Quantitative and Microstructural Changes of the Blood-Nerve Barrier in Peripheral Neuropathy

Peripheral neuropathy is accompanied by changes in the neuronal environment. The blood-nerve barrier (BNB) is crucial in protecting the neural homeostasis: Tight junctions (TJ) seal paracellular spaces and thus prevent external stimuli from entering. In different models of neuropathic pain, the BNB is impaired, thus contributing to local damage, immune cell invasion and, ultimately, the development of neuropathy with its symptoms. In this study, we examined changes in expression and microstructural localization of two key tight junction proteins (TJP), claudin-1 and the cytoplasmic anchoring ZO-1, in the sciatic nerve of mice subjected to chronic constriction injury (CCI). Via qPCR and analysis of fluorescence immunohistochemistry, a marked downregulation of mRNA as well as decreased fluorescence intensity were observed in the nerve for both proteins. Moreover, a distinct zig-zag structure for both proteins located at cell-cell contacts, indicative of the localization of TJs, was observed in the perineurial compartment of sham-operated animals. This microstructural location in cell-cell-contacts was lost in neuropathy as semiquantified via computational analysis, based on a novel algorithm. In summary, we provide evidence that peripheral neuropathy is not only associated with decrease in relevant TJPs but also exhibits alterations in TJP arrangement and loss in barrier tightness, presumably due to internalization. Specifically, semiquantification of TJP in cell-cell-contacts of microcompartments could be used in the future for routine clinical samples of patients with neuropathy

Pathogen Specific, IRF3-Dependent Signaling and Innate Resistance to Human Kidney 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/IFNβ-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

Tumor Treating Fields (TTFields) Induce Cell Junction Alterations in a Human 3D In Vitro Model of the Blood-Brain Barrier

In a recent study, we showed in an in vitro murine cerebellar microvascular endothelial cell (cerebEND) model as well as in vivo in rats that Tumor-Treating Fields (TTFields) reversibly open the blood–brain barrier (BBB). This process is facilitated by delocalizing tight junction proteins such as claudin-5 from the membrane to the cytoplasm. In investigating the possibility that the same effects could be observed in human-derived cells, a 3D co-culture model of the BBB was established consisting of primary microvascular brain endothelial cells (HBMVEC) and immortalized pericytes, both of human origin. The TTFields at a frequency of 100 kHz administered for 72 h increased the permeability of our human-derived BBB model. The integrity of the BBB had already recovered 48 h post-TTFields, which is earlier than that observed in cerebEND. The data presented herein validate the previously observed effects of TTFields in murine models. Moreover, due to the fact that human cell-based in vitro models more closely resemble patient-derived entities, our findings are highly relevant for pre-clinical studies

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

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. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. 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 4 (from cerebral cortex) and cerebEND 5 (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- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 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

Charakterisierung der Virulenzeigenschaften und Wirt-Pathogen-Interaktionen von asymptomatischer Bakteriurie (ABU) Escherichia coli Isolaten

Urinary tract infection (UTI) is one of the most serious health problems worldwide. It accounts for a million hospital visits annually in the United States. Among the many uropathogenic bacteria, uropathogenic Escherichia coli (UPEC) is the most common causative agent of UTI. However, not all E. coli that inhabit the urinary tract can cause UTI. Some of them thrive for long periods of time in the urinary bladder without causing overt symptoms of infection. This carrier state is called asymptomatic bacteriuria (ABU). E. coli ABU isolates can live in the host without inducing host response due to deletions, insertions and point mutations in the genome leading to the attenuation of virulence genes. They therefore behave in the same way as commensals. Since bacteria that inhabit the urinary tract are said to originate from the lower intestinal tract and ABU behave in a similar way as commensals, this study compared various phenotypic and genotypic characteristics of ABU and commensal E. coli fecal isolates. The two groups did not show a strict clustering with regards to phylogenetic lineage since there appears to be overlaps in their distribution in some clonal complexes. In addition, it was observed that the UPEC virulence genes were more frequently inactivated in ABU than in fecal isolates. Hence, ABU tend to have less functional virulence traits compared to the fecal isolates. The ABU model organism E. coli 83972 which is known not only for its commensal behavior in the urinary bladder but its ability to outcompete other bacteria in the urinary tract is currently being used as prophylactic treatment in patients who have recurrent episodes of UTI at the University Hospital in Lund, Sweden. The pilot studies showed that upon deliberate long-term colonization of the patients with E. coli 83972, they become protected from symptomatic UTI. In this study, the phenotypic and genotypic characteristics of eight re-isolates taken from initially asymptomatically colonized patients enrolled in the deliberate colonization study who reported an episode of symptoms during the colonization period were investigated. Two out of the eight re-isolates were proven to be a result of super infection by another uropathogen. Six re-isolates, on the other hand, were E. coli 83972. The urine re-isolates confirmed to be E. coli 83972 were phenotypically heterogeneous in that they varied in colony size as well as in swarming motility. Four of these re-isolates were morphologically homogenous and similar to the parent isolate E. coli 83972 whereas one of them appeared phenotypically heterogenous as a mixture of smaller and normal-sized colonies. Still another re-isolate phenotypically resembled small colony variants. Meanwhile, three of the six re-isolates did not differ from the parent isolate with regards to motility. On the other hand, three exhibited a markedly increased motility compared to the parent isolate. Transcriptome analysis demonstrated the upregulation of a cascade of genes involved in flagellar expression and biosynthesis in one of the three motile re-isolates. However, upon further investigation, it was found out that the expression of flagella had no effect on bacterial adhesion to host cells in vitro as well as to the induction of host inflammatory markers. Thus, this implies that the increased motility in the re-isolates is used by the bacteria as a fitness factor for its benefit and not as a virulence factor. In addition, among the various deregulated genes, it was observed that gene regulation tends to be host-specific in that there is no common pattern as to which genes are deregulated in the re-isolates. Taken together, results of this study therefore suggest that the use of E. coli 83972 for prophylactic treatment of symptomatic UTI remains to be very promising.Harnwegsinfektionen (HWI) sind weltweit ein ernstes Gesundheitsproblem, auf welches allein in den USA jährlich ca. eine Million Krankenhausbesuche entfallen. Innerhalb der Gruppe uropathogener Bakterien stellen die uropathogenen Escherichia coli (UPEC) die wichtigsten Verursacher akuter Harnwegserkrankungen dar. Interessanterweise führen nicht alle E. coli Varianten, die den Harnweg besiedeln, zwangsläufig zu HWI. Einige von ihnen sind in der Lage, die Harnblase über einen langen Zeitraum zu kolonisieren ohne Symptome einer HWI auszulösen. Dieses Phänomen wird als asymptomatische Bakteriurie (ABU) bezeichnet. Die Eigenschaft von E. coli ABU-Isolaten innerhalb des Wirtsorganismus leben zu können, ohne eine deutliche Wirtsabwehrreaktion hervorzurufen, ist unter anderem bedingt durch Deletionen, Insertionen und Punktmutationen im bakteriellen Genom und der daraus resultierenden Inaktivierung einiger Virulenzgene. Ihre Lebensweise ist daher mit der kommensaler Organismen vergleichbar. Da die den Harnweg besiedelnden Bakterien mit hoher Wahrscheinlichkeit ihren Ursprung im unteren Darmtrakt haben und sich die ABU-Isolate ähnlich der Kommensalen verhalten, wurden in dieser Arbeit zahlreiche phäno- und genotypische Charakteristika von ABU-Isolaten mit denen kommensaler E. coli-Fäkalisolate verglichen. Für diese beiden Gruppen konnte hinsichtlich ihrer phylogenetischen Abstammung keine strikte Clusterbildung festgestellt werden, da ihre Verteilung in einigen klonalen Komplexen Überlappungen aufwies. Es zeigte sich jedoch, dass die UPEC-Virulenzgene in den ABU-Isolaten häufiger inaktiviert vorlagen als in den Fäkalisolaten. Demzufolge scheinen die ABU-Isolate weniger funktionale Virulenzeigenschaften zu besitzen als die Fäkalisolate. Der Modell-ABU Stamm E. coli 83972 ist sowohl für sein kommensales Verhalten in der menschlichen Blase bekannt, als auch für seine Fähigkeit, andere Bakterien aus dem Harntrakt zu verdrängen. Er wird gegenwärtig am Universitätsklinikum in Lund (Schweden) als prophylaktisches Therapeutikum bei der Behandlung von Patienten mit rezidivierenden HWI eingesetzt. In Pilotstudien konnte gezeigt werden, dass eine vorsätzliche Langzeit-Kolonisierung von Patienten mit E. coli 83972 zum Schutz vor symptomatischen HWI führt. In der vorliegenden Arbeit wurden die phäno- und genotypischen Charakteristika von acht Patienten-Reisolaten dieser Langzeitstudie untersucht. Die Reisolate wurden aus zunächst asymptomatisch kolonisierten Patienten isoliert, die allerdings im Verlauf der Langzeit-Kolonisierung über eine Reihe von Symptomen klagten. Zwei dieser acht Reisolate waren nachweislich das Resultat einer Superinfektion mit einem anderen uropathogenen Bakterium. Die restlichen sechs Reisolate konnten jedoch als E. coli 83972 identifiziert werden. Für diese sechs Reisolate war eine phänotypische Heterogenität zu beobachten, die sich zum einen in variierender Koloniegröße, zum anderen in unterschiedlichem Schwärmverhalten zeigte: Vier der Reisolate entsprachen morphologisch dem Ausgangsstamm E. coli 83972, wohingegen ein Reisolat phänotypisch abweichend als Mischung von kleineren und normal-großen Kolonien in Erscheinung trat. Ein weiteres Reisolat ähnelte phänotypisch sogenannten „Small Colony Variants“. Das Schwärmverhalten betreffend unterschieden sich indessen drei der sechs Reisolate vom Ausgangsstamm. Sie zeigten im Vergleich eine erhöhte Motilität. In einem dieser drei motilen Reisolate konnte mittels Transkriptomanalyse die hochregulierte Expression einer Reihe von Genen, welche für die Flagellenexpression und -biosynthese verantwortlich sind, aufgezeigt werden. Weiterführende in vitro-Untersuchungen ergaben jedoch, dass diese erhöhte Flagellenexpression weder einen verstärkenden Effekt auf die bakterielle Adhäsion an die Wirtszellen hat, noch in der Wirtszelle die Bildung von Entzündungsmarkern induziert. Dieses Ergebnis impliziert, dass die erhöhte Motilität der Reisolate als Fitnessfaktor und nicht als Virulenzfaktor zu betrachten ist. Ferner führte die genauere Analyse der deregulierten Gene zu der Annahme, dass die Genregulation in den Reisolaten wirtsspezifisch ist, da sich kein übereinstimmendes Muster bezüglich der Deregulation abzeichnete. Unter Berücksichtigung aller Ergebnisse dieser Studie lässt sich abschließend sagen, dass die Verwendung des E. coli Stammes 83972 als prophylaktisches Therapeutikum bei der Behandlung von symptomatischen HWI weiterhin als sehr vielversprechend angesehen werden kann

Stretch and/or oxygen glucose deprivation (OGD) in an in vitro traumatic brain injury (TBI) model induces calcium alteration and inflammatory cascade

The blood-brain barrier (BBB), made up of endothelial cells of capillaries in the brain, maintains the microenvironment of the central nervous system. During ischemia and traumatic brain injury (TBI), cellular disruption leading to mechanical insult results to the BBB being compromised. Oxygen glucose deprivation (OGD) is the most commonly used in vitro model for ischemia. On the other hand, stretch injury is currently being used to model TBI in vitro. In this paper, the two methods are used alone or in combination, to assess their effects on cerebrovascular endothelial cells cEND in the presence or absence of astrocytic factors. Applying severe stretch and/or OGD to cEND cells in our experiments resulted to cell swelling and distortion. Damage to the cells induced release of lactate dehydrogenase enzyme (LDH) and nitric oxide (NO) into the cell culture medium. In addition, mRNA expression of inflammatory markers interleukin (I L)-6, IL-1$\alpha$ chemokine (C-C motif) ligand 2 (CCL2) and tumor necrosis factor (TNF)-$\alpha$ also increased. These events could lead to the opening of calcium ion channels resulting to excitotoxicity. This could be demonstrated by increased calcium level in OGD-subjected cEND cells incubated with astrocyte-conditioned medium. Furthermore, reduction of cell membrane integrity decreased tight junction proteins claudin-5 and occludin expression. In addition, permeability of the endothelial cell monolayer increased. Also, since cell damage requires an increased uptake of glucose, expression of glucose transporter glut1 was found to increase at the mRNA level after OGD. Overall, the effects of OGD on cEND cells appear to be more prominent than that of stretch with regards to TJ proteins, NO, glutl expression, and calcium level. Astrocytes potentiate these effects on calcium level in cEND cells. Combining both methods to model TBI in vitro shows a promising improvement to currently available models

Glucocorticoids and endothelial cell barrier function

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

Senescence and associated blood-brain barrier alterations in vitro

Progressive deterioration of the central nervous system (CNS) is commonly associated with aging. An important component of the neurovasculature is the blood-brain barrier (BBB), majorly made up of endothelial cells joined together by intercellular junctions. The relationship between senescence and changes in the BBB has not yet been thoroughly explored. Moreover, the lack of in vitro models for the study of the mechanisms involved in those changes impede further and more in-depth investigations in the field. For this reason, we herein present an in vitro model of the senescent BBB and an initial attempt to identify senescence-associated alterations within

Quantitative and Microstructural Changes of the Blood-Nerve Barrier in Peripheral Neuropathy

Peripheral neuropathy is accompanied by changes in the neuronal environment. The blood-nerve barrier (BNB) is crucial in protecting the neural homeostasis: Tight junctions (TJ) seal paracellular spaces and thus prevent external stimuli from entering. In different models of neuropathic pain, the BNB is impaired, thus contributing to local damage, immune cell invasion and, ultimately, the development of neuropathy with its symptoms. In this study, we examined changes in expression and microstructural localization of two key tight junction proteins (TJP), claudin-1 and the cytoplasmic anchoring ZO-1, in the sciatic nerve of mice subjected to chronic constriction injury (CCI). Via qPCR and analysis of fluorescence immunohistochemistry, a marked downregulation of mRNA as well as decreased fluorescence intensity were observed in the nerve for both proteins. Moreover, a distinct zig-zag structure for both proteins located at cell-cell contacts, indicative of the localization of TJs, was observed in the perineurial compartment of sham-operated animals. This microstructural location in cell-cell-contacts was lost in neuropathy as semiquantified via computational analysis, based on a novel algorithm. In summary, we provide evidence that peripheral neuropathy is not only associated with decrease in relevant TJPs but also exhibits alterations in TJP arrangement and loss in barrier tightness, presumably due to internalization. Specifically, semiquantification of TJP in cell-cell-contacts of microcompartments could be used in the future for routine clinical samples of patients with neuropathy