792 research outputs found

    Wound Repair and Regeneration

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    Wounds are a largely unrecognized, spiraling epidemic that affect millions of people world-wide. They are complex and involve temporal and spatial involvement of many different cell types and tissue processes. Recent advances in our understanding of wound repair and regeneration, as well as the many novel and exciting approaches aimed at healing chronic/acute wounds and reducing scar formation, make this a pertinent time for a Special Issue aimed at overviewing this important field. The goal of this book is to provide a summary of the field, describe its impact, as well as introduce the recent advances in understanding the mechanisms that underpin wound healing and scar formation. The articles include in this book highlight new developments in therapeutic approaches for wound repair including the use of nanomedicine and biomaterials to deliver cells and/or drugs to promote healing. Cellular responses that underpin angiogenesis, inflammation, proliferation and remodeling, as well as advances in cytoskeletal interactions in keratinocytes and fibroblast cell functions. Wound remodeling and scar formation including the roles of growth factors, cytokines and stem cells are included

    Transient tear hyperosmolarity disrupts the neuroimmune homeostasis of the ocular surface and facilitates dry eye onset

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    Dry eye disease (DED) is a highly prevalent ocular surface disorder with neuroimmune pathophysiology. Tear hyperosmolarity (THO), a frequent finding in affected patients, is considered a key element in DED pathogenesis, yet existing animal models are based on subjecting the ocular surface to the more complex desiccating stress − decreased tear production and/or increased evaporation − instead of strict hyperosmolar stress. Here we characterized a murine model of THO that does not involve desiccating stress, thus allowing us to dissect the contribution of THO to DED. Our results showed that THO is sufficient to disrupt neuroimmune homeostasis of the ocular surface in mice, and thus reproduce many sub‐clinical DED findings. THO activated nuclear factor‐κB signalling in conjunctival epithelial cells and increased dendritic cell recruitment and maturation, leading to more activated (CD69+) and memory (CD62lo CD44hi) CD4+ T‐cells in the eye‐draining lymph nodes. Ultimately, THO impaired the development of ocular mucosal tolerance to a topical surrogate antigen in a chain of events that included epithelial nuclear factor‐κB signalling and activation of transient receptor potential vanilloid 1 as the probable hypertonicity sensor. Also, THO reduced the density of corneal intraepithelial nerves and terminals, and sensitized the ocular surface to hypertonicity. Finally, the adoptive transfer of T‐cells from THO mice to naïve recipients under mild desiccating stress favoured DED development, showing that THO is enough to trigger an actual pathogenic T‐cell response. Our results altogether demonstrate that THO is a critical initiating factor in DED development.Fil: Guzmán Fonseca, Oscar Mauricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Miglio Rodríguez, Maximiliano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Keitelman, Irene Angélica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Shiromizu, Carolina Maiumi. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Sabbione, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fuentes, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Trevani, Analía Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Galletti, Jeremías Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

    The Transcriptional Regulation Of Flagellin-Induced Innate Protection Of The Cornea: Role Of Irf1 And Atf3

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    Pre-exposure of the cornea to TLR5 ligand flagellin induces profound mucosal innate protection against infections by reprogramming gene expression. This study explored the flagellin-induced modifications of transcription factor expression and function, specifically of IRF1 and ATF3 in corneal epithelial cells to elucidate the transcriptional mechanisms underlying the protective function of flagellin on the cornea. Initially we used Superarray to screen for transcription factors and identified Interferon Regulatory Factor (IRF) 1 and Activating Transcription Factor (ATF) 3 as the most drastically affected genes by flagellin pretreatment in P. aeruginosa challenged human corneal epithelial cells (CEC). However, flagellin pretreatment had opposite effects on IRF1 (inhibition) and ATF3 (enhancement) gene expression in response to P. aeruginosa, and other IRFs were not affected. To find the functional target gene of IRF1, we knocked-down IRF1 using siRNA and identified the pleiotropic chemokine CXCL10, but not IL12-p35 or iNOS, as a specific target. We then attempted to understand the role of IRF1 and CXCL10 in mucosal innate protection against infections in vivo. Flagellin augmented the P. aeruginosa-induced expression of CXCL10 in CECs in WT, but not in IRF1-/- mice at 6 hpi. IRF1 deficiency markedly increased the severity of P. aeruginosa keratitis and significantly attenuated flagellin-elicited protection compared to WT controls at 3 dpi. CXCL10 neutralization in the cornea of WT mice displayed similar pathogenesis to that of IRF1-/- mice. To understand the regulation of CXCL10 expression in vivo, we used neutralizing antibodies to target IFNγ and its producer NK cells. The neutralization of IFNγ receptor or NK cells prevented flagellin-augmented IRF1 and CXCL10 expression and increased the susceptibility to P. aeruginosa infection in mouse corneas. Together, our results indicate that IRF1 plays a role in the corneal innate immune response by regulating CXCL10 expression and that IFNγ-producing NK cells induces epithelial expression of IRF1, thus significantly contributing to the protection of the cornea from P. aeruginosa infection. Next we sought understand the functional role of ATF3 in mucosal innate protection against infections in vivo. Consistent with in vitro results, flagellin augmented the P. aeruginosa-induced epithelial expression of ATF3 in CECs in WT at 6 hpi. ATF3 deficiency markedly increased the severity of P. aeruginosa keratitis, indicated by significantly increased bacterial count, neutrophil infiltration and CXCL2 expression. In addition, ATF3-null mice displayed compromised flagellin-induced corneal protection compared to PBS-pretreated ATF3-/- littermates at 3 dpi. Together, our results indicate that ATF3 plays a role in the corneal innate immune response by regulating inflammation and bacterial clearance, and ATF3 is indispensible to maintain the protective properties of flagellin against bacterial keratitis. In conclusion, flagellin pretreatment of corneal epithelial cells reprograms the expression of epithelial IRF1 and ATF3, via different mechanisms, resulting in the enhancement of the innate immune response of the cornea. Both transcription factors play an critical role in protecting the cornea from bacterial invasion and regulating inflammation, and may serve as important markers for the development of therapeutic drugs to treat and/or prevent bacterial keratitis

    Lipopolysaccharides (LPSs)

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    The cytoplasm of Gram-negative bacteria is bound by three layers: an inner membrane, a layer of peptidoglycan, and an outer membrane. The outer membrane is an asymmetric lipidic bilayer, with phospholipids on its inner surface and lipopolysaccharides (LPSs) on the outside, with the latter being the major component of the outer leaflet and covering nearly three-quarters of the total outer cell surface. All LPSs possess the same general chemical architecture independently of bacterial activity (pathogenic, symbiotic, commensal), ecological niche (human, animal, soil, plant, water), or growth conditions. Endotoxins are large amphiphilic molecules consisting of a hydrophilic polysaccharide component and a covalently bound hydrophobic and highly conserved lipid component, termed lipid A (the endotoxin subunit). The polysaccharide component can be divided into two subdomains: the internal and conserved core region as well as the more external and highly variable O-specific chain, also referred to as the O-antigen due to its immunogenic properties. LPSs are endotoxins, one of the most potent class of activators of the mammalian immune system; they can be released from cell surfaces of bacteria during multiplication, lysis, and death. LPS can act through its biological center (lipid A component) on various cell types, of which macrophages and monocytes are the most important

    Macrophages and cytokines in the early defence against herpes simplex virus

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    Herpes simplex virus (HSV) type 1 and 2 are old viruses, with a history of evolution shared with humans. Thus, it is generally well-adapted viruses, infecting many of us without doing much harm, and with the capacity to hide in our neurons for life. In rare situations, however, the primary infection becomes generalized or involves the brain. Normally, the primary HSV infection is asymptomatic, and a crucial element in the early restriction of virus replication and thus avoidance of symptoms from the infection is the concerted action of different arms of the innate immune response. An early and light struggle inhibiting some HSV replication will spare the host from the real war against huge amounts of virus later in infection. As far as such a war will jeopardize the life of the host, it will be in both interests, including the virus, to settle the conflict amicably. Some important weapons of the unspecific defence and the early strikes and beginning battle during the first days of a HSV infection are discussed in this review. Generally, macrophages are orchestrating a multitude of anti-herpetic actions during the first hours of the attack. In a first wave of responses, cytokines, primarily type I interferons (IFN) and tumour necrosis factor are produced and exert a direct antiviral effect and activate the macrophages themselves. In the next wave, interleukin (IL)-12 together with the above and other cytokines induce production of IFN-γ in mainly NK cells. Many positive feed-back mechanisms and synergistic interactions intensify these systems and give rise to heavy antiviral weapons such as reactive oxygen species and nitric oxide. This results in the generation of an alliance against the viral enemy. However, these heavy weapons have to be controlled to avoid too much harm to the host. By IL-4 and others, these reactions are hampered, but they are still allowed in foci of HSV replication, thus focusing the activity to only relevant sites. So, no hero does it alone. Rather, an alliance of cytokines, macrophages and other cells seems to play a central role. Implications of this for future treatment modalities are shortly considered

    Inflammation In The Pathogenesis Of Diabetic Retinopathy

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    The general purpose of these studies is to investigate inflammation in diabetic retinopathy in an effort to identify key intervention points to develop as treatments. Firstly, we showed that the neuropeptide VIP displayed protective immunoregulatory effects on retinal endothelial cells cultured under high glucose conditions. This effect was carried out, in part through the VPAC2 receptor. Next, we studied the β-adrenergic receptor agonist, Compound 49b, and its effect on the pro-resolving RvD1 pathway. Compound 49b was previously shown to suppress both inflammatory and apoptotic responses in DR. We demonstrated that Compound 49b rescued the high glucose-induced decrese in RvD1 and its receptors in diabetic animals and retinal endothelial cell culture, by upregulating 15-LOX enzyme expression. We also studied the phosphorylation of NF-κB p65 in two retinal cell types exposed to high glucose. High glucose conditions stimulated phosphorylation of NF-κB p65 at Thr-254, Ser-276, Ser-468, Ser-529, Thr-435 in retinal endothelial cells and Thr-254, Ser-281, Ser-311, Ser-468, Thr-435 in Müller cells. IL-4, an anti-inflammatory cytokine, suppressed phosphorylation at Thr-254, Ser-311, Thr-435 in retinal endothelial cells and Thr-254, Ser-276, Ser-281, Thr-435 in Müller cells. Futhermore, IL-4 also reduced related downstream NF-κB regulated molecules IL-8, TNF-α, and upregulated IL-10. The influence of type 1 vs type 2 immune backgrounds on DR-related damage using a model of retinal ischemia-reperfusion was studied in C57BL/6 and BALB/c mice. Notably, both neuronal and vascular degeneration were significantly less in BALB/c compared to B6 mice. Furthermore, key inflammatory molecules IL-1β, TNF-α, NF-κB, ICAM-1 and VEGF were downregulated in BLAB/c mice, as well. Collectively, we have shown the extensive role that inflammation plays in diabetic retinopathy pathogenesis. More importantly, the innate type 1/type 2 paradigm suggests that the potential of anti-inflammatory treamtents and pro-resolving lipid mediators in suppressing pathogenesis of DR. We expect our findings in pathogenesis of inflammation to contibute to development of anti-inflammatory and pro-resolving treatments for diabetic retinopathy

    CFTR Involvement in Cell Migration and Epithelial Restitution

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    Over the past decade, research has shown that cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in epithelial cell migration and wound healing. Experiments with airway epithelium, ovarian epithelial cells, placental epithelium and epidermal keratinocytes demonstrated that CFTR function is necessary to achieve maximum migration rates during restitution and in certain cancer cells, CFTR activity contributes to tumor cell invasion. Multiple mechanisms appear to underlie the motility‐promoting actions of CFTR, and although many details remain to be established, our present understanding indicates that processes such as electrotaxis (galvanotaxis), integrin‐mediated cell adhesion and lamellipodia protrusion are dependent on normal CFTR function. In this chapter, the role of CFTR in epithelial cell migration and its implications in cystic fibrosis (CF) will be reviewed with emphasis on the underlying mechanisms that may explain observations made in various epithelial tissues, particularly in airways. Ultimately, a better understanding of CFTR involvement in epithelial repair may lead to new therapeutic approaches to improve barrier function and reduce airway infection and inflammation associated with CF

    Inflammatory Mechanisms of Chemokine Receptor 7 expression in Metastatic Squamous Cell Carcinoma of the Head and Neck (SCCHN)

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    The microenvironment of aerodigestive cancers contains tumor promoting inflammatory signals often involved in innate immunity. SCCHN is an epithelial malignancy characterized by the secretion of inflammatory mediators that can promote tumorigenesis and lymph node metastasis. The chemokine receptor CCR7 is a key molecule whose aberrant expression in SCCHN has been linked to pro-survival, invasive and metastatic pathways. Indeed, the selective upregulation of CCR7 in metastatic SCCHN tumors has been previously described. However, the mechanisms of CCR7 expression have not yet been elucidated. Inflammatory cytokines are known to upregulate CCR7 in immune cells through downstream NF-κB dependent mechanisms. In addition, antimicrobial peptides such as human β-defensin 3 (HBD3) are capable of promoting an inflammatory microenvironment and may possess tumor-promoting properties. Given the frequent overexpression NF-κB in SCCHN and its association with a more aggressive SCCHN phenotype, I hypothesized that NF-κB may be a key mediator of invasive and metastatic disease by promoting CCR7 expression in SCCHN tumors. Indeed, I identified and studied four potential NF-κB binding sites in the promoter region upstream of the CCR7 gene and report on their relative contribution to CCR7 expression in metastatic SCCHN. Furthermore, I demonstrate that HBD3 induces CCR7 expression in dendritic cells as well as primary SCCHN tumors in an NF-κB-dependent fashion. Interestingly, HBD3 stimulation provides anti-apoptotic signals to SCCHN cells, as evidenced by tumor resistance to cisplatin-induced cell death. As presented in this dissertation, these findings suggest that HBD3 represents a novel, NF-κB-regulated mediator of CCR7 expression and anti-apoptotic pathways, which may be exploited by developing SCCHN tumors to enhance their growth, survival and evolution into a metastatic phenotype. NF-κB appears to be a key regulator of basal and inducible CCR7 expression. The observed NF-κB induction of CCR7 and its subsequent downstream pathways provide clinically important therapeutic targets to control the progression and metastasis of SCCHN tumors

    Role of Reactive Gliosis and Neuroinflammation in Experimental Glaucoma

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    Le glaucome est la principale cause de cécité irréversible dans le monde. Chez les patients atteints de cette pathologie, la perte de la vue résulte de la mort sélective des cellules ganglionnaires (CGR) de la rétine ainsi que de la dégénérescence axonale. La pression intraoculaire élevée est considérée le facteur de risque majeur pour le développement de cette maladie. Les thérapies actuelles emploient des traitements pharmacologiques et/ou chirurgicaux pour diminuer la pression oculaire. Néanmoins, la perte du champ visuel continue à progresser, impliquant des mécanismes indépendants de la pression intraoculaire dans la progression de la maladie. Il a été récemment démontré que des facteurs neuroinflammatoires pourraient être impliqués dans le développement du glaucome. Cette réponse est caractérisée par une régulation positive des cytokines pro-inflammatoires, en particulier du facteur de nécrose tumorale alpha (TNFα). Cependant, le mécanisme par lequel le processus neuroinflammatoire agit sur la mort neuronale reste à clarifier. L’hypothèse principale de ce doctorat propose que les facteurs pro-inflammatoires comme le TNFα et la phosphodiestérase 4 (PDE4) interagissent avec les mécanismes moléculaires de la mort neuronale, favorisant ainsi la survie et la protection des CGRs au cours du glaucome. Dans la première partie de ma thèse, J’ai utilisé un modèle in vivo de glaucome chez des rats Brown Norway pour montrer que l’expression du TNFα est augmentée après l'induction de l'hypertension oculaire. L'hypothèse spécifique de cette étude suggère que les niveaux élevés de TNFα provoquent la mort des CGRs en favorisant l'insertion de récepteurs AMPA perméables au calcium (CP-AMPAR) à la membrane cytoplasmique. Pour tester cette hypothèse, j’ai utilisé un inhibiteur sélectif de la forme soluble du TNFα, le XPro1595. L'administration de cet agent pharmacologique a induit une protection significative des somas et des axones des neurones rétiniens. L'évaluation de la perméabilité au cobalt a montré que le TNFα soluble est impliqué dans l'insertion de CP-AMPAR à la membrane des CGRs lors du glaucome. L’exposition des neurones à une pression oculaire élevée est à l’origine de la hausse de la densité membranaire des CP-AMPARs, grâce à une diminution de l’expression de la sous-unité GluA2. La présence de GluA2 au sein du récepteur ne permet pas l’entrée du calcium à l’intérieur de la cellule. L'administration intraoculaire d’antagonistes spécifiques des CP-AMPARs promeut la protection des somas et des axones des CGRs. Ces résultats montrent que les CP-AMPARs jouent un rôle important dans la pathologie du glaucome. Dans la deuxième partie de ma thèse, j’ai caractérisé l'effet neuroprotecteur d’un inhibiteur de la PDE4, l’ibudilast, dans notre modèle de glaucome. L'hypothèse spécifique s’oriente vers une atténuation de la réponse neuroinflammatoire et de la gliose par l’administration d’ibudilast, favorisant ainsi la protection neuronale. Les résultats montrent que dans les rétines glaucomateuses, l’ibudilast diminue la gliose et l'expression de plusieurs facteurs tels que le TNFα, l'interleukine-1β (IL-1β), l’interleukine-6 (IL-6) et le facteur inhibiteur de la migration des macrophages (MIF). Chez les rats glaucomateux, nous avons observé une expression notable de PDE4A dans les cellules de Müller, qui est en corrélation avec l'accumulation de l’AMP cyclique (AMPc) dans ces cellules après un traitement d’ibudilast. Finalement, nous avons démontré que la protection des CGRs via l’administration d’ibudilast est un mécanisme dépendent de l’AMPc et de la protéine kinase A (PKA). En conclusion, les résultats présentés dans cette thèse identifient deux mécanismes différents impliqués dans la perte des CGRs au cours du glaucome. Ces mécanismes pourraient fournir des perspectives potentielles pour le développement de nouvelles stratégies de traitement du glaucome.Glaucoma is the leading cause of irreversible blindness worldwide. Loss of vision in glaucoma results from the selective death of retinal ganglion cells (RGCs) and axonal degeneration. Elevated intraocular pressure (IOP) is the major risk factor for developing glaucoma, and current therapies have focused on pharmacological or surgical strategies to lower IOP. However, visual field loss continues to progress in spite of effective pressure control, indicating that mechanisms other than elevated IOP contribute to disease progression. Recent data demonstrate a neuroinflammatory component in glaucoma, characterized by upregulation of proinflammatory cytokines, most notably tumor necrosis factor α (TNFα). However, the mechanism by which the neuroinflammatory response acts on RGC death needs to be clarified. The main hypothesis of this thesis is that targeting pro-inflammatory factors including TNFα and phosphodiesterase-type 4 (PDE4), interferes with molecular mechanisms that contribute to RGC death and this will thus successfully promote neuronal protection. In the first part of my thesis, I used an in vivo glaucoma model in Brown Norway rats to show that TNFα is upregulated early after induction of ocular hypertension. The specific hypothesis of this study is that high levels of TNFα promote RGC death by mediating the membrane insertion of Ca2+-permeable AMPA receptors (CP-AMPARs). I blocked TNFα function with XPro1595, a selective inhibitor of soluble TNFα. Administration of XPro1595 effectively protected RGC soma and axons. The cobalt permeability assay was used to show that soluble TNFα triggers the membrane insertion of CP-AMPAR in RGCs of glaucomatous retinas. This CP-AMPAR activation is caused by the downregulation of GluA2 which occurs when neurons are exposed to elevated IOP. Finally, intraocular administration of specific CP-AMPAR antagonists promoted RGC soma and axon protection. Taken together, these results show that CP-AMPARs play an important role in in the pathology of glaucoma. In the second part of my thesis, I characterized the neuroprotective effect of ibudilast, an inhibitor of PDE4, in the Brown Norway glaucoma model. We hypothesized that ibudilast promotes neuron protection by attenuating gliosis and the neuroinflammatory response. The results show that in glaucomatous retinas, ibudilast attenuates gliosis and the expression of TNFα, interleukin-1β (IL-1β), interleukin-6 (IL-6) and macrophage migration inhibitory factor (MIF). Interestingly, elevated IOP leads to substantial expression of PDE4A in Müller cells, which correlates with the accumulation of cAMP in these cells after ibudilast treatment. Lastly, ibudilast promoted RGC soma and axons protection through the activation of the cAMP/PKA pathway. In conclusion, the findings presented in this thesis identify two different mechanisms underlying RGC loss in glaucoma. These mechanisms can potentially provide new insights to develop novel strategies for the treatment of glaucom
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