Alteracions motores i neurals en un model de colitis induïda per Trichinella spiralis en rata

Consultable des del TDXTítol obtingut de la portada digitalitzadaL'objectiu d'aquest estudi ha estat caracteritzar la colitis induïda amb Trichinella spiralis en rata i investigar les semblances que comparteix amb la colitis ulcerosa. S'ha utilitzat la tècnica del bany d'òrgans aïllat per l'estudi de l'activitat mecànica, els microelèctrodes intracel·lulars per l'estudi de l'activitat elèctrica i tècniques d'immunohistoquímca i/o immunofluorescència pel marcatge de neurotransmissors i la caracterització de determinats tipus cel·lulars. Les rates infectades presenten 2 períodes de disminució d'ingesta i pes. El primer es dóna entre el dia 1 i 4 post infecció (PI) i el segon entre els dies 11 i 15 PI, a partir del dia 15 les rates es recuperen sense arribar als valors observats en els animals control. Les rates infectades presenten a més a més leucocitosi amb neutrofília i eosinofília del dia 6 al 21 PI. La consistència de les femtes disminueix i la presència de mucus augmenta del dia 2 al 20 PI. Les troballes histològiques en aquest model consisteixen en: atròfia epitelial, edema, hiperplàsia de la mucosa i edema de la submucosa a dia 2 PI; microúlceres i larves en la mucosa i edema submucós sever amb augment de l'infiltrat inflamatori a dia 6 PI; a dia 14 PI s'observa regeneració de la mucosa i presència d'algunes larves; a dia 30 PI no s'observa cap tipus de lesió inflamatòria. El nombre de cèl·lules immunoreactives a l'OX-6, que reconeix l'antigen del complex major d'histocompatibilitat de tipus II (MHCII), es troba dramàticament incrementat al plexe mientèric i submucós de les rates infectades (6-14 PI). El nombre de cèl·lules immunoreactives a l'ED1, que s'expressa en monòcits i macròfags de rata, es troba molt incrementat en ambdós plexes submucosos dels animals infectats. La inflamació colònica induïda amb T. spiralis també es caracteritza per un increment en l'expressió de la iNOS a l'epiteli i infiltrat inflamatori de la mucosa i submucosa, i s'associa a la disminució de la motilitat colònica observada en les rates infectades. Les tires circulars del còlon d'aquestes rates presenten a més a més una resposta disminuïda a l'acetilcolina i/o al clorur potàssic; en canvi, la depleció del calci intracel·lular disminueix més marcadament la contracció induïda per acetilcolina en les rates infectades. Aquesta troballa suggereix que la utilització del calci extracel·lular pot estar alterada durant la inflamació colònica per T. spiralis. El potencial de membrana de les cèl·lules musculars llises del còlon inflamat es troba més hiperpolaritzat que en animals control. Els potencials d'unió de tipus inhibitori (IJPs) tenen una durada disminuïda en el còlon inflamat, fet que suggereix una disminució en l'alliberament d'òxid nítric des de les motoneurones inhibidores del plexe mientèric. La disminució en el nombre de neurones immunoreactives a la nNOS observada en el plexe mientèric de rates infectades, pot explicar la disminució observada en la durada dels IJPs. El model de colitis induïda per l'administració intra rectal de larves de T. spiralis presenta semblances amb la colitis ulcerosa, tant pel que fa el curs clínic com les troballes histològiques i les alteracions funcionals. La presència de macròfags ED1 positius que expressen MHC II podrien mediar els canvis neurals i alteracions musculars que s'observen en aquest model; per altre costat, l'increment en l'expressió de la iNOS contribueix a l'increment de l'activtat d'aquest enzim i dóna lloc a la formació de quantitats molt grans d'òxid nítric. L'increment en la síntesi d'òxid nítric és un factor determinant per l'agreujament de la inflamació contribuint al desenvolupament de dany tissular, i pot ser considerat un factor rellevant en la disminució de motilitat i contractilitat que observem en aquest model.We intended to study to what extent T. spiralis-induced colitis shares similarities with UC and may thus be useful as a model of such human disease Colitis was induced by an intra-colonic enema of T. spiralis larvae. We examined the temporal relationship between the severity of inflammation and the altered contractility of the underlying circular muscle as well as the role of NANC inhibitory pathways in the disruption of the motility pattern. Colitis was induced by intrarectal administration of T. spiralis larvae. Responses to acetylcholine (ACh) and increased extracellular potassium as well as the effect of tetrodotoxin (TTX, 1 µM), L-nitro-N-arginine (L-NOARG, 1 mM) and apamin (1 µM) were determined in vitro in the organ bath with circular muscle strips from sham and infected rats at days 2-30 postinfection (PI). Microelectrode recordings were performed to study the putative changes in electrical activity of colonic smooth muscle cells. Whole mounts preparations of the SMP and MP were used for immunofluorescence studies. Altered stool consistency was found from day 1 to 21 PI; leukocytosis peaked on days 6-21 PI. Edema and cell infiltration were found in mucosa and submucosa (day 2-14 PI). Contractility displayed a disorganized pattern with decreased high amplitude low frequency (HALF) contractions. A progressive fading of spontaneous activity was observed and was partly restored in strips devoid of submucosa. iNOS immunoreactivity increased in epithelial and infiltrating cells (days 2-14 PI). Responses to ACh and KCl were decreased at all days PI compared to sham. Intracellular calcium depletion had a greater inhibitory effect in inflamed tissue (6-14 PI). The effect of TTX, L-NOARG and apamin on the spontaneous contractions was found to be altered in all infected rats, i.e. their effects were transient and milder. Inflamed tissue showed lower resting membrane potential and a decreased duration of inhibitory junction potentials induced by electrical stimulation. PGP9.5-immunoreactive neurons were decreased in the myenteric plexus (MP) and were significantly reduced in the submucous plexus (SMP) at 14 days PI. Substance P innervation of submucosal blood vessels was also reduced after infection. No changes in vasoactive intestinal peptide, choline acetyl transferase and CGRP immunoreactivity were observed. These data suggest that the decreased contractility of colonic circular smooth muscle induced by the intrarectal T. spiralis infection results from NO generated from mucosal and submucosal iNOS, the impairment of the excitation-contraction coupling, from a persistent hyperpolarization of smooth muscle cells and from impaired NANC inhibitory neurotransmission. The reduction in NOS neurons appears to underlie changes in gut motility. Macrophages are found extensively in the enteric plexuses and may play a pivotal role in the neuromuscular alterations. This model of colitis bears some traits with changes observed in ulcerative colitis and might thus be useful to study the dismotility observed in this human disease

Therapeutic Effect of a Novel Phosphatidylinositol-3-Kinase δ Inhibitor in Experimental Epidermolysis Bullosa Acquisita

Epidermolysis bullosa acquisita (EBA) is a rare, but prototypical, organ-specific autoimmune disease, characterized and caused by autoantibodies against type VII collagen (COL7). Mucocutaneous inflammation, blistering, and scarring are the clinical hallmarks of the disease. Treatment of EBA is difficult and mainly relies on general immunosuppression. Hence, novel treatment options are urgently needed. The phosphatidylinositol-3-kinase (PI3K) pathway is a putative target for the treatment of inflammatory diseases, including EBA. We recently discovered LAS191954, an orally available, selective PI3Kδ inhibitor. PI3Kδ has been shown to be involved in B cell and neutrophil cellular functions. Both cell types critically contribute to EBA pathogenesis, rendering LAS191954 a potential drug candidate for EBA treatment. We, here, demonstrate that LAS191954, when administered chronically, dose-dependently improved the clinical phenotype of mice harboring widespread skin lesions secondary to immunization-induced EBA. Direct comparison with high-dose corticosteroid treatment indicated superiority of LAS191954. Interestingly, levels of circulating autoantibodies were unaltered in all groups, indicating a mode of action independent of the inhibition of B cell function. In line with this, LAS191954 also hindered disease progression in antibody transfer-induced EBA, where disease develops dependent on myeloid, but independent of B cells. We further show that, in vitro, LAS191954 dose-dependently impaired activation of human myeloid cells by relevant disease stimuli. Specifically, immune complex-mediated and C5a-mediated ROS release were inhibited in a PI3Kδ-dependent manner. Accordingly, LAS191954 also modulated the dermal–epidermal separation induced in vitro by co-incubation of immune complexes with polymorph nuclear cells, thus pointing to an important role of PI3Kδ in EBA effector functions. Altogether, these results suggest a new potential mechanism for the treatment of EBA and potentially also other autoimmune bullous diseases

image_2_Therapeutic Effect of a Novel Phosphatidylinositol-3-Kinase δ Inhibitor in Experimental Epidermolysis Bullosa Acquisita.TIF

<p>Epidermolysis bullosa acquisita (EBA) is a rare, but prototypical, organ-specific autoimmune disease, characterized and caused by autoantibodies against type VII collagen (COL7). Mucocutaneous inflammation, blistering, and scarring are the clinical hallmarks of the disease. Treatment of EBA is difficult and mainly relies on general immunosuppression. Hence, novel treatment options are urgently needed. The phosphatidylinositol-3-kinase (PI3K) pathway is a putative target for the treatment of inflammatory diseases, including EBA. We recently discovered LAS191954, an orally available, selective PI3Kδ inhibitor. PI3Kδ has been shown to be involved in B cell and neutrophil cellular functions. Both cell types critically contribute to EBA pathogenesis, rendering LAS191954 a potential drug candidate for EBA treatment. We, here, demonstrate that LAS191954, when administered chronically, dose-dependently improved the clinical phenotype of mice harboring widespread skin lesions secondary to immunization-induced EBA. Direct comparison with high-dose corticosteroid treatment indicated superiority of LAS191954. Interestingly, levels of circulating autoantibodies were unaltered in all groups, indicating a mode of action independent of the inhibition of B cell function. In line with this, LAS191954 also hindered disease progression in antibody transfer-induced EBA, where disease develops dependent on myeloid, but independent of B cells. We further show that, in vitro, LAS191954 dose-dependently impaired activation of human myeloid cells by relevant disease stimuli. Specifically, immune complex-mediated and C5a-mediated ROS release were inhibited in a PI3Kδ-dependent manner. Accordingly, LAS191954 also modulated the dermal–epidermal separation induced in vitro by co-incubation of immune complexes with polymorph nuclear cells, thus pointing to an important role of PI3Kδ in EBA effector functions. Altogether, these results suggest a new potential mechanism for the treatment of EBA and potentially also other autoimmune bullous diseases.</p

image_3_Therapeutic Effect of a Novel Phosphatidylinositol-3-Kinase δ Inhibitor in Experimental Epidermolysis Bullosa Acquisita.TIF

<p>Epidermolysis bullosa acquisita (EBA) is a rare, but prototypical, organ-specific autoimmune disease, characterized and caused by autoantibodies against type VII collagen (COL7). Mucocutaneous inflammation, blistering, and scarring are the clinical hallmarks of the disease. Treatment of EBA is difficult and mainly relies on general immunosuppression. Hence, novel treatment options are urgently needed. The phosphatidylinositol-3-kinase (PI3K) pathway is a putative target for the treatment of inflammatory diseases, including EBA. We recently discovered LAS191954, an orally available, selective PI3Kδ inhibitor. PI3Kδ has been shown to be involved in B cell and neutrophil cellular functions. Both cell types critically contribute to EBA pathogenesis, rendering LAS191954 a potential drug candidate for EBA treatment. We, here, demonstrate that LAS191954, when administered chronically, dose-dependently improved the clinical phenotype of mice harboring widespread skin lesions secondary to immunization-induced EBA. Direct comparison with high-dose corticosteroid treatment indicated superiority of LAS191954. Interestingly, levels of circulating autoantibodies were unaltered in all groups, indicating a mode of action independent of the inhibition of B cell function. In line with this, LAS191954 also hindered disease progression in antibody transfer-induced EBA, where disease develops dependent on myeloid, but independent of B cells. We further show that, in vitro, LAS191954 dose-dependently impaired activation of human myeloid cells by relevant disease stimuli. Specifically, immune complex-mediated and C5a-mediated ROS release were inhibited in a PI3Kδ-dependent manner. Accordingly, LAS191954 also modulated the dermal–epidermal separation induced in vitro by co-incubation of immune complexes with polymorph nuclear cells, thus pointing to an important role of PI3Kδ in EBA effector functions. Altogether, these results suggest a new potential mechanism for the treatment of EBA and potentially also other autoimmune bullous diseases.</p

image_4_Therapeutic Effect of a Novel Phosphatidylinositol-3-Kinase δ Inhibitor in Experimental Epidermolysis Bullosa Acquisita.TIF

<p>Epidermolysis bullosa acquisita (EBA) is a rare, but prototypical, organ-specific autoimmune disease, characterized and caused by autoantibodies against type VII collagen (COL7). Mucocutaneous inflammation, blistering, and scarring are the clinical hallmarks of the disease. Treatment of EBA is difficult and mainly relies on general immunosuppression. Hence, novel treatment options are urgently needed. The phosphatidylinositol-3-kinase (PI3K) pathway is a putative target for the treatment of inflammatory diseases, including EBA. We recently discovered LAS191954, an orally available, selective PI3Kδ inhibitor. PI3Kδ has been shown to be involved in B cell and neutrophil cellular functions. Both cell types critically contribute to EBA pathogenesis, rendering LAS191954 a potential drug candidate for EBA treatment. We, here, demonstrate that LAS191954, when administered chronically, dose-dependently improved the clinical phenotype of mice harboring widespread skin lesions secondary to immunization-induced EBA. Direct comparison with high-dose corticosteroid treatment indicated superiority of LAS191954. Interestingly, levels of circulating autoantibodies were unaltered in all groups, indicating a mode of action independent of the inhibition of B cell function. In line with this, LAS191954 also hindered disease progression in antibody transfer-induced EBA, where disease develops dependent on myeloid, but independent of B cells. We further show that, in vitro, LAS191954 dose-dependently impaired activation of human myeloid cells by relevant disease stimuli. Specifically, immune complex-mediated and C5a-mediated ROS release were inhibited in a PI3Kδ-dependent manner. Accordingly, LAS191954 also modulated the dermal–epidermal separation induced in vitro by co-incubation of immune complexes with polymorph nuclear cells, thus pointing to an important role of PI3Kδ in EBA effector functions. Altogether, these results suggest a new potential mechanism for the treatment of EBA and potentially also other autoimmune bullous diseases.</p