INTRODUCTION: Injury to the facial nerve can lead to functional and aesthetic sequelae in patients. Though surgical interventions are available to restore lost motor and sensory function, outcomes are often suboptimal due to inadequate or disorganized axonal regeneration. While engineering improvements to the standard of care are underway, gaps remain in our molecular understanding of peripheral nerve injury to translate these efforts clinically. Over the last few decades, advancements in intravital imaging such as the development of fluorescent reporter mice and use of multiphoton excitation techniques have allowed for markedly enhanced characterization of biological phenomena at higher resolutions, at greater depths, and for longer timescales. Challenges in reliably and serially imaging in vivo within murine models have been overcome through the development of chronic imaging windows in various settings of the body. However, there are very few techniques available presently for imaging the peripheral nerve microenvironment and no prior work detailing use in the facial nerve setting. OBJECTIVE: Longitudinal studies employing intravital imaging techniques carry potential to improve understanding of peripheral nerve regeneration and function. Using multiphoton microscopy and fluorescent reporter mice, we propose a prototype, surgical protocol of implantation, and initial safety and efficacy testing of a facial nerve window to enable chronic imaging for enhanced characterization of the peripheral nerve microenvironment. METHODS: A stainless-steel implant with an affixed glass coverslip and aluminum external fixation component was developed for implantation in a transgenic reporter mouse model to enable chronic intravital imaging of the facial nerve buccal and marginal mandibular branches. A qualitative observational study and clinical assessment scoring study was performed post-surgical implantation to monitor behavior, physical appearance, weight loss, and reactivity to animal handling over the typical time-course of nerve regeneration. Segments of facial nerve branches were harvested from control and window-implanted mice and imaged using widefield epifluorescence microscopy for axon quantification to determine any adverse effects from window compression onto axonal fibers. Two-photon microscopy (2PM) and Simulated Raman Scattering (SRS) were also performed through the window to visualize axon tracts, myelin sheaths, and surrounding collagen matrix in wild-type and transgenic mice models. RESULTS: Qualitative serial observational studies and assessment scoring indicated no obvious functional deficits over the time-course of typical nerve regeneration and normal scores for weight, behavior, physical appearance, and reactivity. Neural histomorphometric analysis indicated no significant difference in mean myelinated axon count of buccal (mean ± SD; control buccal, 947.6 ± 129.9; window-implanted buccal, 799.3 ± 128.6; p = .136) and marginal mandibular branches (control marginal mandibular, 801.3 ± 145.1; window-implanted marginal mandibular, 738.0 ± 197.2; p = .599) between control and window-implanted mice, suggesting that neuropathy was not induced from the window itself. High-resolution images of nerve morphology in healthy and injured transgenic and wild-type mice were obtained using 2PM and SRS. CONCLUSION: Herein, we describe a novel and replicable platform for longitudinal intravital imaging of murine facial nerve. Future studies will evaluate viability of this model for imaging the facial nerve microenvironment, particularly Schwann cell-axon interactions, in the setting of severe nerve injury over a period of several weeks to months. Improved understanding gained through such studies of the structural peripheral nerve microenvironment may allow for advancements in viral vector therapeutics, nerve graft scaffold design, as well as advanced injury diagnostics and tracking. / 2022-06-02T00:00:00Z

La peau est un « avant poste » fascinant du système immunitaire. Elle forme une barrière entre l'environnement extérieur et l’organisme. Elle est aussi le point d'entrée pour les agents pathogènes, contre lesquels le système immunitaire organise des réponses adaptatives. Les acteurs de l'immunité innée de la peau contrôlent l'invasion des pathogènes et perçoivent également des changements environnementaux physiques et chimiques directs. Plusieurs composants du système immunitaire, tels que des cellules dendritiques (DCs), les macrophages (MΦ) et les mastocytes (MCs), participent à l'éradication des pathogènes et à l'initiation des réponses mémoires adaptatives. Ce qui permet une mobilisation rapide des cellules T effectrices ainsi que la sécrétion des anticorps par les cellules B à la suite d’une seconde exposition aux agents pathogènes. Les MCs qui sont des cellules résidentes du derme, jouent un rôle déterminant dans la libération de signaux d’alertes et sont classiquement considérés comme des cellules effectrices de la réaction allergique cutanée liée à l'IgE. Plusieurs observations récentes indiquent que les MCs seraient aussi impliqués dans les processus immunorégulateurs lors de l'initiation des réponses immunitaires adaptatives, dans le maintien de la tolérance périphérique aux composants de la peau et dans la régénération de la peau au cours des processus de cicatrisation. Cependant, les interactions entre les MCs et d'autres cellules immunitaires innées et adaptatives recrutées dans des conditions inflammatoires cutanées n'ont pas été élucidées en détail. Dans ce travail, nous décrivons l'utilisation d'une nouvelle souris possédant des MCs fluorescents (RMB), dans laquelle nous avons marqué les MCs FcεRI+ avec un marqueur fluorescent rouge tomato (TdT) et avec un système d'ablation conditionnelle basé sur l'expression concurrente du récepteur de la toxine diphtérique (DTR). Avec ces souris RMB, nous avons visualisé la dynamique des MCs et nous avons suivi les interactions entre les MCs et les lymphocytes T régulateurs (Tregs) après l'activation des MCs par l'IgE, dans une réaction inflammatoire typique de l'anaphylaxie cutanée passive (PCA). Dans un second volet d’étude, nous avons évalué le rôle des MCs lors d'un modèle expérimental de la greffe de peau de l'oreille, afin de révéler leur influence dans la cinétique de rejet ou prise de greffe du transplant. Nous avons constaté que 1) l'activation et la dégranulation des MCs induites par le pontage du récepteur FcεRI via des IgE couplées à un antigène multivalent sont les seules responsables de la réaction de PCA, et induisent le recrutement de Tregs ayant une grande motilité sur le site de l'inflammation. Nous avons constaté dans ces conditions, que les MCs restent immobiles, et que les Tregs établissent des contacts dynamiques avec les MCs dans le derme. 2) En outre, nous avons mis en place un modèle pour identifier les paramètres moléculaires de l'interaction MC-Treg et avons constaté que le complexe de l'antigène avec l'IgE peut être présenté aux Tregs en association avec les molécules du complexe majeur d'histocompatibilité de classe II, permettant la formation des contacts stables MC-Treg. 3) En utilisant un modèle de transplantation de la peau in vivo, nous avons montré que l'ablation conditionnelle des MCs conduit à une accélération du rejet du greffon dans le cas d'une transplantation en présence d’une disparité d’antigènes d’histocompatibilité mineurs depuis une souris mâle sur une souris femelle. Nous avons également constaté un impact inattendu de l'ablation des MCs dans la greffe de peau en l’absence de disparité antigénique d'une souris femelle sur une souris femelle, conduisant à un rejet rapide. Les MCs semblent donc être essentiels pour la cicatrisation et la régénération tissulaire après greffe. (...) / The skin is a fascinating outpost of the immune system. It performs a barrier function between the outside environment and the inner body and is also a port of entry for pathogens against which the immune system mounts adapted responses. The skin innate immune defenses control pathogen invasion and perceive also direct physical and chemical environmental changes. Several component of the immune system such as dendritic cells (DC), macrophages (MΦ) and mast cells (MC) participate in initial pathogen clearance and in initiating adaptive memory responses, allowing rapid mobilization of effector T cells and secretion of B cellderived antibodies after secondary pathogen challenge. MCs residing in the dermis exert a determinant alert function through the liberation of various factors and are classically considered as effector cells in the IgE-mediated cutaneous allergic reaction. As emerging now, MC are also involved in immunoregulatory processes during the initiation of adaptive immune responses, the maintenance of peripheral tolerance to skin components and skin regeneration during wound healing. Yet, the crosstalks between MCs and other innate and adaptive immune cells recruited during cutaneous inflammatory conditions have not been elucidated in detail. Here, we report the use of a novel Mast cell fluorescent reporter mouse (RMB), in which we tagged FcεRI+ MCs, with red fluorescence marker tomato (Tdt) and with a conditional ablation system based on concurrent diphtheria toxin receptor (DTR) expression. Using these RMB mice, we visualized MC dynamics and monitored MC interactions with regulatory T lymphocytes (Tregs) after IgE-mediated activation of MCs, in a typical passive cutaneous anaphylaxis (PCA) inflammatory reaction. Using another setting, we further assessed the role of MC during experimental ear skin grafting to reveal their potential influence in skin grafting and rejection. We found that 1) the activation and degranulation of MCs induced by FcεRI crosslinking by multivalent IgE is solely responsible for the PCA reaction and induces the recruitment of highly motile regulatory T cells (Tregs) to the site of inflammation. In these conditions, we found that MC remain sessile and Tregs establish dynamic contacts with MC in the dermis. 2) Further we set up a model system to reveal the molecular requirement for MC-Treg interaction and found that antigen complexed with IgE were able to be presented to Treg in association with major histocompatibility complex class II molecules allowing the formation of stable MC-Treg contacts. 3) Using in vivo skin transplantation model, we showed that conditional ablation of MCs leads to an acceleration of skin transplant rejection in sex-mismatched model (male skin transplant to female). We also found an unexpected impact of MC conditional ablation in sex-matched skin graft (female skin transplant to female) leading to rapid rejection, implying that MCs are essential for the wound healing reaction and the regeneration of tissue continuity after grafting. The aforementioned results point out to an important immunoregulatory role of MC beyond their classically described activator functions in inflamed tissues. The fact that MC constantly interact with Treg during inflammatory processes suggest that MCs could participate in skin homeostasis by exerting tolerogenic functions. These functions remain to be elucidated at the molecular level as presented in the discussion.

<p>The effect of direction thrombin inhibition on acute deep vein thrombosis (DVT) stability has not been defined and could contribute to pulmonary embolism (PE) risk. Direct thrombin inhibitors (DTIs) effectively inhibit free and clot-bound thrombin, which could potentiate thrombus instability through disruption of platelet, fibrin, and FXIIIa stabilizing mechanisms. This could manifest as increased thrombus embolization. A clinically relevant mouse model of DVT stability could further our understanding of venous thrombosis pathophysiology and define the effect of direct thrombin inhibition on PE. We hypothesized that acute DTI administration would decrease acute DVT stability and potentially increase PE risk. Platelets were labeled <em>in vivo</em>, femoral vein thrombosis was induced using FeCl₃, and lepirudin (8U/g) was administered <em>after</em> clot formation. Using intravital videomicroscopy (IVM), real time embolization was quantified as a measurement of thrombus stability. Thrombus stability increased in the control group and decreased in the lepirudin-treated group over two hours. The decrease in α₂-antiplasmin (α₂-AP) content within lepirudin-treated thrombi, compared to control thrombi, could possibly contribute to the observed decrease in thrombus stability. Continued growth and embolization established the dynamic nature of formed thrombi. In both groups, emboli were detected in the pulmonary artery circulation. Therefore, we successfully developed a mouse model of venous thrombus stability, which imitated the clinical progression of DVT to PE. DTI administration in the acute DVT setting could decrease thrombus stability, demonstrated through increased embolization and PE. This model could be useful in examining the effect of other antithrombotics and risk factors settings on DVT stability.</p> / Master of Science (MSc)

Yes / Ex vivo wounded human skin organ culture is an invaluable tool for translationally relevant preclinical wound healing research. However, studies incorporating this system are still underutilized within the field because of the low throughput of histological analysis required for downstream assessment. In this study, we use intravital fluorescent dye to lineage trace epidermal cells, demonstrating that wound re‐epithelialization of human ex vivo wounds occurs consistent with an extending shield mechanism of collective migration. Moreover, we also report a relatively simple method to investigate global epithelial closure of explants in culture using daily fluorescent dye treatment and en face imaging. This study is the first to quantify healing of ex vivo wounds in a longitudinal manner, providing global assessments for re‐epithelialization and tissue contraction. We show that this approach can identify alterations to healing with a known healing promoter. This methodological study highlights the utility of human ex vivo wounds in enhancing our understanding of mechanisms of human skin repair and in evaluating novel therapies to improve healing outcome. / University of Manchester Strategic Fund; Wellcome Trust; BBSRC; Ministry of Higher Education, Malaysia Universiti; Sains Malaysia

A úlcera gástrica é uma doença crônica, de alta prevalência, e a eficácia dos tratamentos farmacológicos disponíveis é limitada pela alta incidência de efeitos adversos. Neste trabalho é mostrado o mecanismo de ação terapêutica e os efeitos toxicológicos da molécula indol-tiazolidínica LYSO-7 em diferentes modelos experimentais de úlcera gástrica. Camundongos Swiss machos foram tratados com veículo, LYSO-7 (5, 25 ou 50 mg/kg, v.o.) ou bezafibrato (25 ou 50 mg/kg, v.o.) 1 hora antes da administração oral de Et/HCl (60%/0,03 M) ou indometacina (100 mg/kg). Em outro conjunto de ensaios, animais foram pré-tratados com GW9962, um antagonista PPAR&#947; (2 mg/kg, i.p.); anticorpo anti-granulócito (50 &#181;L, i.p.), ou L-NAME (70 mg/kg, i.p) 1 hora antes dos tratamentos com veículo ou LYSO-7. Uma hora após administração da solução de Et/HCl, os neutrófilos foram quantificados no sangue e medula óssea, a rede microcirculatória gástrica foi estudada em in situ, utilizando a técnica de microscopia intravital; o tecido gástrico foi utilizado para quantificar a percentagem de área lesada, atividade da MPO, a expressão gênica e proteica de PPAR&#947;, expressão proteica de iNOS e eNOS, e a atividade das enzimas catalase, SOD, GPx, GR e GST. Uma hora após a administração de indometacina, o tecido gástrico foi removido para avaliar a eficácia do tratamento e a secreção de mediadores inflamatórios. Ensaio de úlcera crônica, induzida por ácido acético, foi realizado em camundongos Balb/c WT ou ANXA1-/-, aplicando-se 20&#181;L de ácido acético na camada subserosa do estômago e 24 horas após a indução, os animais foram tratados, uma vez ao dia, durante sete dias com LYSO-7 (50 mg/kg), bezafibrato (50 mg/kg) ou veículo. Foram realizados ensaios com macrófagos recrutados para o peritônio pela ação do tioglicolato de sódio (3%, i.p.) e com neutrófilos recrutados pela ação do glicogênio de ostra (1%, i.p.). Ensaios de toxicologia aguda, crônica e mutagenicidade também foram realizados. Os resultados obtidos mostram que o tratamento com LYSO-7 reduz a área lesada, o influxo de neutrófilos e a estase da rede microcirculatória provocada pela administração de Et/HCl. Os efeitos protetores foram revertidos em animais pré-tratados com GW9962, indicando a participação do PPAR&#947; no efeito. O influxo de neutrófilos é determinante para a lesão, uma vez que a depleção destas células reduziu a ulceração gástrica, e indica que o bloqueio da mobilização de neutrófilos da medula óssea para o sangue e destes para o tecido lesado pela LYSO-7 pode ser um mecanismo de ação gastroprotetora desta molécula. A reversão da estase vascular na microcirculação, mas não o influxo de neutrófilos, é mediado pelo NO, pois o pré-tratamento com L-NAME aboliu os efeitos da LYSO-7 no restabelecimento do fluxo sanguíneo da microcirculação. Este efeito pode ser dependente da maior e menor expressão proteica de eNOS e iNOS, respectivamente. A LYSO-7 foi capaz de alterar favoravelmente a atividade das enzimas antioxidantes no tecido gástrico. Ainda, a LYSO-7 diminuiu a área lesada e reduziu a concentração de TNF&#945; e aumentou a de IL-10 no tecido gástrico lesado pela indometacina. Na resolução do processo inflamatório, o tratamento com LYSO-7 diminuiu a percentagem de área lesada, aumentou a apoptose de neutrófilos e a eferocitose de neutrófilos por macrófagos peritoneais, inibiu a secreção de TNF&#945; e aumentou a secreção de IL-10, TFG-1&#946; e VEGF para o sobrenadante de macrófagos em fagocitose. A resolução de lesão gástrica, bem como a indução da fagocitose pela LYSO-7 foi reduzida em animais ANXA1-/-. As investigações destes últimos dados mostraram a relação da ANXA1 e PPAR&#947;, já que a expressão do receptor é reduzida em macrófagos obtidos de animais depletados de ANXA1. Os estudos toxicológicos mostraram que a LYSO-7 apresenta baixa toxicidade aguda e crônica in vivo, além de não ocasionar mutagenicidade em eritrócitos da medula óssea. Os dados obtidos mostram que a molécula LYSO-7 atua como agonista PPAR&#947; na modulação da úlcera gástrica e modula a migração de neutrófilos e o fluxo sanguíneo na microcirculação. A transativação e transrepressão de eNOS e iNOS, respectivamente, o bloqueio da migração de neutrófilos para a lesão e a inibição da atividade de enzimas oxidativa, ativação de enzimas antioxidantes no epitélio gástrico e a inibição da secreção de mediadores inflamatórios parecem ser os mecanismos de ação da LYSO-7 na citoproteção gástrica. Adicionalmente, a LYSO-7 atua na resolução do processo inflamatório promovendo downregulation na secreção de mediadores inflamatórios, aumento na apoptose de neutrófilos e eferocitose de neutrófilos apoptóticos. / Gastric ulcer is a chronic disease that presents high prevalence, and effectiveness of pharmacological treatments available is limited by several adverse effects. In this study is shown the mechanism of action and toxicological effects of the molecule indole-thiazolidine LYSO-7 in different models of gastric ulcer. Male Swiss mice were treated with vehicle LYSO-7 (5, 25, or 50 mg/kg, p.o.) or bezafibrate (25 or 50 mg/kg, p.o.) 1 hour before the oral administration of Et/HCl (60%/0.03 M) or indomethacin (100 mg/kg). In another set of assays, animals were pre-treated with GW9962, a PPAR&#947; antagonist (2 mg/kg, i.p.), anti-granulocyte antibody (50 &#181;L, i.p.) or L-NAME (70 mg/kg, i.p.) 1 hour before the treatment with vehicle or LYSO-7. One hour after administration of the Et/HCl solution, neutrophils were quantified in the blood and bone marrow, the gastric microcirculatory network was studied in situ by intravital microscopy, in the gastric tissue were quantified the percentage of injured area, MPO activity, PPAR&#947; gene and protein expression, iNOS and eNOS protein expression, and catalase, SOD, GPx, GR and GST activity. One hour after indomethacin administration, gastric tissue was removed to verify the efficacy of LYSO-7 on inflammatory mediator secretion. Chronic ulcer assay induced by acetic acid was carried out in Balb/c WT or ANXA1-/-, applying 20&#181;L of acetic acid in the subserosal layer of the stomach and 24 hours after induction, animals were treated during seven days, once a day, with LYSO-7 (50 mg/kg), bezafibrate (50 mg/kg) or vehicle. Assays were performed with macrophages recruited to the peritoneum by sodium thioglycollate (3%, i.p.) and neutrophils by oyster glycogen (1%, i.p.). Acute and chronic toxicological and mutagenicity assays were also conducted. The results obtained show that LYSO-7 treatment decrease the injured area, neutrophil influx and microcirculatory stasis evoked by Et/HCl administration. Protective effects were reversed in animals pretreated with GW9962, indicating the involvement of PPAR&#947;. Neutrophil influx is a determinant of the gastric lesion, once the depletion of these cells decreased the gastric damage, indicating that in the neutrophil mobilization blockade from the bone marrow to blood and to injured tissue may be a gastroprotective mechanism of LYSO-7. The vascular stasis reversion in the microcirculation is mediated by NO, but not the neutrophil influx, since the pretreatment with L-NAME abolished the effects of LYSO-7 on blood flow. This effect was dependent on increase and decrease of eNOS and iNOS protein expression, respectively. LYSO-7 positively altered the activity of antioxidant enzymes in the gastric tissue. Furthermore, LYSO-7 reduced the injured area and the concentration of TNF&#945; and increased IL-10 in the gastric tissue in the indomethacin-induced ulcer model. In the resolution of inflammation, LYSO-7 treatment decreased the percentage of the injured area, increased the neutrophils apoptosis and the efferocytosis of apoptotic neutrophils by peritoneal macrophages, inhibited the TNF&#945; release and increased the secretion of IL-10, IL-1&#946; and VEGF in the supernatant of phagocytosis assay. The resolution of gastric lesions, as well as, the induction of phagocytosis by LYSO-7 was reduced in animals ANXA1-/-. This data shown the relation of PPAR&#947; and ANXA1, as PPAR&#947; expression is reduced in macrophages obtained from ANXA1-/- animals. Toxicological studies showed that LYSO-7 has low acute and chronic toxicity in vivo, and did not cause mutagenicity in bone marrow erythrocytes. The data obtained show that LYSO-7 acts as PPAR&#947; in the modulation of gastric ulcer and modulate neutrophil migration and blood flow in the microcirculation. The transactivation and transrepression of eNOS and iNOS, respectively, blocking the neutrophil influx into the injury, antioxidant enzymes activation in the gastric epithelium and inhibition of inflammatory mediators release seem to be the mechanisms action of LYSO-7 in gastric cytoprotection. Additionally, LYSO-7 operates in the resolution of inflammation promoting downregulation in the secretion of inflammatory mediators and increases the neutrophil apoptosis and efferocytosis of apoptotic neutrophils.

A úlcera gástrica é uma doença crônica, de alta prevalência, e a eficácia dos tratamentos farmacológicos disponíveis é limitada pela alta incidência de efeitos adversos. Neste trabalho é mostrado o mecanismo de ação terapêutica e os efeitos toxicológicos da molécula indol-tiazolidínica LYSO-7 em diferentes modelos experimentais de úlcera gástrica. Camundongos Swiss machos foram tratados com veículo, LYSO-7 (5, 25 ou 50 mg/kg, v.o.) ou bezafibrato (25 ou 50 mg/kg, v.o.) 1 hora antes da administração oral de Et/HCl (60%/0,03 M) ou indometacina (100 mg/kg). Em outro conjunto de ensaios, animais foram pré-tratados com GW9962, um antagonista PPAR&#947; (2 mg/kg, i.p.); anticorpo anti-granulócito (50 &#181;L, i.p.), ou L-NAME (70 mg/kg, i.p) 1 hora antes dos tratamentos com veículo ou LYSO-7. Uma hora após administração da solução de Et/HCl, os neutrófilos foram quantificados no sangue e medula óssea, a rede microcirculatória gástrica foi estudada em in situ, utilizando a técnica de microscopia intravital; o tecido gástrico foi utilizado para quantificar a percentagem de área lesada, atividade da MPO, a expressão gênica e proteica de PPAR&#947;, expressão proteica de iNOS e eNOS, e a atividade das enzimas catalase, SOD, GPx, GR e GST. Uma hora após a administração de indometacina, o tecido gástrico foi removido para avaliar a eficácia do tratamento e a secreção de mediadores inflamatórios. Ensaio de úlcera crônica, induzida por ácido acético, foi realizado em camundongos Balb/c WT ou ANXA1-/-, aplicando-se 20&#181;L de ácido acético na camada subserosa do estômago e 24 horas após a indução, os animais foram tratados, uma vez ao dia, durante sete dias com LYSO-7 (50 mg/kg), bezafibrato (50 mg/kg) ou veículo. Foram realizados ensaios com macrófagos recrutados para o peritônio pela ação do tioglicolato de sódio (3%, i.p.) e com neutrófilos recrutados pela ação do glicogênio de ostra (1%, i.p.). Ensaios de toxicologia aguda, crônica e mutagenicidade também foram realizados. Os resultados obtidos mostram que o tratamento com LYSO-7 reduz a área lesada, o influxo de neutrófilos e a estase da rede microcirculatória provocada pela administração de Et/HCl. Os efeitos protetores foram revertidos em animais pré-tratados com GW9962, indicando a participação do PPAR&#947; no efeito. O influxo de neutrófilos é determinante para a lesão, uma vez que a depleção destas células reduziu a ulceração gástrica, e indica que o bloqueio da mobilização de neutrófilos da medula óssea para o sangue e destes para o tecido lesado pela LYSO-7 pode ser um mecanismo de ação gastroprotetora desta molécula. A reversão da estase vascular na microcirculação, mas não o influxo de neutrófilos, é mediado pelo NO, pois o pré-tratamento com L-NAME aboliu os efeitos da LYSO-7 no restabelecimento do fluxo sanguíneo da microcirculação. Este efeito pode ser dependente da maior e menor expressão proteica de eNOS e iNOS, respectivamente. A LYSO-7 foi capaz de alterar favoravelmente a atividade das enzimas antioxidantes no tecido gástrico. Ainda, a LYSO-7 diminuiu a área lesada e reduziu a concentração de TNF&#945; e aumentou a de IL-10 no tecido gástrico lesado pela indometacina. Na resolução do processo inflamatório, o tratamento com LYSO-7 diminuiu a percentagem de área lesada, aumentou a apoptose de neutrófilos e a eferocitose de neutrófilos por macrófagos peritoneais, inibiu a secreção de TNF&#945; e aumentou a secreção de IL-10, TFG-1&#946; e VEGF para o sobrenadante de macrófagos em fagocitose. A resolução de lesão gástrica, bem como a indução da fagocitose pela LYSO-7 foi reduzida em animais ANXA1-/-. As investigações destes últimos dados mostraram a relação da ANXA1 e PPAR&#947;, já que a expressão do receptor é reduzida em macrófagos obtidos de animais depletados de ANXA1. Os estudos toxicológicos mostraram que a LYSO-7 apresenta baixa toxicidade aguda e crônica in vivo, além de não ocasionar mutagenicidade em eritrócitos da medula óssea. Os dados obtidos mostram que a molécula LYSO-7 atua como agonista PPAR&#947; na modulação da úlcera gástrica e modula a migração de neutrófilos e o fluxo sanguíneo na microcirculação. A transativação e transrepressão de eNOS e iNOS, respectivamente, o bloqueio da migração de neutrófilos para a lesão e a inibição da atividade de enzimas oxidativa, ativação de enzimas antioxidantes no epitélio gástrico e a inibição da secreção de mediadores inflamatórios parecem ser os mecanismos de ação da LYSO-7 na citoproteção gástrica. Adicionalmente, a LYSO-7 atua na resolução do processo inflamatório promovendo downregulation na secreção de mediadores inflamatórios, aumento na apoptose de neutrófilos e eferocitose de neutrófilos apoptóticos. / Gastric ulcer is a chronic disease that presents high prevalence, and effectiveness of pharmacological treatments available is limited by several adverse effects. In this study is shown the mechanism of action and toxicological effects of the molecule indole-thiazolidine LYSO-7 in different models of gastric ulcer. Male Swiss mice were treated with vehicle LYSO-7 (5, 25, or 50 mg/kg, p.o.) or bezafibrate (25 or 50 mg/kg, p.o.) 1 hour before the oral administration of Et/HCl (60%/0.03 M) or indomethacin (100 mg/kg). In another set of assays, animals were pre-treated with GW9962, a PPAR&#947; antagonist (2 mg/kg, i.p.), anti-granulocyte antibody (50 &#181;L, i.p.) or L-NAME (70 mg/kg, i.p.) 1 hour before the treatment with vehicle or LYSO-7. One hour after administration of the Et/HCl solution, neutrophils were quantified in the blood and bone marrow, the gastric microcirculatory network was studied in situ by intravital microscopy, in the gastric tissue were quantified the percentage of injured area, MPO activity, PPAR&#947; gene and protein expression, iNOS and eNOS protein expression, and catalase, SOD, GPx, GR and GST activity. One hour after indomethacin administration, gastric tissue was removed to verify the efficacy of LYSO-7 on inflammatory mediator secretion. Chronic ulcer assay induced by acetic acid was carried out in Balb/c WT or ANXA1-/-, applying 20&#181;L of acetic acid in the subserosal layer of the stomach and 24 hours after induction, animals were treated during seven days, once a day, with LYSO-7 (50 mg/kg), bezafibrate (50 mg/kg) or vehicle. Assays were performed with macrophages recruited to the peritoneum by sodium thioglycollate (3%, i.p.) and neutrophils by oyster glycogen (1%, i.p.). Acute and chronic toxicological and mutagenicity assays were also conducted. The results obtained show that LYSO-7 treatment decrease the injured area, neutrophil influx and microcirculatory stasis evoked by Et/HCl administration. Protective effects were reversed in animals pretreated with GW9962, indicating the involvement of PPAR&#947;. Neutrophil influx is a determinant of the gastric lesion, once the depletion of these cells decreased the gastric damage, indicating that in the neutrophil mobilization blockade from the bone marrow to blood and to injured tissue may be a gastroprotective mechanism of LYSO-7. The vascular stasis reversion in the microcirculation is mediated by NO, but not the neutrophil influx, since the pretreatment with L-NAME abolished the effects of LYSO-7 on blood flow. This effect was dependent on increase and decrease of eNOS and iNOS protein expression, respectively. LYSO-7 positively altered the activity of antioxidant enzymes in the gastric tissue. Furthermore, LYSO-7 reduced the injured area and the concentration of TNF&#945; and increased IL-10 in the gastric tissue in the indomethacin-induced ulcer model. In the resolution of inflammation, LYSO-7 treatment decreased the percentage of the injured area, increased the neutrophils apoptosis and the efferocytosis of apoptotic neutrophils by peritoneal macrophages, inhibited the TNF&#945; release and increased the secretion of IL-10, IL-1&#946; and VEGF in the supernatant of phagocytosis assay. The resolution of gastric lesions, as well as, the induction of phagocytosis by LYSO-7 was reduced in animals ANXA1-/-. This data shown the relation of PPAR&#947; and ANXA1, as PPAR&#947; expression is reduced in macrophages obtained from ANXA1-/- animals. Toxicological studies showed that LYSO-7 has low acute and chronic toxicity in vivo, and did not cause mutagenicity in bone marrow erythrocytes. The data obtained show that LYSO-7 acts as PPAR&#947; in the modulation of gastric ulcer and modulate neutrophil migration and blood flow in the microcirculation. The transactivation and transrepression of eNOS and iNOS, respectively, blocking the neutrophil influx into the injury, antioxidant enzymes activation in the gastric epithelium and inhibition of inflammatory mediators release seem to be the mechanisms action of LYSO-7 in gastric cytoprotection. Additionally, LYSO-7 operates in the resolution of inflammation promoting downregulation in the secretion of inflammatory mediators and increases the neutrophil apoptosis and efferocytosis of apoptotic neutrophils.

L’objectif principal de ce travail de thèse a été de caractériser les effets de l’anti-VEGF Bevacizumab (Avastin) sur le réseau vasculaire tumoral in vivo, au cours du temps, à l’aide du modèle de la chambre dorsale chez la souris nude. Les images du réseau vasculaire tumoral acquises par microscopie intravitale ont été analysées par un algorithme de traitement d’images développé au sein de notre équipe, permettant de mettre en évidence les modifications morphologiques induites par le traitement et d’isoler des paramètres discriminants de la « normalisation » vasculaire, par comparaison à un réseau vasculaire sain. La période de « normalisation » vasculaire détectée par notre outil a été confortée par l’analyse de la fonctionnalité des vaisseaux sanguins au cours du temps, in vivo et par une analyse immunohistochimique des vaisseaux sanguins tumoraux et du tissu tumoral. A travers des essais préliminaires in vivo, en regard des résultats de ce travail concernant une fenêtre de "normalisation", nous avons cherché à vérifier l'hypothèse d'un bénéfice d'un traitement anti-VEGF préalablement à la thérapie photodynamique (PDT) sur des tumeurs de glioblastome xénogreffées en sous-cutané et en chambre dorsale. L'efficacité de la PDT est décrite comme étant dépendante d'une d'oxygénation tumorale suffisante et d'une distribution maximale de l'agent photosensibilisant au coeur des tumeurs. Parallèlement à ces travaux, nous avons cherché en équipe pluridiscilinaire à développer un modèle mathématique de la réponse au bevacizumab à partir de données biologiques réelles obtenues sur le même modèle in vivo et permettant pour l'avenir de simuler les réponses à différentes doses et différentes durées de traitement, toujours à des fins d'optimisation des protocoles thérapeutiques / The main aim of this work was to characterize the effects of the anti-VEGF Bevacizumab (Avastin) on the tumor vascular network, in vivo, over time, thanks to the skin fold chamber model on the nude mouse. Images of the vascular network obtained using intravital microscopy were analyzed par a dedicated image processing algorithm developed within our research team, allowing to highlight the morphological modifications induced by the treatment and to isolate discriminating parameters of the vascular "normalization", by comparison to healthy vascular networks. Le vascular "normalization" period detected with our tool was comforted by the analysis of the functionality of the blood vessels over time, in vivo and by an immunohistochemical analysis of the blood vessels and of the tumor tissue. In preliminary in vivo experiments, we tried to verify the hypothesis of the benefits of an anti-VEGF treatment prior to photodynamic therapy (PDT) on glioblastoma xenografts implanted subcutaneously or in the skin fold chamber. The efficacy of PDT is described as being dependent on tumor oxygenation and on the distribution of the photosensitizing agent within the tumor. In paralel to this work, we tried as a pluridisciplinary team to develop a mathematical model of the tumor response to bevacizumab using biological data obtained on the same in vivo model et that will allow in the future to simulate the response for different doses and different treatment durations, for the optimization of therapeutic protocols

<p>The gastric mucosa is frequently exposed to endogenously secreted hydrochloric acid of high acidity. Gastric mucosal defense mechanisms are arranged at different levels of the gastric mucosa and must work in unison to maintain its integrity. </p><p>In this thesis, several mechanisms underlying gastric mucosal resistance to strong acid were investigated in anesthetized rats and mice. The main findings were as follows:</p><p>Only when acid secretion occurred did the pH gradient in the mucus gel withstand back-diffusion of luminal acid (100 mM or 155 mM HCl), and keep the juxtamucosal pH (pH_jm) neutral. Thus, when no acid secretion occurred and the luminal pH was 0.8-1, the pH gradient was destroyed. </p><p>Bicarbonate ions, produced concomitant with hydrogen ions in the parietal cells during acid secretion and blood-borne to the surface epithelium, were carried transepithelially through a DIDS-sensitive transport. </p><p>Prostaglandin-dependent bicarbonate secretion seemed to be less important in maintaining a neutral pH_jm. </p><p>Removal of the loosely adherent mucus layer did not influence the maintenance of the pH_jm. Hence, only the firmly adherent mucus gel layer, approximately 80µm thick, seemed to be important for the pH_jm. </p><p>Staining of the mucus gel with a pH-sensitive dye revealed that secreted acid penetrated the mucus gel from the crypt openings toward the gastric lumen only in restricted paths (channels). One crypt opening was attached to one channel, and the channel was irreversibly formed during acid secretion. </p><p>Gastric mucosal blood flow increased on application of strong luminal acid (155 mM HCl). This acid-induced hyperemia involved the inducible but not the neural isoform of nitric oxide synthase. These results suggest a novel role for iNOS in gastric mucosal protection and indicate that iNOS is constitutively expressed in the gastric mucosa. </p><p>It is concluded that a pH gradient in the gastric mucus gel can be maintained during ongoing acid secretion, since the acid penetrates the mucus only in restricted channels and bicarbonate is carried from the blood to the lumen via a DIDS-sensitive transporter.</p>

The gastric mucosa is frequently exposed to endogenously secreted hydrochloric acid of high acidity. Gastric mucosal defense mechanisms are arranged at different levels of the gastric mucosa and must work in unison to maintain its integrity. In this thesis, several mechanisms underlying gastric mucosal resistance to strong acid were investigated in anesthetized rats and mice. The main findings were as follows: Only when acid secretion occurred did the pH gradient in the mucus gel withstand back-diffusion of luminal acid (100 mM or 155 mM HCl), and keep the juxtamucosal pH (pHjm) neutral. Thus, when no acid secretion occurred and the luminal pH was 0.8-1, the pH gradient was destroyed. Bicarbonate ions, produced concomitant with hydrogen ions in the parietal cells during acid secretion and blood-borne to the surface epithelium, were carried transepithelially through a DIDS-sensitive transport. Prostaglandin-dependent bicarbonate secretion seemed to be less important in maintaining a neutral pHjm. Removal of the loosely adherent mucus layer did not influence the maintenance of the pHjm. Hence, only the firmly adherent mucus gel layer, approximately 80µm thick, seemed to be important for the pHjm. Staining of the mucus gel with a pH-sensitive dye revealed that secreted acid penetrated the mucus gel from the crypt openings toward the gastric lumen only in restricted paths (channels). One crypt opening was attached to one channel, and the channel was irreversibly formed during acid secretion. Gastric mucosal blood flow increased on application of strong luminal acid (155 mM HCl). This acid-induced hyperemia involved the inducible but not the neural isoform of nitric oxide synthase. These results suggest a novel role for iNOS in gastric mucosal protection and indicate that iNOS is constitutively expressed in the gastric mucosa. It is concluded that a pH gradient in the gastric mucus gel can be maintained during ongoing acid secretion, since the acid penetrates the mucus only in restricted channels and bicarbonate is carried from the blood to the lumen via a DIDS-sensitive transporter.

INTRODUCCIÓN: El tratamiento de la enfermedad inflamatoria intestinal se ha basado en el uso de aminosalicilatos corticoides e inmunosupresores, que ayudan a controlar los brotes de la enfermedad, aunque no consiguen su curación. El bloqueo de las moléculas de adhesión endotelial, que permiten el reclutamiento de leucocitos desde el lecho vascular hacia el área inflamada, podría ser una diana terapéutica útil en esta enfermedad.OBJETIVOS: 1. Determinar el valor de las moléculas de adhesión endotelial como nuevas dianas terapéuticas en la enfermedad inflamatoria del intestino. 2. Evaluar el posible valor terapéutico de su modulación con anticuerpos monoclonales bloqueantes de las moléculas de adhesión endotelial, péptidos trébol y ciclosporina A.MATERIAL Y MÉTODOS: El modelo experimental utilizado ha sido el de la colitis inducida por DSS en el ratón, que remeda la colitis ulcerosa en humanos. Para cuantificar la expresión in vivo de las moléculas de adhesión endotelial se ha recurrido a la técnica del doble anticuerpo marcado y para evaluar su expresión in vitro. a los cultivos primarios de células endoteliales de vena de cordón umbilical humano. Asimismo, para analizar las interacciones leucocito-endotelio en las vénulas del colon se ha utilizado la microscopía intravital de fluorescencia.RESULTADOS Y CONCLUSIONES: 1. En la colitis experimental inducida por DSS existe un aumento en la expresión de las moléculas de adhesión endotelial ICAM-1, VCAM-1 y MAdCAM-1 y de las interacciones leucocito-endotelio (roadamiento y adhesión) en las vénulas postcapilares del colon. 2. VCAM-1 es la principal molécula responsable del incremento de la adhesión leucocitaria al endotelio y juega un papel fundamental en la fisiopatología de la enfermedad inflamatoria intestinal experimental. 3. El inmunobloqueo sostenido de VCAM-1 tiene efecto terapéutico ya que mejora el curso de la colitis. En cambio, la inmunoneutralización de ICAM-1 o de MAdCAM-1 no altera el curso clínico en este modelo experimental. 4. El aumento de la expresión de las moléculas de adhesión endotelial VCAM 1 y MAdCAM-1 en el colon es proporcional a la gravedad de la colitis inducida. 5. El péptido trébol TFF2 es útil en el tratamiento de la colitis experimental, siendo su administración tópica superior a la sistémica 6 TFF2 produce de forma indirecta, una reducción en la expresión de las moléculas de adhesión endotelial y en el reclutamiento leucocitario que contribuye a su efecto beneficioso. 7. La ciclosporina A mejora la colitis experimental inducida por DSS en el ratón. 8. El efecto terapéutico de la ciclosporina A se debe, en parte, a que reduce la producción de citocinas proinflamatorias y la expresión de las moléculas de adhesión del endotelio, actuando directamente sobre la célula endotelial, lo que decrece el reclutamiento leucocitario hacia el intestino inflamado.SUMARIO: 1- VCAM-1 es la molécula de adhesión endotelial más importante en este modelo experimental de colitis que remeda la colitis ulcerosa en humanos- 2 El inmunobloqueo de VCAM-1, en contraste con el de ICAM-1 o MAdCAM-1, podría atenuar la respuesta inflamatoria sin alterar los mecanismos inmunes fisiológicos ya que esta molécula no está implicada en la recirculación fisiológica de los leucocitos. 3. Los trabajos que componen esta Tesis Doctoral constituyen la base racional para investigar el efecto de los anticuerpos anti-VCAM-1 en los pacientes con colitis ulcerosa. / INTRODUCTION: Treatment of inflammatory bowel disease is currently based on the use of aminosalicylates, corticosteroids and immunomodulators, which can control flares of disease but are not curative. Blockade of endothelial adhesion molecules involved in the recruitment of leukocytes from the vascular bed to the inflammatory site might be a useful therapeutic strategy in this disease.AIM: To assess the usefulness of modulation of endothelial adhesion molecules with monoclonal antibodies, trefoil peptides (TFF2), and cyclosporin A (CsA) as a therapeutic strategy for experimental colitis. MATERIAL AND METHODS: Dextran sulphate sodium (DSS)-induced colitis, a murine model that mimics human ulcerative colitis, was used. Cell adhesion molecule expression was measured by the dual radiolabeled antibody technique. Leukocyte-endothelial cell interactions were determined in colonic venules by fluorescence intravital microscopy.RESULTS AND CONCLUSIONS: 1. VCAM-1 plays a central role in leukocyte recruitment in colitis since blockade of this adhesion molecule has a higher therapeutic effect than immunoneutralization of ICAM-1 or MAdCAM-1 in this experimental model.2. TFF2 is useful in the treatment of colitis, and topical administration is superior to the systemic route. Indirect reduction in adhesion molecule expression and leukocyte recruitment into the inflamed intestine contributes to the beneficial effect of TFF2 treatment.3. CsA A ameliorates DSS-induced colitis in mice. Reduced adhesion molecule expression resulting from diminished pro-inflammatory cytokine production and from a direct effect of CsA in endothelial cells decreases leukocyte recruitment into the inflamed intestine, contributing to this protective effect. SUMMARY: 1. VCAM-1 is the most important endothelial adhesion molecule in an experimental model of colitis that mimics human ulcerative colitis. 2. VCAM-1 immunoblockade, in contrast to ICAM-1 or MAdCAM-1, can attenuate the inflammatory response without altering the physiological immune mechanisms since this molecule is not involved in the physiological recirculation of leukocytes. 3. The results of this Doctoral Thesis constitute the rationale to investigate the effects of anti-VCAM-1 antibodies in patients with ulcerative colitis.