Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation. Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.
Glucocorticoids (GCs) are steroid hormones that have inflammatory and immunosuppressive effects on a wide variety of cells. They are used as therapy for inflammatory disease and as a common agent against edema. The blood brain barrier (BBB), comprising microvascular endothelial cells, serves as a permeability screen between the blood and the brain. As such, it maintains homeostasis of the central nervous system (CNS). In many CNS disorders, BBB integrity is compromised. GC treatment has been demonstrated to improve the tightness of the BBB. The responses and effects of GCs are mediated by the ubiquitous GC receptor (GR). Ligand-bound GR recognizes and binds to the GC response element located within the promoter region of target genes. Transactivation of certain target genes leads to improved barrier properties of endothelial cells. In this review, we deal with the role of GCs in endothelial cell barrier function. First, we describe the mechanisms of GC action at the molecular level. Next, we discuss the regulation of the BBB by GCs, with emphasis on genes targeted by GCs such as occludin, claudins and VE-cadherin. Finally, we present currently available GC therapeutic strategies and their limitations.
Asymptomatic bacteriuria established by intravesical inoculation of Escherichia coli 83972 is protective in patients with recurrent urinary tract infections. In this randomized, controlled crossover study a total of 3 symptomatic urinary tract infection episodes developed in 2 patients while they carried E. coli 83972. We examined whether virulence reacquisition by symptom isolates may account for the switch from asymptomatic bacteriuria to symptomatic urinary tract infection.
Asymptomatic bacteriuria (ABU) is a condition where bacteria stably colonize the urinary tract, in a manner closely resembling commensalism at other mucosal sites. The patients carry >10(5) CFU/ml for extended periods of time and rarely develop symptoms. Contrasting the properties of ABU strains to those of uropathogenic isolates causing symptomatic infection is therefore highly relevant to understand mechanisms of bacterial adaptation. The prototype ABU strain Escherichia coli 83972 has a smaller genome than uropathogenic E. coli (UPEC) strains with deletions or point mutations in several virulence genes, suggesting that ABU strains undergo a programmed reductive evolution within human hosts. This study addressed if these observations can be generalized. Strains causing ABU in outpatients or hospitalized patients after catheterization or other invasive procedures were compared to commensal E. coli isolates from the intestinal flora of healthy individuals. Notably, clonal complex 73 (CC73) was a prominent phylogenetic lineage dominated by ABU isolates. ABU isolates from outpatients and hospitalized patients had a similar overall virulence gene repertoire, which distinguished them from many commensals, but typical UPEC virulence genes were less frequently attenuated in hospital strains than in outpatient strains or commensals. The decreased virulence potential of outpatient ABU isolates relative to that of ABU strains from hospitalized patients supports the hypothesis that loss of expression or decay of virulence genes facilitates long-term carriage and adaptation to host environments.
Bacterial pathogens with the ability to cause persistent infection have different strategies to withstand the induction of host immune responses and to successfully establish long-term colonization. In case of asymptomatic bacteriuria and other persistent infections, prolonged growth in the host is accompanied with genomic alterations that result in e.g., bacterial attenuation thus contributing to bacterial adaptation to their host niche and a reduced activation of host immune responses. The accumulating amount of information regarding bacterial adaptation during persistent infection helps to increase our understanding of driving forces of bacterial adaptation in vivo as well as of factors that contribute to symptomatic infection.
The mucosal immune system identifies and fights invading pathogens, while allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen discrimination are poorly understood, as is the contribution of human genetic variation in disease susceptibility. We describe here a new, IRF3-dependent signaling pathway that is critical for distinguishing pathogens from normal flora at the mucosal barrier. Following uropathogenic E. coli infection, Irf3(-/-) mice showed a pathogen-specific increase in acute mortality, bacterial burden, abscess formation and renal damage compared to wild type mice. TLR4 signaling was initiated after ceramide release from glycosphingolipid receptors, through TRAM, CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in nuclear translocation of IRF3 and activation of IRF3/IFN?-dependent antibacterial effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored glycosphingolipid receptors. Relevance of this pathway for human disease was supported by polymorphic IRF3 promoter sequences, differing between children with severe, symptomatic kidney infection and children who were asymptomatic bacterial carriers. IRF3 promoter activity was reduced by the disease-associated genotype, consistent with the pathology in Irf3(-/-) mice. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response.
Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al.). Human tissue samples are available only in a restricted number of laboratories or companies. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest. Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND (from cerebral cortex) and cerebEND (from cerebellar cortex), were isolated according to this procedure in the Förster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- and estrogen-treatment as well as to pro-infammatory mediators, such as TNFalpha. Moreover, we have studied the pathology of multiple sclerosis and hypoxia on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.
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