Fluorescein-labeled Dextran Research Articles

The Hormonal Control of Uterine Luminal Fluid Secretion and Absorption

The secretion of uterine luminal fluid initially provides a transport and support medium for spermatozoa and unimplanted embryos, while the absorption of uterine luminal fluid in early pregnancy results in the closure of the lumen and allows blastocysts to establish intimate contact with the uterine epithelium. We have established an in vivo perfusion technique of the lumen to study the hormonal control of the events in the peri-implantation period. Fluorescein-labelled dextran was included in the perfusion medium to monitor fluid movements and the concentrations of Na(+) and CI(-) ions in the effluent were monitored. Using an established regimen of steroid treatment of ovariectomized rats mimicking early pregnancy, oestradiol caused fluid secretion, while progesterone resulted in an amiloride-sensitive fluid absorption. Fluid absorption peaked at about the expected time of implantation. The effect of progesterone could be inhibited by treatment with a high dose of oestradiol, by the anti-progestin RU486, and by the presence of an intra-uterine contraceptive device. Studies of expression of Na(+) and CI(-) channels (ENaC, CFTR) indicated that these channels were subject to tissue-specific regulation within the uterus, but more work is required to determine their role and the factors controlling their abundance and localization in early pregnancy.

Cytosolic delivery of macromolecules: 4. Head group-dependent membrane permeabilization by pH-sensitive detergents

Three tertiary amine-based detergents with zero, one, or two hydroxyl groups at various positions in their head group were characterized for their ability to promote the cytosolic delivery of macromolecules. Critical micellar concentrations (CMC) and membrane-bound pKa values of the lipid constructs increased with increasing head group polarity, ranging from 1–5 μM and 5.9 to 6.3, respectively. Fluorescence resonance energy transfer (FRET) and calcein leakage experiments revealed that when the amine group is protonated introduction of –OH moieties to detergent head groups enhanced their ability to interact with and permeabilize anionic, endosome-mimicking vesicles. Different formulations of a diethanolamine-based lipid (DEL) were further evaluated for pH-dependent hemolytic activity and ability to promote cytosolic delivery of macromolecules in vitro. Intact liposomes containing DEL at its maximum limit of incorporation were less efficient than DEL-containing micelles in promoting hemoglobin leakage from human erythrocytes at acidic pH. In HeLa cells, DEL-containing detergent micelles facilitated efficient cytosolic release of endocytosed macromolecules such as fluorescein-labeled dextran of MW 10 kDa. This observation was further corroborated by a functional assay based on antisense-mediated up-regulation of enhanced green fluorescent protein (EGFP). Taken together, our findings emphasize the key role of polar head groups and micellar architecture of pH-sensitive detergents in mediating endosomal permeabilization and the efficient cytosolic delivery of macromolecules.

PH Responsive Decomposable Layer-by-Layer Nanofilms and Capsules on the Basis of Tannic Acid

Tannic acid (TA) was assembled in alternation with two different polycations, strong poly(dimethyldiallylamide) (PDDA) and weak poly(allylamine) (PAH), using a layer-by-layer technique. Their deposition at different pH was confirmed by QCM, UV−vis spectroscopy, and surface charge measurements. TA/polyelectrolyte multilayer films and capsules have pH-dependent structural properties different from those of commonly used poly(styrenesulfonate)/poly(allylamine) (PSS/PAH) compositions. The lowest speed of TA/polycation multilayer dissolution was found at the conditions close to those used for film preparation. Permeability for fluorescein-labeled dextrans into tannic acid/polycation capsules with a five bilayer wall composition was investigated as a function of pH using confocal microscopy. It was found that minimal permeability occurs at pH 5−7 and maximal permeability at very high and very low pH, providing new opportunities for capsule loading as compared with an established procedure for PSS/PAH microcapsu...

Transport study of macromolecules through microporous poly(2-hydroxyethyl methacrylate) sponges

Drug transport through polymeric membranes is an important consideration in designing drug delivery system s using polymer matrices. In this study, the transport properties of macroporous sponges formed from poly(2-hydroxyethyl methacrylate) (PHEMA) have been studied. In addition to the in vitro study of insulin release from the PHEMA sponges, the permeation of glucose and macromolecules, such as fluorescein-labelled dextrans, albumin and immunoglobulin G (IgG), through the PHEMA sponges has been investigated. The results suggest that glucose and insulin travel freely through PHEMA sponges and IgG transport is minimized. The microstructure of the PHEMA networks have been characterized using scanning electron microscopy (SEM), in which the PHEMA sponges were observed to contain a multitude of pores and water channels in the porous structure. A biocompatibility study was performed by implanting the sponges into rats for one year. From histological evaluation of the explanted sponges, we observed that all sponges implanted had no e ff ects on the surrounding tissues. In addition, the appearance of the ingrowth of foreign body granulation tissue with neovascularization was also observed. These results indicate that it may be possible to use PHEMA sponges for an implantable device, which controls drug transport.

Inhibition of Choroidal Neovascularization by a Peptide Inhibitor ofthe Urokinase Plasminogen Activator and Receptor System in a Mouse Model

To determine the role played by the urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) system in choroidal neovascularization (CNV) and whether inhibition of this system can suppress the extent of CNV in an animal model. Choroidal neovascularization was induced in mice by laser photocoagulation using the slitlamp delivery system. Reverse transcriptase-polymerase chain reaction and immunocytochemical analysis were performed on the retina choroids of these animals to examine the expression of uPAR. For 2 weeks following laser treatment, animals were injected intraperitoneally with a novel peptide inhibitor of the uPA-uPAR system (100 mg/kg twice a day every day, every other day, and once a week). Control laser-treated animals receive an intraperitoneal injection of phosphate-buffered saline every day. Following treatment, animals were perfused with fluorescein-labeled dextran, eyes were removed, and the areas of new vessels were examined in the retina-choroid whole mounts by fluorescence microscopy and quantitated using image analysis software. In this study, uPAR was found to be up-regulated in the choroidal tissues of mice with laser-induced CNV. The uPAR was localized to the endothelial cells of the fibrovascular tissue within the CNV complex. Systemic administration of the peptide inhibitor of the uPA-uPAR system resulted in a significant reduction of CNV (up to 94%). The response was found to be frequency-of-dose dependent. No toxic effects or tissue destruction was noted following the peptide treatment. Our results strongly suggest that up-regulation of the uPA-uPAR system is an important step during CNV, and significant inhibition of CNV was seen when cell surface-associated uPA-uPAR activity was prevented with the peptide inhibitor. Clinical Relevance Inhibition of the protease system (uPA-uPAR) may prove to be a potential novel antiangiogenic therapy for CNV as seen in age-related macular degeneration.

Ca2+-dependent and -independent mechanisms of calmodulin nuclear translocation

Translocation from the cytosol to the nucleus is a major response by calmodulin (CaM) to stimulation of cells by Ca2+. However, the mechanisms involved in this process are still controversial and both passive and facilitated diffusion have been put forward. We tested nuclear translocation mechanisms in electroporated HeLa cells, rat cortical neurons and glial cells using novel calmodulin and inhibitor peptide probes and confocal microscopy. Passive diffusion of calmodulin across the nuclear membrane was measured in conditions in which facilitated transport was blocked and was compared to that of a similarly sized fluorescein-labeled dextran. Wheat germ agglutinin, which blocks facilitated transport but not passive diffusion, inhibited the nuclear entry of both wild-type and Ca2+-binding-deficient mutant calmodulin both in low and elevated [Ca2+]. Ca2+-dependent nuclear translocation was prevented by a membrane-permeant CaM inhibitor, the mTrp peptide, which indicated that it was specific to Ca2+/CaM. Diffusion of free CaM and Ca2+/CaM was considerably slower than the observed nuclear translocation by facilitated transport. Our data show that the majority of CaM nuclear entry occurred by facilitated mechanisms in all cell types examined, in part by a Ca2+-independent and in part by a Ca2+-dependent translocation mechanism.

Essential role of receptor for advanced glycation end products (RAGE) in the inflammatory response and mucosal injury after hemorrhagic shock (HS)

Introduction: Activation of RAGE by alarm ligands including HMGB1 stimulates the inflammatory response. We hypothesized that HS activates RAGE in injured tissues, specifically the intestinal epithelium. We previously showed that soluble(s) RAGE blocks ligand-RAGE interactions and prevents HS-induced mucosal damage/inflammation. We utilized RAGE deficient (KO) animals to definitively test the hypothesis that RAGE mediates intestinal injury following HS. Methods: RAGE KO mice and controls (n = 5/group) underwent 3h HS followed by 24h resuscitation. Intestinal mucosal permeability to fluorescein-labeled dextran (FD4) was determined using everted gut sacs; bacterial translocation (BT) to mesenteric lymph nodes was quantified. Serum IL-6 and IL-10 levels were measured by ELISA. In vitro, Caco-2 enterocytic monolayers grown in bicameral diffusion chambers were subjected to 16h hypoxia (0.2%O2) and reoxygenation. Monolayers were pretreated with sRAGE or anti-RAGE antibody; FD4 permeability was measured. Using ANOVA, p<0.05 significant. Results: RAGE KO mice post-HS had significant reductions in both HS-induced injury and inflammation vs controls. Permeability (pL/min/cm) was 0.0620 ± 0.008 vs 0.130 ± 0.02, 52%-(p<.001), BT(cfu/g) was 102 ± 50 vs 1280 ± 407, 92%-(p<.001). IL-6, a pro-inflammatory cytokine, was suppressed in HS RAGE KO. IL-10, an anti-inflammatory cytokine, was increased in HS RAGE KO vs controls. In vitro, pretreatment with sRAGE or anti-RAGE antibody prior to hypoxia dramatically reduced monolayer permeability to FD4 after 24h reoxia vs hypoxia/reoxia alone (17.5 ± 2.9, 18.3 ± 3.9 vs 44.65 ± 5.4-p<0.001). Conclusions: These studies identify RAGE as an essential receptor for inflammatory response induction and gut mucosal injury following HS. Preventing the RAGE-ligand interaction is critical for maintaining gut barrier function after HS; RAGE may represent a new therapeutic target to prevent end-organ damage in HS.

Simvastatin Inhibits Leukocyte Accumulation and Vascular Permeability in the Retinas of Rats with Streptozotocin-Induced Diabetes

Leukocytes play important roles in the pathogenesis of diabetic retinopathy. Recently, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors have been reported to exert various effects in addition to their lipid-lowering ability. We investigated the effects of simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on leukocyte-induced diabetic changes in retinas. Diabetes was induced in Long-Evans rats with streptozotocin, and simvastatin administration was begun immediately after the induction of diabetes. Two weeks of treatment with simvastatin suppressed significantly the number of leukocytes adhering to retinal vessel endothelium and the number of leukocytes accumulated in the retinal tissue by 72.9% and 41.0%, respectively (P < 0.01). The expression of intercellular adhesion molecule-1 (ICAM-1) and the CD18 (the common beta-chain of ICAM-1 ligands) were both suppressed with simvastatin. The amount of vascular endothelial growth factor in the retina was attenuated in the simvastatin-treated group. To evaluate the effects of simvastatin on leukocyte-induced endothelial cell damage, vascular permeability in the retina was measured with fluorescein-labeled dextran. Treatment with simvastatin markedly reduced retinal permeability (P = 0.014). This suggests that simvastatin attenuates leukocyte-endothelial cell interactions and subsequent blood-retinal barrier breakdown via suppression of vascular endothelial growth factor-induced ICAM-1 expression in the diabetic retina. Simvastatin may thus be useful in the prevention of diabetic retinopathy.

Passage of cell-penetrating peptides across a human epithelial cell layer in vitro.

Cell barriers are essential for the maintenance and regulation of the microenvironments of the human body. Cell-penetrating peptides have simplified the delivery of bioactive cargoes across the plasma membrane. Here, the passage of three cell-penetrating peptides (transportan, the transportan analogue transportan 10, and penetratin) across a Caco-2 human colon cancer cell layer in vitro was investigated. The peptides were internalized into epithelial Caco-2 cells as visualized by indirect fluorescence microscopy and quantified by fluorimetry. Studies of peptide outflow from cells showed that the peptides were in equilibrium across the plasma membrane. The ability of the peptides to cross a Caco-2 cell layer was tested in a two-chambered model system. After 120 min, 7.0%, 2.8% and 0.6% of added transportan, transportan 10 and penetratin respectively was detected in the lower chamber. Both transportan and transportan 10 reversibly decreased the trans-epithelial electrical resistance of the barrier model, with minimum values after 60 min of 46% and 60% of control respectively. Penetratin did not affect the resistance of the cell layer to the same extent. Although transportan markedly increased the passage of ions, the paracellular flux of 4.4 kDa fluorescein-labelled dextran was limited. In conclusion, the results indicate that the transportan peptides pass the epithelial cell layer mainly by a mechanism involving a transcellular pathway.

RPE cells modulate subretinal neovascularization, but do not cause regression in mice with sustained expression of VEGF.

Previous studies using models of choroidal neovascularization (CNV) in which the angiogenic stimulus is not sustained, have concluded that the retinal pigmented epithelium (RPE) causes regression of neovascularization (NV). However, the withdrawal of angiogenic stimuli may actually be the major modulator of NV, and RPE cells may simply be responding to withdrawal of the angiogenic stimuli or something released by NV because of the withdrawal. In this study, the long-term course of NV and the behavior of the RPE in rhodopsin/VEGF transgenic mice, in which there is a sustained angiogenic stimulus, was investigated. Hemizygous mice from the V-6 line were killed at 0.75, 1, 3, 6, and 12 months after birth, and at each time point mRNA for VEGF, VEGF-R1, and VEGF-R2 was measured by RT-PCR. Some mice were perfused with fluorescein-labeled dextran and retinal flatmounts were examined by fluorescence microscopy. Light and electron microscopy was performed on Epon-embedded eyes. The mRNA levels for VEGF, VEGF-R1, and VEGF-R2 remained constant from the earliest to the latest time point. Retinal flatmounts showed numerous small areas of subretinal NV at 3 weeks and at 1 month, and there were a similar number of larger lesions. By 6 months, many of the individual NV lesions had grown together to form large networks of new vessels. At 12 months, NV networks were similar to those at 6 months, but some of the vessels were not perfused. Light microscopy showed serous retinal detachments overlying NV lesions in mice up to 3 months of age, but at 6 and 12 months, the RPE completely surrounded new vessels and formed tight junctions to reestablish the outer blood-retinal barrier, and there were no serous detachments. Electron microscopy showed that compared with more acute NV lesions, chronic lesions contained thinner endothelial cells, similar to those of the choriocapillaris in that they had scant cytoplasm and numerous fenestrations, or pinocytotic vesicles with thick basement membrane surrounded by extracellular matrix (ECM). Bruch's membrane remained intact. Despite persistent high expression of VEGF and its receptors, NV stopped growing and reached a plateau in older V-6 mice. RPE cells modulated the NV by surrounding it and reestablishing the blood-retinal barrier, but did not cause regression, although some vessels in chronic lesions were not perfused. These data do not support the conclusion of several previously reported studies, that RPE cells cause regression of CNV.

Do connexin 43 gap-junctional hemichannels activate and cause cell damage during ATP depletion of renal-tubule cells?

We review our evidence in favour of the hypothesis that gap-junctional hemichannels (GJH) are activated by depletion of adenosine triphosphate (ATP) in human renal proximal tubule cells in primary culture (hPT cells). Undocked GJH permit fluxes of ions and hydrophilic molecules up to 1 kDa, and thus their opening can cause alterations of cell composition conducive to cell damage. We show that hPT cells express connexin 43 (Cx43) (at the mRNA and protein levels) and that the protein is expressed on the plasma membrane. Moderate levels of pharmacological depletion of ATP increased plasma-membrane permeability, as shown by loading with the hydrophilic dye 5/6 carboxyfluorescein (CF, 376 Da) and other low-molecular weight dyes, but not with fluorescein-labelled dextran (>1500 Da). Roles for endocytosis and activation of purinergic-receptor channels were experimentally ruled out. Moderate ATP depletion also caused necrosis, assessed by cell permeabilization to propidium iodide. Prolonged exposure to gadolinium reduced both the dye loading and the necrosis induced by ATP depletion, i.e. it protected the cells. Cx43 overexpressed in insect cells, purified to homogeneity and reconstituted in proteoliposomes formed hemichannels that are activated by dephosphorylation of Ser368, a residue in a protein-kinase-C consensus phosphorylation sequence near the end of the C-terminal domain. (1) ATP depletion of hPT cells induces a Gd3+-sensitive permeability of the plasma membrane to hydrophilic dyes with a cut-off size consistent with Cx43 GJH. (2) ATP depletion also increases the percentage of necrotic cells, an effect also reduced by Gd3+. (3) The experiments with purified Cx43 reconstituted in liposomes suggest that dephosphorylation of Ser368 is sufficient to activate GJH, although other mechanisms may be involved in some cells.

DNA electrophoresis in dilute polymer solutions: a nonbinary mechanism.

The dynamical behavior of the neutral polymer (dextran, M(w)=2 x 10(6)) is investigated during DNA electrophoresis in a dilute solution. Using a fluorescence recovery after photobleaching setup, we measured the velocity of fluorescein-labeled dextran induced by the migration of the DNA. We found that each DNA molecule drags a large number of dextrans with it. We show that DNA-dextran interactions are not only binary but long range and indirect. We conclude that the DNA-dextran complex creates a hydrodynamic field that entrains polymers far from the DNA during electrophoresis.

Semiconductor quantum dot/albumin complex is a long-life and highly photostable endosome marker

For the purpose of selecting the efficient dispersion condition of hydrophilic semiconductor quantum dots (QDs) in biological buffers, the dispersion of the QDs mixed with a serum albumin from 9 different species or an ovalbumin was compared by a fluorescence intensity analysis. The QDs mixed with sheep serum albumin (SSA) showed the highest fluorescence of all when the mixtures were dissolved in Dulbecco’s MEM. QD/SSA complexes were accumulated in the endosome/lysosome of Vero cells and the fluorescence could be detected over a 5-day post-incubation period. The photostability of QD/SSA complexes associated with the endosomes was detectable, at least, 30 times as long as that of fluorescein-labeled dextran involved in endosomes. QD/SSA complex, therefore, can be used as a long-life and highly photostable endosome marker.

Spatial variation of plasma flow in the oxazolone-stimulated microcirculation.

In cutaneous lymphocytic inflammation, enhanced regional blood flow is suggested by persistent erythema and warmth. Direct assessment of the microcirculation, however, has been limited by tissue edema and skin thickness. To assess the microcirculatory adaptations to the epicutaneous antigen oxazolone, we studied the first pass kinetics and microvascular topography of the inflammatory skin microcirculation using a specially adapted epi-illumination intravital microscopy system. The fluorescence intravital videomicroscopy and streaming image acquisition of fluorescein-labeled dextran (approximately 500,000 MW) injections were used to assess changes in plasma flow. Direct plasma tracer injections of both the oxazolone-stimulated and control microcirculation demonstrated comparable transit times (leading edge and intensity-weighted peak times) from the carotid artery to the superficial vascular plexus (p > 0.05). In contrast to transit times, continuous infusion of the plasma tracer demonstrated a significant increase in the delivery of the fluorescein-labeled dextran to the oxazolone-stimulated microcirculation. Quantitative morphometry of intravital microscopic images demonstrated a 2.2-fold increase in the mean diameter of vessels in the superficial vascular plexus (p < 0.01). Further, fluorescence intensity mapping indicated that the increase was associated with increased perfusion of focal regions of the superficial vascular plexus (p < 0.001). These results indicate that the oxazolone-stimulated adaptations of the inflammatory microcirculation include both microvascular dilatation and the redistribution of plasma flow.

Identification and Molecular Cloning of a Heparosan Synthase fromPasteurella multocida Type D

Pasteurella multocida Type D, a causative agent of atrophic rhinitis in swine and pasteurellosis in other domestic animals, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported previously that the capsule was removed by treating microbes with heparin lyase III. We molecularly cloned a 617-residue enzyme, pmHS, which is a heparosan (nonsulfated, unepimerized heparin) synthase. Recombinant Escherichia coli-derived pmHS catalyzes the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA. Other structurally related sugar nucleotides did not substitute. Synthase activity was stimulated about 7-25-fold by the addition of an exogenous polymer acceptor. Molecules composed of approximately 500-3,000 sugar residues were produced in vitro. The polysaccharide was sensitive to the action of heparin lyase III but resistant to hyaluronan lyase. The sequence of the pmHS enzyme is not very similar to the vertebrate heparin/heparan sulfate glycosyltransferases, EXT1 and 2, or to other Pasteurella glycosaminoglycan synthases that produce hyaluronan or chondroitin. The pmHS enzyme is the first microbial dual-action glycosyltransferase to be described that forms a polysaccharide composed of beta4GlcUA-alpha4GlcNAc disaccharide repeats. In contrast, heparosan biosynthesis in E. coli K5 requires at least two separate polypeptides, KfiA and KfiC, to catalyze the same polymerization reaction.

Compartments in the lamprey embryonic brain as revealed by regulatory gene expression and the distribution of reticulospinal neurons

The vertebrate neural tube consists of a series of neuromeres along its anteroposterior axis. Between amphioxus that possesses no neuromeres and gnathostomes, the lamprey occupies a critical position in the phylogeny for the origin of the segmented brain. To clarify the rhombomeric configuration of the Japanese lamprey, Lampetra japonica, we injected rhodamine- and fluorescein-labeled dextrans into the larval spinal cord, and retrogradely labeled the reticulospinal neurons. We also isolated prosomere marker genes from the embryonic cDNA library of L. japonica, and performed in situ hybridization on the embryonic brain. Of the genes examined, LjOtxA, LjPax6, LjPax2/5/8, LjDlx1/6, and LjTTF-1 were expressed in clearly demarcated polygonal domains. In the telencephalon, LjDlx1/6, LjPax6, and a putative paralogue of LjEmx were expressed in different domains; the LjEmx paralogue was expressed in the dorsal region, and LjDlx1/6 and LjPax6 in a complimentary fashion of the middle part. These expression patterns implied existence of a tripartite configuration of the lamprey telencephalon similar to that in gnathostomes. All these evidences strongly suggest that the segmental and compartmental architecture of the vertebrate brain was already established before the divergence of agnathans and gnathostomes.

Evidence that the cell wall of Bacillus subtilis is protonated during respiration.

Several independent experiments suggest that cell walls of Bacillus subtilis are protonated during growth. When cells were grown in the presence of fluorescein-labeled dextran to saturate the cell walls, centrifuged, and suspended in PBS, fluorescence-activated cell sorter analyses revealed the bacteria were only poorly fluorescent. In contrast, when the bacteria were purged with N(2) to dissipate protonmotive force (pmf) fluorescence became intense. Upon reconstitution of the pmf with phenazine methosulfate, glucose, and oxygen, fluorescence declined. Another approach used pH-dependent chemical modification of cell walls. The walls of respiring B. subtilis cells were amenable to carboxylate modification by [(14)C]ethanolamine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The carbodiimide activation of carboxylate groups occurs only in acidic conditions. Upon dissipation of pmf the walls were refractory to chemical modification. Ammonium groups can be condensed with FITC in alkaline medium, but the condensation is very slow in acidic buffers. It was found that the derivatization of the walls with FITC could occur in the absence of pmf. The use of pH-dependent fluorophores and pH-dependent chemical modification reactions suggest that cell walls of respiring B. subtilis cells have a relatively low pH environment. This study shows a bacterium has a protonated compartment. Acidification of cell walls during growth may be one means of regulating autolytic enzymes.

Focal topographic changes in inflammatory microcirculation associated with lymphocyte slowing and transmigration.

Microcirculation is the primary mechanism for delivering lymphocytes to inflammatory tissues. Blood flow within microvessels ensures a supply of lymphocytes at the blood-endothelial interface. Whether the structure of the inflammatory microcirculation facilitates lymphocyte transmigration is less clear. To illuminate the microcirculatory changes associated with lymphocyte transmigration, we used intravital videomicroscopy to examine the dermal microcirculation after application of the epicutaneous antigen oxazolone. Intravascular injection of fluorescein-labeled dextran demonstrated focal topographic changes in the microcirculation. These focal changes had the appearance of loops or hairpin turns in the oxazolone-stimulated skin. Changes were maximal at 96 h and coincided with peak lymphocyte recruitment. To determine whether these changes were associated with lymphocyte transmigration, lymphocytes obtained from efferent lymph of draining lymph nodes at 96 h were fluorescently labeled and reinjected into inflammatory microcirculation. Epifuorescence intravital video microscopy demonstrated focal areas were associated with lymphocyte slowing and occasional transmigration. In contrast, focal loops and lymphocyte slowing were rarely observed in the contralateral control microcirculation. Results suggest that structural adaptations in inflammatory microcirculation represented by focal topographic changes may contribute to regulation of tissue entry by recirculating lymphocytes.

Proinflammatory Cytokines Inhibit Secretion in Rat Bile Duct Epithelium

Background & Aims: Cholestatic disorders often are associated with portal inflammation, but whether or how inflammation contributes to cholestasis is unknown. Thus we studied the effects of proinflammatory cytokines on bile duct epithelia secretory mechanisms. Methods: Isolated bile duct units (IBDUs) were cultured with interleukin (IL)-6, interferon γ, tumor necrosis factor (TNF)-α, and IL-1 alone or in combination. Ductular secretion was measured using video-optical planimetry. Bicarbonate and Cl − transport were assessed microfluorimetric measuring pH i (BCECF) and [Cl −] i transients (MEQ). Expression of Cl −/HCO 3 − exchanger (AE-2), cystic fibrosis transmembrane conductance regulator (CFTR), and the secretin receptor (SR) were assessed by ribonuclease protection assay. Cellular cyclic adenosine monophosphate (cAMP) levels were studied by enzymatic immunoassay. Paracellular permeability was assessed using fluorescein-labeled dextrans (FD) in cholangiocyte monolayers (NRC-1). Results: Although not effective when given alone, each combination of IL-6, interferon γ, IL-1, and TNF-α inhibited secretion in IBDU. Cytokines inhibited cAMP formation, AE-2 activity, and cyclic AMP-dependent Cl − efflux, but not that induced by purinergic agonists. AE-2 gene expression was unaffected by proinflammatory cytokines, whereas CFTR and SR expression was increased. In addition, paracellular transit of FD across NRC-1 monolayers was increased. Conclusions: Inflammatory cytokines inhibit cAMP-dependent fluid secretion in cholangiocytes and impair the barrier functions of biliary epithelia. These changes may represent the molecular mechanisms by which inflammation leads to ductular cholestasis in vivo.

Validation of a new live cell strain system: characterization of plasma membrane stress failure

Motivated by our interest in lung deformation injury, we report on the validation of a new live cell strain system. We showed that the system maintains a cell culture environment equivalent to that provided by conventional incubators and that its strain ouput was uniform and reproducible. With this system, we defined cell deformation dose (i.e., membrane strain amplitude)-cell injury response relationships in alveolar epithelial cultures and studied the effects of temperature on them. Deformation injury occurred in the form of reversible, nonlethal plasma membrane stress failure events and was quantified as the fraction of cells with uptake and retention of fluorescein-labeled dextran (FITC-Dx). The undeformed control population showed virtually no FITC-Dx uptake at any temperature, which was also true for cells strained by 3%. However, when the membrane strain was increased to 18%, ~5% of cells experienced deformation injury at a temperature of 37 degrees C. Moreover, at that strain, a reduction in temperature to 4 degrees C resulted in a threefold increase in the number of cells with plasma membrane breaks (from 4.8 to 15.9%; P < 0.05). Cooling of cells to 4 degrees C also lowered the strain threshold at which deformation injury was first seen. That is, at a 9% substratum strain, cooling to 4 degrees C resulted in a 10-fold increase in the number of cells with FITC-Dx staining (0.7 vs. 7.5%, P < 0.05). At that temperature, A549 cells offered a 50% higher resistance to shape change (magnetic twisting cytometry measurements) than at 37 degrees C. We conclude that the strain-injury threshold of A549 cells is reduced at low temperatures, and we consider temperature effects on plasma-membrane fluidity, cytoskeletal stiffness, and lipid trafficking as responsible mechanisms.