layer in culture.

Early basement membrane formation following the grafting of cultured epidermal sheets detached with thermolysin or Dispase.

The basement membrane zone is important for graft adhesion and stability. The aim of the present study was to visualize the regeneration of the basement membrane and determine the sequential appearance of its constituents in the early postgrafting period of cultured human epidermal sheets. A keratinocyte single cell suspension, devoid of dermal fibroblast contamination, was obtained from human skin by a two-step tissue digestion method with thermolysin and trypsin. After culturing, epidermal sheets were generated, detached enzymatically by incubating with thermolysin (for 20-30 min) or Dispase (for 45-60 min), and deposited on a muscular graft bed of athymic mice. Immunohistochemistry and ultrastructural analyses were performed on biopsies harvested 2, 4 and 21 days postgrafting. Bullous pemphigoid antigens and laminin were detected at the dermo-epidermal junction, showing an almost continuous line 2 days postgrafting. Type IV collagen was generally absent at this time, but it was detected 4 days postgrafting. Type VII collagen was labelled as a discontinuous line of increasing intensity from 2 to 21 days postgrafting. Ultrastructural analysis revealed hemidesmosomes and a discontinuous lamina densa 2 days postgrafting, and a complete basement membrane with a continuous lamina densa, hemidesmosomes and anchoring fibrils 21 days postgrafting. The sequence of appearance of major basement membrane components was similar for cultured sheets detached with thermolysin or Dispase. However, it differed from that of other wound healing models. Results are discussed in terms of the variable keratinocyte migration requirement between various wound healing models.

Improvement of human keratinocyte isolation and culture using thermolysin.

We propose a modification of the conventional keratinocyte isolation method which has shown a significant improvement in the purity, colony forming efficiency (c.f.e.) and growth capacity of the isolated epidermal cell population. This method utilized thermolysin since it selectively digests the dermo-epidermal junction. Following separation from the dermis, the epidermis was digested with trypsin to obtain a single cell suspension. Compared with the conventional procedure, this isolation method was shorter and resulted in (i) cells displaying a higher colony forming efficiency, (ii) cells reaching confluence 1-3 days earlier, (iii) cells not contaminated by fibroblasts, (iv) a cell population containing all the basal layer keratinocytes. These cells were suitable for the establishment of primary cultures and could be subcultured. Such cell populations should be advantageous in studies of epithelial-mesenchymal interactions in which keratinocyte populations, free of fibroblasts, are desirable. In the treatment of extensively burned patients using cultured epidermal sheets, the main problem remains the time required for their production. Thus, the absence of fibroblast overgrowth of the keratinocyte cultures and the significantly reduced time to obtain confluent cultures and epidermal sheets with our method have very important implications for the treatment of large burn wounds.

Role and innocuity of Tisseel, a tissue glue, in the grafting process and in vivo evolution of human cultured epidermis.

Cultured epidermal sheets are currently used for burn wound treatment but reported results on graft take are variable. This study was designed to evaluate the role and influence of Tisseel, a fibrin glue, in the take of cultured human epidermal sheets in an athymic mouse model. On days 4, 10 and 21 post-grafting, histology, electron microscopy and immunofluorescence staining confirmed the presence of a human epithelium and the development of a basement membrane. Tisseel was detectable on day 4 only, but overall treated and untreated grafts were similar. The use of Tisseel enhanced the mechanical stability of these fragile grafts, increased the percentage of graft take, and its innocuity on the in vivo evolution of cultured epidermal sheets was demonstrated. For these reasons, we think that Tisseel may be advantageous in a clinical setting.

Optimization of murine keratinocyte culture for the production of graftable epidermal sheets.

The aim of the present study was to optimize murine epidermal cell cultures in order to obtain graftable sheets. Newborn (1-3 days old) Balb/c mice skin were used to optimize culture media and plating cell concentration, then epidermal sheet production, and grafting. Epidermal cells were plated at various concentrations in different culture media containing low (0.1 mM) or high (greater than 1 mM) Ca2+ levels. After a 3 day culture at the 10(4) cells/cm2 plating cell concentration, the percentage of differentiated cells was more than 80% in the high Ca2+ culture medium and less than 50% with bulky cells in the low Ca2+ culture medium. Under these conditions confluence was not obtained. At the 10(5) cells/cm2 seeding inoculum, the percentage of confluence increased to 95-100% during the first 72 h of culture in both high and low Ca2+ culture media. Three-day-old culture showed stratified multilayer epidermal sheets in the high calcium medium, and monolayer epidermal sheets were present in the low calcium medium after seeding keratinocytes in fibronectin precoated flasks. Seven days after plating, post confluent cultures were composed of a high percentage of differentiated cells (90%) with an increase in shedding cells in the medium. Considering the above morphological observations, sheets obtained with 10(5) cells/cm2 in MCDB-153 (A), DME-HAM (B) or GMEM (C) media after 3 days in culture were grafted. Twenty days after grafting, histological analysis of biopsies showed an epidermal structure and organization comparable to normal murine epidermis without hair follicles. Epidermal transplants showed a complete basement membrane, hemidesmosomes, and tonofilament bundles. Sheets obtained after seven day culture in all media showed lower coverage of the wound bed. These studies point out the importance of the plating cell and Ca2+ concentrations, and the culture time for murine keratinocyte confluence and differentiation to obtain graftable epidermal sheets.

[Esthetic and functional scar sequelae: plastic reconstruction of soft tissue]. / Séquelles cicatricielles esthétiques et fonctionnelles: correction plastique des tissus mous.

The irregularities of a scarred area result of the destruction or alteration of the soft tissues. Functional consequences may occur if the scar tissue directly apply on a bony structure without any protection against shocks. Finally an underlying osteitis or unconsolidated fracture cannot be cured unless a good skin coverage and sufficient local vascularization are obtained. Secondary correction of the scar deformations intend to create a new subcutaneous layer. This can be done with adjacent adipous tissue, or by transfer of a well vascularized tissue, such as a muscle, if the underlying structures make it necessary. Some recent techniques are illustrated, especially tissue expansion and pediculated or free microvascular muscle transfer.

[Incomplete section of a superficial flexor tendon in zone II: successive complications and quadriga syndrome]. / Section incomplète d'un tendon fléchisseur superficiel en zone II: complications successives et syndrome du quadrige.

Partial section of the flexor tendon may be asymptomatic and heal without sequelae. However, in the absence of primary treatment, they may lead to subsequent complications such as: tendon locking due to incarceration of a partially detached slip of tendon, jump phenomena due to irregularity of the surface or the volume of the tendon, which induces a sudden jerk as the tendon passe through the retinaculum, and lastly, secondary tendon rupture. These complications all occurred in the case reported here, who also presented with syndrome of the quadriga. Locking of the intact deep flexor tendon by the ruptured superficial tendon interfered with the movements of the other fingers. Resection of the ruptured tendon was able to restore complete function. Opinions diverge concerning the need to suture partial sections of the flexor tendons, but the authors agree on the great importance of meticulous surgical exploration and immediate controlled mobilisation, which ensures the best functional results.

Mouse thymic virus-mediated immunosuppression: association with decreased helper T cells and increased suppressor T cells.

Mouse thymic virus (MTV) is a herpesvirus which, when administered to newborn mice, induces an extensive but temporary thymic necrosis associated with immunosuppression. In the present study, the T cell subsets in the thymus of MTV infected newborn C57Bl/6 mice were evaluated at 4, 7, 14, 28, 56, and 84 days after infection, using labeled monoclonal anti-CD4 and anti-CD8 antibodies with two-color flow cytometry. At 7 and 14 days, the percentages of CD4+8- and CD4+8+ cells were significantly decreased whereas the percentage of CD4-8+ cell was increased. At days 28 and 56 percentages had returned to normal. These results indicate that the virus has an affinity for CD4+ T cells (helper cells and their precursors). Increased percentage of CD4-8+ T cells (suppressor cells) is also associated with depressed immune functions in MTV infected newborn mice.