Anna Börjesson

The effect of intestinal ischemia and reperfusion injury on ICAM-1 expression, endothelial barrier function, neutrophil tissue influx, and protease inhibitor levels in rats.

Multiple organ dysfunction syndrome (MODS) is mediated by complex mechanisms in which interactions between activated leukocytes and endothelial cells play a central role. ICAM-1 (intercellular adhesion molecule-1) mediates firm adhesion and transendothelial migration of activated leukocytes from postcapillary venules into the tissue. The present study evaluated the ICAM-1 expression in various organs after 40 min of intestinal ischemia and 1, 3, 6, 12 h of reperfusion (I/R) in the rat, using a dual monoclonal antibody technique (n = 36). Endothelial barrier permeability, using the vascular leakage of radiolabeled human serum albumin was also assessed (n = 12). Neutrophil sequestration in the lungs was quantitated by myeloperoxidase activity and plasma protease inhibitor levels were measured with electroimmunoassay. Significant regional differences were found in ICAM-1 expression between organs, both constitutively and after I/R-injury. The highest constitutive levels were observed in the liver and lungs, followed by the kidneys. The constitutive ICAM-1 expression in the intestines and in the heart was about 1/20 compared with that found in the liver and lungs. The brain and muscle had levels of about 1/150 of that in the liver and lungs. After intestinal I/R, significant increases (17–45%) were found in the lungs, intestines, brain, heart, and muscle. Albumin leakage index (ALI) in all examined organs and myeloperoxidase activity in the lungs increased after I/R-injury. Serum levels of albumin and most protease inhibitors decreased significantly after I/R challenge. Intestinal I/R results in an increase of systemic ICAM-1 expression with marked organ variability. The upregulation of ICAM-1 could represent a crucial step in the adherence- and migration process of activated leukocytes and potentially in the development of tissue injury.

Phagocytic and intestinal endothelial and epithelial barrier function during the early stage of small intestinal ischemia and reperfusion injury.

ABSTRACT The effects of intestinal ischemia and reperfusion (I/R) on small intestinal mucosal endothelial and epithelial barrier integrity and phagocytic function were assessed in rats subjected to 20‐ or 40‐min mesenteric ischemia and a 3‐h reperfusion. The results showed that human serum albumin (125I‐HSA) flux through the endothelial layer to the interstitial space increased as did 125I‐HSA clearance from blood to the gut lumen and 131I‐HSA flux from the gut lumen to the interstitial space in rats with I/R. E.coli adhering to microvilli, invading and passing into the microvessels, were noted on the small intestinal mucosa in animals subjected to 40‐min ischemia and a 3‐h reperfusion. Phagocytic function increased, especially in the small intestinal wall, lungs, liver, and spleen in the groups with I/R, correlating with the length of ischemia. The results imply that both endothelial and epithelial barrier integrity is impaired in the early phase after I/R and that the epithelial barrier more effectively restricts macromolecular leakage compared with the endothelial barrier. I/R impairs the intestinal barrier not only by causing tissue hypoxia but also by activating the phagocytic system and aggravating barrier damage, which finally may result in bacterial translocation and remote organ dysfunction.

Intratumoral genome diversity parallels progression and predicts outcome in pediatric cancer

Genetic differences among neoplastic cells within the same tumour have been proposed to drive cancer progression and treatment failure. Whether data on intratumoral diversity can be used to predict clinical outcome remains unclear. We here address this issue by quantifying genetic intratumoral diversity in a set of chemotherapy-treated childhood tumours. By analysis of multiple tumour samples from seven patients we demonstrate intratumoral diversity in all patients analysed after chemotherapy, typically presenting as multiple clones within a single millimetre-sized tumour sample (microdiversity). We show that microdiversity often acts as the foundation for further genome evolution in metastases. In addition, we find that microdiversity predicts poor cancer-specific survival (60%; P=0.009), independent of other risk factors, in a cohort of 44 patients with chemotherapy-treated childhood kidney cancer. Survival was 100% for patients lacking microdiversity. Thus, intratumoral genetic diversity is common in childhood cancers after chemotherapy and may be an important factor behind treatment failure.

Treatment with lexipafant ameliorates the severity of pancreatic microvascular endothelial barrier dysfunction in rats with acute hemorrhagic pancreatitis

SummaryConclusion: Treatment with lexipafant reduced the severity of pancreatitis-associated endothelial barrier compromise, also associated with a decrease in systemic concentrations of interleukin (IL) 1. Thus, the present findings imply that platelet-activating factor (PAF) may play an important role in the pathogenesis of pancreatic endothelial dysfunction by signaling and triggering the production and release of certain cytokines.Background: Pancreatic capillary endothelial barrier dysfunction is an initial and characteristic feature of acute pancreatic injury and pancreatitis. PAF, a proinflammatory mediator and an intercellular signaling substance, has been considered to be involved in the inflammatory reaction and the systemic endothelial dysfunction of acute pancreatitis.Methods: The development of pancreatic capillary endothelial barrier dysfunction was monitored by tissue edema and exudation of plasma albumin into the interstitium, 3 and 12 h after induction of acute pancreatitis by intraductal infusion of 5% sodium taurodeoxycholate in rats. Pancreatic leukocyte recruitment was reflected by measuring myeloperoxidase activity. Serum levels of IL-1β and IL-6 were determined by an enzyme-linked immunosorbent assay (ELISA).Results: Pretreatment with lexipafant, a potent PAF receptor antagonist, significantly reduced the pancreatitis-induced increase in pancreatic endothelial barrier dysfunction, pancreatic leukocyte recruitment, and serum levels of IL-1β, although a difference persisted between animals with sham operation and pancreatitis.

Neuroblastoma Patient-Derived Orthotopic Xenografts Retain Metastatic Patterns and Geno- and Phenotypes of Patient Tumours.

Neuroblastoma is a childhood tumour with heterogeneous characteristics and children with metastatic disease often have a poor outcome. Here we describe the establishment of neuroblastoma patient‐derived xenografts (PDXs) by orthotopic implantation of viably cryopreserved or fresh tumour explants of patients with high risk neuroblastoma into immunodeficient mice. In vivo tumour growth was monitored by magnetic resonance imaging and fluorodeoxyglucose–positron emission tomography. Neuroblastoma PDXs retained the undifferentiated histology and proliferative capacity of their corresponding patient tumours. The PDXs expressed neuroblastoma markers neural cell adhesion molecule, chromogranin A, synaptophysin and tyrosine hydroxylase. Whole genome genotyping array analyses demonstrated that PDXs retained patient‐specific chromosomal aberrations such as MYCN amplification, deletion of 1p and gain of chromosome 17q. Thus, neuroblastoma PDXs recapitulate the hallmarks of high‐risk neuroblastoma in patients. PDX‐derived cells were cultured in serum‐free medium where they formed free‐floating neurospheres, expressed neuroblastoma gene markers MYCN, CHGA, TH, SYP and NPY, and retained tumour‐initiating and metastatic capacity in vivo. PDXs showed much higher degree of infiltrative growth and distant metastasis as compared to neuroblastoma SK‐N‐BE(2)c cell line‐derived orthotopic tumours. Importantly, the PDXs presented with bone marrow involvement, a clinical feature of aggressive neuroblastoma. Thus, neuroblastoma PDXs serve as clinically relevant models for studying and targeting high‐risk metastatic neuroblastoma.

Effects of anticoagulant treatment on intestinal ischaemia and reperfusion injury in rats

Background:  In recent years it has become increasingly clear that a cross‐talk between the inflammatory response and blood coagulation exists, although many of the underlying mechanisms remain unclear. In the present study we investigated the potential anti‐inflammatory properties of two different anticoagulant compounds, i.e. active‐site inactivated FVIIa (FVIIai) and fondaparinux sodium, a selective FXa inhibitor, administered as pretreatment in a model of intestinal I/R in rats.

Neuroblastoma patient-derived orthotopic xenografts reflect the microenvironmental hallmarks of aggressive patient tumours

Treatment of high-risk childhood neuroblastoma is a clinical challenge which has been hampered by a lack of reliable neuroblastoma mouse models for preclinical drug testing. We have previously established invasive and metastasising patient-derived orthotopic xenografts (PDXs) from high-risk neuroblastomas that retained the genotypes and phenotypes of patient tumours. Given the important role of the tumour microenvironment in tumour progression, metastasis, and treatment responses, here we analysed the tumour microenvironment of five neuroblastoma PDXs in detail. The PDXs resembled their parent tumours and retained important stromal hallmarks of aggressive lesions including rich blood and lymphatic vascularisation, pericyte coverage, high numbers of cancer-associated fibroblasts, tumour-associated macrophages, and extracellular matrix components. Patient-derived tumour endothelial cells occasionally formed blood vessels in PDXs; however, tumour stroma was, overall, of murine origin. Lymphoid cells and lymphatic endothelial cells were found in athymic nude mice but not in NSG mice; thus, the choice of mouse strain dictates tumour microenvironmental components. The murine tumour microenvironment of orthotopic neuroblastoma PDXs reflects important hallmarks of aggressive and metastatic clinical neuroblastomas. Neuroblastoma PDXs are clinically relevant models for preclinical drug testing.

Protective Effects of N-Acetyl- L -Cysteine and Platelet Activating Factor Inhibition Are Not Linked to Intercellular Adhesion Molecule-1 Expression after Intestinal Ischemia and Reperfusion Injury in Rats

Background: Intestinal ischemia and reperfusion (I/R) injury may result in development of the systemic inflammatory response syndrome (SIRS). The interactions between activated leukocytes and endothelial cells, mediated by adhesion molecules, seem to be pivotal in these conditions, leading as they do to extravasation of circulating leukocytes within the inflamed tissue. The intercellular adhesion molecule-1 (ICAM-1) mediating firm adhesion of activated leukocytes is upregulated in many organs after I/R injury, but the regulatory mechanisms are complex and have not been fully investigated. Methods: We evaluated whether ICAM-1 expression was linked with a potential protective effect of N-acetyl- l -cysteine (NAC) and the platelet activating factor (PAF) inhibitor (Lexipafant), administered 15 r min after the start of reperfusion, in a model of intestinal ischemia (40 r min) and reperfusion (12 r h) in the rat. Results: ICAM-1 expression increased significantly in the ileum, colon, lungs and pancreas after intestinal I/R. Treatments with NAC and the PAF inhibitor did not affect this response. An increased endothelial albumin-leakage was observed in the same organs after I/R. Treatment with NAC reduced the endothelial leakage of albumin in the ileum, colon and lungs, whereas administration of the PAF inhibitor alone demonstrated a protective effect only in the ileum. Furthermore, neutrophil sequestration in the lungs and IL-1 # levels in plasma increased significantly after I/R, and these changes were markedly reduced by both treatment regimes. Conclusion: The protective effect of NAC and the PAF inhibitor Lexipafant in intestinal I/R injury is not due to a decreased expression of ICAM-1.BACKGROUND Intestinal ischemia and reperfusion (I/R) injury may result in development of the systemic inflammatory response syndrome (SIRS). The interactions between activated leukocytes and endothelial cells, mediated by adhesion molecules, seem to be pivotal in these conditions, leading as they do to extravasation of circulating leukocytes within the inflamed tissue. The intercellular adhesion molecule-1 (ICAM-1) mediating firm adhesion of activated leukocytes is upregulated in many organs after I/R injury, but the regulatory mechanisms are complex and have not been fully investigated. METHODS We evaluated whether ICAM-1 expression was linked with a potential protective effect of N-acetyl-L-cysteine (NAC) and the platelet activating factor (PAF) inhibitor (Lexipafant), administered 15 min after the start of reperfusion, in a model of intestinal ischemia (40 min) and reperfusion (12 h) in the rat. RESULTS ICAM-1 expression increased significantly in the ileum, colon, lungs and pancreas after intestinal I/R. Treatments with NAC and the PAF inhibitor did not affect this response. An increased endothelial albumin-leakage was observed in the same organs after I/R. Treatment with NAC reduced the endothelial leakage of albumin in the ileum, colon and lungs, whereas administration of the PAF inhibitor alone demonstrated a protective effect only in the ileum. Furthermore, neutrophil sequestration in the lungs and IL-1beta levels in plasma increased significantly after I/R, and these changes were markedly reduced by both treatment regimes. CONCLUSION The protective effect of NAC and the PAF inhibitor Lexipafant in intestinal I/R injury is not due to a decreased expression of ICAM-1.

Effects of N-Acetylcysteine on Pulmonary Macrophage Activity after Intestinal Ischemia and Reperfusion in Rats / with Invited Commentaries

Background/Aims: Intestinal ischemia and reperfusion (I/R) is considered to be a critical and triggering event in the development of distal organ dysfunction after a variety of insults. It appears that activated leukocytes, especially polymorphonuclear granulocytes (PMNs), and reactive oxygen species are important mediators in the process. In the present study, the aim was to evaluate the behavior of pulmonary macrophages, acute lung injury and pulmonary endothelial permeability after intestinal I/R, together with potential alterations in pulmonary endothelial and epithelial ultrastructure and cellular membrane system integrity. Methods: Intestinal ischemia for 40 min was followed by reperfusion for 12 h in the rat. Macrophage uptake of radiolabeled bacteria, levels of pulmonary blood content assessed by radiolabeled red blood cells and pulmonary endothelial permeability of radiolabeled albumin, as well as pulmonary endothelial and epithelial ultrastructure and cellular membrane system integrity by the use of scanning electron microscopy and a tracer was evaluated after 12 h reperfusion. Treatment with the free radical scavenger N-acetylcysteine (NAC) administered prior to reperfusion was evaluated. Results: Overactivation of pulmonary macrophages was noted after intestinal I/R, as was a significant decrease in pulmonary blood content. No increase in pulmonary albumin leakage or increase in pulmonary water content was found after intestinal I/R as compared to controls. Treatment with NAC prevented against intestinal I/R-induced overactivation of pulmonary macrophages and a decrease in pulmonary blood content. Conclusion: Reactive oxygen species may be involved in the regulation of pulmonary macrophage function and pulmonary circulation after intestinal I/R.

Multimodal management - of value in fulminant acute pancreatitis?

Background: The multiple organ dysfunction syndrome (MODS) is the major cause of morbidity and mortality associated with acute pancreatitis. Presently, therapy is merely organ supportive as no effective therapy against underlying causative pathophysiological mechanisms exists. Aims: To evaluate the effect of treatment with a platelet-activating factor inhibitor (PAFI), a monoclonal antibody against platelet endothelial cell adhesion molecule 1 (PECAM-1-MAb) and an oxygen free radical scavenger (N-acetylcystein; NAC), alone or in combination, on systemic organ dysfunction in experimental acute pancreatitis. Methods: Severe acute pancreatitis was induced in rats by the intraductal administration of taurodeoxycholate. Treatment was given after 1 or 3 h, and evaluations were performed 6 h after induction. Organ dysfunction was evaluated by means of endothelial integrity impairment expressed as endothelial barrier leakage index. Results: Severe acute pancreatitis caused a significant impairment in endothelial integrity in all organs studied and decreased levels of protease inhibitors compared to controls. The endothelial barrier impairment was significantly ameliorated by all treatment modalities, either given early or later. Combinations of NAC and the PECAM-1-MAb or the PECAM-1-MAb and the PAFI were the only schedules to restore endothelial barrier integrity to normal levels in most of the organs studied. Conclusion: Combination therapy with NAC and PECAM-1-MAb and/or PAFI may offer effective, causative-directed supplements to organ-supportive therapy of MODS in severe acute pancreatitis.