RESUMEN
A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.
Asunto(s)
Afinidad de Anticuerpos , Antígenos de Neoplasias/inmunología , Moléculas de Adhesión Celular/inmunología , Inmunoglobulinas/metabolismo , Receptor EphA2/química , Receptores de Antígenos/metabolismo , Serina Endopeptidasas/química , Animales , Especificidad de Anticuerpos , Antígenos de Neoplasias/química , Autoantígenos/química , Autoantígenos/metabolismo , Proteínas Portadoras , Moléculas de Adhesión Celular/química , Molécula de Adhesión Celular Epitelial , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Inmunoglobulinas/química , Inmunoglobulinas/inmunología , Técnicas In Vitro , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Biblioteca de Péptidos , Receptor EphA2/inmunología , Receptores de Antígenos/química , Receptores de Antígenos/inmunología , Serina Endopeptidasas/inmunología , Tiburones/inmunologíaRESUMEN
BACKGROUND: Ischemia-reperfusion edema is a common early complication after lung transplantation where the hypoxia-induced vascular endothelial growth factor (VEGF)-A plays a pivotal role. It remains unclear whether a VEGF blockade is beneficial in lung transplantation. METHODS: VEGF-A blockade was investigated in an orthotopic rat model of lung transplantation. VEGF-A antibody was added into the preservation solution alone (α-VEGF D/-), in the preservation solution and systemically to the recipient before reperfusion (α-VEGF D/R), or applied to the recipient alone before reperfusion (α-VEGF -/R). Forty-eight hours after lung transplantation, left lungs were collected and wet-to-dry ratio, Western blotting, RT-PCR, and immunohistology were performed. RESULTS: VEGF-A blockade in α-VEGF D/-, α-VEGF D/R, and α-VEGF -/R resulted in neutralization of tissue VEGF-A. Reperfusion edema was only reduced in α-VEGF D/R and α-VEGF D/- groups versus Perfadex controls. Some α-VEGF -/R rats showed a hyperinflammation leading to increased pro-inflammatory cytokine expressions as well as increased edema. Whereas generally the α-VEGF D/- group showed decreased inflammation, the combination with anti-VEGF treatment to the recipient resulted in a pro-inflammatory and a pro-apoptotic phenotype. Short-term survival, however, was not significantly different in all groups as compared to the controls. In the α-VEGF (D/R) or (D/-) groups, animals mainly died from arterial thromboembolisms and in the α-VEGF (-/R) group, hyperinflammation was the main cause of death. CONCLUSION: VEGF-A directly contributes to the formation of a reperfusion edema, which might be reduced by its blockade. However, the α-VEGF effect on the endothelial integrity might also favor arterial thrombosis formation.
Asunto(s)
Edema/metabolismo , Trasplante de Pulmón , Daño por Reperfusión/metabolismo , Tromboembolia/patología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Apoptosis , Arterias/patología , Modelos Animales de Enfermedad , Hipoxia , Inflamación , Masculino , Soluciones Preservantes de Órganos , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Trombosis , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The lung is, more than other solid organs, susceptible for ischemia reperfusion injury after orthotopic transplantation. Corticosteroids are known to potently suppress pro-inflammatory processes when given in the post-operative setting or during rejection episodes. Whereas their use has been approved for these clinical indications, there is no study investigating its potential as a preservation additive in preventing vascular damage already in the phase of ischemia. To investigate these effects we performed orthotopic lung transplantations (LTX) in the rat. Prednisolone was either added to the perfusion solution for lung preservation or omitted and rats were followed for 48 hours after LTX. Prednisolone preconditioning significantly increased survival and diminished reperfusion edema. Hypoxia induced vasoactive cytokines such as VEGF were reduced. Markers of leukocyte invasiveness like matrix metalloprotease (MMP)-2, or common pro-inflammatory molecules like the CXCR4 receptor or the chemokine (C-C motif) ligand (CCL)-2 were downregulated by prednisolone. Neutrophil recruitment to the grafts was only increased in Perfadex treated lungs. Together with this, prednisolone treated animals displayed significantly reduced lung protein levels of neutrophil chemoattractants like CINC-1, CINC-2α/ß and LIX and upregulated tissue inhibitor of matrix metalloproteinase (TIMP)-1. Interestingly, lung macrophage invasion was increased in both, Perfadex and prednisolone treated grafts, as measured by MMP-12 or RM4. Markers of anti-inflammatory macrophage transdifferentiation like MRC-1, IL-13, IL-4 and CD163, significantly correlated with prednisolone treatment. These observations lead to the conclusion that prednisolone as an additive to the perfusion solution protects from hypoxia triggered danger signals already in the phase of ischemia and thus reduces graft edema in the phase of reperfusion. Additionally, prednisolone preconditioning might also lead to macrophage polarization as a beneficial long-term effect.
Asunto(s)
Antiinflamatorios/administración & dosificación , Trasplante de Pulmón , Prednisolona/administración & dosificación , Disfunción Primaria del Injerto/prevención & control , Animales , Transdiferenciación Celular/efectos de los fármacos , Microambiente Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hipoxia , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/prevención & control , Molécula 1 de Adhesión Intercelular/metabolismo , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Trasplante de Pulmón/efectos adversos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Masculino , Infiltración Neutrófila/efectos de los fármacos , Premedicación , Disfunción Primaria del Injerto/metabolismo , Disfunción Primaria del Injerto/mortalidad , Ratas , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoAsunto(s)
Cuidados Preoperatorios/normas , Manejo de la Vía Aérea , Anestesia por Inhalación , Anestesia Intravenosa , Lista de Verificación , Servicios Médicos de Urgencia , Humanos , Complicaciones Intraoperatorias/terapia , Pacientes , Cuidados Posoperatorios , Complicaciones Posoperatorias/terapia , Cuidados Preoperatorios/clasificación , Síndrome de Dificultad Respiratoria/terapia , Mecánica RespiratoriaAsunto(s)
Anestesia General/normas , Anestésicos Generales/administración & dosificación , Cirugía General/normas , Obesidad/cirugía , Guías de Práctica Clínica como Asunto , Apnea Obstructiva del Sueño/cirugía , Alemania , Humanos , Obesidad/complicaciones , Apnea Obstructiva del Sueño/complicacionesAsunto(s)
Fracturas Óseas/terapia , Fracturas Maxilares/terapia , Traumatismo Múltiple/terapia , Médicos , Transporte de Pacientes , Aceleración , Accidentes , Adulto , Presión del Aire , Algoritmos , Aviación , Servicios Médicos de Urgencia , Fracturas del Fémur/terapia , Fracturas Óseas/fisiopatología , Humanos , Masculino , Traumatismo Múltiple/fisiopatología , Pelvis/lesiones , Respiración Artificial , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: The fact that many sepsis therapeutics failed to be translated into the human indicates that there is still a serious need to reassess our models of sepsis research. We aimed to develop a novel modified model of sepsis in the mouse, which simulates the clinical situation more accurately. MATERIALS AND METHODS: Sepsis was induced in C57Bl/6 mice by dissecting the cecum and placing the discontinued organ back into the abdomen (cecum ligation and dissection [CLD]). Septic animals were relaparotomized after 6 h, followed by peritoneal lavage, and antibiotic treatment. Results were compared with shams or the classic colon ligation and puncture (CLP) model. The postoperative lung impairment was assessed using neutrophil invasion as a surrogate. Proinflammatory cytokines were measured by either real-time polymerase chain reaction or Luminex technology, and liver damage was evaluated by aspartate transaminase and alanine transaminase measurements. RESULTS: In CLD animals with relaparotomy after 6 h, lung interleukin (IL) 6, monocyte chemoattractant protein (MCP)-1 messenger RNA levels, and neutrophil invasion were significantly increased. Liver messenger RNA expression in CLD animals was significantly upregulated for IL-6, tumor necrosis factor alpha, IL-10, and MCP-1 compared with sham and CLP animals. Significantly higher levels of alanine transaminase were observed in CLD animals. Finally, systemic inflammation as measured by plasma IL-6, tumor necrosis factor alpha, IL-1ß, IL-10, and MCP-1 was significantly increased in all CLD animals compared with shams, whereas CLP animals only showed an insignificant increase in the latter molecules. CONCLUSIONS: Our modifications to the classic CLP model significantly produced organ inflammation, liver damage, and a similar mortality compared with a clinical setting, with a reliable onset of sepsis.
Asunto(s)
Ciego/cirugía , Modelos Animales de Enfermedad , Sepsis/etiología , Animales , Antibacterianos/administración & dosificación , Disección , Hepatitis/etiología , Laparotomía , Ligadura , Masculino , Ratones , Ratones Endogámicos C57BL , Neumonía/etiologíaRESUMEN
Sepsis is still a leading cause of death on intensive care units. Despite intensive research, only few new therapies have been developed and used in the clinical setting. The fibrin fragment Bß15-42 was already shown to preserve endothelial barrier function by binding to VE-cadherin and thus stabilize the interendothelial junctions. This was accompanied by reduced inflammation. Now we show that treatment with Bß15-42 reduces inflammation in a murine polymicrobial sepsis model. Administration of Bß15-42 reduced proinflammatory cytokine levels in the lung, liver, and blood and decreased neutrophil infiltration into the lung. Analysis alanine aminotransferase and aspartate aminotransferase further indicated reduced liver damage following polymicrobial sepsis. In vitro experiments using endothelial cells and macrophages further revealed that Bß15-42 had no direct effect on Toll-like receptor-mediated inflammation. Therefore, we assume that attenuated inflammation is rather due to sustained vascular integrity and thus suppresses vascular leakage and subsequently leukocyte infiltration during sepsis.
Asunto(s)
Antiinflamatorios/farmacología , Productos de Degradación de Fibrina-Fibrinógeno/farmacología , Fragmentos de Péptidos/farmacología , Sepsis/prevención & control , Animales , Células Cultivadas , Citocinas/metabolismo , Células Endoteliales/efectos de los fármacos , Inmunohistoquímica , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la PolimerasaAsunto(s)
Cuidados Críticos/métodos , Anciano , Creatina Quinasa/sangre , Farmacorresistencia Microbiana , Electrocardiografía , Humanos , Hipotermia Inducida , Intubación Intratraqueal , Masculino , Infarto del Miocardio/diagnóstico , Mioglobina/análisis , Tórax/diagnóstico por imagen , Troponina/sangre , UltrasonografíaRESUMEN
The main goal of adequate organ preservation is to avoid further cellular metabolism during the phase of ischemia. However, modern preservation solutions do rarely achieve this target. In donor organs hypoxia and ischemia induce a broad spectrum of pathologic molecular mechanisms favoring primary graft dysfunction (PGD) after transplantation. Increased hypoxia-induced transcriptional activity leads to increased vascular permeability which in turn is the soil of a reperfusion edema and the enhancement of a pro-inflammatory response in the graft after reperfusion. We hypothesize that inhibition of the respiration chain in mitochondria and thus inhibition of the hypoxia induced mechanisms might reduce reperfusion edema and consecutively improve survival in vivo. In this study we demonstrate that the rotenoid Deguelin reduces the expression of hypoxia induced target genes, and especially VEGF-A, dose-dependently in hypoxic human lung derived cells. Furthermore, Deguelin significantly suppresses the mRNA expression of the HIF target genes VEGF-A, the pro-inflammatory CXCR4 and ICAM-1 in ischemic lungs vs. control lungs. After lung transplantation, the VEGF-A induced reperfusion-edema is significantly lower in Deguelin-treated animals than in controls. Deguelin-treated rats exhibit a significantly increased survival-rate after transplantation. Additionally, a downregulation of the pro-inflammatory molecules ICAM-1 and CXCR4 and an increase in the recruitment of immunomodulatory monocytes (CD163+ and CD68+) to the transplanted organ involving the IL4 pathway was observed. Therefore, we conclude that ischemic periods preceding reperfusion are mainly responsible for the increased vascular permeability via upregulation of VEGF. Together with this, the resulting endothelial dysfunction also enhances inflammation and consequently lung dysfunction. Deguelin significantly decreases a VEGF-A induced reperfusion edema, induces the recruitment of immunomodulatory monocytes and thus improves organ function and survival after lung transplantation by interfering with hypoxia induced signaling.