RESUMO
Hypertensive leg ulcer (HLU) is an inflammatory disease characterized by intense pain, alteration of vascularization, and skin necrosis. The optimal treatment relies on surgical removal of necrotic tissues covered by a split-skin graft. We studied the histomorphology of the lesions and investigated the involvement of inflammatory cells and cytokines to further define the physiopathology of HLU. We report epidermis acanthosis and a preferential occlusion of the precapillary arterioles with infiltration of neutrophils, macrophages, and T lymphocytes in the dermis. OSM, IL-1ß, and IL-6 were overexpressed in the ulcer, whereas the Th17-derived cytokines were not. In vitro, the addition of IL-1ß and OSM promoted acanthosis and destructuring of reconstructed epidermis. Exogenous IL-1ß and OSM synergistically induced epidermal acanthosis in mice. These data show that OSM and IL-1ß are not only a biological characteristic signature of HLU, but these cytokines reflect a specific inflammatory state, directly involved in the pathogenesis. We suggest that anti-cytokine biotherapies could be an alternative strategy to surgery to treat HLU.
Assuntos
Hipertensão/complicações , Interleucina-1beta/metabolismo , Úlcera da Perna/complicações , Úlcera da Perna/patologia , Melanose/complicações , Melanose/patologia , Oncostatina M/metabolismo , Adulto , Idoso , Animais , Diferenciação Celular , Proliferação de Células , Constrição Patológica/complicações , Constrição Patológica/patologia , Epiderme/patologia , Feminino , Humanos , Hipertensão/metabolismo , Hipertensão/patologia , Interleucina-6/metabolismo , Queratina-10/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Antígeno Ki-67/metabolismo , Úlcera da Perna/metabolismo , Leucócitos/patologia , Masculino , Melanose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Modelos Biológicos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologiaRESUMO
Adolescent obesity has risen dramatically in the last few decades. While adult obesity may be osteoprotective, the effects of obesity during adolescence, which is a period of massive bone accrual, are not clear. We used a murine model of induced adolescent obesity to examine the structural, mechanical, and compositional differences between obese and healthy weight bone in 16-week-old female C57Bl6 mice. We also examined the effects of a return to normal weight after skeletal maturity (24 weeks old). We found obese adolescent bone exhibited decreased trabecular bone volume, increased cortical diameter, increased ultimate stress, and increased brittleness (decreased plastic energy to fracture), similar to an aging phenotype. The trabecular bone deficits remained after return to normal weight after skeletal maturity. However, after returning to normal diet, there was no difference in ultimate stress nor plastic energy to fracture between groups as the normal diet group increased ultimate stress and brittleness. Interestingly, compositional changes appeared in the former high-fat diet mice after skeletal maturity with a lower mineral to matrix ratio compared to normal diet mice. In addition there was a trend toward increased fluorescent advanced glycation endproducts in the former high-fat diet mice compared to normal diet mice but this did not reach significance (p < 0.05) due to the large variability. The skeletal consequences of adolescent obesity may have lasting implications for the adult skeleton even after return to normal weight. Given the rates of adolescent obesity, skeletal health should be a concern.
Assuntos
Fraturas Ósseas , Obesidade Infantil , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Osso e Ossos , Osso Esponjoso , Dieta Hiperlipídica/efeitos adversos , Densidade ÓsseaRESUMO
Sebocytes originate from the same lineage as keratinocytes, and both cell types may have similarities in terms of growth and differentiation. We were interested in studying the behaviour of human sebocytes when cultured in conditions validated for epidermal reconstruction. For this purpose, we established a HPV16-E6/7-immortalized human sebocyte cell line (SEBO662) growing in keratinocyte defined media. Postconfluent SEBO662 cells in monolayers express the early sebocyte marker, cytokeratin 7 (K7), do not express Epithelia Membrane Antigen (EMA) and do not exhibit strong lipogenic activity. However, when placed at the air-liquid interface, SEBO662 multilayers spontaneously differentiate into a sebaceous-like structure as shown by the strong polarized expression of the late sebaceous marker EMA, the overexpression of some lipogenic markers and lipid production on the upper side of the epithelium. This work highlights the value of simple 3D models for exhibiting spontaneous differentiation and polarization.
Assuntos
Glândulas Sebáceas/citologia , Biomarcadores/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular , Meios de Cultura Livres de Soro , Células Epidérmicas , Epiderme/metabolismo , Humanos , Queratina-7/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Metabolismo dos Lipídeos , Fenótipo , Glândulas Sebáceas/metabolismoRESUMO
West Nile virus (WNV) is an emerging flavivirus transmitted through mosquito bites and responsible for a wide range of clinical manifestations. Following their inoculation within the skin, flaviviruses replicate in keratinocytes of the epidermis, inducing an innate immune response including the production of antimicrobial peptides (AMPs). Among them, the cathelicidin LL-37 and the human beta-defensin (hBD)-3 are known for their antimicrobial and immunomodulatory properties. We assessed their role during WNV infection of human primary keratinocytes. LL-37 reduced the viral load in the supernatant of infected keratinocytes and of the titer of a viral inoculum incubated in the presence of the peptide, suggesting a direct antiviral effect of this AMP. Conversely, WNV replication was not inhibited by hBD-3. The two peptides then demonstrated immunomodulatory properties whether in the context of keratinocyte stimulation by poly(I:C) or infection by WNV, but not alone. This study demonstrates the immunostimulatory properties of these two skin AMPs at the initial site of WNV replication and the ability of LL-37 to directly inactivate West Nile viral infectious particles. The results provide new information on the multiple functions of these two peptides and underline the potential of AMPs as new antiviral strategies in the fight against flaviviral infections.
Assuntos
Catelicidinas , Queratinócitos , Febre do Nilo Ocidental , beta-Defensinas , Fatores de Restrição Antivirais/imunologia , Catelicidinas/imunologia , Humanos , Queratinócitos/virologia , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental , beta-Defensinas/imunologiaRESUMO
Filaggrin is an epidermal protein involved in skin barrier formation and hydration, whose expression is altered in canine atopic dermatitis (CAD). CAD patients also present an abnormal immune response with an altered expression of antimicrobial peptides (AMPs), such as ß-defensins and cathelicidins. Sphingolipids and glycosaminoglycans (GAGs) have been reported to improve the skin barrier in several animal species, including dogs. Our objective was to evaluate the in vitro effects of a sphingomyelin-rich lipid extract (LE), a hyaluronic acid-rich GAG matrix, and their combination, on the expression of filaggrin and human ß-defensin 2 (hBD-2). Filaggrin expression was quantified in a reconstructed human epidermis (RHE), and hBD-2 in normal human epidermal keratinocyte (NHEK) cultures. LE and GAGs were tested at 0.02 mg/mL, with or without adding a cytokine mix. A significant increase in mean hBD-2, compared to the control (99 pg/mL) was achieved with LE (138 pg/mL) and LE+GAGs (165 pg/mL). Filaggrin increased with GAGs (202% ± 83) and LE (193% ± 44) vs. the stimulated control, but this difference was statistically significant (p < 0.05) only with LE+GAGs (210% ± 39). In conclusion, the tested GAGs and LE enhance filaggrin and AMP expression in vitro, which might benefit CAD patients if applied in vivo.
RESUMO
IL-1 plays a crucial role in triggering sterile inflammation following tissue injury. Although most studies associate IL-1 release by injured cells to the recruitment of neutrophils for tissue repair, the inflammatory cascade involves several molecular and cellular actors whose role remains to be specified. In the present study, we identified dermal fibroblasts among the IL-1R1-expressing skin cells as key sensors of IL-1 released by injured keratinocytes. After in vitro stimulation by recombinant cytokines or protein extracts of lysed keratinocytes containing high concentrations of IL-1, we show that dermal fibroblasts are by far the most IL-1-responsive cells compared to keratinocytes, melanocytes and endothelial cells. Fibroblasts have the property to respond to very low concentrations of IL-1 (from 10 fg/ml), even in the presence of 100-fold higher concentrations of IL-1RA, by increasing their expression of chemokines such as IL-8 for neutrophil recruitment. The capacity of IL-1-stimulated fibroblasts to attract neutrophils has been demonstrated both in vitro using cell migration assay and in vivo using a model of superficial epidermal lesion in IL-1R1-deficient mice which harbored reduced expression of inflammatory mediators and neutrophil skin infiltration. Together, our results shed a light on dermal fibroblasts as key relay cells in the chain of sterile inflammation induced after epidermal lesion.
Assuntos
Dermatite , Interleucina-1 , Camundongos , Animais , Interleucina-1/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-8/metabolismo , Células Endoteliais/metabolismo , Células Cultivadas , Queratinócitos/metabolismo , Dermatite/metabolismo , Fibroblastos/metabolismo , Inflamação/metabolismoRESUMO
Introduction: Although the presence of pathogens in skin wounds is known to delay the wound healing process, the mechanisms underlying this delay remain poorly understood. In the present study, we have investigated the regulatory role of proinflammatory cytokines on the healing kinetics of infected wounds. Methods: We have developed a mouse model of cutaneous wound healing, with or without wound inoculation with Staphylococcus aureus and Pseudomonas aeruginosa, two major pathogens involved in cutaneous wound bacterial infections. Results: Aseptic excision in C57BL/6 mouse skin induced early expression of IL-1ß, TNFα and Oncostatin M (OSM), without detectable expression of IL-22 and IL-17A/F. S. aureus and P. aeruginosa wound inoculation not only increased the expression of IL-1ß and OSM, but also induced a strong cutaneous expression of IL-22, IL-17A and IL-17F, along with an increased number of infiltrating IL-17A and/or IL-22-producing γδ T cells. The same cytokine expression pattern was observed in infected human skin wounds. When compared to uninfected wounds, mouse skin infection delayed the wound healing process. Injection of IL-1α, TNFα, OSM, IL-22 and IL-17 together in the wound edges induced delayed wound healing similar to that induced by the bacterial infection. Wound healing experiments in infected Rag2KO mice (deficient in lymphocytes) showed a wound healing kinetic similar to uninfected Rag2KO mice or WT mice. Rag2KO infected-skin lesions expressed lower levels of IL-17 and IL-22 than WT, suggesting that the expression of these cytokines is mainly dependent on γδ T cells in this model. Wound healing was not delayed in infected IL-17R/IL-22KO, comparable to uninfected control mice. Injection of recombinant IL-22 and IL-17 in infected wound edges of Rag2KO mice re-establish the delayed kinetic of wound healing, as in infected WT mice. Conclusion: These results demonstrate the synergistic and specific effects of IL-22 and IL-17 induced by bacterial infection delay the wound healing process, regardless of the presence of bacteria per se. Therefore, these cytokines play an unexpected role in delayed skin wound healing.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Pseudomonas aeruginosa , Camundongos , Humanos , Animais , Pseudomonas aeruginosa/metabolismo , Interleucina-17/metabolismo , Staphylococcus aureus/metabolismo , Fator de Necrose Tumoral alfa , Oncostatina M , Staphylococcus aureus Resistente à Meticilina/metabolismo , Camundongos Endogâmicos C57BL , Interleucina 22RESUMO
A placebo-controlled randomized pilot study was performed on five postmenopausal women aged from 60 to 75 years. The women applied 320 mg (2 mg/cm(2)) of either placebo or 10% C-ß-D-xylopyranoside-2-hydroxy-propane (C-xyloside) cream to each outer forearm twice daily for 3 months. At the end of the treatment, skin biopsies were collected from application areas on both forearms. Transmission electron microscope examinations revealed skin ultrastructural changes at the dermal epidermal junction (DEJ) after 10% C-xyloside application for 3 months. The morphological appearance of the DEJ showed strong improvements, with more homogeneous and regular lamina densa in the C-xyloside-treated compared to the placebo treated skin areas. The number of zones showing basement membrane re-duplication was indeed strikingly reduced on C-xyloside-treated skin. These ultrastructural results were further confirmed by a statistically significant increase in the expression levels of α6-integrin the and laminin-332, as estimated by immunohistochemistry. Altogether, these data suggest that topical C-xyloside application in vivo may be efficient in inducing a better dermal-epidermal cohesion when such a junction is deficient, as is the case in photo-aged or chronologically aged skin. Moreover, a statistically significant increase in CD44 expression was noted in the epidermis of C-xyloside-treated compared to the placebo treated skin areas.
Assuntos
Derme/crescimento & desenvolvimento , Derme/ultraestrutura , Epiderme/crescimento & desenvolvimento , Epiderme/ultraestrutura , Glicosídeos/farmacologia , Morfogênese/efeitos dos fármacos , Envelhecimento da Pele/patologia , Idoso , Moléculas de Adesão Celular/metabolismo , Derme/efeitos dos fármacos , Método Duplo-Cego , Epiderme/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Integrina alfa6/metabolismo , Pessoa de Meia-Idade , Projetos Piloto , Envelhecimento da Pele/efeitos dos fármacos , CalininaRESUMO
An upward trend in childhood obesity implies a great need to determine its effects, both immediate and long-term. Obesity is osteoprotective in adults, but we know very little about the effects of obesity on the growing skeleton, particularly its ability to adapt to load. The objective of this research is to assess bone mechanoadaptation in adolescent obese mice. Ten mice were fed a high-fat diet (HFD) from 4 to 16 weeks of age, while a control group of the same size received a normal diet (ND). At 14 weeks of age, right tibiae were cyclically loaded with a 12 N peak load for HFD mice and a 9 N peak load for ND mice three times a week for two weeks, resulting in equal peak strains of about 2500 microstrain. At 16 weeks of age, mice were sacrificed, and tibiae and gonadal fat pads were dissected. Fat pads were weighed as an obesity indicator, and tibiae were imaged with microCT to measure bone structure. The left tibiae (nonloaded) were subsequently decalcified, stained with osmium, and scanned to quantify marrow fat. Results showed that HFD mice had larger tibial cross-sectional areas compared to ND mice, as well as greater marrow adiposity. However, there was no significant difference in the amount of bone adaptation in the cortical or trabecular bone between the two groups. This indicates that the bones of HFD and ND mice adapt equally well to loading.
Assuntos
Dieta Hiperlipídica , Obesidade Infantil , Tecido Adiposo , Animais , Osso e Ossos , Dieta Hiperlipídica/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Tíbia/diagnóstico por imagemRESUMO
Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole-mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.
Cells often respond to changes in their environment by producing new molecules and building new cell components, such as proteins, which perform most tasks in the cell, or DNA and RNA, which carry genetic information. Complex tissues such as limbs, which are made up of muscles, tendons, bones and cartilage are difficult to see through, so studying when and where cells in these tissues produce different types of molecules is challenging. New approaches combining advanced three-dimensional microscopy and fluorescent labelling of molecules could provide a way to study these processes within whole animal tissues. One application for this is studying how salamanders regrow lost limbs. When salamanders such as axolotls regrow a limb, some cells in the limb stump form a group called the blastema. The blastema contains cells that are specialized to different purposes. Each cell in the blastema produces many new proteins as well as new DNA and RNA molecules. Fluorescently labeling particular molecules and taking images of the regenerating limb at different times can help to reveal how these new molecules control and coordinate limb regrowth. Duerr et al. developed a three-dimensional microscopy technique to study the production of new molecules in regenerating axolotl limbs. The method labeled molecules of different types with fluorescent markers. As a result, new proteins, RNA and DNA glowed under different colored lights. Duerr et al. used their method to show that nerve damage, which hinders limb regrowth in salamanders, reduces DNA production in the blastema. There are many possible applications of this microscopy method. Since the technique allows the spatial arrangement of the cells and molecules studied to be preserved, it makes it possible to investigate which molecules each cell is making and how they interact across a tissue. Not only does the technique have the potential to reveal much more about limb regrowth at all stages, but the fluorescent markers used can also be easily adapted to many other applications.
Assuntos
Substâncias Macromoleculares/síntese química , Ácidos Nucleicos/síntese química , Proteínas/síntese química , Ambystoma mexicanum , Animais , Regeneração Óssea , Química Click , Processamento de Imagem Assistida por Computador , Técnicas de Cultura de TecidosRESUMO
Loss of melanocytes is the pathological hallmark of vitiligo, a chronic inflammatory skin depigmenting disorder induced by exaggerated immune response, including autoreactive CD8 T cells producing high levels of type 1 cytokines. However, the interplay between this inflammatory response and melanocyte disappearance remains to be fully characterized. Here, we demonstrate that vitiligo skin contains a significant proportion of suprabasal melanocytes, associated with disruption of E-cadherin expression, a major protein involved in melanocyte adhesion. This phenomenon is also observed in lesional psoriatic skin. Importantly, apoptotic melanocytes were mainly observed once cells were detached from the basal layer of the epidermis, suggesting that additional mechanism(s) could be involved in melanocyte loss. The type 1 cytokines IFN-γ and TNF-α induce melanocyte detachment through E-cadherin disruption and the release of its soluble form, partly due to MMP-9. The levels of MMP-9 are increased in the skin and sera of patients with vitiligo, and MMP-9 is produced by keratinocytes in response to IFN-γ and TNF-α. Inhibition of MMP-9 or the JAK/STAT signaling pathway prevents melanocyte detachment in vitro and in vivo. Therefore, stabilization of melanocytes in the basal layer of the epidermis by preventing E-cadherin disruption appears promising for the prevention of depigmentation occurring in vitiligo and during chronic skin inflammation.
Assuntos
Caderinas/metabolismo , Interferon gama/metabolismo , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz/biossíntese , Melanócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vitiligo/metabolismo , Animais , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Melanócitos/patologia , CamundongosRESUMO
Pseudomonas aeruginosa, an opportunistic pathogen involved in skin and lung diseases, possesses numerous virulence factors, including type 2 and 3 secretion systems (T2SS and T3SS) and its flagellum, whose functions remain poorly known during cutaneous infection. Using isogenic mutants deleted from genes encoding each or all of these three virulence factors, we investigated their role in induction of inflammatory response and in tissue invasiveness in human primary keratinocytes and reconstructed epidermis. Our results showed that flagellum, but not T2SS and T3SS, is involved in induction of a large panel of cytokine, chemokine, and antimicrobial peptide (AMP) mRNA in the infected keratinocytes. Chemokine secretion and AMP tissular production were also dependent on the presence of the bacterial flagellum. This pro-inflammatory effect was significantly reduced in keratinocytes infected in presence of anti-toll-like receptor 5 (TLR5) neutralizing antibody. Bacterial invasion of human epidermis and persistence in a mouse model of sub-cutaneous infection were dependent on the P. aeruginosa flagellum. We demonstrated that flagellum constitutes the main virulence factor of P. aeruginosa involved not only in early induction of the epidermis inflammatory response but also in bacterial invasion and cutaneous persistence. P. aeruginosa is mainly sensed by TLR5 during the early innate immune response of human primary keratinocytes.
Assuntos
Epiderme/microbiologia , Flagelos/fisiologia , Inflamação/microbiologia , Queratinócitos/imunologia , Pseudomonas aeruginosa/patogenicidade , Animais , Anticorpos Neutralizantes/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Células Cultivadas , Quimiocinas/genética , Quimiocinas/imunologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Imunidade Inata , Queratinócitos/efeitos dos fármacos , Queratinócitos/microbiologia , Masculino , Camundongos , Mutação , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/ultraestrutura , Receptor 5 Toll-Like/imunologia , Fatores de Virulência/deficiência , Fatores de Virulência/genéticaRESUMO
The development of three-dimensional models of reconstituted mouse epidermis (RME) has been hampered by the difficulty to maintain murine primary keratinocyte cultures and to achieve a complete epidermal stratification. In this study, a new protocol is proposed for the rapid and convenient generation of RME, which reproduces accurately the architecture of a normal mouse epidermis. During RME morphogenesis, the expression of differentiation markers such as keratins, loricrin, filaggrin, E-cadherin and connexins was followed, showing that RME structure at day 5 was similar to those of a normal mouse epidermis, with the acquisition of the natural barrier function. It was also demonstrated that RME responded to skin-relevant proinflammatory cytokines by increasing the expression of antimicrobial peptides and chemokines, and inhibiting epidermal differentiation markers, as in the human system. This new model of RME is therefore suitable to investigate mouse epidermis physiology further and opens new perspectives to generate reconstituted epidermis from transgenic mice.
Assuntos
Citocinas/toxicidade , Epiderme/efeitos dos fármacos , Mediadores da Inflamação/toxicidade , Modelos Biológicos , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proteínas Filagrinas , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Camundongos Endogâmicos C57BL , Morfogênese/efeitos dos fármacos , Receptores de Citocinas/metabolismoRESUMO
Antimicrobial peptides play a major role in innate immunity. The penaeidins, initially characterized from the shrimp Litopenaeus vannamei, are a family of antimicrobial peptides that appear to be expressed in all penaeid shrimps. As of recent, a large number of penaeid nucleotide sequences have been identified from a variety of penaeid shrimp species and these sequences currently reside in several databases under unique identifiers with no nomenclatural continuity. To facilitate research in this field and avoid potential confusion due to a diverse number of nomenclatural designations, we have made a systematic effort to collect, analyse, and classify all the penaeidin sequences available in every database. We have identified a common penaeidin signature and subsequently established a classification based on amino acid sequences. In order to clarify the naming process, we have introduced a 'penaeidin nomenclature' that can be applied to all extant and future penaeidins. A specialized database, PenBase, which is freely available at , has been developed for the penaeidin family of antimicrobial peptides, to provide comprehensive information about their properties, diversity and nomenclature.
Assuntos
Antibacterianos/química , Antibacterianos/classificação , Bases de Dados de Proteínas , Decápodes/química , Peptídeos/química , Peptídeos/classificação , Terminologia como Assunto , Sequência de Aminoácidos , Animais , Antibacterianos/metabolismo , Dados de Sequência Molecular , Peptídeos/metabolismo , Alinhamento de SequênciaRESUMO
Severe burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. However, neither early complete healing nor quality of epithelialization is satisfactory. An alternative approach is to graft isolated keratinocytes. We evaluated paediatric foreskin and auricular skin as donor sources, autologous keratinocyte transplantation, and compared the graft efficiency to the in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. Keratinocytes were isolated from surgical samples by enzymatic digestion. Living cell recovery, in vitro proliferation and epidermal reconstruction capacities were evaluated. Differentiation status was analysed, using qRT-PCR and immunolabelling. Eleven children were grafted with foreskin-derived (boys) or auricular (girls) keratinocyte suspensions dripped onto deep severe burns. The aesthetic and functional quality of epithelialization was monitored in a standardized way. Foreskin keratinocyte graft in male children provides for the re-epithelialization of partial deep severe burns and accelerates wound healing, thus allowing successful wound closure, and improves the quality of scars. In accordance, in vitro studies have revealed a high yield of living keratinocyte recovery from foreskin and their potential in terms of regeneration and differentiation. We report a successful method for grafting paediatric males presenting large severe burns through direct spreading of autologous foreskin keratinocytes. This alternative method is easy to implement, improves the quality of skin and minimizes associated donor site morbidity. In vitro studies have highlighted the potential of foreskin tissue for graft applications and could help in tissue selection with the prospect of grafting burns for girls.
Assuntos
Separação Celular/métodos , Prepúcio do Pênis/citologia , Queratinócitos/citologia , Transplante de Pele , Biomarcadores/metabolismo , Queimaduras/patologia , Diferenciação Celular , Células Cultivadas , Criança , Pré-Escolar , Orelha , Células Epidérmicas , Humanos , Imuno-Histoquímica , Lactente , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração , Células-Tronco/citologia , Transplante Autólogo , CicatrizaçãoRESUMO
OBJECTIVE: Dental extraction in hemophiliacs is associated with a high risk of bleeding. It requires a multidisciplinary approach and stringent protocol. The current trend is to simplify these protocols. In this study we review the efficacy of a protocol using systemic treatment--factors/dihydro-D-arginine vasopressin (DDAVP)--and simplified local hemostatic measures to control bleeding, to limit patient discomfort, and to minimize hospital length of stay. STUDY DESIGN: This retrospective study of 55 dental extractions was performed during 19 interventions in 16 patients with hemophilia A or B to assess the efficacy of a protocol combining general management via the injection of factor concentrates or DDAVP and local hemostasis using biological glue and gelatin packing. Compressive, hemostatic splints, which have been in use by some for many years, are replaced by intermittent tranexamic acid compression during the first 3 days after surgery. RESULTS: We recorded 6 instances of postsurgical bleeding, 4 of which occurred after the compression period. In 2 cases repetition of the local hemostasic measures was required along with the injection of an antihemophilic factor concentrate. In the other 4 cases, the patients' condition reverted to normal following injection of the factor concentrate and the reapplication of the compression. CONCLUSION: The adopted protocol produced a reliable outcome, limiting the duration of the hospital stay to 24 hours in most cases, and improving postsurgical comfort thanks to a combination of systemic treatment and local hemostasic measures including intermittent tranexamic acid compression.
Assuntos
Assistência Odontológica para Doentes Crônicos/métodos , Hemofilia A , Hemorragia Bucal/prevenção & controle , Hemorragia Pós-Operatória/prevenção & controle , Extração Dentária/métodos , Adolescente , Adulto , Idoso , Criança , Protocolos Clínicos , Desamino Arginina Vasopressina/administração & dosagem , Fator IX/administração & dosagem , Fator VIII/administração & dosagem , Hemofilia A/terapia , Técnicas Hemostáticas , Hemostáticos/administração & dosagem , Humanos , Infusões Intravenosas , Injeções Intravenosas , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adesivos Teciduais/uso terapêutico , Ácido Tranexâmico/administração & dosagemRESUMO
Androgens act through non-genomic and androgen receptor (AR)-dependent genomic mechanisms. AR is expressed in the sebaceous gland and the importance of androgens in the sebaceous function is well established. However, the in vitro models used to date have failed to evidence a clear genomic effect (e.g., modification of gene expression profile) of androgens on human sebocyte cells. In order to study the impact of active androgens in sebocytes, we constructed a stable human sebocyte cell line derived from SEBO662 [17] constitutively expressing a fully functional AR. In these SEBO662 AR+ cells, dihydrotestosterone (DHT) induced AR nuclear translocation and the strong modulation of a set of transcripts (RASD1, GREB1...) known to be androgen-sensitive in other androgenic cells and tissues. Moreover, we observed that DHT precociously down-regulated markers for immature follicular cells (KRT15, TNC) and for hair lineage (KRT75, FST) and up-regulated the expression of genes potentially related to sebocyte differentiation (MUC1/EMA, AQP3, FADS2). These effects were fully confirmed at the protein level. In addition, DHT-stimulated SEBO662 AR+, cultured in a low-calcium defined keratinocyte medium without serum or any complement, neosynthesize lipids, including sebum lipids, and store increased amounts of triglycerides in lipid droplets. DHT also induces morphological changes, increases cell size, and treatments over 7 days lead to a time-dependent increase in the population of apoptotic DNA-fragmented cells. Taken together, these results show for the first time that active androgens alone can engage immature sebocytes in a clear lipogenic differentiation process (Graphical abstract). These effects depend on the expression of a functional AR in these cells. This model should be of interest for revisiting the mechanisms of the sebaceous function in vitro and for the design of relevant pharmacological models for drug or compound testing.
Assuntos
Androgênios/metabolismo , Di-Hidrotestosterona/metabolismo , Folículo Piloso/citologia , Receptores Androgênicos/biossíntese , Glândulas Sebáceas/citologia , Transporte Ativo do Núcleo Celular , Apoptose , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Queratinócitos/citologia , Glândulas Sebáceas/metabolismoRESUMO
OBJECTIVE: Severe burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. An alternative approach is to graft isolated keratinocytes. We evaluated foreskin and other anatomic sites as donor sources for autologous keratinocyte graft in children. We studied in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. METHODS: Keratinocytes were isolated from foreskin, auricular skin, chest and abdominal skin by enzymatic digestion. Living cell recovery, in vitro proliferation, epidermal reconstruction capacities and differentiation status were analyzed. RESULTS: In vitro studies revealed the higher yield of living keratinocyte recovery from foreskin and higher potential in terms of proliferative capacity, regeneration and differentiation. Cultured keratinocytes from foreskin express lower amounts of differentiation markers than those isolated from trunk and ear. Histological analysis of reconstituted human epidermis derived from foreskin and inguinal keratinocytes showed a structured multilayered epithelium, whereas those obtained from ear pinna-derived keratinocytes were unstructured. CONCLUSION: Our studies highlight the potential of foreskin tissue for autograft applications in boys. A suitable alternative donor site for autologous cell transplantation in female paediatric burn patients remains an open question in our department. We tested the hypothesis that in vitro studies and RHE reconstructive capacities of cells from different body sites can be helpful to select an optimal site for keratinocyte isolation before considering graft protocols for girls.
Assuntos
Queimaduras/cirurgia , Técnicas de Cultura de Células/métodos , Pavilhão Auricular/citologia , Células Epidérmicas , Prepúcio do Pênis/citologia , Queratinócitos/transplante , Transplante de Pele/métodos , Tronco , Adolescente , Diferenciação Celular , Proliferação de Células , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Transplante AutólogoRESUMO
An expressed sequence tag program was undertaken to isolate genes involved in defense mechanisms of the Pacific oyster, Crassostrea gigas. Putative function could be assigned to 54% of the 1142 sequenced cDNAs. We built a public database where all EST information are accessible through numerous search profiles (http://www.ifremer.fr/GigasBase). Based on sequence similarities we identified 20 genes that may be implicated in immune function. We investigated the expression of four of these genes during bacterial challenge of oysters. Three of them were induced in response to challenge lending support to their involvement in oyster immunity. Moreover, four other genes were highly homologous to components of the NF-kappa B signaling pathway which is involved in innate immune response in Drosophila and mammals. Altogether, our results open a new way to investigate the immune response in mollusks.
Assuntos
Etiquetas de Sequências Expressas , Hemócitos/metabolismo , Imunidade Inata/genética , Ostreidae/genética , Animais , Bactérias/imunologia , Células Sanguíneas/citologia , Moléculas de Adesão Celular/genética , Divisão Celular/genética , DNA Complementar/química , DNA Complementar/genética , Endopeptidases/genética , Ferritinas/genética , Regulação da Expressão Gênica/imunologia , Metalotioneína/genética , Ostreidae/imunologia , Análise de Sequência de DNA , Inibidores Teciduais de Metaloproteinases/genéticaRESUMO
Keratinocyte differentiation program leading to an organized epidermis plays a key role in maintaining the first line of defense of the skin. Epidermal integrity is regulated by a tight communication between keratinocytes and leucocytes, particularly under cytokine control. Imbalance of the cytokine network leads to inflammatory diseases such as psoriasis. Our attempt to model skin inflammation showed that the combination of IL-17A, IL-22, IL-1α, OSM and TNFα (Mix M5) synergistically increases chemokine and antimicrobial-peptide expression, recapitulating some features of psoriasis. Other characteristics of psoriasis are acanthosis and down-regulation of keratinocyte differentiation markers. Our aim was to characterize the specific roles of these cytokines on keratinocyte differentiation, and to compare with psoriatic lesion features. All cytokines decrease keratinocyte differentiation markers, but IL-22 and OSM were the most powerful, and the M5 strongly synergized the effects. In addition, IL-22 and OSM induced epidermal hyperplasia in vitro and M5 induced epidermal thickening and decreased differentiation marker expression in a mouse model, as observed in human psoriatic skin lesions. This study highlights the precise role of cytokines in the skin inflammatory response. IL-22 and OSM more specifically drive epidermal hyperplasia and differentiation loss while IL-1α, IL-17A and TNFα were more involved in the activation of innate immunity.