Background:The absence of well-established immunosuppressed rabbit models poses a significant hurdle in xenograft experiments.Tacrolimus has been identified as a highly promising immunosuppressive agent for rabbits.Ho...Background:The absence of well-established immunosuppressed rabbit models poses a significant hurdle in xenograft experiments.Tacrolimus has been identified as a highly promising immunosuppressive agent for rabbits.However,determining the optimal dosage and route of administration to minimize toxicity while maintaining efficacy remains challenging.Methods:In this study,we investigated the effect of orally administered tacrolimus in rabbits,with an aim to achieve a whole blood target trough level of 3-10 ng/m L,and looked at signs of tissue rejection after the transplantation of a human nerve conduit to repair a severed fibular nerve.An oral dosage range of 0.25-1.5 mg/kg/d was studied for up to 1 year in 63 New Zealand rabbits.Results:We demonstrated the feasibility of long-term grafting in rabbits while maintaining safe immunosuppression,with side effects mainly limited to diarrhea.Customizing the administered dose proved crucial for graft efficacy and low toxicity,which translated into 100%individual survival.We suggest an oral tacrolimus dose of 1.0-1.5 mg/kg depending on individual heterogeneity and recommend to implement a close therapeutic drug monitoring in the rabbits to maintain a whole blood tacrolimus trough level within the range of 5-12 ng/m L,as levels below 5 ng/m L showed signs of inflammation in the graft.Conclusion:The oral administration of tacrolimus enabled efficient immunosuppression of rabbits over a 1-year period without significant side effects or loss of animals.展开更多
New stem cell based therapies are undergoing intense research and are widely investigated in clinical fields including the urinary system. The urinary bladder performs critical complex functions that rely on its highl...New stem cell based therapies are undergoing intense research and are widely investigated in clinical fields including the urinary system. The urinary bladder performs critical complex functions that rely on its highly coordinated anatomical composition and multiplex of regulatory mechanisms. Bladder pathologies resulting in severe dysfunction are common clinical encounter and often cause significant impairment of patient's quality of life. Current surgical and medical interventions to correct urinary dysfunction or to replace an absent or defective bladder are sub-optimal and are associated with notable complications. As a result, stem cell based therapies for the urinary bladder are hoped to offer new venues that could make up for limitations of existing therapies. In this article, we review research efforts that describe the use of different types of stem cells in bladder reconstruction, urinary incontinence and retention disorders. In particular, stress urinary incontinence has been a popular target for stem cell based therapies in reported clinical trials. Furthermore, we discuss the relevance of the cancer stem cell hypothesis to the development of bladder cancer. A key subject that should not be overlooked is the safety and quality of stem cell based therapies introduced to human subjects either in a research or a clinical context.展开更多
The genitourinary tract can be affected by several pathologies which require repair or replacement to recover biological functions.Current therapeutic strategies are challenged by a growing shortage of adequate tissue...The genitourinary tract can be affected by several pathologies which require repair or replacement to recover biological functions.Current therapeutic strategies are challenged by a growing shortage of adequate tissues.Therefore,new options must be considered for the treatment of patients,with the use of stem cells(SCs)being attractive.Two different strategies can be derived from stem cell use:Cell therapy and tissue therapy,mainly through tissue engineering.The recent advances using these approaches are described in this review,with a focus on stromal/mesenchymal cells found in adipose tissue.Indeed,the accessibility,high yield at harvest as well as anti-fibrotic,immunomodulatory and proangiogenic properties make adipose-derived stromal/SCs promising alternatives to the therapies currently offered to patients.Finally,an innovative technique allowing tissue reconstruction without exogenous material,the self-assembly approach,will be presented.Despite advances,more studies are needed to translate such approaches from the bench to clinics in urology.For the 21st century,cell and tissue therapies based on SCs are certainly the future of genitourinary regenerative medicine.展开更多
Covering the entire human body, the skin is considered to be one of the most important organs, since it is the first line of protection against chemical and biological external agents. Although the skin protects tissu...Covering the entire human body, the skin is considered to be one of the most important organs, since it is the first line of protection against chemical and biological external agents. Although the skin protects tissues and organs against external aggression, it can still be unbalanced by various skin diseases, such as psoriasis. This non-contagious inflammatory dermatosis is characterized by the occurrence of erythematous lesions of various sizes covered with whitish scales. This scaling of the skin is the result of a rapid renewal of the epidermis, occurring over five to seven days instead of 28 days. Psoriasis vulgaris, or plaque psoriasis, is the most common form of this disease and is therefore commonly referred to by the term “psoriasis”. This work is a review of the literature on plaque psoriasis, aiming at a better comprehension of the pathology at the histological level, but also to understand the genetic and environmental factors associated with this inflammatory dermatosis.展开更多
Background:Because of its superficial anatomical localization,the cornea is particularly vulnerable to abrasive forces and various traumas,which can lead to significant visual impairments.Upon injury of the corneal ep...Background:Because of its superficial anatomical localization,the cornea is particularly vulnerable to abrasive forces and various traumas,which can lead to significant visual impairments.Upon injury of the corneal epithelium,there are important changes that occur in the composition of the underlying extracellular matrix(ECM).Those changes are perceived by the integrins that recognize the ECM components as their ligand and activate different intracellular signalling pathways,ultimately leading to reepithelialisation and reorganization of the injured epithelium,both of which are necessary in order to restore the visual properties of the cornea.The goal of this study was to analyse the impact of the pharmacological inhibition of specific signal transduction mediators of integrin-dependant signalling pathways on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas(hTECs)as in vitro models.Methods:hTECs were produced by the self-assembly approach and wounded with a 8-mm diameter biopsy punch.Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays.The wounded tissues were then incubated with the WNK1 inhibitor WNK463 and wound healing was monitored over a period of 6 days.Control corneas were incubated with the vehicle alone(DMSO).The impact of WNK1 inhibition on hCECs monolayers was determined using a scratch wound assay.Results:Gene profiling analyses and protein kinases arrays revealed important alterations in the expression and activity of several mediators from the integrin-dependent signalling pathways in response to the ECM changes taking place during corneal wound healing.Among these,WNK1 is considerably activated through phosphorylation during corneal wound healing.The pharmacological inhibition of WNK1 by WNK463 significantly reduced the dynamic of corneal wound closure in our hTECs and hCECs monolayers compared to their respective negative controls.Conclusions:These results allowed the identification of WNK1 kinase as an important player for a proper healing of the cornea.Also,these results allowed for a better understanding of the cellular and molecular mechanisms involved in corneal wound healing and they may lead to the identification of new therapeutic targets in the field of corneal wounds.展开更多
Background:The cornea composes the outer surface of the eye and its transparency is required to allow light transmission to the retina.However,because of its position,the cornea is subjected to chemical and mechanical...Background:The cornea composes the outer surface of the eye and its transparency is required to allow light transmission to the retina.However,because of its position,the cornea is subjected to chemical and mechanical injuries that may lead to blindness.Our studies conducted using the human tissue-engineered cornea(hTEC)as a model provided evidence that the cyclic-AMP-response element binding protein(CREB)pathway is repressed during closure of corneal wounds.Based on these results,we hypothesized that closure of corneal wounds can be enhanced by preventing activation of CREB with the pharmacological inhibitor C646.Our goals were to proceed to the pharmacological inhibition of CREB(I)in vitro using the hTECs as a model,and then(II)in vivo using the rabbit as a model.Methods:The self-assembly approach was used to create hTECs,that were then wounded with an 8-mm diameter biopsy punch to create an epithelial defect.The tissues were then incubated with 10μM of C646(n=8).DMSO was used alone as a negative control(n=4).Closure of the wounds was monitored over a period of 5 days.Besides,the cornea of New Zealand white rabbits was debrided with an ethanol 70%solution to create an epithelial defect of 8-mm diameter.Several concentrations of C646(1,10,100μM et 1 mM)were applied as eye drops 3 times a day for up to 7 days.The wounded corneas(n=4 per concentration)were stained with fluorescein and photographed every day.Results:In vitro pharmacological inhibition of CREB with C646 considerably accelerated wound closure of all treated hTECs(4 days)compared to the control group(7 days).Moreover,the in vivo C646 treatment also accelerated wound healing of the corneas compared to the control group.The most effective concentration of C646 tested was the lowest(1μM),as it considerably enhanced the wound healing process.Conclusions:This study demonstrates that wound healing both in vitro and in vivo can be enhanced by preventing activation of CREB using a pharmacological inhibition approach.Most of all,this experiment suggests mediators from the CREB pathway as potential therapeutic targets on which we may influence to alter the wound healing dynamic of the cornea.We believe this study will lead to significant advancements in the clinical field of corneal defects.展开更多
Background:Damage to the corneal epithelium triggers important changes in the composition of the extracellular matrix(ECM)to which the basal human corneal epithelial cells(hCECs)attach.These changes are perceived by i...Background:Damage to the corneal epithelium triggers important changes in the composition of the extracellular matrix(ECM)to which the basal human corneal epithelial cells(hCECs)attach.These changes are perceived by integrins,a family of trans-membrane receptors that activate different intracellular signalling pathways,ultimately leading to re-epithelialization of the injured epithelium.Our goal is to study the impact of the pharmacological inhibition/activation of specific signal transduction mediators on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas(hTECs)as in vitro models.Methods:hTECs were produced by the self-assembly approach and wounded with a 8-mm biopsy punch.Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays.The wounded tissues were then incubated either with the lysine deficient protein kinase 1(WNK1)inhibitor WNK463,the WNK1 indirect agonist AM1241,or with the vehicle alone(DMSO;negative control)and wound healing was monitored for 6 days.The impact of WNK1 inhibition/activation on hCECs monolayers was determined using scratch wound assays.Results:Gene profiling analyses and protein kinases arrays revealed that expression and activity of several mediators from the integrin-dependent signalling pathways were altered in response to the ECM changes taking place during corneal wound healing.Phosphorylation of the WNK1 kinase turned out to be the most striking activation event occurring during wound healing.Since the pharmacological inhibition of WNK1 by WNK463 significantly reduced the rate of corneal wound closure in our hTECs and hCECs monolayers compared to their respective negative controls,we believe that the pharmacological activation of WNK1 could turn out to be an interesting avenue to accelerate corneal wound closure.Conclusions:These results will contribute to a better understanding of the cellular and molecular mechanisms involved in corneal wound healing.Furthermore,they identified a new function for the WNK1 kinase in corneal wound healing and might lead to the identification of a new therapeutic target in the field of corneal wounds.展开更多
Background:Ocular therapy administrated by ophthalmic drops is advantageous thanks to its simplicity.However,efficiency of active molecules is limited when administered by this method.Indeed,more than 99.9%is discarde...Background:Ocular therapy administrated by ophthalmic drops is advantageous thanks to its simplicity.However,efficiency of active molecules is limited when administered by this method.Indeed,more than 99.9%is discarded due to multiple factors including lacrimal drainage.Low retention time of drugs at the cornea leads to their poor penetration.Our hypothesis is that a drug delivery system based on gold nanoparticles should enhance the efficiency of the drugs.The main objective is to develop new methods to improve active molecules biodisponibility in ocular therapy thanks to a new drug delivery system implying gold nanoparticles.The specific objectives are:(I)to synthesize and characterize ultrastable gold nanoparticles,(II)to establish the drug encapsulation protocol,(III)to develop a separation method of free and encapsulated drugs to allow their quantification,(IV)to study the cytotoxicity of our gold nanoparticles.Methods:Ultrastable gold nanoparticles were synthesized by a new method and their ultrastability toward several harsh conditions was characterized.An encapsulation protocol was settled for several drugs.The separation of free and encapsulated drugs was performed with magnetic beads.The quantification of the encapsulated drugs was performed by HPLC.A MTS assay was performed on 3 corneal epithelial cell populations,exposed or unexposed to gold nanoparticles.Reconstructed corneas were prepared using the self-assembly method.A wound healing experience was performed on those corneas with or without nanoparticles.Results:Gold nanoparticles were synthesized and purified according to our new experimental conditions.They support harsh conditions as several cycles of freeze-drying,heating,salt exposition and ultracentrifugation.For the first time in literature,gold nanoparticle support autoclave sterilisation.The separation method involving magnetic beads was optimized to get rid of non-specific interactions.The encapsulation efficiency varies according to the active molecule.The MTS assay did not show diminution of the cellular viability when in presence of gold nanoparticles.Furthermore,gold nanoparticle exposition did not slow the wound healing of reconstructed corneas.Conclusions:Our new ultrastable gold nanoparticles can have a major impact in nanomedicine.They can support harsh conditions,as autoclave treatment,allowing their sterilisation for in vivo use.We showed that active molecules can be encapsulated in gold nanoparticles.In addition,they do not seem to cause any diminution of cellular viability.These data suggest the possible improvements in ocular therapy thanks to gold nanoparticles.展开更多
Background:Lesion to the retinal pigment epithelium(RPE)is a crucial event in age-related macular degeneration(AMD)development.Although the pathogenesis of this complex disease is poorly understood,sunlight exposure a...Background:Lesion to the retinal pigment epithelium(RPE)is a crucial event in age-related macular degeneration(AMD)development.Although the pathogenesis of this complex disease is poorly understood,sunlight exposure and smoking are major environmental risk factors associated with AMD.High-energy visible blue light(HEV;400-500 nm)is the most energetic and potentially harmful solar wavelengths reaching adults retina.On the other hand,RPE cells can be exposed to a large range of pollutants from cigarette smoke,with polycyclic aromatic hydrocarbons(PAH)being among the most toxic.Some PAH from cigarette smoke can absorb HEV light.This led us hypothesize that in RPE cells,the combination of PAH and HEV could synergize to exacerbate the stress caused by either factor alone.We thus investigate the combined effect of PAH and HEV light in RPE cells.Methods:Confluent RPE immortalized cells(ARPE19)were exposed to nanomolar concentrations of benzo[a]pyrene(BaP)or indeno[1,2,3-cd]pyrene(IcdP).While IcdP efficiently absorbs HEV wavelengths,BaP,the most studied PAH,does not significantly absorb HEV light and was used as a control.BaP or IcdP contaminated ARPE19 were then irradiated with increasing sub-lethal doses of HEV light(150-500 J/cm2)using a setup that mimics the light spectrum normally reaching the retina.Cytotoxicity,apoptosis and reactive oxygen species(ROS)generation were assessed in each condition.Results:In presence of low concentrations of IcdP,sub-lethal amounts of HEV light trigger,in a dose-dependent way,up to 70%of apoptotic cell death.Co-exposure to IcdP and HEV also leads to a synergistic ROS generation in ARPE19 cells,thus inducing oxidative stress.None of these effects were observed with BaP.Efficient inhibition of ROS production by specific antioxidants only decreases death by 20%in cells simultaneously exposed to both IcdP and HEV light.Conclusions:Low concentrations of IcdP synergize with HEV light to induce phototoxicity in ARPE19 cells.An increased oxidative stress results from the interaction between both agents and partially explains the enhanced HEV phototoxicity in IcdP contaminated ARPE19 cells.This suggests that another major mechanism is involved in the synergetic toxicity.For smokers,this synergy between HEV and PAH may accelerate RPE cells loss and contribute to their greater risk of developing AMD.展开更多
Background:Uveal melanoma(UM)is the most common primary eye tumor in adults,and the most frequent site of malignant transformation of the melanocytes after the skin.It spreads to the liver in half of the cases,and the...Background:Uveal melanoma(UM)is the most common primary eye tumor in adults,and the most frequent site of malignant transformation of the melanocytes after the skin.It spreads to the liver in half of the cases,and the death rate following the report of metastasis is 92%at 2 years.Hepatic tropism of UM cannot simply be explained by the blood circulatory system organization,and illustrates the“seed and soil”hypothesis that describes an interaction between tumor cells(seed)and a specific microenvironment(soil).We decided to focus our study on the synergic interaction between UM cells and hepatic stellate cells(HSC),whose role has been previously described in the metastatic progression of colon and pancreatic cancers.Furthermore,HSC have been found surrounding UM liver metastasis,and the UM secretome contains activating cytokines of hepatic stromal cells.Our hypothesis is that HSC provide a specific microenvironment in the liver enhancing the growth of UM cells and increasing their therapeutic resistance.Using an in vitro 3D model and an original xenograft mouse model,we aim to decipher the mechanisms of UM metastatic progression,in order to elaborate new therapeutic strategies.Methods:First,using an agar coating,spheroids were generated with UM cells and were allowed to grow for 72 h.These tumor spheroids were then embedded in Matrigel and the HSC conditioned medium was used to evaluate the impact of the HSC secretome on UM invasion.Next,an original in vivo xenograft mouse model was generated,in which metastatic UM cells were injected alone or with human HSC in the spleen of immunodeficient mice.This model allows us to evaluate in 3-6 weeks the metastatic potential of each cell population,and thus to determine the cooperation between HSC and UM cells in the liver.Results:The HSC conditioned medium increased the invasion of UM spheroids compared to non-conditioned medium in our in vitro model.In addition,UM cells inoculated in the mouse spleen alone or with human HSC were able to metastasize to the liver,and the host HSC were also recruited by UM metastases.Conclusions:Our preliminary results strongly suggest that the secretome of HSC provides a permissive microenvironment for UM metastatic progression.We now have to confirm these results by characterizing the secretome of HSC,in order to identify cytokines or growth factors that increase the invasion of the liver by UM.Our models can be used to test the efficacy of new therapeutic strategies targeting the UM microenvironment.展开更多
Background:The goal of this project was to analyze the relationship between cell morphology and proteases/proteases inhibitors(PIs)secretion profile in fuchs endothelial corneal dystrophy(FECD)corneal endothelial cell...Background:The goal of this project was to analyze the relationship between cell morphology and proteases/proteases inhibitors(PIs)secretion profile in fuchs endothelial corneal dystrophy(FECD)corneal endothelial cells(CECs).Methods:Cell morphology was determined using a circularity index(4π×area/perimeter2)for each CECs population extracted from surgical FECD specimens(N=2)and healthy Eye bank corneas(N=3).CECs were cultured 28 days post-confluency.Supernatant was collected and analysed using Proteome Profiler Array detecting 35 proteases and 32 PIs(R&D Systems).Proteome signal was analyzed using Image Studio Lite and correlated with the population’s circularity index.Results:Calculation of circularity index reported different morphologies among FECD populations(0.59±0.18 and 0.64±0.17)and healthy populations(0.44±0.18,0.66±0.13 and 0.71±0.11).Proteome arrays revealed the presence of 10 proteases(ADAMTS1,Cathepsin A,B,D,and X/Z/P,DPPIV/CD26,MMP-2,3 and 12,uPA/Urokinase)and 10 PIs(Protease Nexin II,Cystatin B and C,EMMPRIN/CD147,Latexin,Lipocalin-1,Serpin E1,TFPI,TFPI-2,TIMP-1,2 and 4).Healthy and FECD specimens showed similar variation patterns according to morphology for secretion of ADAMTS1,MMP-3 and 12.However,opposing patterns between healthy and FECD populations were observed for Cathepsin B and D.Moreover,some proteins did not show variation according to phenotype in healthy CECs,but did in FECD CECs:Cathepsin A,Cystatin C,TFPI-2 and total TIMPs.For the other proteins,secretion did not vary according to morphology or no specific pattern was distinguishable.Conclusions:To conclude,our results suggest that cell phenotype is linked to the secretion of certain proteases/PIs in both groups.However,there seems to be differences in secretion of particular proteases and PIs between FECD and healthy specimens as morphology did not have a similar influence.These differences might initiate an imbalance between proteases and PIs explaining the irregular thickening of the Descemet membrane seen in FECD.展开更多
Background:The goal of this study was to engineer an epithelialized and endothelialized pigmented choroidal substitute using the self-assembly approach of tissue engineering.Methods:Cells from human choroids were isol...Background:The goal of this study was to engineer an epithelialized and endothelialized pigmented choroidal substitute using the self-assembly approach of tissue engineering.Methods:Cells from human choroids were isolated and cultured.Culture purity was assessed using immunostaining(CD31,HMB45,vimentin,keratins 8/18).To engineer the choroid,fibroblasts were cultured in the presence of serum and ascorbic acid to promote extracellular matrix(ECM)assembly.Endothelial cells,melanocytes or RPE cells were separately seeded on the stromal substitutes.Choroidal substitutes were further characterized by histology,mass spectrometry,immunostaining,and compared to native human choroids.Results:The technique used to isolate choroidal cells yielded pure cultures of fibroblasts,melanocytes and vascular endothelial cells.The stromal substitutes engineered using the self-assembly approach were composed of collagen(types I,VI,XII and XIV),proteoglycans(decorin,lumican)and other ECM proteins.Protein expression was confirmed using immunostaining.Endothelial cells spontaneously assembled into capillary-like structures and vascular networks when cocultured with fibroblast-containing ECM sheets.Conclusions:This study shows that the self-assembly approach of tissue engineering can be used to reconstruct a choroid using native cells.This model represents a unique tool to better understand the crosstalk between the different choroidal cell types and cell-ECM interactions.展开更多
Background:Cells are influenced by their environment.In vivo,the corneal endothelium is subjected to intraocular pressure(IOP).The purpose of this project was to evaluate in vitro,the effect of the IOP on the formatio...Background:Cells are influenced by their environment.In vivo,the corneal endothelium is subjected to intraocular pressure(IOP).The purpose of this project was to evaluate in vitro,the effect of the IOP on the formation of tight junctions in the corneal endothelium.Methods:Cultivated corneal endothelial cells(P2-P3;n=6 populations)were seeded on devitalized on corneas(n=10 pairs).Native corneas and devitalized corneas were respectively used as positive(n=2 pairs)and negative controls(n=3 pairs).Corneas were placed in artificial anterior chambers and subjected to a hydrostatic pressure between 0.3 and 0.4 psi during 4-5 days.Unpressured control corneas were maintained in cell culture dishes.Pictures of the corneas were taken following the experiment to assess stromal transparency.Morphology,corneal thickness and distribution of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporter were evaluated by electron microscopy,histological staining and immunofluorescences.Results:Pressure treated corneas were more transparent than the controls.Thickness was accordingly reduced by 38.4%±4.9%for cultivated endothelium and 32.2%±2.7%for native endothelium.Negative controls change in transparency and thickness were marginal.Pressure treated cells showed none or at most marginal difference in morphology and expression of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporters and failed to recreate a phenotype similar to native corneas.Pressure however increased cortical localisation of the protein ZO-1 in both cultivated and native endothelium.Conclusions:These results suggest that anterior chamber hydrostatic pressure may enhance endothelial functionality by modulating the distribution of tight junction’s proteins.展开更多
Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression ...Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression by non-hematopoietic cells is scarce. In the present study, gene and protein expression of this integrin subunit was characterized in proliferating and quiescent human RPE cells. Immunofluorescent studies confirm that the α4 subunit is expressed in vitro by RPE cells, a result that has been validated by immunofluorescence and FACS analyses. The accumulation of the α4 integrin at cell-cell junctions in post-confluent RPE cell cultures negatively correlated with the level of expression of the mRNA transcript. Accordingly, transient transfection analyses reveal that the α4 promoter activity is considerably reduced when RPE cells form a confluent monolayer. Moreover, transfection of recombinant constructs bearing 5’-deletions of the α4 promoter segment allows the localization of strong negative regulatory elements on the -76 to -300 region of the α4 gene suggesting that its expression is intimately linked to the proliferative state of primary cultured RPE cells.展开更多
Background:Sharing biological material and clinical data from patients with uveal melanoma.Methods:Uveal melanoma is the most common intraocular malignancy in the adult population.Because uveal melanoma is primarily a...Background:Sharing biological material and clinical data from patients with uveal melanoma.Methods:Uveal melanoma is the most common intraocular malignancy in the adult population.Because uveal melanoma is primarily a sporadic cancer and familial cases are rare,it is difficult to prevent or detect it.Despite effective treatment of ocular tumors,more than 50%of patients develop incurable liver metastases mainly in the 5-10 years following the detection of the primary tumor.This cancer is relatively rare and the obtained biopsies are very small.About 20 samples are taken each year in Quebec.This provincial infrastructure is made of biological material from donors with uveal melanoma and a large clinical database.Collected tumor biopsies are used for culturing cell lines and the creation of a DNA/RNA library used for genomic and genetic studies.Results:This infrastructure plays an important role in the achievement of various research programs for a better understanding of genetic and environmental factors involved in the development of melanoma and the spread of metastasis.It allows collaboration with other researchers at a provincial,national and international level in order to make progress in basic and clinical research on uveal melanoma.Conclusions:The biological material and clinical data of this infrastructure are available upon request to VHRN members whose research project was approved by the ethics committee of the institution.展开更多
Background:This infrastructure delivers biological material necessary for several research projects to Vision Health Research Network investigators(VHRN).Methods:Héma-Québec is the organism in charge obtaini...Background:This infrastructure delivers biological material necessary for several research projects to Vision Health Research Network investigators(VHRN).Methods:Héma-Québec is the organism in charge obtaining consent and retrieving donor eyes for patient treatment or for research.In Quebec City,donor eyes are sent to the eye bank of the“Centre Universitaire d’Ophtalmologie”(CUO)of Saint-Sacrement hospital.Technicians at the eye bank evaluate the quality of the tissues.Those unfit for graft are transferred to the infrastructure where the coordinator encodes samples prior to their distribution.Results:Between 2013 and 2017,27 fundamental investigators,clinical investigators and collaborators supported by 60 students,trainees and laboratory assistants used this infrastructure to move forward their projects.Since 2013,results from those projects generated 21 scientific publications and 232 presentations.The infrastructure helped VHRN investigators obtain near 4 million dollars in grants from many organisms(CIHR,NSERC,Foundations,etc.).These grants allowed recruitment and formation of highly qualified personnel.Last year(April 2016 to March 2017),189 corneas and 23 eyes transited through the infrastructure.Conclusions:This infrastructure is available for all investigators that are members of the VHRN.Many original projects have been elaborated thanks to the human ocular tissues provided by this infrastructure.These projects will advance our knowledge in vision health.A better understanding of eye functions will lead to new treatments for eye diseases.展开更多
Background:The aim of this in vitro study was to compare side-by-side two models of human bilayered tissue-engineered skin substitutes(hbTESSs)designed for the treatment of severely burned patients.These are the scaff...Background:The aim of this in vitro study was to compare side-by-side two models of human bilayered tissue-engineered skin substitutes(hbTESSs)designed for the treatment of severely burned patients.These are the scaffold-free self-assembled skin substitute(SASS)and the human plasma-based skin substitute(HPSS).Methods:Fibroblasts and keratinocytes from three humans were extracted from skin biopsies(N=3)and cells from the same donor were used to produce both hbTESS models.For SASS manu-facture,keratinocytes were seeded over three self-assembled dermal sheets comprising fibroblasts and the extracellular matrix they produced(n=12),while for HPSS production,keratinocytes were cultured over hydrogels composed of fibroblasts embedded in either plasma as unique biomaterial(Fibrin),plasma combined with hyaluronic acid(Fibrin-HA)or plasma combined with collagen(Fibrin-Col)(n/biomaterial=9).The production time was 46-55 days for SASSs and 32-39 days for HPSSs.Substitutes were characterized by histology,mechanical testing,PrestoBlue™-assay,immunofluorescence(Ki67,Keratin(K)10,K15,K19,Loricrin,type IV collagen)and Western blot(type I and IV collagens).Results:The SASSs were more resistant to tensile forces(p-value<0.01)but less elastic(p-value<0.001)compared to HPSSs.A higher number of proliferative Ki67+cells were found in SASSs although their metabolic activity was lower.After epidermal differentiation,no significant difference was observed in the expression of K10,K15,K19 and Loricrin.Overall,the production of type I and type IV collagens and the adhesive strength of the dermal-epidermal junction was higher in SASSs.Conclusions:This study demonstrates,for the first time,that both hbTESS models present similar in vitro biological characteristics.However,mechanical properties differ and future in vivo experiments will aim to compare their wound healing potential.展开更多
Cutaneous nociception is essential to prevent individuals from sustaining injuries.According to the conventional point of view,the responses to noxious stimuli are thought to be exclusively initiated by sensory neuron...Cutaneous nociception is essential to prevent individuals from sustaining injuries.According to the conventional point of view,the responses to noxious stimuli are thought to be exclusively initiated by sensory neurons,whose activity would be at most modulated by keratinocytes.However recent studies have demonstrated that epidermal keratinocytes can also act as primary nociceptive transducers as a supplement to sensory neurons.To enlighten our understanding of cutaneous nociception,this review highlights recent and relevant findings on the cellular and molecular elements that underlie the contribution of epidermal keratinocytes as nociceptive modulators and noxious sensors,both under healthy and pathological conditions.展开更多
基金the Canadian Institutes of Health Research(CIHR),Grant/Award Number:PJT-175016the Fonds de recherche du Québec(FRQ)through the research centre grant for the CHU de Québec-UniversitéLaval Research Center,Grant/Award Number:30641+2 种基金the Quebec Cell,Tissue and Gene Therapy Network—ThéCellthe FRQS,the Fondation du CHU de Québec-UniversitéLavalNeuro Québec。
文摘Background:The absence of well-established immunosuppressed rabbit models poses a significant hurdle in xenograft experiments.Tacrolimus has been identified as a highly promising immunosuppressive agent for rabbits.However,determining the optimal dosage and route of administration to minimize toxicity while maintaining efficacy remains challenging.Methods:In this study,we investigated the effect of orally administered tacrolimus in rabbits,with an aim to achieve a whole blood target trough level of 3-10 ng/m L,and looked at signs of tissue rejection after the transplantation of a human nerve conduit to repair a severed fibular nerve.An oral dosage range of 0.25-1.5 mg/kg/d was studied for up to 1 year in 63 New Zealand rabbits.Results:We demonstrated the feasibility of long-term grafting in rabbits while maintaining safe immunosuppression,with side effects mainly limited to diarrhea.Customizing the administered dose proved crucial for graft efficacy and low toxicity,which translated into 100%individual survival.We suggest an oral tacrolimus dose of 1.0-1.5 mg/kg depending on individual heterogeneity and recommend to implement a close therapeutic drug monitoring in the rabbits to maintain a whole blood tacrolimus trough level within the range of 5-12 ng/m L,as levels below 5 ng/m L showed signs of inflammation in the graft.Conclusion:The oral administration of tacrolimus enabled efficient immunosuppression of rabbits over a 1-year period without significant side effects or loss of animals.
基金funding from the Science Technology Development Fund (STDF), Egypt
文摘New stem cell based therapies are undergoing intense research and are widely investigated in clinical fields including the urinary system. The urinary bladder performs critical complex functions that rely on its highly coordinated anatomical composition and multiplex of regulatory mechanisms. Bladder pathologies resulting in severe dysfunction are common clinical encounter and often cause significant impairment of patient's quality of life. Current surgical and medical interventions to correct urinary dysfunction or to replace an absent or defective bladder are sub-optimal and are associated with notable complications. As a result, stem cell based therapies for the urinary bladder are hoped to offer new venues that could make up for limitations of existing therapies. In this article, we review research efforts that describe the use of different types of stem cells in bladder reconstruction, urinary incontinence and retention disorders. In particular, stress urinary incontinence has been a popular target for stem cell based therapies in reported clinical trials. Furthermore, we discuss the relevance of the cancer stem cell hypothesis to the development of bladder cancer. A key subject that should not be overlooked is the safety and quality of stem cell based therapies introduced to human subjects either in a research or a clinical context.
基金Supported by the‘Fonds de Recherche du Québec-Santé(FRQS)(C.C.)the Canadian Institutes of Health Research(to S.B.as PI and J.F.as co-PI),No.258229the Quebec Cell,Tissue and Gene Therapy Network-ThéCell(a thematic network supported by the FRQS).
文摘The genitourinary tract can be affected by several pathologies which require repair or replacement to recover biological functions.Current therapeutic strategies are challenged by a growing shortage of adequate tissues.Therefore,new options must be considered for the treatment of patients,with the use of stem cells(SCs)being attractive.Two different strategies can be derived from stem cell use:Cell therapy and tissue therapy,mainly through tissue engineering.The recent advances using these approaches are described in this review,with a focus on stromal/mesenchymal cells found in adipose tissue.Indeed,the accessibility,high yield at harvest as well as anti-fibrotic,immunomodulatory and proangiogenic properties make adipose-derived stromal/SCs promising alternatives to the therapies currently offered to patients.Finally,an innovative technique allowing tissue reconstruction without exogenous material,the self-assembly approach,will be presented.Despite advances,more studies are needed to translate such approaches from the bench to clinics in urology.For the 21st century,cell and tissue therapies based on SCs are certainly the future of genitourinary regenerative medicine.
文摘Covering the entire human body, the skin is considered to be one of the most important organs, since it is the first line of protection against chemical and biological external agents. Although the skin protects tissues and organs against external aggression, it can still be unbalanced by various skin diseases, such as psoriasis. This non-contagious inflammatory dermatosis is characterized by the occurrence of erythematous lesions of various sizes covered with whitish scales. This scaling of the skin is the result of a rapid renewal of the epidermis, occurring over five to seven days instead of 28 days. Psoriasis vulgaris, or plaque psoriasis, is the most common form of this disease and is therefore commonly referred to by the term “psoriasis”. This work is a review of the literature on plaque psoriasis, aiming at a better comprehension of the pathology at the histological level, but also to understand the genetic and environmental factors associated with this inflammatory dermatosis.
文摘Background:Because of its superficial anatomical localization,the cornea is particularly vulnerable to abrasive forces and various traumas,which can lead to significant visual impairments.Upon injury of the corneal epithelium,there are important changes that occur in the composition of the underlying extracellular matrix(ECM).Those changes are perceived by the integrins that recognize the ECM components as their ligand and activate different intracellular signalling pathways,ultimately leading to reepithelialisation and reorganization of the injured epithelium,both of which are necessary in order to restore the visual properties of the cornea.The goal of this study was to analyse the impact of the pharmacological inhibition of specific signal transduction mediators of integrin-dependant signalling pathways on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas(hTECs)as in vitro models.Methods:hTECs were produced by the self-assembly approach and wounded with a 8-mm diameter biopsy punch.Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays.The wounded tissues were then incubated with the WNK1 inhibitor WNK463 and wound healing was monitored over a period of 6 days.Control corneas were incubated with the vehicle alone(DMSO).The impact of WNK1 inhibition on hCECs monolayers was determined using a scratch wound assay.Results:Gene profiling analyses and protein kinases arrays revealed important alterations in the expression and activity of several mediators from the integrin-dependent signalling pathways in response to the ECM changes taking place during corneal wound healing.Among these,WNK1 is considerably activated through phosphorylation during corneal wound healing.The pharmacological inhibition of WNK1 by WNK463 significantly reduced the dynamic of corneal wound closure in our hTECs and hCECs monolayers compared to their respective negative controls.Conclusions:These results allowed the identification of WNK1 kinase as an important player for a proper healing of the cornea.Also,these results allowed for a better understanding of the cellular and molecular mechanisms involved in corneal wound healing and they may lead to the identification of new therapeutic targets in the field of corneal wounds.
文摘Background:The cornea composes the outer surface of the eye and its transparency is required to allow light transmission to the retina.However,because of its position,the cornea is subjected to chemical and mechanical injuries that may lead to blindness.Our studies conducted using the human tissue-engineered cornea(hTEC)as a model provided evidence that the cyclic-AMP-response element binding protein(CREB)pathway is repressed during closure of corneal wounds.Based on these results,we hypothesized that closure of corneal wounds can be enhanced by preventing activation of CREB with the pharmacological inhibitor C646.Our goals were to proceed to the pharmacological inhibition of CREB(I)in vitro using the hTECs as a model,and then(II)in vivo using the rabbit as a model.Methods:The self-assembly approach was used to create hTECs,that were then wounded with an 8-mm diameter biopsy punch to create an epithelial defect.The tissues were then incubated with 10μM of C646(n=8).DMSO was used alone as a negative control(n=4).Closure of the wounds was monitored over a period of 5 days.Besides,the cornea of New Zealand white rabbits was debrided with an ethanol 70%solution to create an epithelial defect of 8-mm diameter.Several concentrations of C646(1,10,100μM et 1 mM)were applied as eye drops 3 times a day for up to 7 days.The wounded corneas(n=4 per concentration)were stained with fluorescein and photographed every day.Results:In vitro pharmacological inhibition of CREB with C646 considerably accelerated wound closure of all treated hTECs(4 days)compared to the control group(7 days).Moreover,the in vivo C646 treatment also accelerated wound healing of the corneas compared to the control group.The most effective concentration of C646 tested was the lowest(1μM),as it considerably enhanced the wound healing process.Conclusions:This study demonstrates that wound healing both in vitro and in vivo can be enhanced by preventing activation of CREB using a pharmacological inhibition approach.Most of all,this experiment suggests mediators from the CREB pathway as potential therapeutic targets on which we may influence to alter the wound healing dynamic of the cornea.We believe this study will lead to significant advancements in the clinical field of corneal defects.
文摘Background:Damage to the corneal epithelium triggers important changes in the composition of the extracellular matrix(ECM)to which the basal human corneal epithelial cells(hCECs)attach.These changes are perceived by integrins,a family of trans-membrane receptors that activate different intracellular signalling pathways,ultimately leading to re-epithelialization of the injured epithelium.Our goal is to study the impact of the pharmacological inhibition/activation of specific signal transduction mediators on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas(hTECs)as in vitro models.Methods:hTECs were produced by the self-assembly approach and wounded with a 8-mm biopsy punch.Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays.The wounded tissues were then incubated either with the lysine deficient protein kinase 1(WNK1)inhibitor WNK463,the WNK1 indirect agonist AM1241,or with the vehicle alone(DMSO;negative control)and wound healing was monitored for 6 days.The impact of WNK1 inhibition/activation on hCECs monolayers was determined using scratch wound assays.Results:Gene profiling analyses and protein kinases arrays revealed that expression and activity of several mediators from the integrin-dependent signalling pathways were altered in response to the ECM changes taking place during corneal wound healing.Phosphorylation of the WNK1 kinase turned out to be the most striking activation event occurring during wound healing.Since the pharmacological inhibition of WNK1 by WNK463 significantly reduced the rate of corneal wound closure in our hTECs and hCECs monolayers compared to their respective negative controls,we believe that the pharmacological activation of WNK1 could turn out to be an interesting avenue to accelerate corneal wound closure.Conclusions:These results will contribute to a better understanding of the cellular and molecular mechanisms involved in corneal wound healing.Furthermore,they identified a new function for the WNK1 kinase in corneal wound healing and might lead to the identification of a new therapeutic target in the field of corneal wounds.
文摘Background:Ocular therapy administrated by ophthalmic drops is advantageous thanks to its simplicity.However,efficiency of active molecules is limited when administered by this method.Indeed,more than 99.9%is discarded due to multiple factors including lacrimal drainage.Low retention time of drugs at the cornea leads to their poor penetration.Our hypothesis is that a drug delivery system based on gold nanoparticles should enhance the efficiency of the drugs.The main objective is to develop new methods to improve active molecules biodisponibility in ocular therapy thanks to a new drug delivery system implying gold nanoparticles.The specific objectives are:(I)to synthesize and characterize ultrastable gold nanoparticles,(II)to establish the drug encapsulation protocol,(III)to develop a separation method of free and encapsulated drugs to allow their quantification,(IV)to study the cytotoxicity of our gold nanoparticles.Methods:Ultrastable gold nanoparticles were synthesized by a new method and their ultrastability toward several harsh conditions was characterized.An encapsulation protocol was settled for several drugs.The separation of free and encapsulated drugs was performed with magnetic beads.The quantification of the encapsulated drugs was performed by HPLC.A MTS assay was performed on 3 corneal epithelial cell populations,exposed or unexposed to gold nanoparticles.Reconstructed corneas were prepared using the self-assembly method.A wound healing experience was performed on those corneas with or without nanoparticles.Results:Gold nanoparticles were synthesized and purified according to our new experimental conditions.They support harsh conditions as several cycles of freeze-drying,heating,salt exposition and ultracentrifugation.For the first time in literature,gold nanoparticle support autoclave sterilisation.The separation method involving magnetic beads was optimized to get rid of non-specific interactions.The encapsulation efficiency varies according to the active molecule.The MTS assay did not show diminution of the cellular viability when in presence of gold nanoparticles.Furthermore,gold nanoparticle exposition did not slow the wound healing of reconstructed corneas.Conclusions:Our new ultrastable gold nanoparticles can have a major impact in nanomedicine.They can support harsh conditions,as autoclave treatment,allowing their sterilisation for in vivo use.We showed that active molecules can be encapsulated in gold nanoparticles.In addition,they do not seem to cause any diminution of cellular viability.These data suggest the possible improvements in ocular therapy thanks to gold nanoparticles.
文摘Background:Lesion to the retinal pigment epithelium(RPE)is a crucial event in age-related macular degeneration(AMD)development.Although the pathogenesis of this complex disease is poorly understood,sunlight exposure and smoking are major environmental risk factors associated with AMD.High-energy visible blue light(HEV;400-500 nm)is the most energetic and potentially harmful solar wavelengths reaching adults retina.On the other hand,RPE cells can be exposed to a large range of pollutants from cigarette smoke,with polycyclic aromatic hydrocarbons(PAH)being among the most toxic.Some PAH from cigarette smoke can absorb HEV light.This led us hypothesize that in RPE cells,the combination of PAH and HEV could synergize to exacerbate the stress caused by either factor alone.We thus investigate the combined effect of PAH and HEV light in RPE cells.Methods:Confluent RPE immortalized cells(ARPE19)were exposed to nanomolar concentrations of benzo[a]pyrene(BaP)or indeno[1,2,3-cd]pyrene(IcdP).While IcdP efficiently absorbs HEV wavelengths,BaP,the most studied PAH,does not significantly absorb HEV light and was used as a control.BaP or IcdP contaminated ARPE19 were then irradiated with increasing sub-lethal doses of HEV light(150-500 J/cm2)using a setup that mimics the light spectrum normally reaching the retina.Cytotoxicity,apoptosis and reactive oxygen species(ROS)generation were assessed in each condition.Results:In presence of low concentrations of IcdP,sub-lethal amounts of HEV light trigger,in a dose-dependent way,up to 70%of apoptotic cell death.Co-exposure to IcdP and HEV also leads to a synergistic ROS generation in ARPE19 cells,thus inducing oxidative stress.None of these effects were observed with BaP.Efficient inhibition of ROS production by specific antioxidants only decreases death by 20%in cells simultaneously exposed to both IcdP and HEV light.Conclusions:Low concentrations of IcdP synergize with HEV light to induce phototoxicity in ARPE19 cells.An increased oxidative stress results from the interaction between both agents and partially explains the enhanced HEV phototoxicity in IcdP contaminated ARPE19 cells.This suggests that another major mechanism is involved in the synergetic toxicity.For smokers,this synergy between HEV and PAH may accelerate RPE cells loss and contribute to their greater risk of developing AMD.
文摘Background:Uveal melanoma(UM)is the most common primary eye tumor in adults,and the most frequent site of malignant transformation of the melanocytes after the skin.It spreads to the liver in half of the cases,and the death rate following the report of metastasis is 92%at 2 years.Hepatic tropism of UM cannot simply be explained by the blood circulatory system organization,and illustrates the“seed and soil”hypothesis that describes an interaction between tumor cells(seed)and a specific microenvironment(soil).We decided to focus our study on the synergic interaction between UM cells and hepatic stellate cells(HSC),whose role has been previously described in the metastatic progression of colon and pancreatic cancers.Furthermore,HSC have been found surrounding UM liver metastasis,and the UM secretome contains activating cytokines of hepatic stromal cells.Our hypothesis is that HSC provide a specific microenvironment in the liver enhancing the growth of UM cells and increasing their therapeutic resistance.Using an in vitro 3D model and an original xenograft mouse model,we aim to decipher the mechanisms of UM metastatic progression,in order to elaborate new therapeutic strategies.Methods:First,using an agar coating,spheroids were generated with UM cells and were allowed to grow for 72 h.These tumor spheroids were then embedded in Matrigel and the HSC conditioned medium was used to evaluate the impact of the HSC secretome on UM invasion.Next,an original in vivo xenograft mouse model was generated,in which metastatic UM cells were injected alone or with human HSC in the spleen of immunodeficient mice.This model allows us to evaluate in 3-6 weeks the metastatic potential of each cell population,and thus to determine the cooperation between HSC and UM cells in the liver.Results:The HSC conditioned medium increased the invasion of UM spheroids compared to non-conditioned medium in our in vitro model.In addition,UM cells inoculated in the mouse spleen alone or with human HSC were able to metastasize to the liver,and the host HSC were also recruited by UM metastases.Conclusions:Our preliminary results strongly suggest that the secretome of HSC provides a permissive microenvironment for UM metastatic progression.We now have to confirm these results by characterizing the secretome of HSC,in order to identify cytokines or growth factors that increase the invasion of the liver by UM.Our models can be used to test the efficacy of new therapeutic strategies targeting the UM microenvironment.
文摘Background:The goal of this project was to analyze the relationship between cell morphology and proteases/proteases inhibitors(PIs)secretion profile in fuchs endothelial corneal dystrophy(FECD)corneal endothelial cells(CECs).Methods:Cell morphology was determined using a circularity index(4π×area/perimeter2)for each CECs population extracted from surgical FECD specimens(N=2)and healthy Eye bank corneas(N=3).CECs were cultured 28 days post-confluency.Supernatant was collected and analysed using Proteome Profiler Array detecting 35 proteases and 32 PIs(R&D Systems).Proteome signal was analyzed using Image Studio Lite and correlated with the population’s circularity index.Results:Calculation of circularity index reported different morphologies among FECD populations(0.59±0.18 and 0.64±0.17)and healthy populations(0.44±0.18,0.66±0.13 and 0.71±0.11).Proteome arrays revealed the presence of 10 proteases(ADAMTS1,Cathepsin A,B,D,and X/Z/P,DPPIV/CD26,MMP-2,3 and 12,uPA/Urokinase)and 10 PIs(Protease Nexin II,Cystatin B and C,EMMPRIN/CD147,Latexin,Lipocalin-1,Serpin E1,TFPI,TFPI-2,TIMP-1,2 and 4).Healthy and FECD specimens showed similar variation patterns according to morphology for secretion of ADAMTS1,MMP-3 and 12.However,opposing patterns between healthy and FECD populations were observed for Cathepsin B and D.Moreover,some proteins did not show variation according to phenotype in healthy CECs,but did in FECD CECs:Cathepsin A,Cystatin C,TFPI-2 and total TIMPs.For the other proteins,secretion did not vary according to morphology or no specific pattern was distinguishable.Conclusions:To conclude,our results suggest that cell phenotype is linked to the secretion of certain proteases/PIs in both groups.However,there seems to be differences in secretion of particular proteases and PIs between FECD and healthy specimens as morphology did not have a similar influence.These differences might initiate an imbalance between proteases and PIs explaining the irregular thickening of the Descemet membrane seen in FECD.
文摘Background:The goal of this study was to engineer an epithelialized and endothelialized pigmented choroidal substitute using the self-assembly approach of tissue engineering.Methods:Cells from human choroids were isolated and cultured.Culture purity was assessed using immunostaining(CD31,HMB45,vimentin,keratins 8/18).To engineer the choroid,fibroblasts were cultured in the presence of serum and ascorbic acid to promote extracellular matrix(ECM)assembly.Endothelial cells,melanocytes or RPE cells were separately seeded on the stromal substitutes.Choroidal substitutes were further characterized by histology,mass spectrometry,immunostaining,and compared to native human choroids.Results:The technique used to isolate choroidal cells yielded pure cultures of fibroblasts,melanocytes and vascular endothelial cells.The stromal substitutes engineered using the self-assembly approach were composed of collagen(types I,VI,XII and XIV),proteoglycans(decorin,lumican)and other ECM proteins.Protein expression was confirmed using immunostaining.Endothelial cells spontaneously assembled into capillary-like structures and vascular networks when cocultured with fibroblast-containing ECM sheets.Conclusions:This study shows that the self-assembly approach of tissue engineering can be used to reconstruct a choroid using native cells.This model represents a unique tool to better understand the crosstalk between the different choroidal cell types and cell-ECM interactions.
文摘Background:Cells are influenced by their environment.In vivo,the corneal endothelium is subjected to intraocular pressure(IOP).The purpose of this project was to evaluate in vitro,the effect of the IOP on the formation of tight junctions in the corneal endothelium.Methods:Cultivated corneal endothelial cells(P2-P3;n=6 populations)were seeded on devitalized on corneas(n=10 pairs).Native corneas and devitalized corneas were respectively used as positive(n=2 pairs)and negative controls(n=3 pairs).Corneas were placed in artificial anterior chambers and subjected to a hydrostatic pressure between 0.3 and 0.4 psi during 4-5 days.Unpressured control corneas were maintained in cell culture dishes.Pictures of the corneas were taken following the experiment to assess stromal transparency.Morphology,corneal thickness and distribution of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporter were evaluated by electron microscopy,histological staining and immunofluorescences.Results:Pressure treated corneas were more transparent than the controls.Thickness was accordingly reduced by 38.4%±4.9%for cultivated endothelium and 32.2%±2.7%for native endothelium.Negative controls change in transparency and thickness were marginal.Pressure treated cells showed none or at most marginal difference in morphology and expression of ZO-1,n-cadherin,b-catenin,NaK ATPase pump and HCO3-cotransporters and failed to recreate a phenotype similar to native corneas.Pressure however increased cortical localisation of the protein ZO-1 in both cultivated and native endothelium.Conclusions:These results suggest that anterior chamber hydrostatic pressure may enhance endothelial functionality by modulating the distribution of tight junction’s proteins.
文摘Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression by non-hematopoietic cells is scarce. In the present study, gene and protein expression of this integrin subunit was characterized in proliferating and quiescent human RPE cells. Immunofluorescent studies confirm that the α4 subunit is expressed in vitro by RPE cells, a result that has been validated by immunofluorescence and FACS analyses. The accumulation of the α4 integrin at cell-cell junctions in post-confluent RPE cell cultures negatively correlated with the level of expression of the mRNA transcript. Accordingly, transient transfection analyses reveal that the α4 promoter activity is considerably reduced when RPE cells form a confluent monolayer. Moreover, transfection of recombinant constructs bearing 5’-deletions of the α4 promoter segment allows the localization of strong negative regulatory elements on the -76 to -300 region of the α4 gene suggesting that its expression is intimately linked to the proliferative state of primary cultured RPE cells.
文摘Background:Sharing biological material and clinical data from patients with uveal melanoma.Methods:Uveal melanoma is the most common intraocular malignancy in the adult population.Because uveal melanoma is primarily a sporadic cancer and familial cases are rare,it is difficult to prevent or detect it.Despite effective treatment of ocular tumors,more than 50%of patients develop incurable liver metastases mainly in the 5-10 years following the detection of the primary tumor.This cancer is relatively rare and the obtained biopsies are very small.About 20 samples are taken each year in Quebec.This provincial infrastructure is made of biological material from donors with uveal melanoma and a large clinical database.Collected tumor biopsies are used for culturing cell lines and the creation of a DNA/RNA library used for genomic and genetic studies.Results:This infrastructure plays an important role in the achievement of various research programs for a better understanding of genetic and environmental factors involved in the development of melanoma and the spread of metastasis.It allows collaboration with other researchers at a provincial,national and international level in order to make progress in basic and clinical research on uveal melanoma.Conclusions:The biological material and clinical data of this infrastructure are available upon request to VHRN members whose research project was approved by the ethics committee of the institution.
文摘Background:This infrastructure delivers biological material necessary for several research projects to Vision Health Research Network investigators(VHRN).Methods:Héma-Québec is the organism in charge obtaining consent and retrieving donor eyes for patient treatment or for research.In Quebec City,donor eyes are sent to the eye bank of the“Centre Universitaire d’Ophtalmologie”(CUO)of Saint-Sacrement hospital.Technicians at the eye bank evaluate the quality of the tissues.Those unfit for graft are transferred to the infrastructure where the coordinator encodes samples prior to their distribution.Results:Between 2013 and 2017,27 fundamental investigators,clinical investigators and collaborators supported by 60 students,trainees and laboratory assistants used this infrastructure to move forward their projects.Since 2013,results from those projects generated 21 scientific publications and 232 presentations.The infrastructure helped VHRN investigators obtain near 4 million dollars in grants from many organisms(CIHR,NSERC,Foundations,etc.).These grants allowed recruitment and formation of highly qualified personnel.Last year(April 2016 to March 2017),189 corneas and 23 eyes transited through the infrastructure.Conclusions:This infrastructure is available for all investigators that are members of the VHRN.Many original projects have been elaborated thanks to the human ocular tissues provided by this infrastructure.These projects will advance our knowledge in vision health.A better understanding of eye functions will lead to new treatments for eye diseases.
基金funded by the Instituto de Salud Carlos III through the project PI17/02083(co-funded by the European Regional Development Fund“A way to make Europe”)the RegionalGovernment of Andalusia(PIGE-0242-2019)+2 种基金the Canadian Institutes for Health Research(CIHR)(FDN-143213 and IC-132948)the Fondation des Pompiers du Québec pour les Grands Brûlés(FPQGB)the Quebec Network for Cell,Tissue and Gene Therapy-ThéCell(a thematic network supported by the Fonds de Recherche du Québec-Santé[FRQS]).
文摘Background:The aim of this in vitro study was to compare side-by-side two models of human bilayered tissue-engineered skin substitutes(hbTESSs)designed for the treatment of severely burned patients.These are the scaffold-free self-assembled skin substitute(SASS)and the human plasma-based skin substitute(HPSS).Methods:Fibroblasts and keratinocytes from three humans were extracted from skin biopsies(N=3)and cells from the same donor were used to produce both hbTESS models.For SASS manu-facture,keratinocytes were seeded over three self-assembled dermal sheets comprising fibroblasts and the extracellular matrix they produced(n=12),while for HPSS production,keratinocytes were cultured over hydrogels composed of fibroblasts embedded in either plasma as unique biomaterial(Fibrin),plasma combined with hyaluronic acid(Fibrin-HA)or plasma combined with collagen(Fibrin-Col)(n/biomaterial=9).The production time was 46-55 days for SASSs and 32-39 days for HPSSs.Substitutes were characterized by histology,mechanical testing,PrestoBlue™-assay,immunofluorescence(Ki67,Keratin(K)10,K15,K19,Loricrin,type IV collagen)and Western blot(type I and IV collagens).Results:The SASSs were more resistant to tensile forces(p-value<0.01)but less elastic(p-value<0.001)compared to HPSSs.A higher number of proliferative Ki67+cells were found in SASSs although their metabolic activity was lower.After epidermal differentiation,no significant difference was observed in the expression of K10,K15,K19 and Loricrin.Overall,the production of type I and type IV collagens and the adhesive strength of the dermal-epidermal junction was higher in SASSs.Conclusions:This study demonstrates,for the first time,that both hbTESS models present similar in vitro biological characteristics.However,mechanical properties differ and future in vivo experiments will aim to compare their wound healing potential.
文摘Cutaneous nociception is essential to prevent individuals from sustaining injuries.According to the conventional point of view,the responses to noxious stimuli are thought to be exclusively initiated by sensory neurons,whose activity would be at most modulated by keratinocytes.However recent studies have demonstrated that epidermal keratinocytes can also act as primary nociceptive transducers as a supplement to sensory neurons.To enlighten our understanding of cutaneous nociception,this review highlights recent and relevant findings on the cellular and molecular elements that underlie the contribution of epidermal keratinocytes as nociceptive modulators and noxious sensors,both under healthy and pathological conditions.
