The use of autologous cells harvested and subsequently transplanted in an intraoperative environment constitutes a new approach to promote regeneration. Usually cells are isolated by selection methods such as fluorescence- or magnetic- activated cell sorting with residual binding of the antibodies or beads. Thus, cell-based therapies would benefit from the development of new devices for cell isolation that minimally manipulate the target cell population. In the clinic, 5 to 10 percent of fractures do not heal properly and CD31+ cells have been identified as promising candidates to support bone regeneration. The aim of this project was to develop and prototype a simple system to facilitate the enrichment of CD31+ cells from whole blood. After validating the specificity of a commercially available aptamer for CD31, we combined this aptamer with traditional magnetic bead strategies, which led to enrichment of CD31+ cells with a purity of 91±10%. Subsequently, the aptamer was attached to agarose beads (Ø = 100-165 um) that were incorporated into a column-based system to enable capture and subsequent release of the CD31+ enriched cells. Different parameters were investigated to allow a biophysical-based cell release from beads, and a simple mixing was found sufficient to release initially bound cells from the optimized column without the need for any chemicals that promote disassociation. The system led to a significant enrichment of CD31+ cells (initial population: 63±9%, released: 87±3%) with excellent cell viability (released: 97±1%). The composition of the released CD31+ fraction indicated an enrichment of the monocyte population. The angiogenic and osteogenic potential of the released cell population were confirmed in vitro. These results and the simplicity of this system highlight the potential of such approach to enable cell enrichment strategies in intraoperative settings.

Introduction: Primary cutaneous diffuse large B-cell lymphoma leg-type (PCLBCL-LT) is recognized by the World Health Organization (WHO) classification as a rare and aggressive disease accounting for 5 to 10% of primary cutaneous lymphoma. It displays original clinical features occurring mostly in the elderly and preferentially involving the legs but date the genetic specificities of this entities, as compared to other B-cell aggressive lymphoma is unknown. Methods: To determine whether the mutational profile of primary cutaneous diffuse large B-cell lymphoma leg-type (PCLBCL-LT) is unique by comparison with other diffuse large B-cell lymphoma (DLBCL) subtypes, we analyzed a total cohort of 28 PCLBCL-LT cases by next generation sequencing with a Lymphopanel designed for DLBCL. We also analyzed 12 pairs of tumor and control DNA samples by whole exome sequencing which led us to perform resequencing of three selected genes not included in the Lymphopanel: TBL1XR1, KLHL6 and IKZF3. To pinpoint specificities, comparison with a cohort of DLBCL and Primary central nervous System lymphoma (PCNSL) analyzed by the same targeted panel was performed. Results: Our study clearly identifies an original mutational landscape of PCLBCL-LT with a very restricted set of highly recurrent mutations (>40%) involving MYD88 (p.L265P variant), PIM1 and CD79B. Other genes involved in B-cell signaling, NFKB activation or DNA modeling were found altered notably TBL1XR1 (33%), MYC (26%) CREBBP (26%) and IRF4 (21%) or HIST1H1E(41%). MYD88L265P variant was associated with copy number variations or copy neutral loss of heterozygosity in 60% of cases. The most frequent genetic losses involved CDKN2A/2B, TNFAIP3/A20, PRDM1, TCF3 and CIITA. The high recurrence of specific gene mutations such as MYD88 and the absence of mutations of KMT2D or FOXO1 are distinct features from nodal DLBCLs of either GC or ABC subtypes. Interestingly PCLBCL-LT exhibits a mutational pattern that is closer to PCNSL than to ABC-type nodal DLBCL but also has distinctive features, such as a very high mutational rate for PIM1, a higher rate of CD79B mutations and other original mutations such as CREBBP, MYC and IRF4 mutations (figure 1). Figure 1. Heat map of mutation frequencies by DLBCL subtypes. Mutation frequencies in each subtype are indicated for each gene of the Lymphopanel. Mutation frequencies per gene and per subtype are shown here as the percentage of the total number of patients. Statistical significance of gene mutation enrichment in a given subtype is indicated in the False Discovery Rate (FDR) column. The horizontal line separates genes with FDR < 0.05, considered as significant, from genes with FDR ≥ 0.05. Bold text indicates FDR values <0.05. Mutation rates in nodal Activated B-Cell (ABC), primary mediastinal B-cell (PMBL) and germinal center B-cell (GCB) subtypes have been published in Dubois et al. CCR 2016. Mutation rates in primary central nervous system lymphoma (PCNSL) have been obtained by the same methodology. Keywords: activated B-cell-like (ABC); cutaneous B-cell lymphoma (CBCL).

Injectable gelatin hydrogels formed with bioorthogonal click chemistry (ClickGel) are cell-responsive ECM mimics for in vitro and in vivo biomaterials applications. Gelatin polymers with pendant norbornene (GelN) or tetrazine (GelT) groups can quickly and spontaneously crosslink upon mixing, allowing for high viability of encapsulated cells, establishment of 3D elongated cell morphologies, and biodegradation when injected in vivo.

The complex arrangement of the extracellular matrix (ECM) produced by cells during tissue growth, healing and remodelling is fundamental to tissue function. In connective tissues, it is still unclear how both cells and the ECM become and remain organized over length scales much larger than the distance between neighbouring cells. While cytoskeletal forces are essential for assembly and organization of the early ECM, how these processes lead to a highly organized ECM in tissues such as osteoid is not clear. To clarify the role of cellular tension for the development of these ordered fibril architectures, we used an in vitro model system, where pre-osteoblastic cells produced ECM-rich tissue inside channels with millimetre-sized triangular cross sections in ceramic scaffolds. Our results suggest a mechanical handshake between actively contracting cells and ECM fibrils: the build-up of a long-range organization of cells and the ECM enables a gradual conversion of cell-generated tension to pre-straining the ECM fibrils, which reduces the work cells have to generate to keep mature tissue under tension.

Mycobacterium marinum is an atypical and non-tuberculosis mycobacterium that mainly leads to cutaneous infections. Infections occur through inoculation of the organism through injury to the skin in the presence of contaminated water or fish. The patient often presents with unspecific symptoms and the evolution, in the absence of adequate treatment, is characterized by an expansion of the cutaneous lesion and a spread to deep structures. Infections of tendon sheaths and joints are described, rarely osteomyelitis. Sure diagnosis is hard to obtain and is established from the medical history and microbiological examination. There are no specific therapeutic guidelines. Double or triple antibiotherapy is often effective and should be continued several months after complete resolution of clinical signs. Surgical debridement is required in cases of invasive or resistant infections. We report the case of a young immunocompetent fishmonger with a rare osteocutaneous M. marinum infection of the elbow. Treatment included large surgical excision of infected skin and bone areas and a triple antibiotics administration. Reconstruction have been ensured by a radial collateral artery perforator-based propeller flap, satisfying appropriates functional and cosmetical concerns of this anatomical region. Surgery and appropriate antibiotics treatment were effective and allowed healing of an invasive cutaneous and bone M. marinum infection. (C) 2015 Elsevier Masson SAS. All rights reserved.

The reduced basis method recent progress have permitted to make the computations reliable thanks to a posteriori estimators . However, it may not always be possible to use the code to perform all the off-line computations required for an efficient performance of the reduced basis method. We propose here an alternating approach based on a coarse grid finite element, the convergence of which is accelerated through the reduced basis and an improved post processing.

To heal tissue defects, cells have to bridge gaps and generate new extracellular matrix (ECM). Macroporous scaffolds are frequently used to support the process of defect filling and thus foster tissue regeneration. Such biomaterials contain micro-voids (pores) that the cells fill with their own ECM over time. There is only limited knowledge on how pore geometry influences cell organization and matrix production, even though it is highly relevant for scaffold design. This study hypothesized that 1) a simple geometric description predicts cellular organization during pore filling at the cell level and that 2) pore closure results in a reorganization of ECM. Scaffolds with a broad distribution of pore sizes (macroporous starPEG-heparin cryogel) were used as a model system and seeded with primary fibroblasts. The strategies of cells to fill pores could be explained by a simple geometrical model considering cells as tensioned chords. The model matched qualitatively as well as quantitatively by means of cell number vs. open cross-sectional area for all pore sizes. The correlation between ECM location and cell position was higher when the pores were not filled with tissue (Pearson's coefficient ρ = 0.45±0.01) and reduced once the pores were closed (ρ = 0.26±0.04) indicating a reorganization of the cell/ECM network. Scaffold pore size directed the time required for pore closure and furthermore impacted the organization of the fibronectin matrix. Understanding how cells fill micro-voids will help to design biomaterial scaffolds that support the endogenous healing process and thus allow a fast filling of tissue defects.

La gravité de la pemphigoïde cicatricielle (PC) est variable. Le traitement des formes peu ou moyennement sévères repose sur la dapsone. Dans les formes graves, les bolus de cyclophosphamide sont efficaces, mais parfois mal tolérés et nécessitent des hospitalisations répétées. Le mycophénolate mofétil (MMF), administré par voie orale et bien toléré, a montré son efficacité dans le pemphigus et certaines pemphigoïdes bulleuses. Quelques observations encourageantes ont été rapportées dans la PC. Depuis 2000, ce traitement a été proposé à 14 malades de notre service. Nous avons revu rétrospectivement les dossiers.Il s'agissait de 5 hommes et 9 femmes, d'âge moyen 69 ans. Le MMF était prescrit dans 3 circonstances : en relais immédiat du cyclophosphamide chez 7 malades avec atteinte sévère (groupe I) ; en cas de rechute modérément sévère à distance de l'arrêt du cyclophosphamide chez 3 malades (groupe II) ; comme immunosuppresseur de première intention chez 4 malades dont la PC restait évolutive sous dapsone (groupe III), sans toutefois menace immédiate du pronostic visuel ou vital. Le point commun de ces 3 situations était de chercher à contrôler une maladie de gravité moyenne, par un immunosuppresseur oral et bien toléré, dans le but d'améliorer le confort de vie du malade. La dose de MMF utilisée était de 1,5 ou 2 g/j. Le critère d'efficacité du traitement était l'absence de signes cliniques d'évolutivité de la maladie.Le MMF était efficace pour obtenir ou maintenir un bon contrôle de la maladie dans 10 cas sur 14 tant que la dose de dapsone était maintenue à 2 mg/kg/j. Dans 7/10 cas, il était possible dans un deuxième temps de diminuer la dose de la dapsone à des doses mieux tolérées. Dans les 3 autres cas, la maladie rechutait lorsque la dose de la dapsone était diminuée. Dans ces 3 cas, le MMF semblait permettre de passer un cap pour obtenir le contrôle d'une maladie restant évolutive sous dapsone seule, mais n'était pas suffisant à lui seul. Dans 4 cas sur 14, le MMF n'était pas efficace. La tolérance clinique et biologique du MMF était bonne dans 13 cas sur 14.Dans cette étude rétrospective sur dossier, le MMF a été proposé à des malades hétérogènes, mais dont le point commun était d'avoir une maladie de gravité modérée et pour lesquels une amélioration du confort de vie était recherchée. Le MMF semble intéressant pour maintenir un contrôle satisfaisant de la maladie et permettre la baisse de la dose de dapsone dans certaines PC de gravité moyenne. L'utilisation de ce médicament ne peut pas remplacer le cyclophosphamide dans des PC graves avec menace du pronostic visuel ou vital.The severity of cicatricial pemphigoid (CP) varies. First-intent treatment of mild or moderate cases is dapsone. In life or sight-threatening cases, intravenous cyclophosphamide pulses are efficient but may have digestive side effects and imply repeated hospitalizations. Mycophenolate mofetil (MMF) is an oral and well tolerated immunosuppressant agent which has proved its efficacy in pemphigus and some bullous pemphigoid. In CP, encouraging case reports have been previously published. We report herein a retrospective study about 14 patients who have received MMF since 2000.There were 5 men and 9 women, with a mean age of 69 years. MMF was introduced in 3 different clinical situations: immediately in relay to cyclophosphamide in 7 patients with severe CP (group I); in case of a mild-severe relapse at distance from with dranal of cyclophosphamide in 3 patients (group II); as first-intent immunosuppressant agent in 4 patients whose disease was not under control with high-dose dapsone, but not life - or sight-threatening (group III). In all these patients, the disease was invalidating and not controlled by dapsone ± sulfasalazine, but did not threaten life or sight. The aim was to achieve satisfying control of the disease with an oral and well tolerated immunosuppressant agent, and to maintain good quality of life. The dose of MMF was 1.5 or 2 g per day. The criteria of MMF efficacy was the healing of previous lesions and the absence of new progressive lesions.MMF was efficient in obtaining or maintaining a good control of the disease in 10/14 patients, as long as the underlying treatment with dapsone (2 mg/kg/d) was maintained. In 7/10 cases, it was possible to decrease the dapsone dose in order to improve hematological tolerance. In the 3 other cases, a relapse occurred when the dose of dapsone was decreased. MMF was inefficient in controling the disease in 4/14 patients (29 p. 100). Clinical and biological tolerance of MMF was good in 13/14 patients.In this series, MMF was proposed to heterogenous patients, who presented at that time a mild-moderate disease and for whom we wanted in improve the quality of life. MMF seems to be an interesting drug, capable of obtaining or maintaining satisfactory control of the disease and permitting the decrease of dapsone doses in some mild-severe CP. However MMF must not replace cyclophosphamide in severe sight or life-threatening forms of CP.

Fibroblasts as many other cells are known to form, contract, and remodel the extracellular matrix (ECM). The presented study aims to gain an insight into how mechanical boundary conditions affect the production of ECM components, their remodeling, and the feedback of the altered mechanical cell environment on these processes. The influence of cyclic mechanical loading (f = 1 Hz, 10% axial compression) and scaffold stiffness (E = 1.2 and 8.5 kPa) on the mechanical properties of fibroblast-seeded scaffold constructs were investigated in an in vitro approach over 14 days of culture. To do so, a newly developed bioreactor system was employed. While mechanical loading resulted in a clear upregulation of procollagen-I and fibronectin production, scaffold stiffness showed to primarily influence matrix metalloproteinase-1 (MMP-1) secretion and cell-induced scaffold contraction. Higher stiffness of the collagen scaffolds resulted in an up to twofold higher production of collagen-degrading MMP-1. The changes of mechanical parameters like Young's modulus, maximum compression force, and elastic portion of compression force over time suggest that from initially distinct mechanical starting conditions (scaffold stiffness), the construct's mechanical properties converge over time. As a consequence of mechanical loading a shift toward higher construct stiffness was observed. The results suggest that scaffold stiffness has only a temporary effect on cell behavior, while the impact of mechanical loading is preserved over time. Thus, it is concluded that the mechanical environment of the cell after remodeling is depending on mechanical loading rather than on initial scaffold stiffness.

This paper describes the development of a numerical algorithm for investigating the probability of wind-borne compact debris, impacting on the vertical façade of tall buildings. A uniform wind field with constant velocity was employed; turbulence effects were neglected.The trajectory of compact debris was estimated by solving the three-dimensional (3D) equations of motion of the object, immersed in uniform wind field; simulation results were compared to experimental data, derived from literature. Random parameters were selected to estimate the trajectory and to derive "Iso-probability Impact Contours", using Monte Carlo simulation. These contours describe the probability associated with "randomly flying" debris, as they impact against a cladding on the vertical façade, conditional on the initial position of the object relative to the structure. These contours were developed for two different cases, "Quasi-2D" and "Fully 3D", to account for wind directionality.Moreover, a "Universal Probability Curve", describing the probability of impact for compact objects against the façade of a benchmark tall building, was developed in two-dimensional (2D). It was proven that this curve is universal and can be used independently of wind velocity.

BackgroundPemphigus foliaceus is an autoimmune blistering skin disease characterized by the production of pathogenic IgG autoantibodies directed against desmoglein 1.AimTo determine the prevalence of anti-desmoglein 1 antibodies in healthy subjects and their distribution in the different regions of Tunisia and to better identify endemic areas of pemphigus foliaceus.MethodsWe tested, by enzyme-linked immunoserbent assay, sera of 270 normal subjects recruited from different Tunisian areas and 203 related healthy relatives to 90 Tunisian pemphigus foliaceus patients.ResultsSeventy-six patients (84.4%), 20 healthy controls (7.4%), and 32 relatives (15.76%) had anti-desmoglein 1 antibodies. In southern regions where pemphigus foliaceus is associated with a significant sex ratio imbalance (9 female : 1 male in the south vs. 2.3 : 1 in the north) and a lower mean age of disease onset (33.5 in the south vs. 45 years in the north), a higher prevalence of anti-desmoglein 1 antibodies in healthy controls was observed (9.23% vs. 5.71% in the north). Interestingly, the highest prevalence of anti-desmoglein 1 antibodies in healthy relatives (up to 22%) was observed in the most rural southern localities. More than half anti-desmoglein 1-positive healthy controls were living in rural conditions with farming as occupation, which suggests that this activity may expose the subjects to particular environmental conditions.ConclusionThese results show that the endemic features of Tunisian pemphigus foliaceus are focused in these southern areas more than in other areas and that both environmental and genetic factors contribute to the disease.Conflicts of interestNone declared.

BACKGROUND:Pemphigus foliaceus is an autoimmune blistering skin disease that partly results from genetic factors, especially human leucocyte antigen (HLA) class II genes.OBJECTIVES:The aim of the study was to determine the HLA DR/DQ markers of susceptibility and protection in the Tunisian endemic form.METHODS:Genomic DNA from 90 patients with pemphigus foliaceus recruited from all parts of the country and matched by age, sex and geographical origin with 270 healthy individuals, was genotyped.RESULTS:Firstly, when the whole patient population was studied, DRB1*03, DQB1*0302 and DRB1*04 alleles were significantly associated with the disease while a significant decrease of, in particular, DRB1*11 and DQB1*0301 was observed in patients compared with controls. DRB1*0301 was the dominant allele in DR3-positive patients and controls, while DRB1*0402 was found in 42% of DR4-positive patients. Secondly, when the HLA DR/DQ allele distribution was studied after dividing patients according to their geographical origin, the southern group, which consisted exclusively of patients with the endemic form of the disease, showed the same associations as the whole pemphigus foliaceus population, particularly with DRB1*03. In the northern group, only the DRB1*04 and DQB1*0301 alleles were found to be associated. Interestingly, anti-desmoglein 1 antibody-positive healthy controls did not carry susceptibility alleles but, in contrast, most carried negatively associated alleles.CONCLUSIONS:These observations indicate that a particular genetic background characterizes the Tunisian endemic form of pemphigus foliaceus and that HLA class II genes control the pathogenic properties of the autoimmune response rather than the initial breakage of B-cell tolerance.

Desmoglein (Dsg) 1 is a transmembrane glycoprotein of the desmosome allowing cell-cell adhesion between keratinocytes, whose expression is restricted to stratified squamous epithelia-like epidermis. Dsg1 is the target autoantigen of pathogenic autoantibodies produced by pemphigus foliaceus and 50% of pemphigus vulgaris patients in a Dsg1-specific T-cell-dependent pathway. Herewith, we show that mRNA of the DSG1 gene is present in normal human thymus and show by quantitative real-time polymerase chain reaction analysis that the expression of DSG1 transcript increases with age. Although immunoblot analysis on human thymus extracts using different anti-Dsg1 antibodies did not allow to detect the protein, we show by double-immunofluorescence assay that Dsg1 is expressed at protein level by CD19+ CD63+ cells located in the medulla. These data provide another illustration of the thymic expression of a tissue-specific autoantigen involved in an organ-specific autoimmune disease, which may participate in the tolerance acquisition and/or regulation of Dsg1-specific T cells.

Conflicts of interest: none declared. Sir, Pemphigus is an autoimmune blistering disease mediated by pathogenic antibodies directed against desmoglein (Dsg) 1 and Dsg3.1, 2 In Tunisia, pemphigus has particular epidemiological features. It occurs as an endemic form with an incidence rate of 6·7 cases per million per year. Case series suggest that pemphigus, especially the foliaceus form (PF), is frequent among young Tunisian women with an incidence rate reaching 20 cases per million in rural areas. This could be related to a risk factor more prevalent and/or more often expressed in these areas.3, 4 We have reported previously that anti‐Dsg1 antibodies were prevalent in Tunisian healthy subjects, which argues for the role of a still unknown environmental factor in the occurrence of Tunisian endemic pemphigus.5 An epidemiological case–control study demonstrated that several environmental factors were significantly associated to Tunisian endemic pemphigus such as traditional cosmetics, Turkish baths and cutting up raw poultry.6 However, infectious factors have not been yet investigated.

Epidemiological studies performed in different ethnic populations and family studies, notably based on a partial phenotype of the autoimmune process, indicate that genetic factors are involved in the occurrence of pemphigus. However, the precise heritability remains uncertain in the absence of twin concordance rate studies. Among the different strategies available to identify genetic factors participating in autoimmune disease susceptibility, only population studies based on case–control design have been performed in pemphigus. These studies consistently showed that MHC locus, in particular HLA class II alleles, are associated with pemphigus vulgaris and pemphigus foliaceus. Other genes of the MHC locus may also participate in disease susceptibility as shown by studies using microsatellite markers across different regions of the MHC. It is likely that other non-MHC genes are involved in the pathogenesis of pemphigus. In particular, involvement of a polymorphic variant of desmoglein 1 gene was shown to be associated with pemphigus foliaceus and to interact in an epistatic manner with MHC class II genes to contribute to the autoimmune process. Other candidate genes to which a role can be assigned in the disease pathogenesis should be considered to design case–control or family-based association studies. Genome scan studies which require a large number of multiplex families to reach statistical power, should also be considered in the endemic form of pemphigus foliaceus because of the high number of familial cases.