Breakdown Of Tight Junctions Research Articles

Characterisation of the bacterial flora of the larynx

Objectives: Damage to epithelial integrity has been implicated in inflammatory disease and neoplasia. Bacterial species such as Pseudomonas and Helicobacter have been shown to cause disease by tight cell junction breakdown and loss of epithelial integrity in the gut (1) There has been one description of the bacterial flora of the human larynx and its variation between individuals, using traditional culture techniques. (2) Sub‐site variation has not been characterised. We have compared methods of bacterial species identification and laryngeal sub‐site variation.Methods: Bacteria were identified by sequencing of variable regions of the 16S RNA gene, obtained by PCR amplification of DNA from supra‐ and sub‐glottic laryngeal biopsies from pigs. Sequences were aligned with those of known bacteria using the blast nucleotide search engine. Concurrently, bacterial swabs from each larynx subsite were plated on to blood agar. Bacterial colonies isolated by this method were identified by 16S sequencing.Results: A total of 6 Bacterial species were identified in the supra‐glottis compared to eight identified in the sub‐glottis by culture techniques. 10 bacterial species were identified in the supra‐glottis by cloning techniques, including fastidious species.Conclusion: Bacterial species vary between supra and sub‐glottis.Cloning techniques are superior at identifying bacterial species when compared with traditional culture techniques.References 1 Sawada N et al. (2003) Med. Electron Microsc.36,147–1562 Tanaka I et al. (1996)Acta Otoloaryngol.525 (Suppl.), S44–50

Confocal Assessment of the Effects of Fourth-Generation Fluoroquinolones on the Cornea

To evaluate the toxicity of fourth-generation fluoroquinolone antibiotic solutions on the rabbit corneal epithelium. In vivo confocal microscopy was used to assess epithelial structure in 18 rabbits, and tight junction integrity of superficial epithelial cells was evaluated with ZO-1 labeling in 10 rabbits. Eyes were bathed with commercial solutions of moxifloxacin (Vigamox) or gatifloxacin (Zymar) solution for 3 minutes, rinsed with balanced salt solution, and immediately examined. Balanced salt solution rinsing alone served as the control. A decrease in epithelial cell size was observed after treatment with Zymar (P < 0.05, two-way repeated-measures analysis of variance), but not with Vigamox or the control. Normal ZO-1 organization was observed in controls and eyes treated with Vigamox. ZO-1 staining in eyes treated with Zymar was disrupted, patchy, and generally weaker than that in control eyes. After short-term, intensive exposure to Vigamox, corneal epithelial integrity and tight junction organization are maintained. Zymar induces a loss of superficial epithelial cells and breakdown of tight junctions under similar conditions.

Matrix metalloproteinases in early diabetic retinopathy and their role in alteration of the blood–retinal barrier

One of the early features of diabetic retinopathy is the alteration of the blood–retinal barrier (BRB), which may involve the breakdown of endothelial cell tight junctions. The aim of this study was to examine the expression of extracellular proteinases in an animal model of early diabetic retinopathy and to determine their role in the alteration of the BRB. Matrix metalloproteinase (MMP) expression was studied in the retinas of rats with 12 weeks of diabetes. The role of MMPs in regulating tight junction function was investigated in retinal endothelial and pigment epithelial cells by measuring transepithelial electrical resistance (TER). The retinas of diabetic animals demonstrated elevated levels of MMP-2, MMP-9 and MMP-14 messenger RNA. A significant increase in the production of MMP-9 was seen when cells were exposed to high glucose conditions. Both cell types treated with purified MMP-2 or MMP-9 were found to have alterations of tight junction function as shown by decreased TER. Western blot analysis of cell extracts treated with MMP-2 or MMP-9, revealed specific degradation of the tight junction protein, occludin. Results suggest that elevated expression of MMPs in the retina may facilitate an increase in vascular permeability by a mechanism involving proteolytic degradation of the tight junction protein occludin followed by disruption of the overall tight junction complex.

Tight junctions, leaky intestines, and pediatric diseases

Tight junctions (TJs) represent the major barrier within the paracellular pathway between intestinal epithelial cells. Disruption of TJs leads to intestinal hyperpermeability (the so-called "leaky gut") and is implicated in the pathogenesis of several acute and chronic pediatric disease entities that are likely to have their origin during infancy. This review provides an overview of evidence for the role of TJ breakdown in diseases such as systemic inflammatory response syndrome (SIRS), inflammatory bowel disease, type 1 diabetes, allergies, asthma, and autism. A better basic understanding of this structure might lead to prevention or treatment of these diseases using nutritional or other means.

Correction to “Pulmonary Airways: 3-D Reconstruction From Multislice CT and Clinical Investigation”

controls how far from the winning neuron the input signal can reach. In order to find the global optimum of the objective function, the decrease of the size of the radius of the circular region around the position of the winning neuron should be done gradually. We have found out experimentally that a choice of in the range 150 to 250 and in the range 0.99 to 1.0 gives satisfactory results. Regarding the value of the parameter , experiments have shown that it should be near unity and in particular in the range 0.995 to 1.0. It should be emphasized that the algorithm is not critically influenced by the specific chosen values of the aforementioned parameters. The condition given by (11) is necessary to protect the SOM network from generating a signal that is much more different than the weighting vectors of the neurons in the current iteration. The condition is not too strict in the sense that it is not prohibitive of the production of a winner with a measure of match lower than that in the previous iteration. Consequently, it is achieved the escape from local optima of the objective function, since solutions that do not lead to better values of the objective function are accepted too. This approach is similar to the Metropolis process that takes place in simulated annealing [12]. Another aspect of the proposed registration scheme that should be addressed is the utilization of the gradient difference as a measure of match. The selected measure of match provided accurate results for almost all image pairs that were used in the current study. However, it should be stressed that the gradient difference may fail for images characterized by apparent hyperfluorescence. Hyperfluorescence is caused usually by a breakdown or lack of tight vascular junctions in abnormal blood vessels. Any leakage of fluorescein from a retinal vessel or within the retinal tissues indicates an abnormality. For example, capillary microaneurysms, retinal telangiectasias, arterial macroanuerysm, papilledema, and some vascularized tumors exhibit leakage. Finally, it should be noted that the focus of the paper was to show the feasibility of the SOM theory for registering multimodal retinal images. The proposed implementation of the SOM model could be considered as a method for finding the optimum value of an objective function. Under this framework, the proposed registration scheme provides several “degrees of freedom” regarding its parameters. For example, another measure of match (such as the mutual information) could be used, the bifurcation points could be extracted by means of other methods, another transformation (such as bilinear or elastic) could be adopted after the training of the SOM network. VI. CONCLUSION In this paper, a new registration algorithm for registering multimodal retinal images was presented. Two basic novel implementations were introduced. First, the application of the vessel centerline detection and bifurcations extraction process only on the reference image. This step simplifies the registration methodology since candidate points are identified only on the reference image. Second, the novel implementation of the SOM network to define automatic correspondence of the bifurcation points between the reference and the image to be transformed. The proposed algorithm was tested for 24 pairs of multimodal images providing an accuracy of approximately 40 for all retinal pairs.

Differential Expression of Splicing Variants of the Human Caldesmon Gene ( CALD1) in Glioma Neovascularization versus Normal Brain Microvasculature

Caldesmon is a cytoskeleton-associated protein which has not yet been related to neoplastic angiogenesis. In this study we investigated the expression of the caldesmon gene (CALD1) splicing variants and the protein expression level in glioma microvessels versus normal brain microvasculature. To exclude sources of splice variant expression from non-vascular components all possible cellular components present in control and glioma samples were pre-screened by laser-capture microdissection followed by RT-PCR before the cohort study. We discovered differential expression of the splicing variants of CALD1 in the tumor microvessels in contrast to normal brain microvasculature. Missplicing of exons 1, 1 + 4, and 1' + 4 of the gene is exclusively found in glioma microvessels. To exclude the possibility that this missplicing results from splice-site mutations, mutation scanning was performed by a coupled in vitro transcription/translation assay (IVTT). No premature stop mutations were traced by the IVTT. The transcriptional changes consequently resulted in up-regulation at the protein expression level. The up-regulated expression of caldesmon was coincident with the down-regulated expression of tight junction proteins (occludin and ZO-1). The results support the notion that missplicing of the CALD1 gene in glioma microvasculature is an independent epigenetic event regulated at the transcriptional level. The event coexists with tight junction (TJ) breakdown of the endothelial cells in glioma microvasculature. The data reveal a novel mechanism contributing to dysfunctionality of glioma neovascularization.

Testicular pain following vasectomy: a review of postvasectomy pain syndrome.

Testicular pain following vasectomy: a review of postvasectomy pain syndrome.

Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions.

House dust mite (HDM) allergens are important factors in the increasing prevalence of asthma. The lung epithelium forms a barrier that allergens must cross before they can cause sensitization. However, the mechanisms involved are unknown. Here we show that the cysteine proteinase allergen Der p 1 from fecal pellets of the HDM Dermatophagoides pteronyssinus causes disruption of intercellular tight junctions (TJs), which are the principal components of the epithelial paracellular permeability barrier. In confluent airway epithelial cells, Der p 1 led to cleavage of the TJ adhesion protein occludin. Cleavage was attenuated by antipain, but not by inhibitors of serine, aspartic, or matrix metalloproteinases. Putative Der p 1 cleavage sites were found in peptides from an extracellular domain of occludin and in the TJ adhesion protein claudin-1. TJ breakdown nonspecifically increased epithelial permeability, allowing Der p 1 to cross the epithelial barrier. Thus, transepithelial movement of Der p 1 to dendritic antigen-presenting cells via the paracellular pathway may be promoted by the allergen's own proteolytic activity. These results suggest that opening of TJs by environmental proteinases may be the initial step in the development of asthma to a variety of allergens.

Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium.

1. House dust mite (HDM) allergens with cysteine and serine proteinase activity are risk factors for allergic sensitization and asthma. A simple method to fractionate proteinase activity from HDM faecal pellets into cysteine and serine class activity is described. 2. Both proteinase fractions increased the permeability of epithelial cell monolayers. The effects of the serine proteinase fraction were inhibited by 4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The effects of the cysteine proteinase fraction could be inhibited by E-64. No reciprocity of action was found. 3. Treatment of epithelial monolayers with either proteinase fraction caused breakdown of tight junctions (TJs). AEBSF inhibited TJ breakdown caused by the serine proteinase fraction, whereas E-64 inhibited the cysteine proteinase fraction. 4. Agarose gel electrophoresis revealed that the proteinases induced DNA cleavage which was inhibited by the matrix metalloproteinase inhibitor BB-250. Compound E-64 inhibited DNA fragmentation caused by the cysteine proteinase fraction, but was without effect on the serine proteinase fraction. Staining of proteinase-treated cells with annexin V (AV) and propidium iodide (PI) revealed a diversity of cellular responses. Some cells stained only with AV indicating early apoptosis, whilst others were dead and stained with both AV and PI. 5. HDM proteinases exert profound effects on epithelial cells which will promote allergic sensitization; namely disruption of intercellular adhesion, increased paracellular permeability and initiation of cell death. Attenuation of these actions by proteinase inhibitors leads to the conclusion that compounds designed to be selective for the HDM enzymes may represent a novel therapy for asthma.

Loss of endothelial barrier function requires neutrophil adhesion

Background. The inflammatory response is characterized by cytokine-induced up-regulation of endothelial adhesion molecules followed by polymorphonuclear neutrophil (PMN) adhesion and breakdown of tight junctions between cells. The purpose of this investigation was to determine whether PMN adhesion is an essential element in the alteration of endothelial permeability or whether cytokines alone can produce this change. Methods. Human umbilical vein endothelial cells (HUVECs) were exposed to formylated met-leuphe-activated PMNs. In a second series of experiments, PMNs were contained in a microporous membrane that allowed passage of secreted cytokines but not cells. Permeability was quantified by using transendothelial electrical resistance (TEER, ohm · cm 2,) whereas expressions of two cell adhesion molecules (endothelial leukocyte adhesion molecule-1 [ELAM-1] and intercellular adhesion molecule-1 [ICAM-1]) were measured by flow cytometry (% shift). Cytokine production was monitored with enzyme-linked immunosorbant assays (picograms per milliliter). Results. Stimulated PMNs secreted comparable amounts of cytokines whether allowed access to HUVECs or trapped in a microporous membrane (interleukin-1α, 5.88 ± 2.38 versus 3.65 ± 1.84 pg/ml; tumor necrosis factor-α, 10.27 ± 3.21 versus 6.61 ± 1.82 pg/ml). Up-regulation of ELAM-1 and ICAM-1 was observed whether PMNs were free or restricted (52.97% ± 2.14% versus 75.32% ± 4.19% and 71.66% ± 7.37% versus 73.66% ± 4.32%, respectively). TEER was unchanged in controls and when PMNs were membrane restricted. In contrast, TEER decreased precipitously (51% ± 5.9% of control, p < 0.05) if PMNs were allowed access to HUVECs. Conclusions. Cytokine secretion by PMNs is independent of endothelial contact and is sufficient to upregulate adhesion molecules. However, PMN adhesion is essential for the loss of endothelial barrier function, which leads to diapedesis of activated PMNs and eventual tissue injury.

C. difficile toxin A increases intestinal permeability and induces Cl- secretion

Mucosal sheets of guinea pig ileum mounted in Ussing chambers were used to determine effects of highly purified Clostridium difficile toxin A on intestinal structure and barrier function in the absence of recruited neutrophils and blood flow. With the use of standard electrophysiological and morphological techniques, our results indicate that 5 micrograms/ml mucosal toxin A induces substantial alteration in epithelial permeability and in structure of absorptive cells. Transepithelial fluxes of mannitol (3.6 A) and inulin (11.5 A) increased 20-80 min after toxin A exposure (63 +/- 13 vs. 149 +/- 14 and 2.33 +/- 0.43 vs. 7.03 +/- 1.0 nmol.cm-2.h-1 for mannitol and inulin; controls vs. toxin exposed, respectively, both P less than 0.01). Toxin A exposure diminished short-circuit current (153 +/- 8 vs. 77 +/- 6 microA/cm2 for control and toxin exposed, respectively, P less than 0.001), decreased transepithelial electrical resistance (61.6 +/- 3 vs. 50.1 +/- 3.9 omega.cm2 for control and toxin exposed, respectively, P less than 0.05), increased passive permeation of Na+ (19.1 +/- 0.9 vs. 27.4 +/- 0.9 mumol.cm-2.h-1 for control and toxin exposed, respectively), and induced a Cl- secretory response (net flux 3.65 +/- 3.0 vs. -4.62 +/- 2.6 mumol.cm-2.h-1, for control and toxin exposed, respectively, P less than 0.05) over the 2-h experimental time course. Toxin A-induced structural alterations of villus tip absorptive cells were strikingly similar to those induced by the actin-binding agent cytochalasin D. Specifically, cells displayed constricted subjunctional zones, flared microvillus brush borders, condensation of microfilaments in the zone of the perijunctional actomyosin ring, and breakdown of intercellular tight junctions.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathogenic Events During Infection of the Human Nasopharynx with Neisseria meningitidis and Haemophilus influenzae

The human nasopharynx is the natural habitat and reservoir for Neisseria meningitidis and Haemophilus influenzae type b (Hib). Meningococcal and Hib pathogenesis was studied in human nasopharyngeal tissue in organ culture. Inocula of greater than or equal to 10(6) meningococci or cfu of Hib were required for consistent production of infection in these cultures. By 24 hours meningococci and Hib grew to 10(8)-10(10) cfu/mL in culture supernatants, while 10(4)-10(7) cfu per organ culture were tissue associated. These studies further indicated that nasopharyngeal mucus contains components that specifically bind Hib; that both meningococci and Hib cause cytotoxicity, resulting in breakdown of tight junctions of epithelial cells, sloughing of ciliated cells, and ciliostasis; that pili are the most important components mediating initial attachment of meningococci to non-ciliated epithelial cells of the human nasopharynx; that Hib expresses both pilus and nonpilus adhesions that facilitate attachment to nonciliated cells; and that meningococci and Hib both invade the epithelial surface to reach the submucosa but do so by different routes. Meningococci and Hib have evolved successful, although divergent, mechanisms by which to infect the human nasopharynx.

Filipin and digitonin studies of cell membrane changes during junction breakdown in the dystrophic rat retinal pigment epithelium.

We have previously found that a breakdown of tight junctions in retinal pigment epithelial cells of Royal College of Surgeons' rats is associated with a redistribution of intramembrane particles and Na-K-ATPase activity. Changes in the lipid and sterol composition of membranes can alter their fluidity, permeability and enzyme activity, and may contribute to changes in cell barrier function in the dystrophic epithelium. We have now used filipin and digitonin, which bind to membrane sterols and produce membrane deformations recognizable by freeze-fracture and thin-section electron microscopy, to study the distribution of cholesterol and related 3-B-hydroxysterols in the dystrophic epithelium. The results of these studies show that in the normal pigment epithelium and prior to tight junction breakdown in the dystrophic epithelium, filipin- and digitonin-sterol complexes are rare in the membranes between tight junctions and adhering junctions, and in areas of attachment between the plasma membrane and basal lamina. Complexes are more numerous in the basal infoldings, and most densely packed in the lateral and apical microvillous membranes. During junction breakdown, complexes increase substantially in apical, basal, junctional, and nuclear membranes. Later, after the junctions disappear, complexes decrease. These results indicate that alterations in the expression of membrane sterols accompany the changes in structure and function of tight junctions in the dystrophic retinal pigment epithelium.